Methods using polypeptides having hydrolase activity

TECHNICAL FIELD [0002] The present invention relates to methods of using polypeptides with hydrolase activity, including lipase, saturase, palmitase and / or stearatase activity. The invention also relates to methods for treating fats and oils with peptides and polypeptides, e.g. enzymes with hydrolase activity, e.g. soybean or canola oil.

BACKGROUND OF THE INVENTION [0003] Main industrial applications of hydrolases, e.g. lipases, saturases, palmitases and / or stearatases, include the food and beverage industries, they are used as anti-staining agents for bakery products, and in the production of margarine and other spreads with natural butter aromas; in waste disposal systems; and in the pharmaceutical industry in which they are used as digestive aids.

[0004] Processed oils and fats are a major component of food, food additives and food processing aids, and are also important renewable raw materials for the chemical industry. They are available in large quantities from the processing of oilseeds from plants such as rice (bran), corn, oilseed rape, canola oilseed rape, sunflower, olive, oil palm or soybean. Other sources of valuable oils and fats include fish, catering waste and rendered animal fats. These fats and oils are a mixture of triacylglycerides or lipids, i.e. fatty acids (KT) esterified on the glycerol backbone. Each oil or fat contains a wide range of different lipid structures, determined by the KT content and their regiochemical distribution on the glycerol backbone. These properties of individual lipids determine the physical properties of pure triacylglyceride. Thus, the content of triacylglyceride in fat or oil largely determines the physical, chemical and biological properties of this oil. The value of lipids increases significantly as a function of their purity. High purity can be achieved by fractional chromatography or fractional distillation, isolating the desired triacylglyceride from a mixed background of a fat or oil source. However, this is expensive and yields are often limited by the low levels of natural occurrence of triacylglyceride. In addition, the presence of many structurally and physically or chemically similar triacylglycerides in oil often has a negative impact on the product's purity.

[0005] An alternative to the purification of triacylglycerides or other natural source lipids is the synthesis of these lipids. The products of such processes are called structured lipids because they contain a defined set of fatty acids distributed in a specific way on the glycerol backbone. The lipid value can also be significantly increased by controlling the content of fatty acids and their distribution within the lipid. The elimination of fats or oils containing unwanted KT from triglycerides, or the replacement of KT with undesirable properties with fatty acids with better or more desirable chemical, physical or biological properties, increases the value of lipids. In particular, there is a need for lipases that can hydrolyze, e.g., selectively hydrolyze saturated fatty acids ("saturase"), or those that, in particular, can hydrolyze, e.g. selectively hydrolyze palmitic acid ("palmitase") or stearic acid ("stearatase") from the glycerol backbone. Lipases such as saturases, e.g., palmitases and / or stearatases, can be used to achieve such control in which the removed, added or replaced KT is saturated fatty acids, e.g. palmitic acid or stearic acid.

SUMMARY OF THE INVENTION [0006] The present invention provides various methods according to the claims, these methods use polypeptides with hydrolase activity and:

and. encoded by a nucleic acid containing a nucleic acid sequence having a sequence that is at least 85% identical to the full-length sequence of SEQ ID NO: 1 and in which the nucleic acid encodes at least one polypeptide with hydrolase activity and the polypeptide contains mutations D61E, R72K and V163R and in addition contains one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more changes of amino acid residues specified in Table 3, Table 4, Table 9, Table 16 or Table 23 or a sequence complementary thereto; or ii. having a sequence that is at least 85% identical to the full-length sequence of SEQ ID NO: 2, and containing the D61E, R72K and V163R mutations and additionally containing one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more changes in amino acid residues specified in Table 3, Table 4, Table 9, Table 16 or Table 23; or iii. containing an amino acid sequence as shown in SEQ ID NO: 2 but also containing at least one of the modification of D61A amino acid residues; D61E; R72E; R72K; E116; E116Q; E116R; E116T; E116V; S133; I151G; I151A; V163R; D164R, or a combination thereof; or iv. containing an amino acid sequence as shown in SEQ ID NO: 2 but also containing at least one of the modification of the I20L amino acid residue; V62S; G77P; V83C; D88H; Y113G; E116T; E116G; H140K; K146S; I167S; L180E; E194M; A211Q; S212Y; G215C; G215V; G215W; A218H; A218S; V223A; A225M; A225Q, or a combination thereof.

[0007] The invention generally relates to methods using polypeptides with hydrolase activity, including lipase activity. Thus, new classes of lipases are disclosed herein referred to as "saturases," "palmitases," and "stearatases." Also disclosed are polynucleotides encoding polypeptides having saturase activity, e.g., palmitases and / or stearatases, and methods for producing and using these polynucleotides and polypeptides. from aspects disclosed herein are polypeptides, e.g. enzymes with hydrolase activity, e.g. with lipase, saturase, palmitase and / or stearatase activity with thermostable and / or thermotolerant enzymatic (catalytic) activity. Enzyme activities of polypeptides and peptides include (include or consist of) saturase activity or lipase activity, including lipid hydrolysis, acid hydrolysis reactions (e.g. to replace esterified fatty acid with free fatty acid), transesterification reactions (e.g. fatty acid exchanges between triacylglycerides), ester synthesis, ester exchange reactions, and lipid acylhydrolase (LAH) activity. In another aspect, the polypeptides disclosed herein are used to synthesize enantiomerically pure chiral products.

[0008] The polypeptides disclosed herein can be used in a wide variety of pharmaceutical, agricultural and industrial contexts, including for the production of cosmetics and nutraceuticals. In addition, the polypeptides disclosed here can be used in food processing, brewing, bath additives, alcohol production, peptide synthesis, enantioselectivity, dressing hides in the leather industry, waste management and animal waste degradation, silver recovery in the photographic industry, medicine, silk degumming , degradation of biological membranes, conversion of biomass to ethanol, defense against biological weapons, antibacterial and disinfecting agents, personal hygiene products and cosmetics, biotechnological reagents, for increasing the efficiency of wet corn milling starch and as pharmaceuticals such as digestive aids and anti-inflammatory (anti-phlogistic) agents.

[0009] Compositions (e.g., lipases, saturases, palmitases and / or stearatases) and methods for producing low-saturated oils, e.g., oils with low saturated fatty acids, including oils with low palmitic, stearic, myristic, lauric fatty acids are disclosed or butanoic and / or caprylic acid (octanoic acid). Any of the vegetable oil can be treated with the composition or method provided herein, e.g. canola oil, soybean oil or animal oil or fat, e.g. tallow. Any food, edible or baking, frying or cooking products (e.g. sauces, marinades, spices, aerosol oils, margarines, baking oils, mayonnaise, cooking oils, salad oils, spooned dressings and poured dressings, etc., and products made with their participation) may contain vegetable oil or animal fat that has been subjected treating the composition disclosed herein or the method disclosed herein. Vegetable oils modified to be less saturated can be used in any food, edible or baking or cooking products, e.g. sauces, marinades, spices, aerosol oils, margarines, baking oils, mayonnaise, oils cooking oils, salad oils, spooned dressings and poured dressings, etc. Oils such as vegetable oils, e.g. rapeseed oil, canola or soybean oil, and food or baking or cooking product, including sauces, marinades, spices, aerosol oils, margarines, mayonnaise, baking oils, cooking oils, frying oils, salad oils, dressings spooned and poured etc. in which the oil or food product, baking or cooking product has been modified using the disclosed enzyme. In one aspect, these vegetable oils, e.g. canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hemp oil, linseed oil, Limnanthes alba seed oil, olive oil, palm oil, palm oil , peanut oil, rapeseed oil, rice bran oil, cartamus oil, camellia oil, soybean oil, sunflower oil, tall oil, Japanese camellia oil, 'natural' oil varieties with a changed composition of fatty acids produced by genetically modified organisms (GMOs) or obtained as a result of traditional 'breeding selection', such as oils with high oleic acid content, low linolenic acid content or low-saturated oils (rapeseed oil variety high oleic acid canola, low linolenic acid soybean oil or high stearic acid sunflower oil), animal fats (tallow, lard, butter fat and chicken fat), fish oils (olacon oil, fish oil, orange roughy oil, sardine oil, herring oil and menhanden oil), or mixtures of any of the above, and products food or products for baking, frying or cooking, contain oils with a lower content of saturated fatty acids, including oils with a low content of palmitic acid, myristic acid, lauric acid, stearic acid, caprylic acid (octanoic acid) etc. processed using the composition or method disclosed herein.

[0010] Polypeptides, e.g. enzymes and catalytic antibodies, having hydrolase activity, e.g. with lipase, saturase, palmitase and / or stearatase activity, including thermostable and thermotolerant enzymatic activity, and fatty acid specific or fatty acid selective activity, and low or high pH tolerant enzyme activity, and coding polynucleotides these polypeptides, including vectors, host cells, transgenic plants and non-human animals, and methods of making and using these polynucleotides and polypeptides.

[0011] Disclosed herein are isolated, synthetic or recombinant nucleic acids comprising (a) a nucleic acid (polynucleotide) encoding at least one polypeptide, wherein the nucleic acid comprises a sequence having at least about 50%, 51%, 52%, 53%, 54 %, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,

68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%,

83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99%, or greater or full (100%) sequence identity with:

(i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 , SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO: 23, or (ii) a nucleic acid with the sequence SEQ ID NO: 1 containing one or more nucleotide changes (or equivalents thereof) encoding one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two, twenty three, twenty four or more, or all amino acid changes (or their equivalents) as specified in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, wherein the nucleic acid of (i) or (ii) encodes at least one polypeptide with hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity, or encodes a polypeptide or peptide capable of producing a hydrolase specific antibody (e.g. lipases, saturases, palmitases and / or stearatases) (a polypeptide or peptide that acts as an epitope or immunogen), (b) a nucleic acid (polynucleotide) from (a), in which sequence identity is determined: (A) by analysis using a sequence comparison algorithm or visually, or (B) in an area of ​​at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150,175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more residues, or along the entire length of a cDNA, transcript (mRNA) or gene, (c) nucleic acid (polypeptide) from (a) or (b), where the sequence comparison algorithm is the BLAST version 2.2.2 algorithm with the filter setting blastall -p blastp -d "pataa no." -FF, and all other options are set by default, (d) nucleic acid (polynucleotide) encoding at least one polypeptide or peptide with hydrolase activity, e.g. having lipase, saturase, palmitase and / or stearatase activity, wherein the nucleic acid comprises a sequence that hybridizes under stringent conditions to a (a), (b) or (c) nucleic acid complement, where stringent conditions include a washing step in which the composition comprises washing in 0.2X SSC at a temperature of about 65 ° C for about 15 minutes, (e) a nucleic acid (polynucleotide) encoding at least one polypeptide with hydrolase activity, e.g. with lipase, saturase, palmitase and / or stearatase activity, wherein the polypeptide has the sequence SEQ ID NO: 2, or enzymatically active fragments thereof, containing at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two, twenty three, twenty four, or more or all amino acid changes (or their equivalents) as specified in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, (f) nucleic acid (polynucleotide) encoding at least one polypeptide with activity hydrolases, e.g. having lipase, saturase, palmitase and / or stearatase activity, wherein the polypeptide has the sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO : 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20 or enzyme-active fragments thereof, (g) (A) nucleic acid (polynucleotide) according to any of the points (a) to (f) and encoding a polypeptide having at least one conservative amino acid substitution and retaining its hydrolase activity, e.g. lipase, saturase, palmitase and / or stearatase activity, or, (B) nucleic acid according to item (g) (A), wherein at least one conservative amino acid substitution involves substitution of the amino acid with another amino acid with similar characteristics; or conservative substitution includes: replacing the aliphatic amino acid with another aliphatic amino acid; threonine replacement or vice versa; replacing the acid residue with another acid residue; replacing the residue containing the amide group with another residue containing the amide group; exchanging the basic residue for another basic residue; or replacing an aromatic residue with another aromatic residue, (h) a nucleic acid (polynucleotide) according to any one of (a) to (g) encoding a polypeptide having hydrolase activity, e.g. with lipase, saturase, palmitase and / or stearatase activity, but lacking a signal sequence, (i) a nucleic acid (polynucleotide) according to any one of (a) to (h) encoding a polypeptide with hydrolase activity, e.g. with lipase, saturase, palmitase and / or stearatase activity, further comprising a heterologous sequence, (j) a nucleic acid (polynucleotide) according to item (i), wherein the heterologous sequence comprises or consists of a coding sequence: (A) heterologous signal sequence, (B) sequence from (A), wherein the heterologous signal sequence is from a heterologous enzyme, or (C) tag, epitope, targeting peptide, susceptible sequence, detectable moiety or enzyme, or (k) a nucleic acid sequence (polynucleotide) completely (fully) complementary to the sequence from any one of (a) to (j). [0012] In one aspect, a synthetic or recombinant nucleic acid encodes a polypeptide or peptide with hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity that is thermostable. Polypeptides and peptides encoded by the nucleic acids disclosed herein, or any polypeptide or peptide disclosed herein, may retain enzymatic or binding activity (e.g. substrate binding) under conditions covering a temperature range between about -100 ° C to about -80 ° C, about -80 ° C to about -40 ° C, about -40 ° C to about -20 ° C, about -20 ° C to 0 ° C, about 0 ° C to about 5 ° C, about 5 ° C to about 15 ° C, about 15 ° C to about 25 ° C, about 25 ° C to about 37 ° C, about 37 ° C to about 45 ° C, about 45 ° C to about 55 ° C, about 55 ° C to about 70 ° C, about 70 ° C to about 75 ° C, about 75 ° C to about 85 ° C, about 85 ° C to about 90 ° C, about 90 ° C to about 95 ° C, about 95 ° C to about 100 ° C, about 100 ° C to about 105 ° C, about 105 ° C to about 110 ° C, about 110 ° C to about 120 ° C, or 95 ° C, 96 ° C, 97 ° C, 98 ° C, 99 ° C, 100 ° C, 101 ° C, 102 ° C, 103 ° C, 104 ° C, 105 ° C, 106 ° C, 107 ° C, 108 ° C, 109 ° C, 110 ° C, 111 ° C, 112 ° C, 113 ° C, 114 ° C, U5 ° C or more. Heat-stable polypeptides retaining hydrolase activity are disclosed herein, e.g. lipase, saturase, palmitase and / or stearatase activity at temperatures in the ranges described above, at pH about 3.0, pH about 3.5, pH about 4.0, pH about 4.5, pH about 5.0, pH about 5.5, pH about 6.0, pH about 6.5, pH about 7.0, pH about 7.5, pH about 8.0, pH about 8.5, pH about 9.0, pH about 9 , 5, pH about 10.0, pH about 10.5, pH about 11.0, pH about 11.5, pH about 12.0 or more.

[0013] In one aspect, the polypeptides disclosed herein may be thermotolerant and may retain hydrolase activity, e.g. lipase, saturase, palmitase and / or stearatase activity after exposure to a temperature in the range of about -100 ° C to about -80 ° C, about -80 ° C to about -40 ° C, about -40 ° C to about -20 ° C, about -20 ° C to about 0 ° C, about 0 ° C to about 5 ° C, about 5 ° C to about 15 ° C, about 15 ° C to about 25 ° C, about 25 ° C to about 37 ° C, about 37 ° C to about 45 ° C, about 45 ° C to about 55 ° C, about 55 ° C to about 70 ° C, about 70 ° C to about 75 ° C, about 75 ° C to about 85 ° C, about 85 ° C to about 90 ° C, about 90 ° C to about 95 ° C, about 95 ° C to about 100 ° C, about 100 ° C to about 105 ° C, about 105 ° C to about 110 ° C, about 110 ° C to about 120 ° C, or 95 ° C, 96 ° C, 97 ° C, 98 ° C, 99 ° C, 100 ° C, 101 ° C, 102 ° C, 103 ° C, 104 ° C, 105 ° C, 106 ° C, 107 ° C, 108 ° C, 109 ° C , 110 ° C, 111 ° C, 112 ° C, 113 ° C, 114 ° C, 115 ° C or more.

[0014] The disclosed thermotolerant polypeptides retain hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity, after exposure to temperature in the ranges described above, at a pH of about 3.0, about pH 3.5, about pH 4.0 , about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 9.0, about pH 9.5, about pH 10.0, about pH 10.5, about pH 11.0, about pH 11.5, about pH 12.0 or more.

[0015] The disclosed isolated, synthetic or recombinant nucleic acids contain a sequence that hybridizes under stringent conditions to the nucleic acid disclosed herein, e.g. of the exemplary nucleic acid disclosed herein comprising the sequence given as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 , SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO: 23, or the sequence given as SEQ ID NO: 1 containing one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more, or all residue changes (SEQ ID NO: 1 sequence modifications) listed in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, or fragments or subsequences thereof, and sequences (fully) complementary thereto . In one aspect, the nucleic acid encodes a polypeptide with hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity. Nucleic acid may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300 in length, 350, 400, 450, 500, 550, 600, 650, 700 or more residues or the full length of a gene or transcript containing SEQ ID NO: 1, and having the sequence given as SEQ ID NO: 1 containing one, two, three, four, five six seven eight nine ten, eleven or twelve or more or all residue changes (amino acid sequence modifications) in SEQ ID NO: 1 listed in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23; and sequences (fully) complementary to it. In one aspect, stringent conditions include a washing step comprising washing in 0.2X SSC at a temperature of about 65 ° C for about 15 minutes.

[0016] A disclosed nucleic acid probe, e.g., a probe for identifying a nucleic acid encoding a polypeptide with hydrolase activity, e.g. with lipase, saturase, palmitase and / or stearatase activity, contains a probe containing or consisting of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more further principles disclosed herein of a sequence, or fragments or subsequences thereof, wherein the probe identifies the nucleic acid by binding or hybridizing. The probe may comprise an oligonucleotide containing at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80 or about 60 to 100 sequential bases having a sequence comprising the sequence disclosed herein, or fragments or subsequences thereof. The probe may contain an oligonucleotide consisting of at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80 or about 60 to 100 sequential bases from the nucleic acid sequence disclosed herein, or a sub-sequence thereof.

[0017] The disclosed pair of amplification primer sequences for amplifying a nucleic acid encoding a polypeptide having hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity, comprises a pair of primers containing or consisting of a pair of primers capable of amplifying the nucleic acid containing the disclosed here the sequence, or fragments or subsequences thereof. One or each element of the amplification primer pair of sequences may contain an oligonucleotide containing at least about 10 to 50 sequential bases of that sequence.

[0018] The disclosed methods for amplifying a nucleic acid encoding a polypeptide having hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity, include amplifying the template nucleic acid using a pair of primer sequences of amplification reactions capable of amplifying the nucleic acid sequence disclosed herein, or fragments or sub-sequences thereof.

[0019] The disclosed expression cassettes contain the nucleic acid or subsection disclosed herein. In one aspect, the expression cassette may contain a nucleic acid that is operably linked to a promoter. The promoter may be a viral, bacterial, mammalian or plant promoter. In one aspect, the plant promoter may be a potato, rice, corn, wheat, tobacco or barley promoter. The promoter may be a constitutive promoter. The constitutive promoter may contain CaMV35S. In another aspect, the promoter may be an inducible promoter. In one aspect, the promoter may be a tissue-specific, environmentally regulated or developmentally-controlled promoter. Thus, the promoter may be, e.g., seed-specific, leaf-specific, root-specific, stem-specific, or be an organ-induced promoter. In one aspect, the expression cassette may further comprise a plant or plant virus expression vector.

[0020] The disclosed gene cloning carriers include the expression cassette disclosed herein (eg, a vector) or the nucleic acid disclosed herein. The gene carrier for cloning can be a viral vector, plasmid, phage, phagemid, cosmid, phosmid, bacteriophage or artificial chromosome. The viral vector may include an adenovirus vector, a retroviral vector, or adeno-associated viral vector. The gene carrier for cloning may include an artificial bacterial chromosome (BAC), a plasmid, a vector derived from bacteriophage P1 (PAC), an artificial yeast chromosome (YAC) or an artificial mammalian chromosome (MAC).

[0021] The transformed cells disclosed comprise a nucleic acid disclosed herein or an expression cassette disclosed herein (eg, a vector), or a gene cloning carrier disclosed herein. In one aspect, the transformed cell may be a bacterial cell, mammalian cell, fungal cell, yeast cell, insect cell or plant cell. In one aspect, the plant cell may be a potato, wheat, rice, corn, tobacco or barley cell. The transformed cell may be any of the host cells known to those skilled in the art, including a prokaryotic cell, eukaryotic cell, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells or plant cells.

Exemplary bacterial cells include any species of the genera Escherichia, Bacillus, Streptomyces, Salmonella, Pseudomonas and Staphylococcus, including, e.g., Escherichia coli, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, Pseudomonas fluorescens. Exemplary fungal cells include any species of the genus Aspergillus. Exemplary yeast cells include any species of the genera Pichia, Saccharomyces, Schizosaccharomyces or Schwanniomyces, including Pichia pastoris, Saccharomyces cerevisiae or Schizosaccharomyces pombe. Exemplary insect cells include any species of the Spodoptera or Drosophila species, including Drosophila S2 and Spodoptera Sf9. Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line.

[0022] The transgenic plants disclosed contain the nucleic acid disclosed herein or the expression cassette disclosed herein (eg, a vector). The transgenic plant may be a corn plant, potato plant, tomato plant, wheat plant, oil seed plant, rapeseed plant, soybean plant, rice plant, barley plant or tobacco plant.

[0023] The disclosed transgenic seeds contain the nucleic acid disclosed herein or the expression cassette disclosed herein (eg, a vector). Transgenic seeds may be rice grains, corn grains, wheat grains, oilseeds, rapeseeds, soybeans, oil palm seeds, sunflower seeds, sesame seeds, peanuts or tobacco seeds.

[0024] The disclosed isolated, synthetic or recombinant polypeptides exhibit hyrolase activity, e.g. lipase, saturase, palmitase and / or stearatase activity, or are polypeptides capable of producing a hydrolase specific immune response, e.g. lipase, saturase, palmitase and / or stearatase (e.g. with an epitope); and in alternative aspects, the peptides and polypeptides disclosed herein comprise the sequence:

(a) showing at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or

100% (full) sequence identity with:

(i) the amino acid sequence of SEQ ID NO: 2, or enzymatically active fragments thereof, and comprising at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen , sixteen, seventeen, eighteen, nineteen, twenty, twenty-one twenty-two, twenty-three, twenty-four or more, or all changes in amino acid residues (or equivalent) listed in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, or (ii) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ

ID NO: 20, wherein the polypeptide or peptide of (i) or (ii) has hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity, or the polypeptide or peptide is capable of producing a hydrolase specific antibody (e.g. . lipase, saturase, palmitase and / or stearatase) (a polypeptide or peptide that acts as an epitope or immunogen), (b) the polypeptide or peptide of (a), in which sequence identity was determined: (A) by analysis using a sequence comparison algorithm or visually, or (B) in an area of ​​at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, 150, 200, 250, 300 or more amino acid residues, or along the entire length of the polypeptide, peptide or enzyme, and / or its enzymatically active subsection (fragments), (c) the polypeptide or peptide of (b), wherein the sequence comparison algorithm is the BLAST version 2.2.2 algorithm with the filter setting blastall -p blastp -d "pataa no." -FF, and all other options are set by default;

(d) the amino acid sequence encoded by the nucleic acid disclosed herein, wherein the polypeptide has (i) hydrolase activity, e.g., lipase, saturase, palmitase, and / or stearatase activity, or (ii) exhibits immunogenic activity that is is capable of producing an antibody that binds specifically to a polypeptide having the sequence given in (a), and / or its enzymatically active sub-sequences (fragments);

(e) the amino acid sequence according to any one of (a) to (d), and which comprises at least one conservative amino acid residue substitution, and the polypeptide or peptide retains hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity;

(f) the amino acid sequence of (e), wherein the conservative substitution involves the replacement of an aliphatic amino acid with another aliphatic amino acid; threonine replacement or vice versa; replacing the acid residue with another acid residue; replacing the residue containing the amide group with another residue containing the amide group; exchanging the basic residue for another basic residue; or replacing the aromatic residue with another aromatic residue, or combinations thereof, (g) the amino acid sequence of (f), wherein the aliphatic residue includes alanine, valine, leucine, isoleucine or a synthetic equivalent thereof; the acid residue includes aspartic acid, glutamic acid or a synthetic equivalent thereof; the amide containing residue includes asparagine, glutamine or a synthetic equivalent thereof; the basic residue includes lysine, arginine, histidine or a synthetic equivalent thereof; or the aromatic residue includes phenylalanine, tyrosine, tryptophan or a synthetic equivalent thereof;

(h) a polypeptide according to any one of (a) to (f) with a hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity, but lacking a signal sequence, (i) a polypeptide according to any one of (a) to (h) having hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity further comprising a heterologous sequence;

(j) a polypeptide according to item (i), wherein the heterologous sequence comprises or consists of:

(A) heterologous signal sequence, (B) sequence from (A), wherein the heterologous signal sequence is derived from a heterologous enzyme, and / or (C) tag, epitope, targeting peptide, cleavage sequence, detectable moiety or enzyme; or (m) contain an amino acid sequence encoded by any of the nucleic acid sequences disclosed herein.

[0025] Exemplary polypeptide or peptide sequences disclosed herein include SEQ ID NO: 2, and its sub-sequences and variants, e.g. at least about 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more in length, or the entire length of the enzyme, all containing one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more or all amino acid residue changes (amino acid sequence modifications of SEQ ID NO: 2) listed in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23. Exemplary polypeptide or peptide sequences disclosed herein include the sequence encoded by the nucleic acid disclosed herein. Exemplary polypeptide or peptide sequences disclosed herein include polypeptides or peptides specifically bound by the antibody disclosed herein. In one aspect, the polypeptide disclosed herein has at least one hydrolase activity, e.g., lipase, saturase, palmitase, and / or stearatase activity. In one aspect, this activity is regioselective and / or chemoselective activity.

[0026] In one aspect, an isolated, synthetic or recombinant polypeptide may comprise a polypeptide lacking a signal sequence (peptide) disclosed herein, e.g., which lacks its homologous signal sequence, and in one aspect, comprises a heterologous signal sequence (peptide). In one aspect, an isolated, synthetic or recombinant polypeptide may include a polypeptide disclosed herein comprising a heterologous signal sequence, such as a heterologous hydrolase or non-hydrolase signal sequence (e.g., non-lipase, non-saturase or non-palmitase). In one aspect, the chimeric proteins consist of a first domain comprising the signal sequence disclosed herein and at least a second domain. This protein may be a fusion protein. The second domain may contain an enzyme. This enzyme may be a hydrolase (e.g., lipase, saturase, palmitase and / or stearatase) disclosed herein, or other hydrolase.

[0027] In one aspect, the hydrolase activity (e.g., lipase, saturase, palmitase, and / or stearatase) includes specific activity at a temperature of about 37 ° C in the range of about 100 to about 1000 units per milligram of protein. In another aspect, the hydrolase activity (e.g., lipase, saturase, palmitase and / or stearatase) includes specific activity from about 500 to about 750 units per milligram of protein. Alternatively, the hydrolase activity includes specific activity at 37 ° C in the range of from about 500 to about 1200 units per milligram of protein. In one aspect, the hydrolase activity includes specific activity at 37 ° C in the range of from about 750 to about 1000 units per milligram of protein. In another aspect, thermotolerance includes maintaining at least half of the specific hydrolase activity at 37 ° C after heating to elevated temperature. Alternatively, thermotolerance may include maintaining specific activity at 37 ° C in the range of from about 500 to about 1200 units per milligram of protein after heating to elevated temperature.

[0028] In one aspect, the isolated, synthetic or recombinant polypeptides disclosed herein contain at least one glycosylation site. In one aspect, glycosylation can be glycosylation via N-glycosidic binding. In one aspect, the polypeptide may be glycosylated after expression in P. pastoris or S. pombe or in plants such as oilseeds e.g. soybean, canola, rice, sunflower, or genetically modified (GMO) varieties of these plants.

[0029] In one aspect, the polypeptide may retain hydrolase activity (e.g., lipase, saturase, palmitase, and / or stearatase) under conditions including about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, or pH 4.0 or lower. In another aspect, the polypeptide may retain hydrolase activity (e.g., lipase, saturase, palmitase, and / or stearatase) under conditions including about pH 7, pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5 , pH 10, pH 10.5, pH 11, pH 11.5, pH 12.0 or higher.

[0030] In one aspect, the protein preparation comprises the polypeptide disclosed herein, wherein the protein preparation comprises a liquid, solid or gel.

[0031] In one aspect, the heterodimers disclosed herein comprise the polypeptide and the second domain. In one aspect, the second domain may be a polypeptide and the heterodimer may be a fusion protein. In one aspect, the second domain may be an epitope or tag. In one aspect, the homodimers disclosed herein comprise the polypeptide disclosed herein.

[0032] In one aspect, the immobilized polypeptides disclosed herein exhibit hyrolase activity (e.g., lipase, saturase, palmitase, and / or stearatase), wherein the polypeptide comprises a polypeptide disclosed herein, a polypeptide encoded by a nucleic acid or polypeptide disclosed herein, and a polypeptide disclosed herein second domain. In one aspect , the polypeptide disclosed herein can be immobilized on / in the cell, follicle, liposome, membrane, membrane, metal, resin, polymer, ceramics, glass, microelectrode, graphite particle, bead, gel, plate, crystal, tablet, pill , capsule, powder, agglomerate, surface, porous structure, matrix or capillary tube or materials such as grains, husks (scales), bark, skin, hair, enamel, bone, crust (shell) and materials derived therefrom. The polynucleotides, polypeptides and enzymes disclosed herein may be prepared in the form of solid preparations, such as a powder, lyophilized preparation, granules, tablet, cube, crystal, capsule, pill, pellet, or in liquid form, such as an aqueous solution, aerosol, gel, paste, thick suspension, water / oil emulsion, cream, capsule, or vesicular or micellar suspension.

[0033] The disclosed food supplements for an animal comprise a polypeptide disclosed herein, e.g. a polypeptide encoded by a nucleic acid disclosed herein. In one aspect, the polypeptide in addition to food can be glycosylated. The disclosed templates providing an edible enzyme comprise the polypeptide disclosed herein, e.g., a polypeptide encoded by the nucleic acid disclosed herein. In one aspect, the delivery matrix comprises pellets. In one aspect, the polypeptide may be glycosylated. In one aspect, the hydrolase activity is thermotolerant. In another aspect, the hydrolase activity is thermostable.

[0034] Disclosed methods for isolating or identifying a polypeptide with hydrolase activity (e.g. lipases, saturases, palmitases and / or stearatases) consists of the following steps: (a) providing the antibody disclosed herein; (b) providing a sample containing polypeptides; and (c) contacting the sample of step (b) with the antibody of step (a) under conditions in which the antibody can specifically bind to the polypeptide, thereby isolating or identifying the polypeptide having hydrolase activity (e.g., lipase, saturase, palmitases and / or stearatases).

[0035] The disclosed methods for producing an anti-hydrolase antibody include administering to a non-human animal the nucleic acid disclosed herein or the polypeptide disclosed herein or a subsection thereof in an amount sufficient to obtain a humoral immune response and thereby generate an anti-hydrolase antibody. Disclosed herein are methods of making an anti-hydrolase antibody comprising administering to a non-human animal the nucleic acid disclosed herein or the polypeptide disclosed herein or a sufficient amount thereof to obtain an immune response.

[0036] The disclosed methods for producing a recombinant polypeptide include the steps of: (a) providing a nucleic acid disclosed herein operably linked to a promoter; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, and thereby production of a recombinant polypeptide. In one aspect, the method may further comprise transforming the host cell with the nucleic acid of step (a) followed by the expression of the nucleic acid of step (a), thereby producing a recombinant polypeptide in the transformed cell.

[0037] Disclosed methods for identifying a polypeptide having hydrolase activity (e.g. lipases, saturases, palmitases and / or stearatases) include the following steps: (a) providing the polypeptide disclosed herein; or a polypeptide encoded by the nucleic acid disclosed herein; (b) providing a substrate for hydrolase; and (c) contacting the polypeptide or fragment or variant thereof from step (a) with the substrate from step (b) and detecting a decrease in the amount of the substrate or an increase in the amount of the reaction product, wherein the decrease in the amount of the substrate or the increase in the amount of the reaction product allows the polypeptide to be detected. hydrolase activities (e.g., lipase, saturase, palmitase and / or stearatase).

[0038] The disclosed methods for identifying a substrate for hydrolase include the following steps: (a) providing the polypeptide disclosed herein; or a polypeptide encoded by the nucleic acid disclosed herein; (b) providing a test substrate; and (c) contacting the polypeptide of step (a) with the test substrate from step (b) and detecting a decrease in the amount of the substrate or an increase in the amount of the reaction product, wherein the decrease in the amount of the substrate or the increase in the amount of the reaction product identifies the tested substrate as a substrate for hydrolase ( e.g. lipases, saturases, palmitases and / or stearatases).

[0039] The disclosed methods for determining whether a test compound specifically binds to a polypeptide comprise the following steps: (a) expressing a nucleic acid or vector containing the nucleic acid under conditions that allow translation of the nucleic acid into the polypeptide, wherein the nucleic acid comprises the nucleic acid disclosed herein, or providing a polypeptide disclosed herein; (b) providing a test compound; (c) contacting the polypeptide with a test compound; and (d) determining whether the test compound of step (b) specifically binds to the polypeptide.

[0040] Disclosed methods for identifying a modulator of hydrolase activity (e.g. lipases, saturases, palmitases and / or stearatases) include the following steps: (a) providing a polypeptide disclosed herein or a polypeptide encoded by a nucleic acid disclosed herein; (b) providing a test compound; (c) contacting the polypeptide of step (a) with the test compound from step (b) and measuring the hydrolase activity, wherein the change in the hydrolase activity measured in the presence of the test compound compared to the activity in the absence of the test compound shows that the test compound modulates hydrolase activity. In one aspect, the hydrolase activity (e.g. lipase, saturase, palmitase and / or stearatase) can be measured by providing a substrate for hydrolase and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, or an increase in the amount of substrate or a decrease in the amount of reaction product. A decrease in the amount of substrate or an increase in the amount of reaction product in the presence of a test compound compared to the amount of a substrate or reaction product without a test compound identifies the test compound as a hydrolase activator. An increase in the amount of substrate or a decrease in the amount of reaction product in the presence of the test compound compared to the amount of the substrate or reaction product without the test compound identifies the test compound as an inhibitor of hydrolase activity.

[0041] The disclosed computer systems comprise a processor and a data storage device, said data storage device having a stored in its memory polypeptide sequence or nucleic acid sequence (e.g., a polypeptide encoded by the nucleic acid disclosed herein). In one aspect, the computer system may further include a sequence comparison algorithm and a data storage device having at least one reference sequence stored in its memory. In another aspect, the sequence comparison algorithm includes a computer program that indicates polymorphisms. In one aspect, the computer system may further include an identifier that identifies one or more features in said sequence. The disclosed computer readable media comprises the stored polypeptide sequence or nucleic acid sequence disclosed in memory.

[0042] The disclosed methods for identifying a certain feature in a sequence include the steps of: (a) reading the sequence using a computer program that identifies one or more features in the sequence, said sequence comprising the polypeptide sequence or nucleic acid sequence disclosed herein; and (b) identifying one or more features in this sequence using a computer program.

[0043] Methods of comparing the first sequence with the second sequence have been disclosed, comprising the following steps: (a) reading the first sequence and the second sequence by using a computer program that compares the sequences, the first sequence comprising the polypeptide sequence or nucleic acid sequence disclosed herein ; and (b) determining the differences between the first sequence and the second sequence using a computer program. The step of determining the differences between the first sequence and the second sequence may further include the step of identifying the polymorphisms. In one aspect, the method may further include an identifier that identifies one or more features in the sequence. In another aspect, the method may include reading the first sequence using a computer program and identifying one or more features in the sequence.

[0044] Disclosed methods for isolating or recovering nucleic acid encoding a polypeptide having hyrolase activity (e.g. the lipase, saturase, palmitase and / or stearatase) from the sample include the following steps: (a) providing a pair of amplification primer sequences for amplifying the nucleic acid encoding a polypeptide having hydrolase activity, wherein the pair of primers is capable of amplifying the nucleic acid disclosed herein; (b) isolating the nucleic acid from the sample or treating the sample in such a way that the nucleic acid contained in the sample becomes available for hybridization for a pair of primer amplification reactions; and, (c) combining the nucleic acid of step (b) with a pair of primers for amplifying the reaction of step (a) and amplifying the nucleic acid from the sample, thereby isolating or recovering the nucleic acid encoding the polypeptide having the hydrolase activity from the sample. The disclosed sample is an environmental sample, e.g. a water sample, liquid sample, soil sample, air sample, or biological sample, e.g., bacterial cells, protozoal cells, insect cells, yeast cells, plant cells, fungal cells or mammalian cells. One of each component of the amplification primer primer sequence may include an oligonucleotide containing at least about 10 to 50 or more sequential bases with the sequence disclosed herein.

[0045] Disclosed methods for increasing the thermotolerance or thermostability of a hydrolase polypeptide include glycosylation of the hydrolase polypeptide, wherein the polypeptide comprises at least thirty adjacent amino acids from the polypeptide disclosed herein; or a polypeptide encoded by the nucleic acid sequence disclosed herein, thereby increasing the thermotolerance or thermostability of the hydrolase polypeptide. In one aspect, the specific activity of the hydrolase may be thermostable or thermotolerant at temperatures ranging from over about 37 ° C to about 95 ° C.

[0046] Disclosed methods of overexpressing a recombinant hydrolase polypeptide (e.g. (lipases, saturases, palmitases and / or stearatases) in a cell include the expression of a vector comprising the nucleic acid disclosed herein or the nucleic acid sequence disclosed herein, wherein sequence identity is determined by analysis using a sequence comparison algorithm or visually, while overexpression is accomplished by using a promoter with high activity, a bicistronic vector or by amplification of vector genes.

[0047] The detergent compositions disclosed comprising a polypeptide disclosed herein or a polypeptide encoded by a nucleic acid disclosed herein exhibit hydrolase activity, eg, lipase, saturase, palmitase and / or stearatase activity. In one aspect, the hydrolase may be surface-inactive hydrolase. In another aspect, the hydrolase may be surface active hydrolase.

[0048] The disclosed methods for washing an object include the following steps: (a) providing a composition comprising a polypeptide having hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity, said polypeptide comprising: the polypeptide or polypeptide disclosed herein encoded by the disclosed here nucleic acid; (b) delivery of the object; and (c) contacting the polypeptide of step (a) and the object of step (b) under conditions in which the composition can wash the object.

[0049] The disclosed methods for producing a transgenic plant include the following steps: (a) introducing a heterologous nucleic acid sequence into the plant cell, wherein the heterologous nucleic sequence comprises the nucleic acid sequence disclosed herein, thereby forming a transformed plant cell; and (b) forming a transgenic plant from the transformed cell. In one aspect, step (a) may further include the introduction of a heterologous nucleic acid sequence by electroporation or microinjection of plant cell protoplasts. In another aspect, step (a) may further comprise introducing a heterologous nucleic acid sequence directly into plant tissue by bombardment with DNA-coated particles. Alternatively, step (a) may further include the introduction of a heterologous nucleic acid sequence into the plant cell DNA using an Agrobacterium tumefaciens host. In one aspect, the plant cell may be a potato, corn, rice, wheat, tobacco or barley cell.

[0050] The disclosed methods for expressing a heterologous nucleic acid sequence in a plant cell include the following steps: (a) transforming the plant cell with a heterologous nucleic acid sequence operably linked to a promoter, the heterologous nucleic acid sequence comprising the nucleic acid disclosed herein; (b) allowing the plant to grow under conditions in which the heterologous nucleic acid sequence is expressed in the plant cell.

[0051] In one embodiment, the first method of biocatalytic synthesis of structured lipid comprises the following steps: (a) providing a polypeptide (e.g. lipases, saturases, palmitases and / or stearatases); (b) providing a composition containing triacylglyceride (TAG); (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions in which the polypeptide hydrolyzes the acyl residue at the Sn2 position of triacylglyceride (TAG), thereby forming 1,3-diacylglyceride (DAG); (d) providing an ester R1; (e) providing R1-specific hydrolase and (f) contacting 1,3-DAG from step (c) with the R 1 ester from step (d) and R1 specific hydrolase from step (e) under conditions in which the specific hydrolase for R1 catalyses the esterification of the Sn2 position and thus the obtaining of a structured lipid. The hydrolase may be a Sn2-specific lipase. The structured lipid may include an alternative cocoa butter (CBA), synthetic cocoa butter, natural cocoa butter, 1,3-dipalmitoyl-2-oleoyl glycerol (POP), 1,3-distearoyl-2-oleoyl glycerol (SOS), 1-palmitoyl-2 -oleoyl-3-stearoylglycerol (POS) or 1-oleoyl-2,3-dimyrystoylglycerol (OMM).

[0052] In one embodiment, the second method of biocatalytic synthesis of structured lipid comprises the following steps: (a) providing a hydrolase (e.g. lipases, saturases, palmitases and / or stearatases); (b) providing a composition containing triacylglyceride (TAG); (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions in which the polypeptide hydrolyzes the acyl residue at the Sn1 or Sn3 position of the triacylglyceride (TAG), thereby producing 1,2-DAG or 2,3 -DAG; and (d) facilitating migration of the acyl group on the 1,2-DAG or 2,3-DAG molecule from step (c) under kinetic controlled conditions, thereby obtaining a composition comprising 1,3-DAG.

[0053] The latter method may further include providing R1-ester and R1-specific lipase, and contacting 1,3-DAG of step (d) with R1-ester and R1-specific lipase under conditions where R1-specific lipase catalyzes esterifying the Sn2 position and thereby producing a structured lipid. The hydrolase, e.g. lipase, saturase, palmitase and / or stearatase may be an Snl or Sn3 specific enzyme. The structured lipid may include any vegetable oil, e.g. soybean oil, canola oil, alternative cocoa butter (CBA), synthetic cocoa butter, natural cocoa butter, 1,3-dipalmitoyl-2-oleoyl glycerol (POP), 1,3-distearoyl-2-oleoyl glycerol (SOS), 1 palmitoilo -2-oleoyl-3-stearoylglycerol (POS) or 1-oleoyl-2,3-dimyrystoylglycerol (OMM).

[0054] The ester R1 may contain a moiety with a lower degree of saturation than the hydrolysed acyl residue, in which case the lipid produced in this way is structured with a lower saturated fat or oil than the original TAG. Ester R1 may contain one or more omega-3 fatty acids, omega-6 fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, phosphate groups, phytosterol esters and oryzanol. In particular, the R1 ester may contain a moiety selected from the group consisting of alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid, dihomogamma-linolenic acid, arachidonic acid, oleic acid, palmitoleoleic acid, choline, serine, beta-sitosterol , cumestrol, diethylstilbestrol and oryzanol.

[0055] In one aspect of this second method, step (d) further includes the use of ion exchange resins. Kinetically controlled conditions may include non-equilibrium conditions that result in the production of an end product with a 1.3-DAG to 2.3-DAG ratio greater than 2: 1. This composition of step (b) may contain a fluorogenic fatty acid (KT). The composition of step (b) may contain an umbeliferone ester KT. The final product may be enantiomerically pure.

[0056] In one embodiment, the method of producing a lower saturation fat or oil comprises the steps of: (a) providing a polypeptide (hydrolase, e.g. lipase, saturase, palmitase and / or stearatase); (b) providing an oil or fat, and (c) bringing the polypeptide of step (a) into contact with the oil or fat of step (b) under conditions where the hydrolase may modify this oil or fat, e.g., remove at least one saturated fatty acid e.g. palmitic, stearic, lauric, caprylic (octane) acid, etc. The modification may include hydrolase catalyzed fat or oil hydrolysis. Hydrolysis can mean complete or partial hydrolysis of fat or oil. The hydrolyzed oil may contain a glycerol ester of polyunsaturated fatty acid, which may replace removed saturated fatty acid, or fish, animal or vegetable oil. Vegetable oil may include olive oil, canola oil, sunflower, palm, soy or lauric oil or rice bran oil or a combination thereof.

[0057] In one embodiment, the method of producing a lower saturation fat or oil which may contain necessary unsaturated fatty acids comprises the following steps: (a) providing a polypeptide (e.g. lipases, saturases, palmitases and / or stearatases); (b) providing a composition containing triacylglyceride (TAG); (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions in which the polypeptide hydrolyzes the acyl residue at the Sn1 or Sn3 position of the triacylglyceride (TAG), thereby producing 1,2-DAG or 2, 3-DAG; and (d) facilitating migration of the acyl group on the 1,2-DAG or 2,3-DAG molecule from step (c) under kinetically controlled conditions, thereby forming 1,3-DAG.

[0058] The method may further comprise providing an RI ester and an R1 specific lipase, and bringing the 1,3-DAG of step (d) into contact with an RI ester and an R1 specific lipase under conditions where the R1 specific lipase catalyzes the esterification. Sn2 position, thereby forming the rice lipid structure. The ester R1 may contain a lower saturation moiety than a hydrolyzed acyl residue, in which case the structured lipid thus produced will be a fat or oil with a lower saturation than the original TAG. Ester R1 may contain omega-3 fatty acid (alpha linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)), omega-6 fatty acid (gamma-linolenic, dihomo-gamma-linolenic (DGLA) or arachidonic), monounsaturated acid fatty (oleic, palmitoleic, etc.), phosphate groups (choline and serine), phytosterol esters (beta-sitosterol, coumestrol and diethylstilbestrol) and oryzanol. The hydrolase disclosed herein, e.g. lipase, saturase, palmitase and / or stearatase may be an Snl or Sn3 specific enzyme. Lower saturation fat or oil can be produced by the hydrolysis of any algae oil, vegetable oil or fat or animal oil as described above, e.g. oil from Neochloris oleoabundans, oil from Scenedesmus dimorphus, oil from Euglena gracilis, oil from Phaeodactylum tricornmutum, oil from Pleurochrysis carterae, oil from Prymnesium parvum, oil from Tetraselmis chui, oil from Tetraselmis suecica, oil from Isochrysina oil, from Isochrys oil Botryococcus brauń oil, Dunaliella tertiolecta oil, Nannochloris oil, Spirulina oil, Chlorophycease oil (green algae) and Bacilliarophy oil, canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hemp oil, linseed oil, Limnanthes alba seed oil, olive oil, palm oil, palm oil, peanut oil, rapeseed oil, rice bran oil, cardamom oil, camellia oil, soybean oil, sunflower oil, tall oil, Japanese camellia oil, varieties of "natural" oils with altered fatty acid compositions through genetically modified organisms (GMOs) or as a result of traditional "breeding" such as oils with high oleic acid content, low linolenic acid or low saturated oils (high acid canola oil oleic, soybean oil with low linolenic acid or sunflower oils with high stearic acid); animal fats (tallow, lard, butter fat and chicken fat), fish oils (olacon oil, fish oil, orange rough oil, sardine oil, herring oil and menhanden oil), or mixtures of any of the foregoing. The fat or oil with lower saturation obtained in this way may be used in food or in baking, frying or cooking products containing oils or fats with a lower content of fatty acids, including oils with a low content of palmitic acid, oleic acid, lauric acid, stearic acid, caprylic acid (octanoic acid) etc. processed using the composition or method disclosed herein.

[0059] The disclosed method for refining a lubricant comprises the following steps: (a) providing a composition comprising a hydrolase disclosed herein (e.g., lipase, saturase, palmitase and / or stearatase); (b) providing a lubricant; and (c) treating the lubricant with a hydrolase under conditions in which the hydrolase disclosed herein (e.g., lipase, saturase, palmitase and / or stearatase) can selectively hydrolyze oils contained in the lubricant, thereby leading to its refining. The lubricant may be hydraulic oil.

[0060] The disclosed fabric treatment method comprises the following steps: (a) providing a composition comprising a hydrolase disclosed herein (e.g., lipase, saturase, palmitase and / or stearatase), wherein the hydrolase can selectively hydrolyze carboxylic acid esters; (b) providing a fabric; and (c) treating the fabric with a hydrolase under conditions in which the hydrolase can selectively hydrolyze carboxylic acid esters and thereby process the fabric. Fabric treatment may include improving the grip and arrangement of the final fabric, dyeing, achieving flame retardancy, achieving hydrophobicity, obtaining optical brightness or resin finish. The fabric may contain cotton, viscose, rayon, lyocell, flax, flax fibers, ramie fibers, all mixtures thereof, or mixtures thereof with polyesters, wool, polyamide acrylic fibers or poly and acrylic fibers. The disclosed fabric, yarn or fiber contains a hydrolase disclosed herein, which may be adsorbed, absorbed or immobilized on the surface of the fabric, yarn or fiber.

[0061] The disclosed method of removing or reducing the size of a stain from a food or oil includes contacting the hydrolase disclosed herein (e.g., lipase, saturase, palmitase and / or stearatase) with a stain from food or oil under conditions where the hydrolase can hydrolyze the oil or fat contained in the stain. Hydrolase disclosed herein (e.g. lipase, saturase, palmitase and / or stearatase) may show increased stability against denaturation caused by surfactants and heat deactivation. The hydrolase disclosed herein (e.g., lipase, saturase, palmitase and / or stearatase) may be in the form of a detergent solution or a washing solution.

[0062] The disclosed dietary composition comprises a hydrolase disclosed herein (eg, lipase, saturase, palmitase and / or stearatase). The dietary composition may further comprise a nutritional base containing fat. Hydrolase can be activated by the bile salt. The dietary composition may further comprise a cow's milk infant formula. Hydrolase can hydrolyze long chain fatty acids.

[0063] The disclosed method for reducing fat content in milk or vegetable based dietary compositions comprises the following steps: (a) providing a composition comprising a hydrolase disclosed herein (e.g., lipase, saturase, palmitase and / or stearatase); (b) providing a composition comprising milk or vegetable oil, and (c) treating the composition of step (b) with a hydrolase under conditions in which the hydrolase can hydrolyze the oil or fat contained in the composiq. The disclosed dietary composition for human or non-ruminant animals contains a nutritional base, wherein the base contains fat and does not contain or does not contain a small amount of hydrolase, and an effective amount of hydrolase disclosed herein (e.g., lipases, saturases, palmitases and / or stearatase) to increase fat absorption and accelerate the growth of a human or non-ruminant animal.

[0064] In one embodiment, the method of catalyzing the interesterification reaction to obtain new triacylglycerides comprises the following steps: (a) providing a composition comprising the polypeptide (e.g. lipase, saturase, palmitase and / or stearatase), wherein the polypeptide can catalyze the interesterification reaction; (b) providing a mixture of triacylglycerides and free fatty acids; (c) treating the mixture of step (b) with a polypeptide under conditions in which the polypeptide can catalyze the exchange of free fatty acids with triacylglyceride acyl groups and thereby generate new triacylglycerides enriched in added fatty acids. This polypeptide may be a Snl, 3 specific lipase.

[0065] In one embodiment, the interesterification method used to produce an oil with a low content of trans fatty acids and a low content of medium length fatty acids comprises the following steps: (a) providing a mixture for interesterification reaction containing a stearic acid source material selected from the group consisting of stearic acid, stearic acid monoesters with low molecular weight monohydric alcohols and mixtures thereof, (b) providing liquid vegetable oil; (c) providing the polypeptide (e.g. lipases, saturases, palmitases and / or stearatases), said polypeptide having 1,3-specific lipase activity; (d) interesterification of the material being the source of stearic acid and triacylglyceride contained in vegetable oil, (e) separating the component and interest-free free fatty acids from the glyceride component of the interesterification mixture to obtain an interesterified margarine oil product and a fatty acid mixture containing fatty acids, acid monoesters fatty or a mixture thereof, released from vegetable oil, and (f) hydrogenating the fatty acid mixture. In one embodiment of the interesterification process, the interesterification reaction lasts until a substantial equilibrium of the ester groups occurs at the 1-, 3- positions of the glyceride component with the components of the reaction mixture containing non-glyceride fatty acids.

[0066] The disclosed method of making a composition comprising 1-palmitoyl-3-stearoyl-2-monooleine (POSt) and 1,3-distearoyl-2-monooleine (StOSt) involves providing the polypeptide disclosed herein (e.g. lipase, saturase, palmitase and / or stearatase), wherein the polypeptide is capable of lipase-catalyzed 1.3-binding, 1,3-dipalmitoyl-2-monooleine (POP) interesterification (POP) with stearic acid or tristearin, and contacting said polypeptide with a composition containing said POP in the presence of a stearin source such as stearic acid or tristearin, to obtain a product enriched in 1-palmitoyl-3-stearoyl-2-monooleine (POSt) or 1,3-distearoyl-2-monooleine (StOSt).

[0067] The disclosed method of alleviating or preventing lipopolysaccharide (LPS) -induced toxicity includes administering to a patient a pharmaceutical composition containing the hydrolase disclosed herein (e.g., lipase, saturase, palmitase and / or stearatase). The disclosed method of endotoxin detoxification includes contacting the endotoxin with the hydrolase disclosed herein (e.g., lipase, saturase, palmitase, and / or stearatase). The disclosed method for deacylating a 2 'or 3' fatty acid chain from lipid A involves contacting lipid A with the polypeptide disclosed herein.

[0068] The disclosed methods for changing the substrate specificity or substrate preference of the parent lipase enzyme (fatty acid hydrolase) having the amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 2 include the step of producing (introducing) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more amino acid residue mutations in SEQ ID NO: 2, as given in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, and thereby producing a new hydrolase enzyme with a modified amino acid sequence and altered substrate specificity or substrate preference compared to the parent lipase enzyme (fatty acid hydrolase) having the sequence SEQ ID NO: 2. In one aspect, the substrate specificity or substrate preference of a new lipase enzyme (fatty acid hydrolase) includes a privileged or increased hydrolysis of palmitic acid from oil, or the substrate specificity or substrate preference of a new lipase enzyme (fatty acid hydrolase) includes a privileged or increased hydrolysis of stearic acid from oil .

In one aspect, the modified amino acid sequence (compared to the "parent" SEQ ID NO: 2) contains at least one amino acid modification A48C; D49R; D61A; D61E; R72E; R72K; V83M; R85Y; E95K; E116A; El 161; E116L; E116N; E116Q; E116R; E116T; E116V; S133A; A144I; E149H; A150I; I151G; I15IA; P162G; P162K; V163R; D164R; R172H; R172L; or A225S, or a combination thereof, or their combination and / or at least one codon modification (GCG) 35 (GCT); (GGC) 45 (GGA); (GCG) 92 (GCT), (GTG) 102 (GTT); (AGC) 108 (AGT); (CTA) 117 (CTT); (CTG) 124 (TTG); (CGG) 126 (AGG); (GTC) 128 (GTG); (AGT) 133 (TCT); (TTC) 135 (TTT); (GTG) 183 (GTT); (ACC) 188 (ACG), or their equivalent, or a combination thereof, and the substrate specificity or substrate preference of the new lipase enzyme (fatty acid hydrolase) includes privileged or increased hydrolysis of palmitic acid from oil. In one aspect, the modified amino acid sequence (compared to the "parent" SEQ ID NO: 2) includes I20L; V62S; G77P; V83C; D88H; Y113G; E116T; E116G; H140K; K146S; I167S; L180E; E194M; A211Q; S212Y; G215C; G215V; G215W; A218H; A218S; V223A; A225M; A225Q, or a combination thereof, and the substrate specificity or substrate preference of the new lipase enzyme (fatty acid hydrolase) includes preferential or increased hydrolysis of stearic acid from oil.

[0070] The disclosed methods for producing an enzyme having a substrate specificity or substrate preference, including a preferential or increased hydrolysis of palmitic acid from oil, consists of the following steps: (a) providing a parent hydrolase enzyme (e.g. lipase, saturase, palmitase and / or stearatase) having substrate specificity or substrate preference including preferential hydrolysis of palmitic acid from oil, wherein the parent hydrolase enzyme (e.g., lipase, saturase, palmitase and / or stearatase) has the sequence disclosed herein; and (b) introducing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more amino acid residue modifications into the parent hydrolase enzyme (e.g. lipases, saturases, palmitases and / or stearatases), wherein the amino acid residue modifications correspond to the amino acid sequence mutations of SEQ ID NO: 2, as shown in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, and thereby producing an enzyme showing substrate specificity or substrate preference including preferential or increased hydrolysis of palmitic acid from oil.

[0071] The disclosed methods for producing an enzyme having substrate specificity or substrate preference, including a preferential or increased hydrolysis of stearic acid from oil, consists of the following steps: (a) providing a parent hydrolase enzyme (e.g. lipase, saturase, palmitase and / or stearatase) having substrate specificity or substrate preference including preferential hydrolysis of stearic acid from the oil, wherein the parent hydrolase enzyme (e.g., lipase, saturase, palmitase and / or stearatase) has the sequence disclosed herein; and (b) introducing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more amino acid residue modifications into the parent hydrolase enzyme (e.g. lipases, saturases, palmitases and / or stearatases), wherein the amino acid residue modifications correspond to the amino acid sequence mutations of SEQ ID NO: 2, as shown in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, and thereby producing an enzyme that has substrate specificity or substrate preference including preferential or increased hydrolysis of stearic acid from the oil.

[0072] The disclosed methods for producing the fatty acid hydrolase enzyme (e.g., lipase, saturase, palmitase and / or stearatase) exhibiting a substrate specificity or substrate preference, including preferential hydrolysis of a particular fatty acid, consists of the following steps (a) to provide the enzyme sequence given herein fatty acid hydrolases (e.g. lipases, saturases, palmitases and / or stearatases); (b) generating (introducing) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more nucleic acid base residue mutations, said mutations corresponding to the sequence changes defined in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23; and (c) examining the activity of the newly formed enzyme in terms of substrate specificity or substrate preference including preferential hydrolysis of a particular fatty acid, thereby creating a new fatty acid hydrolase enzyme (e.g. lipase, saturase, palmitase and / or stearatase) exhibiting substrate specificity or preference substrate including the privileged hydrolysis of a particular fatty acid. In one aspect, the fatty acid hydrolase enzyme (e.g., lipase, saturase, palmitase, and / or stearatase) comprises the sequence given as SEQ ID NO: 2. In one aspect, the fatty acid is linolenic acid, linoleic acid, oleic acid, palmitic acid or stearic acid.

[0073] The disclosed methods for producing the fatty acid hydrolase enzyme (e.g., lipase, saturase, palmitase and / or stearatase) exhibiting a substrate specificity or substrate preference, including preferential hydrolysis of a particular fatty acid, comprises the steps of (a) providing an acid sequence disclosed herein nucleic acid encoding fatty acid hydrolase enzyme (e.g. lipases, saturases, palmitases and / or stearatases); (b) generating (introducing) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more nucleic acid base residue mutations, said mutations corresponding to the sequence changes defined in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23; and (c) expressing the produced nucleic acid to obtain a new fatty acid hydrolase enzyme (e.g. lipase, saturase, palmitase and / or stearatase), thereby producing an enzyme of fatty acid hydrolase (e.g., lipase, saturase, palmitase and / or stearatase) having substrate specificity or a substrate preference including preferential hydrolysis of a particular fatty acid.

[0074] In one aspect, the sequence coding for the fatty acid hydrolase enzyme (e.g., lipase, saturase, palmitase, and / or stearatase) comprises the sequence given as SEQ ID NO: 1. In one aspect, the fatty acid is linolenic acid, linoleic acid, oleic acid, palmitic acid or stearic acid. In one aspect, the substrate specificity or substrate preference of a new fatty acid hydrolase enzyme (e.g. lipase, saturase, palmitase and / or stearatase) refers to palmitic acid compared to the substrate specificity or substrate preference for stearic acid for the parent fatty acid hydrolase enzyme (e.g. lipase, saturase, palmitase and / or stearatase), or substrate specificity or substrate preference a new fatty acid hydrolase enzyme (e.g. lipase, saturase, palmitase and / or stearatase) refers to stearic acid compared to the substrate specificity or substrate preference for palmitic acid for the parent fatty acid hydrolase enzyme (e.g. lipase, saturase, palmitase and / or stearatase).

[0075] The disclosed lipases comprise the amino acid sequence given as SEQ ID NO: 2, but also including at least a modification of the A48C amino acid residues; D49R; D61A; D61E; R72E; R72K; V83M; R85Y; E95K; E116; E116I; E116L; E116N; EU6Q; E116R; Ε116Τ; E116V; S133; A144I; E149H; A150I; I151G; I151A; P162G; P162K; V163R; D164R; R172H; R172L; or A225S, or their equivalent, or a combination thereof, and / or at least one codon modification (GCG) (GCT); (GGC) 45 (GGA); (GCG) 92 (GCT), (GTG) 102 (GTT);

(AGC) 108 (AGT); (CTG) 117 (CTT); (CTG) 124 (TTG); (CGG) 126 (AGG); (GTC) 128 (GTG);

(AGT) 133 (TCT); (TTC) 135 (TTT); (GTG) 183 (GTT); (ACC) 188 (ACG) or an equivalent or a combination thereof.

[0076] In one aspect, the substrate specificity or substrate preference of the new lipase includes the privileged or increased hydrolysis of fatty acid from oil compared to the "parent" sequence of SEQ ID NO: 2. In one aspect, fatty acid is linolenic acid, linoleic acid, oleic, palmitic or stearic acid.

[0077] The details of one or more embodiments are given in the accompanying drawings and the description below. Other features, objectives and advantages will become apparent after reading the description and drawings, as well as the reservations.

DESCRIPTION OF THE DRAWINGS [0078] The following drawings are for illustration only and are not intended to limit the scope of the claims.

Figure 1 shows a block diagram of a computer system.

Figure 2 is a flowchart illustrating one aspect of the process of comparing a new nucleotide or protein sequence with a sequence database to determine the levels of homology between the new sequence and the sequences in the database.

Figure 3 is a flowchart illustrating one aspect of a computer process to determine if two sequences are homologous.

Figure 4 is a flowchart illustrating one aspect of the identifier process 300 for detecting the presence of a certain feature in a sequence.

Figure 5 illustrates an exemplary method disclosed herein involving the use of lipases for lipid processing, e.g., lipid from soybean oil, to perform selective hydrolysis of palmitic acid to obtain "reduced palmitic acid soybean oil".

Figure 6a illustrates the effect of exemplary GSSM mutations^sm palmitases on the hydrolysis of palmitate and stearate compared to the parent sequence SEQ ID NO: 2, as discussed in detail in Example 4 below. Figure 6b illustrates the effect of exemplary GSSM mutations^sm stearatase for palmitate and stearate hydrolysis compared to the parent SEQ ID NO: 2 sequence, as discussed in detail in Example 4 below.

Figure 7 shows the sequence of SEQ ID NO: 2, with specific mutation positions for palmitate and stearate, in larger bold letters. Underlined mutations (e.g., 61A, E) denote alternative amino acid residue positions (alternative sequences) to improve palmitate hydrolysis. Italicized mutations (e.g., 20L} indicate alternative amino acid residue positions (alternative sequences) to improve stearate hydrolysis. Position 116 is the alternative position for the amino acid residue mutation (alternative sequence for the alternative embodiment) to improve the hydrolysis of both palmitate and stearate.

Figure 8 shows confirmatory soybean oil analysis data for selected clones from the palmitase library.

[0079] Similar reference symbols in various drawings indicate similar elements.

DETAILED DESCRIPTION [0080] The present invention relates to methods of using polypeptides, including lipases, saturases, palmitases and / or stearatases, and the polynucleotides encoding them. Alternative embodiments of these methods include polypeptides, e.g., enzymes, with hydrolase activity, e.g., with lipase, saturase, palmitase and / or stearatase activity, including thermostable and thermotolerant hydrolase activity, and polynucleotides encoding these enzymes. The hydrolase activities of the polypeptides and peptides disclosed herein include lipase activity (lipid hydrolysis), interesterification reactions, ester synthesis, ester exchange reactions, lipid acylhydrolase (LAH) activity, and associated enzymatic activity. For the purposes of this patent application, interesterification reactions may include acid hydrolysis reactions (including fatty acid and triacylglyceride reaction), alcoholization (including alcohol and triacylglyceride reaction), glycerolysis (including glycerol and triacylglyceride reaction) and transesterification reactions (including ester reaction) and triacylglyceride). The polypeptides disclosed herein can be used in a variety of pharmaceutical, agricultural and industrial contexts, including in the manufacture of cosmetics and nutraceuticals. In another aspect, the polypeptides disclosed herein are used to synthesize enantiomerically pure chiral products.

[0081] The enzymes disclosed herein may be highly selective catalysts. They may have the ability to catalyze reactions with stereo-, regio- and chemoselectivity not achievable in conventional chemical synthesis. The enzymes disclosed herein can be universal. Considering different aspects, they can function in organic solvents, operate at extreme pH values ​​(e.g. high pH and low pH), extreme temperatures (e.g. high temperatures and low temperatures), extreme salinity levels (e.g. at high salinity and low salinity), and catalyze reactions with compounds that are structurally unrelated to their natural, physiological substrates.

[0082] In one aspect, the polypeptides disclosed herein include hydrolases with lipase, saturase, palmitase, and / or stearatase activity, and can be used, e.g., in biocatalytic synthesis of structured lipids (lipids containing a specific set of fatty acids distributed in a specific manner on a glycerol backbone) , including any vegetable oil, e.g. canola oil, soybean oil, soybean oil alternatives, cocoa butter alternatives, 1,3-diacylglycerides (DAG), 2-monoacylglycerides (MAG) and triacylglycerides (TAG) such as 1,3-dipalmitoyl-2-oleoylglycerol (POP) ), 1,3-distearoyl-2-oleoylglycerol (StOSt), 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POSt) or 1oleoyl-2,3-dimyrystoylglycerol (OMM), polyunsaturated fatty acids (PUFA), long chain polyunsaturated . such as arachidonic acid, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

[0083] In one embodiment, the enzymes and methods disclosed herein can be used to remove, add or replace any fatty acid from a composition, e.g., to produce an oil with a lower saturated fatty acid content (e.g., "low saturated" oil) or other fatty acid content (e.g. conversion of oil containing "saturated" fatty acids to oil containing alternative "unsaturated" fatty acids).

[0084] Examples of saturated fatty acids that can be removed, added, or "reorganized" on a lipid molecule, e.g., oil, using the enzyme disclosed herein or by carrying out the method disclosed herein include:

acetic acid: CH₃COOH butyric acid: CH₃(CH₂) 2COOH caproic acid: CH₃(CH₂)₄COOH caprylic acid: CH₃(CH₂)₆COOH Capric Acid: CH₃(CH₂)₈COOH undecanoic acid: CH₃(CH₂)₉COOH Lauric acid: (dodecanoic acid): CH₃(CH₂)and₀COOH myristic acid: (tetradecanoic acid): CH₃(CH₂)and₂COOH Pentadecanoic Acid: CH₃(CH₂)and₃COOH Palmitic acid: (hexadecanoic acid): CH₃(CH₂)and₄COOH Margarine acid: CH₃(CH₂)and₅COOH stearic acid (octadecanoic acid): CH₃(CH₂)and₆COOH Peanut acid (eicosanoic acid): CH₃(CH₂) i8COOH behenic acid: CH₃(CH₂)₂₀COOH [0085] Examples of unsaturated omega-3 fatty acids that can be removed, added or "reorganized" on a lipid molecule, e.g., oil, using the enzyme disclosed herein or by carrying out the method disclosed herein include:

α-linolenic acid (ALA): CH3CH2CH = CHCH2CH = CHCH₂CH = CH (CH₂) 7COOH_Stearidic acid (octadecatetraenoic acid):

CH₃CH₂CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CH (CH₂)₄COOH eicosapentaenoic acid (EPA):

CH₃CH₂CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CH (CH₂)₃COOH docosahexaenoic acid (DHA):

CH₃CH₂CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CH (CH₂)₂COOH Examples of unsaturated omega-6 fatty acids that can be removed, added or "reorganized" on a lipid molecule, e.g., oil, using the enzyme disclosed herein or by carrying out the method disclosed herein include:

linoleic acid (9,12-octadecadiene acid):

CH₃(CH₂)₄CH = CHCH₂CH = CH (CH₂)₇COOH Gamma-linolenic acid (6,9,12-octadecatrienic acid):

CH₃(CH₂)₄CH = CHCH₂CH = CHCH₂CH = CH (CH₂)₄COOH eicosadic acid (11,14-eicosadiene):

CH₃(CH₂)₄CH = CHCH₂CH = CH (CH₂)₉COOH-dihomo-gamma-linolenic acid (8,11,14-eicosatrienic acid):

CH₃(CH₂)₄CH = CHCH₂CH = CHCH₂CH = CH (CH₂)₆COOH_arachidonic acid (5,8,11,14-eicosatetraenoic acid):

CH₃(CH₂)₄CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CH (CH₂)₃COOH docosadienoic acid (13.16 docosadienoic acid):

CH₃(CH₂)₄CH = CHCH₂CH = CH (CH₂) nCOOH adren_acid (7,10,13,16-docosatetraenoic acid):

CH₃(CH₂)₄CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CH (CH₂)₅COOH docosaenoic acid (4,7,10,13,16-docosapentaenoic acid):

CH₃(CH₂)₄CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CHCH₂CH = CH (CH₂)₂COOH [0087] Examples of omega-9 fatty acids that can also be removed, added or "reorganized" on a lipid molecule, e.g., oil, using the enzyme disclosed herein or by carrying out the method disclosed herein include:

oleic acid (9-octadecenoic acid): CH₃(CH₂)₇CH = CH (CH2)₇COOH eicosenic acid (11-eicosenic acid) CH₃(CH₂)₇CH = CH (CH₂) 9COOH Meada acid (mead acid) (5,8,11-eicosatrienic acid):

CH₃(CH₂)₇CH = CHCH₂CH = CHCH₂CH = CH (CH₂)₃COOH erucic acid (13-docosenoic acid): CH₃(CH₂)₇CH = CH (CH₂) nCOOH neuronic acid (15-tetracozen acid): CH₃(CH₂)₇CH = CH (CH₂)and₃COOH.

palmitoleic acid: CH₃(CH2) 7 CH = CH (CH₂) 5COOH [0088] In one aspect, new classes of lipases are disclosed herein referred to as "saturases," e.g., "palmitases" and "stearatases." The term "saturase" previously used in the literature referred to an enzyme that saturates specific bonds on the metabolic pathway , e.g. hydrogenation of a double bond (Moise et al., J. Biol. Chem., 2005, 280 (30): 27815-27825). However, new and previously undescribed "saturases" have been disclosed, wherein the saturases described in the present invention hydrolyze saturated fatty acid esters, which hydrolyzed esters may be esters of saturated fatty acids and glycerol, umbeliferol or other alcohols.

[0089] Also previously disclosed herein are undescribed "palmitases" and "stearatases", wherein palmitases and stearatases hydrolyze palmitic acid and stearic acid, respectively, from glycerol backbone, for example. The "saturases" described in the present invention may also be referred to as ", saturohydrolases" Similarly, the "palmitases" described in the present invention may also be referred to as "palmitate hydrolases" and the "stearatases" described in the present invention may also be referred to as "hydrolases stearate. "

[0090] In another aspect, the saturases described in the present invention selectively hydrolyze at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% saturated fatty acids. In another aspect, the palmitases described in the present invention selectively hydrolyze fatty acids such that at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% , 71%, 72%, 73%, 74%, 75%, 76%, 77%,

78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of hydrolyzed fatty acids is palmitic acid. In another aspect, the stearatases described in the present invention selectively hydrolyze fatty acids such that at least 60%, 61%, 62%, 63%, 64%,

65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,

80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99% or 100% of hydrolyzed fatty acids is stearic acid.

In one aspect, as illustrated in Figure 5, enzyme methods can be used to process lipid, e.g., lipid from soybean oil or other vegetable oil, to perform selective hydrolysis of saturated fatty acid, e.g. palmitic or stearic acid (e.g., from oil containing these saturated fatty acids) to form "oil with low (or lower) saturated fatty acids content", e.g. "Oil with reduced palmitic acid", such as "vegetable oil with reduced palmitic acid", e.g. "soybean oil with reduced palmitic acid". The enzymes disclosed herein can also be used to selectively hydrolyze any fatty acid, in particular saturated fatty acids from the glycerol backbone to produce "low (or lower) saturated oil", including selective hydrolysis of saturated fatty acids, e.g. palmitic acid or stearic acid, from the Sn1 or Sn2 position of the glycerol backbone, disclosed as additional to hydrolysis from the Sn3 position (e.g., hydrolysis of palmitic acid from the illustrated Sn3 position in Figure 5).

[0092] In one aspect, an exemplary synthesis of low saturated triglycerides, oils or fats is provided. In this example synthesis, either free fatty acids or fatty acid esters can be used, depending on the enzyme used. In one aspect, hydrolases are used, e.g. lipases, saturases, palmitases and / or stearatase, to remove or hydrolyze saturated fatty acids such as acetic acid, butyric acid, caproic acid, caprylic acid, capric acid, undecanoic acid, lauric acid, myristic acid, pentadecanoic acid, palmitic acid , margaric acid, stearic acid, peanut acid or behenic acid from triglyceride, oil or fat. In one aspect, removed or hydrolyzed fatty acids are replaced with fatty acids that have greater health benefits (such as weaker correlation with cardiovascular disease), or with improved chemical properties (such as oxidative stability or reactivity), or with improved physical properties (such as melting point or mouthfeel). In one aspect, the added fatty acids are unsaturated omega-3 fatty acids such as α-linolenic acid, stearidic acid, eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA), or fatty acids or fatty acids present in fish oil. In one aspect, the added fatty acids are unsaturated omega-6 fatty acids, such as linoleic acid, gamma-linoleic acid, eicosadiene acid, dihomogamma-linoleic acid, arachidonic acid, docosadiene acid, adren acid or docosapentaenoic acid. In one aspect, the added fatty acids are unsaturated omega-9 fatty acids such as oleic acid, eicosenic acid, Meada acid (mead acid, 5,8,11-eicosatrienic acid), erucic acid, neonic acid or palmitoleic acid. In one aspect, the added fatty acids (e.g. omega-3, omega-6 or omega-9) are added by the reaction of fatty acids with triglycerides, oil or fat, after removal or hydrolysis of saturated fatty acids by hydrolases, e.g. lipases, saturases, palmitases and / or stearatases. In one aspect, the added fatty acids (e.g. omega-3, omega-6 or omega-9) are added by reacting fatty acid esters, including glycerol esters, or ethyl or methyl esters, with triglycerides, oil or fat, after removal or hydrolysis of saturated fatty acids by hydrolases, e.g., lipases, saturases, palmitases and / or stearatases, as described herein. In one aspect, the fatty acid addition reaction (e.g., omega-3, omega-6 or omega-9) is catalyzed by a hydrolase or lipase, such as non-specific lipase (including non-regiospecific and non-specific for fatty acids), or a Sn1 specific lipase, 3, or a Snl specific lipase, or a Sn3 specific lipase, or a Sn2 specific lipase, or a fatty acid specific lipase.

[0093] The methods and compositions described herein (hydrolases, e.g. lipases, saturases, palmitases and / or stearatases) can be used to produce nutraceuticals (e.g. polyunsaturated fatty acids and oils), a variety of foods and food additives (e.g. emulsifiers, fat substitutes, margarines and spreads), cosmetics (e.g. emulsifiers, creams), pharmaceuticals and drug carriers (e.g. liposomes, tablets, preparations), and animal feed additives (e.g. polyunsaturated fatty acids (such as linoleic acids).

[0094] In one aspect, the lipases disclosed herein may act on fluorogenic fatty acid esters (KTs), e.g., umbeliferone KT esters. In one aspect, the specificity profiles of lipases for KT formed or modified by the methods provided herein can be obtained by measuring their relative activities to a series of umbeliferone KT esters, such as palmitate, stearate, oleate, laurate, PUFA or butane esters.

[0095] In one aspect, the polypeptide disclosed herein for these reactions (e.g., an antibody or enzyme - e.g., lipase, saturase, palmitase, and / or stearatase) is mobilized, e.g. as given below. In alternative aspects, the methods presented herein do not require an organic solvent, can be carried out at relatively high reaction rates. See. e.g. U.S. Patent No. 5,552,317; US 5 834 259.

[0096] In certain embodiments, the methods and compositions (lipases, saturases, palmitases and / or stearatases) can be used to hydrolyze (including selectively hydrolyze) oils such as fish, animal and vegetable fats, and lipids such as polyunsaturated fatty acids. In one aspect, the polypeptides disclosed herein are used to prepare low-saturated oils, e.g. by removing (hydrolyzing) at least one fatty acid from the oil; and the hydrolysis may be selective hydrolysis, e.g., only removing a specific fatty acid, such as palmitic, stearic or other saturated fatty acid, or only removing fatty acid from one position, e.g., Snl, Sn2 or Sn3. In one aspect, the polypeptides disclosed herein are used to process fatty acids (such as polyunsaturated fatty acids), e.g., fish oil fatty acids, e.g. for use in food or as a food or feed additive (food), or as cooking, frying, baking or cooking oil. In another embodiment, the methods and compositions (lipases, saturases, palmitases and / or stearatases) can be used to perform selective hydrolysis of saturated esters, more effective than hydrolysis of unsaturated esters, to acids or alcohols. In another embodiment, the methods and compositions disclosed herein (lipases, saturases, palmitases and / or stearatases) can be used to treat latex materials for a variety of purposes, e.g., to treat latex materials used in hair fixation compositions to remove odors. In another embodiment, the methods and compositions (lipases, saturases, palmitases and / or stearatases) can be used to treat an animal's lipase deficiency, e.g. in a mammal like man. In another embodiment, the methods and compositions (lipases, saturases, palmitases and / or stearatases) can be used to produce lubricants such as hydraulic oils. In another embodiment, the methods and compositions (lipases, saturases, palmitases and / or stearatases) can be used to make and use detergents. In another embodiment, the methods and compositions (lipases, saturases, palmitases and / or stearatases) can be used in the chemical finishing of fabrics, fibers or yarns. In one aspect, the methods and compositions (lipases, saturases, palmitases and / or stearatases) can be used to reduce the flammability of a fabric using e.g. halogen-substituted carboxylic acid or its ester, i.e., fluorinated, chlorinated or brominated carboxylic acid or its ester . In one aspect, methods for producing lipases from environmental libraries have been presented.

The disclosed "hydrolases" include polypeptides (e.g., antibodies, enzymes) and peptides (e.g., "active sites") with any hydrolase activity, i.e., the polypeptides disclosed herein may exhibit any hydrolase activity, including, e.g., lipase, saturase activity , palmitases and / or stearatases. "Hydrolases" disclosed herein include all polypeptides having any lipase, saturase, palmitase, and / or stearatase activity, including activity involving lipid synthesis or lipid hydrolysis, i.e. the polypeptides disclosed herein may exhibit any lipase, saturase, palmitase and / or stearatase activity. Lipases, saturases, palmitases and / or stearatases disclosed herein include enzymes that are active in lipid bioconversion by catalyzing hydrolysis, alcoholysis, acid hydrolysis, esterification and aminolysis. In one aspect, the hydrolases disclosed herein (e.g., lipases, saturases, palmitases, and / or stearatases) can hydrolyze lipid emulsions. In one aspect, the enzymes disclosed herein can act selectively on the Sn-1, Sn-2 and / or Sn-3 bonds of triacylglycerides, releasing one or more fatty acids from the glycerol backbone. For example, the hydrolase, lipase, saturase, palmitase and / or stearatase activity of the polypeptides disclosed herein includes the synthesis of cocoa butter, polyunsaturated fatty acids (WNKT), 1,3-diacylglycerides (DAG), 2-monoacylglycerides (MAG) and triacylglycerides (TAGs). Lipase, saturase, palmitase and / or stearatase activity exhibited by the polypeptides disclosed herein also includes the production of low-saturated oils, e.g. soybean oil or canola oil, by removing fatty acids, e.g. palmitic, oleic, lauric or stearic acid. In alternative aspects, the enzymes disclosed herein may also hydrolyze and / or isomerize bonds at high temperatures, low temperatures, alkaline pH and acidic pH. In one aspect, the hydrolase e.g. lipase disclosed herein is a saturase that catalyzes hydrolysis, alcoholysis, acid hydrolysis, esterification and aminolysis reactions in which the carboxylic acid or fatty acid in the molecule formed or reacted is a saturated fatty acid such as acid acetic, butyric acid, lauric acid, myristic acid, palmitic acid, stearic acid or peanut acid. In one aspect, the hydrolase disclosed herein e.g. lipase or saturase means palmitase that catalyzes hydrolysis, alcoholysis, acid hydrolysis, esterification and aminolysis reactions in which the carboxylic acid or fatty acid in the molecule formed or reacted is palmitic acid. In one aspect, the hydrolase disclosed herein e.g. lipase or saturase means stearatase, which catalyzes the reactions of hydrolysis, alcoholysis, acid hydrolysis, esterification and aminolysis, in which the carboxylic acid or fatty acid in the molecule formed or reacted is stearic acid.

[0098] Enzymes are disclosed comprising variants of the hydrolase enzymes disclosed herein (e.g., "lipase variant", "saturase variant", "palmitase variant" or "stearatase variant"); these enzymes may contain an amino acid sequence derived from the "precursor" amino acid sequence. The precursor may include naturally occurring hydrolase and / or recombinant hydrolase. The amino acid sequence of the hydrolase variant is "derived" from the amino acid sequence of the precursor hydrolase by substitution, deletion or insertion of one or more amino acids from the precursor amino acid sequence. This modification involves modification of the "precursor DNA sequence" that encodes the precursor lipase amino acid sequence rather than manipulation hydrolase precursor enzyme as such. Suitable methods for this type of precursor DNA sequence manipulation include the methods disclosed herein, as well as methods known to those skilled in the art.

Generation and manipulation of nucleic acids [0099] In one aspect, the present disclosure discloses nucleic acids, including expression cassettes, such as expression vectors encoding polypeptides (e.g., hydrolases, such as lipases, saturases, palmitases and / or stearatases, and antibody). In another aspect, disclosed herein are nucleic acids having the sequence given as SEQ ID NO: 1 and containing at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more, or all changes in base residues described in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23 (or equivalent). Nucleic acids encoding polypeptides having the sequence given as SEQ ID NO: 2 and comprising at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more, or all changes in amino acid residues described are disclosed in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23 (or equivalent).

SEQ ID NO: 1

ATGCTGAAACCGCCTCCCTACGGACGCCTGCTGCGCGAACTGGCCGATATC

CCGGCCATCGTGACGGCACCGTTCCGGGGCGCTGCGAAAATGGGCAAACTG

GCGGATGGCGAGCCGGTACTGGTGCTGCCCGGCTTCCTGGCCGACGACAAC

GCCACCTCGGTGCTGCGCAAGACCTTCGATGTCGCGGGCTTTGCCTGTTCG

GGCTGGGAACGCGGCTTCAACCTCGGCATTCGTGGCGACCTCGTGGACCGG

CTGGTCGACCGGCTGCGGGCGGTGTCGGAGGCGGCCGGTGGTCAGAAGGT

GATCGTGGTCGGCTGGAGCCTCGGCGGCCTCTATGCGCGCGAGCTGGGCCA

CAAGGCGCCCGAACTGATCCGGATGGTCGTCACGCTCGGCAGTCCGTTCGC

GGGCGACCTCCACGCCAACCATGCGTGGAAGATCTACGAGGCGATCAACAG

CCACACGGTCGACAACCTGCCGATCCCGGTCGATTTCCAGATTAAGCCGCC

GGTGCGCACCATCGCGGTGTGGTCGCCGCTCGACGGGGTGGTGGCGCCGG

AGACCTCGGAAGGCTCGCCCGAGCAGTCGGACGAGCGGCTAGAGCTGGCG

GTGACCCACATGGGCTTTGCCGCATCGAAGACCGGGGCCGAGGCTGTGGTC

CGGCTGGTCGCGGCGCGGCTCTAG

SEQ ID N0: 2 (coded by SEQ ID NO: 1):

1 letter code:

MLKPPPYGRLLRELADIPAIVTAPFRGAAKMGKLADGEPVLVLPGFLADDNATSVLR

KTFDVAGFACSGWERGFNLGIRGDLVDRLVDRLRAVSEAAGGQKVIVVGWSLGGL

YARELGHKAPELIRMVVTLGSPFAGDLHANHAWKIYEAINSHTVDNLPIPVDFQIKPP

VRTIAVWSPLDGWAPETSEGSPEQSDERLELAVTHMGFAASKTGAEAWRLVAAR ^L”

3 letter code:

Met Leu Lys Pro Pro Pro Tyr Gly Arg Leu Leu Arg Glii Leu Ala Asp He Pro Ala Ile Val Thr Ala Pro Phe Arg Gly Ala Ala Lys Met Gly Lys Leu Ala Asp Gly Glu Pro Val Leu Val Leu Pro Gly Phe Leu Ala Asp Asp Asn Ala Thr Ser Val Leu Arg Lys Thr Phe Asp Val Ala Gly Phe Ala Cys Ser Gly Trp Glu Arg Gly Phe Asn Leu Gly Ile Arg Gly Asp Leu Val Asp Arg Leu Val Asp Arg Leu Arg Ala Val Ser Glu Ala Ala Gly Gly Gin Lys Val Ile Val Val Gly Trp Ser Leu Gly Gly Leu Tyr Ala Arg Glu Leu Gly His Lys Ala Pro Glu Leu Ile Arg Met Val Val Thr Leu Gly Ser Pro Phe Ala Gly Asp Leu His Ala Asn His Ala Trp Lys He Tyr Glu Ala Ile Asn Ser His Thr Val Asp Asn Leu Pro Ile Pro Val Asp Phe Gin Ile Lys Pro Pro Val Arg Thr He Ala Val Trp Ser Pro Leu Asp Gly Val Val Ala Pro Giu Thr Ser Glu Gly Cheese Pro Glu Gin Cheese Asp Glu Arg Leu Glu Leu Ala Val Thr His Met Gly Phe Ala Ala Cheese Lys Thr Gly Ala Glu Ala Val Val Arg Leu Val Ala Ala Arg Leu

SEQ ID N0: 3:

ATGGGCGGCCACGAGGGCGCGCGGGGCCCCAAAGACGGTCCGCCGGCGATGGTG

ATCCCGGGCTTCCTCGCCCACGACAGGCACACGACAGGATTGCGCCGGGAACTC

GGCGAGGGGGGGTTCAGGGTTCACCCGTGGCGGCAGGGCTGGAAGATGGGAGCG cgtgcggacacgctcgagaaattgaagcgggcagtggaccagtgcGgtcatgac gagccgatgctgctggtcggctggagtctgggcgggctctacgcgagggaggtc gcgcgcgccgagccggatcaggtgcgggcggtggtcactcttggttccccggtgt cgggcgaccggcgccgctacaccaacgtgtggaagctGtacgaatgggtggcgg gtcacccggtggacgacccgccgatccccgacaaggaggaaaagccgccggtgc

CGAGCCTGGCTTTGTGGTCGGCGGATGACGGGATCGTCGGCGCCCCGTCGGCGCG cgggactcagttatctcacgacaaggcggtcgagatgcgaagggtttccgggggtggccgggtg

SEQ ID N0: 4 (coded by SEQ ID NO: 3):

maghqgargpkdoppamvipgflahdrhttrlrrelaeagfrvhpwrqgwnmga radtleklkravdqcghdepillvgwslgglyarevaraepdqvrawtlgspvsg

DRRRYTNVWKLYEWVAGHPVDDPPIPDKEEKPPVPTLALWSADDGIVGAPSARGtQ

LSHDKAVEMRTSHMGFAMSAKSARFVVAEIVKFLKKTEGSESHD

SEQ ID NO: 5:

GTGAGCGAGAAAGGCGCACCCAAGGGAAGGCAGCGGCTGAAGGAGATCGGCGC

GCrrCTGTTCCACGCGCCTCGCAGCTTGGGCCATCTGGGCGCGCGCGGCCCCAAG

GACGGTCCTCCGGTGATGGTCATCCCGGGATTCCTCGCGCACGACTTGCATACGA

CGCAGTTGCGCCGGGCGCTCGCGAAGGCAGGCTTCCGAGTGCATCCGTGGCGGC

AGGGGATGAACCTTGGAGCGCGCGCCGATACGCTCGAAATTCTGAAGCGCGCGG

TGGATTCCTGCGGCTCGAGCGAGCCGATGCTGCTCGTCGGCTGGAGCCTGGGCGG

TCTCTATGCCCGGGAGATCGCGCGTGCGGAGCCGGACCGGGTGCGGGCGGTGGT

GACGATGGGATCGCCGGTGTGGGGCGACCGCAGGCGCTACACCAACGTGTGGAA

GCTGTACGAACGGATTGCCGGCCATCCGGTCGACAAGCCGCCGATCCCGGACAA

GAGCCAGAAGCCGCCGGTGCCGACTCTGGCTTTGTGGTCGCAGCATGATGGCATC

GTCGGCGCGCCCTCGGCGAGAGGGACGAAGAAGACCCGCGACAAGGCGGTCGC

CATCGACACGACTCAĆATGGGGTTTGCCATGTCGCCCAAGACGACGCGCGCGGC

AGTGCGTGAGATCGTGGGCTTTTTGAATGAAGTCGAAGGCGGTTCGTCACCCCGG

GCGTGA

SEQ ID NO: 6 (coded by SEQ ID N0: 5):

MSEKGAPKGRQRLKEIGALLFHAPRSLGHLGARGPKDGPPVMVIPGFLAHDLHTTQL

RRALAKAGFRVHPWRQGMNLGARADTLEILKRAVDSCGSSEPMLLVGWSLGGLYA

REIARAEPDRVRAVVTMGSPVWGDRRRYTNVWKLYERIAGHFVDKPPIPDKSQKPP

VPTLALWŚQHDGIVGAPSARGTKKTRDKAVAIDTTHMGFAMSPKTTRAAVREIVGF

LNEYEGGSSPRA

SEQ ID NO: 7:

ATGAGGCTGCGCGAGGGGGGCGCGCTCGTATCGCGGGCCTATCGCGCCTTCGGG

CGCCTCGGCGAGCGCGGCCCGGCGGACGGGCCGCCGCTGATGGTGATCCCGGGC

TTCCTCGCCACCGATCGCACCACTTTGGGGCTGCAGCGGGCGCTGGCCAAGGGCG

GCTACAAGGfGACCGGATGGGGCATGGGCCTCAACAGCGGCGTCACCGAAGACA

TAGTCGAĆCGCATCGCCGCTCGGGTCGAAAGGTTTGGAGCCGGCCGCAAAGTGA

TCCTCGTCGGCfGGAGCCTCGGCGGACTCTACGCGCGCGTGGTCGCGCAGGAGC gggcggatctcgtcgacaaggtggtcacgctcggćtcgcccttttcgggcgacag

GCGCĆGCAACAACAATGTCTGGCGGCTCTACGAGTTCGTC

GCCGGCCATCCGGTCAACAGCCCGGCGATCGACAAGGACCCCGAGGTGAAGCCG

CCGGTGCCGACGCTCGCTATCTGGTCGCGGCGCGACGGCATCGTCTGTCCGGCGG

GCGCGCGCGGGCGGGAGGGAGAGCGCGACGCCGAGCTCGAGCTCGACTGCAGC

CACATGGGCTTTGCGGTCAGCGCCAGGGCTTATCCCAAGATCGTGGAGGCGGTG

CGGGCGTTTCCGGAAAACATCCGTTCGCGCTGA

SEQ ID N0: 8 (coded by SEQ ID NO: 7):

MRLREGGALVSRAYRAFGRLGERGPADGPPLMVIPGFLATDRTTLGLQRALAKGGY

KVTGWGMGLNSGVTEDIVDRIAARVERFGAGRKVILVGWSLGGLYARWAQERPD

LVDKVVTLGSPFSGDRRRNNNVWRLYEFVAGHPVNSPPIDKDPEVKPPVPTLAIWSR

RDGIVSPAGARGREGERDAELELDĆSHMGFAVSARAYPKIVEAVRAFPENIRSR

SEQ ID NO: 9:

ATGAAGCCGCCGCGCGGATGGATGAAGATCCGGGAGGCGGGCTCGCTCCTCGCG

CGCTTCTACCGCGCGTTCGGCAAGCTCGAGCCGCGCGGGCCGGCGGACGGGCCG

AAGCTGATGGTGATCCCGGGTTTCCTCGGGGGCGACAGGACGACGCTCGGGCTG

CAGCGAGCGCTGGCCGGCGGCGGCTACCGGGTCGCCGGCTGGGGGCTGGGGGTG aaccgcggcgtttcggaggacgtggtcgaccggatcggccagcaagtcgcgcgg ttcggggcgggcgagaaggtgatcctggtcggctggagccttggcgggctttat gcgcgcgtggtggcgcaggagcggcccgacctcgtcgagaaggtggtgaccttg

GGCTCGCCGTTTTCGGGCGACCGGCGGCGCAACAACAATGTGTGGCGGCTCTATG

AGTGGGTGGCTGGGCATCCGGTGAACGATCCGCCGATCGACAAGGACCCGGCGA

AGAAGCCCCCGGTGCCGACGCTCGCGATCTGGTCGCGGCGTGATGGGATCGTGG

CGGTCGAAGGCGCGCGGGGGCGGCCGGAGGAGCGGGATGCCGAGCTGGAGATC

GATTGCAGCCACATGGGGTTTGGGGTCAGCGGCAAGGCGTTTCCCCGAATCGTA

GAGGCGGTGAAGGGGTTCTAA

SEQ ID NO: 10 (coded by SEQ ID NO: 9):

MKPPPGWMKIREAGSLLARFYRAFGKLEPRGPADGPKLMVIPGFLAGDRTTLGLQR

ALAGGGYRVAGWGLGVNRGVSEDVVDRIGQQVARFGAGEKVILVGWSLGGLYAR

VVAQERPDLVEKVVTLGSPFSGDRRRNNNVWRLYEWVAGHPVNDPPIDKDPAKKPP

VPTLAIWSRRDGIVAVEGARGRPEERDAELEIDCSHMGFGVSGKAFPRIVEAVKGF

SEQ ID NO: 11:

GTGTTGGTGCTGCCGGCGTTCCTCGCCAACGACCTTCCCACTTCGCTTCTCCGCAG

GACGCTGAAGGCGAACGGGTTTCGCCCGTTCGGCTGGGCGAACGGTTTCAACTTA

GGTGCACGGCCGGACACGCTCCAGCGCCTGAGCGCACGGCTCGATGCGGTGGTT

CAGGAAGGGGGCAGGCCGGTTGCATTGATCGGCTGGAGCCTTGGCGGGCTTTAT

GCCCGAGAGCTGGCGAAACGCAGGTCGGCTGAGGTGTCGGCAGTGATCACGCTC ggcacgcccttctcggttgacctgagacgcaacaacgcctggaagctgtacgag ctcatcaacgatcatcctgtcgatggccgcccgggg

AGCCACCCGTCCGAACCTTCGCTTTGTGGTCGCGTCGCGACGGGATCGTAGCGCC

CGCGAGCGCGCACGGCATGGAGGGCGAGTTCGACCAGGCGATCGAGCTGCAGTG

CACGCACAACGAGATGGTCAGTGATCCGGAGGCCCTCTCCACGATCGTTACCTTG

CTGCGGGAAAATGTTGGCTCCTGA

SEQ ID N0: 12 (coded by SEQ ID NO: 11):

MLVLPAFLANDLPTSLLRRTLKANGFRPFGWANGFNLGARPDTLQRLSARLDAVVQ

EAGRPVALIGWSLGGLYARELAKRRSAEVSAVITLGTPFSVDLRRNNAWKLYELIND

HPVDAPPLDVQVDAKPPVRTFALWSRRDG1VAPASAHGMEGEFDQAIELQCTHNEM

VSDPEALSTIVTLLRENVGS

SEQ ID NO: 13:

GTGAATACAGCCGACCTATTGAAGCCACCACCCGCAAGCATGACAGTTCTCGAG

GCGAGAGCGCTGCTGGACATATGGAAGATGAGCGGCCCATTGGCGCGCTTGCTA

TTCAAAAAGAACTCGCCCTGGCGCAAACAACGGGTTĆTCGTAATACCTGGCTTTG

GCGCTGATGATCGCTACACCTGGCCGTTGCGCAATTTCGTCeAGGCACAGGGCTA

TGCCACGACTGGCTGGGGCCTGGGCACCAACAAGGCAGGTCTCAATATGCCGCA

TCAACTATCCGACGTCCACCCCAGATGGAAGCTAAAACCCAAGACGCCGTACCG

TGGTGAGGCGGGCGTACCTTACGTGATTGACCGCTTGATGGAACGGTTTGACGAA

TTGGCATCGACGGATCCGCAACCCATCGCACTTATAGGTTGGAGTCTGGGTGGTT

TCATGGCCCGTGAAGTTGCCCGAGAGCGCCCAAACCAGGTGAGTCAGGTTATTA

CCCTCGGTTCTCCTGTCATCGGAGGCCCAAAATACACCCTCGCTGCATCGGCTTT

CATCCGGCGCAAATACGATTTGGACTGGGTGGAGCAAGTGATCGCGGAGCGGGA

AGATCGCCCCATTACTGTTCCTATTACAGCAATaGTCAGCCAGTGTGATGGCATC gtcggatattcagcggcaatcgatcaccacagtcccgctgtgcagcatttacata

TGGATGTTGCCCATTTGGGCfTTCGTTACAACACGAGGGTTTGGTCAGAAATCGC

CAATGCGCTCAACTCTTTAGAGGTGGAGAAGGAGCGTGTTTAG

SEQ ID NO: 14 (coded by SEQ ID NO: 13):

MNTADLLKPPPASMTVLEARALLDICKMSAPLARLLFKKNSPWRKQRVLVIPGFGA

DDRYTWPLRNFVQAQGYATTGWGLGTNKAGLNMPHQLSDVHPRWKLKPKTPYRG

EAGVPYVIDRLIERFDELASTDPQPIALIGWSLGGFMAREVARERPNQVSQVITLGSPV

IGGPKYTLAASAFIRRKYDLDWVEQVIAEREDRPITVPITAIVSQSDGIVGYSAAIDHH

SPAVQHLHMDVAHLGFPYNTRVWSEIANALNSLEVEKERV

SEQ ID NO: 15:

ATGGAGCTCGCCAAGGTCACCGCCCTGATGAAGGCCACCGCCCTCGAGATCGCG

ATCCTCACCGGCCACCTCGTCCTCTACCCCTCCGGGATCGTGGCCGAGCGCCTCG

CGGCCGCCCCCTCTTCACCGTCCTCCCCGTCCGCGGGCCCGACGGGCCGACGTCC

GGTCGTCCTGCTGCACGGTTTCGTGGACAACCGCTCGGTCTTCGTCCTGCTGCGC

CGTGCGCTCACCCGGAGCGGCCGTGACTGCGTCGAGTCGCTCAACTACTCGCCGC

TCACCTGCGACCTGCGGGCCGCCGCCGAACTGCTGGGGCGCCGGGTGGACGAGA

TCCGCGCCCGGACCGGACACGCCGAGGTCGACATCGTCGGCCACAGCCTGGGCG

GGCTCATCGCCCGTTATTACGTACAGCGTCTCGGCGGTGACAGCCGGGTGCGCAC

CCTGGTCATGCTCGGCACCCCGCACTCCGGCACCACCGTGGCCCGGCTCGCCGAC

GCGCATCCGCTGGTGCGGCAGATGCGGCCGGGTTCGGAGGTGGTGCGGGAGCTC gcGgcgccctcgcccggctgccgtacccggttcgtgagctcacgggggccacccgggggccacgg

GTGCACAACGTCCGGGTCAGCGGGATCGGTCATCTCGCGCTGCCGGTCCATCCCA

CGGTGGCGGCCGGGGTCCGGGAGGCCCTCGACGCGAGCGGCGCGGGGGTCCCGG

GGGTGCGGGAGGAGGGGCCCGGCGCCGGCGCCGTGGCGTGA

SEQ ID NO: 16 (coded by SEQ ID NO: 15):

MELAKVTALMKATALEIAILTGHLVLYPSGIVAERLAAAPSSPSSPSAGPTGRRPVVL

LHGFVDNRSVFVLLRRALTRSGRDCVESLNYSPLTCDLRAAAELLGRRVDEIRARTG

HAEVDIVGHSLGGLIARYYVQRLGGDSRVRTLVMLGTPHSGTTVARLADAHPLVRQ

MRPGSEVLREtAAPSPGCRTRFVSFWSDLDQVMVPVDTACLDHPDLLVHNVRVSGI ghlalpvhptvaaGvrealdasgagvpgvreegpgagava SEO ID NO: 17:

GTGGCCGCCGCGGACAGCGGGACGGCGGAAGGGCAAAGGCTTCGGCCGCCGAG

CCtGTtCCTGATGCTGGCCGAGGCGAGGGGCTTGCTCGAACTGAACTCGAGCCTG

TTGTTGTĆGCCGCTGTTGTTGCGGGCGCCGAAGGGCGACGGACATCCGGTGCTGG

CGCTGCCGGGCTTTCTCGCCAGCGATCTGTCGATGGCGCCGATGCGGCGCTATCT

GAAAGAAGTCGGCTAGGATGCCCATGCGTGGAACATGGGCCGCAATCTCGGCGG cgtcgcgtgcaagcgcgaagccttgcgcgacgtgttgcggcgccttacagccagggtagggggctggtag

GATCTCGCTItGCAGGCGCCCGACATGGTGCGTTCCGTGATCACGCTCGGGAGTC

CGTTTGCCAGCGACATCAGGGCGACCAACGCCACGCGGCTCTACGAGGCGCTGT

CGGGAGAAAGGGTCGACGAĆAATCCGGAGTTAACAGCGGCGATCGCCGGCGACC

TGCCGGTGCCGGCGACCTCGATCTATTCCCGTACCGACGGTATCGTGAACTGGCA

CACCAGCCTGCTGCGTCCrrCCGCAACGGCTGAAAAGATCGAGGTTTACTTCGCC

AGCCATATCGGGCTCGGCGTCAACCCGGCAGCGCTGTGGGCGGTGGCCGACCGC

CTGGCGCAGCCCGAGGGGGAATTTAAGCATTTTGACCGGTCGGGTCCCTTTGCCA

TTGCCTATGGCCCCCCTGAAAATGCACAATCCTGA

SEQ ID NO: 18 (coded by SEQ ID NO: 17):

MAAADSGTAEGQRLRPPSLFLMLAEARGLLELNSSLLLSPLLLRAPKGDGHPVLALP

GFLASDLSMAPMRRYLKELGYDAHAWNMGRNLGGVASKREALRDLLRRIYSQTGR

KVSLVGWSLGGVYARDLALQAPDMVRSVITLGSPFASDIRATNATRLYEALSGERV

DDNPELTAAIAGDLPVPATSIYSRTDGIVNWHTSLLRPSATAENIEVYFASHIGLGVNP

AALWAVADRLAQPEGEFKHFDRSGPFAIAYGPPENAQS

SEQ ID NO: 19:

ATGCCGGAGCGAAACGAAGCGCAGGCCCCGCCGCGTCTTCGTCCGCCGGGGCTC

GGGCTGTTCCTCGCCGAAGCGCGGGGCATTTTCGAGCTCAACGCGAGCCTGTTGC

TGTCGCCGCTTCTGTTGCGCGCGCCGCGCGGCGACGGCCATCCGGTGCTGGCGTT

GCCGGGCTTTCTTGCCAGTGATCTATCGATGGCGCCGTTGCGCCGCTACCTCACC

GAGCTCGGCTACGACAGCCACGGCTGGCGCATGGGGCGCAATGTCGGCGGCATC gcgaagatgcggatcgggctgctcgagcggctcacgcagatccatggcgagtgc

GGCCGCAAGGTCTCGATTGTGGGCTGGAGTCTCGGCGGCGTCTATGCGCGCGACC tcgcgttgcaggcgcccgagatggtgcgctacgtcgtcaccctcggcagcccctt

CGCCAGCGACGTCCGCGCCACCAATGCGACGCGGCTCTATGAGGCGATGTCGGG

CGAAACGGTCGGCGACAATGTCGACCTCGTGCAGGCGATTGCCGGCGACCTGCC

GGTTCCCGTGACCTCGATCTATTCGAAGAGCGACGGCATCGTGAACTGGCGGACC

TGGCTGGTGGGCGGGTCCGCGACCGCCGAGAATATCGAGGTCTATTTCGCGAGCC atgtcggcatcggcgtcaatccggccgcgctgtgggcgatcgcggagcggctgg

CCCAGCGGGAAGGCGAATTCCGCCCCTTCGACCGGTCCGGTCCTTTTGCCATTGC

ĆTAĆGCGCCCCCGGAACAGGCACAAfCGATCTGA

SEQ ID NO: 20 (coded by SEQ ID NO: 19):

MPERNEAQAPPRLRPPGLGLFLAEARGIFELNASLLLSPLLLRAPRGDGHPVLALPGF

LASDLSMAPLRRYLTELGYDTHAWRMGRNVGGIAKMRIALLERLTQIHAECGRKVS

IVGWSLGGVYARDLALQAPEMVRYWTLGSPFASDVRATNATRLYEAMSGETVGD

NVDLVQAIAGDLPVPVTSIYSKSDGIVNWRTCLLRPSATAENIEVYFASHVGIGVNPA

ALWAIADRLAQREGEFRPFDRSGPFA1AYAPPEQAQSI [0100] Methods of discovering new hydrolase sequences using the nucleic acids described herein have been disclosed. Methods for modifying the nucleic acids disclosed herein by, e.g., GSSM techniques have been disclosed^sm and GeneReassembly^SM. The nucleic acids disclosed herein can be produced, isolated and / or manipulated e.g. by cloning and expression of cDNA libraries, amplification of information or genomic DNA by PCR, etc.

[0101] The primary source of selected exemplary polypeptides and nucleic acids are:

<td>SEQ ID NO:</td><td>Source</td>

<td> 1,2</td><td>Obtained from an environmental sample</td>

<td> 3,4</td><td>Obtained from an environmental sample</td>

<td> 5, 6</td><td>Obtained from an environmental sample</td>

<td> 7,8</td><td>Obtained from an environmental sample</td>

<td> 9, 10</td><td>Obtained from an environmental sample</td>

<td> 11, 12</td><td>Obtained from an environmental sample</td>

<td> 13, 14</td><td>Obtained from an environmental sample</td>

<td> 15, 16</td><td>Bacteria</td>

<td> 17, 18</td><td>Obtained from an environmental sample</td>

<td> 19, 20</td><td>Obtained from an environmental sample</td>

[0102] In the practical implementation of the methods described herein, homologous genes can be modified by manipulating template nucleic acid as described herein. The subject-matter of the invention can be implemented in conjunction with any of the methods, protocols or devices known in the art that are well described in the scientific and patent literature.

General techniques [0103] Nucleic acids, including RNA, RNAi (e.g., siRNA, miRNA), antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or their hybrids, nucleic acids isolated from various sources, modified by genetic engineering, have been disclosed, amplified and / or expressed / recombinantly generated. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for desired activity (e.g. hydrolase activity (e.g. lipase, saturase, palmitase and / or stearatase). Any recombinant expression system can be used, including expression systems based on bacterial, mammalian, yeast, fungal, insect or plant cells.

[0104] Alternatively, these nucleic acids can be synthesized in vitro using well-known chemical synthesis techniques, as described, e.g., in Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acids Res. 25: 3440-3444; Frenkel (1995) Free Radie. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; in U.S. Patent No. 4,458,066.

[0105] Nucleic acid manipulation techniques such as, e.g., subcloning, use of labeling probes (e.g., labeling using random primers using a Klenow fragment polymerase, nick translation, amplification), sequencing, hybridization, etc. are well described in the scientific and patent literature, see e.g. Sambrook (ed.), MOLECULAR CLONING: A LABORATORY MANUAL (ed. 2), volumes 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel (ed.), John Wiley &amp; Sons, Inc., New York (1997);

LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen (ed.), Elsevier, New York (1993).

[0106] Another useful method of obtaining and manipulating nucleic acids used to implement the methods described herein is cloning from genomic samples and, if desired, screening and re-cloning of inserts isolated or amplified, e.g. from genomic clones or cDNA clones. Nucleic acid sources used in the methods provided herein include genomic or cDNA libraries contained, e.g., in mammalian artificial chromosomes (MAC), see for example. U.S. Patent Nos. 5,721,118; 6,025 155; human artificial chromosomes, see e.g. Rosenfeld (1997) Nat. Genet. 15: 333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); artificial chromosomes derived from phage P1, see e.g. Woon (1998) Genomics 50: 306-316; vectors derived from Pl phage (PAC), see e.g., Kern (1997) Biotechniques 23: 120-124; cosmids, recombinant viruses, phages or plasmids.

[0107] The terms "nucleic acid" or "nucleic acid sequence" may include an oligonucleotide, nucleotide, polynucleotide or fragment of any of them, DNA or RNA (e.g., mRNA, rRNA, tRNA, RNAi) of genomic or synthetic origin, which may be single-stranded or double-stranded, and may represent a sense or antisense strand, peptide nucleic acid (PNA), or any DNA-like or RNA-like material of natural or synthetic origin, including, e.g. RNAi (double-stranded "interfering" RNA), ribonucleoproteins (e.g., iRNPs). This term includes nucleic acids, i.e. oligonucleotides, containing known analogues of natural nucleotides. This term also includes structures similar to nucleic acid structures with a synthetic backbone, see e.g. Mata (1997) Toxicol. Appl. Pharmacol. 144: 189-197; Strauss-Soukup (1997) Biochemistry 36: 8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156.

As used herein, the term "promoter" includes all sequences capable of directing transcription of a coding sequence in a cell, e.g., a plant cell. Thus, the promoters disclosed herein used in the disclosed constructs include cis transcriptional control elements and regulatory sequences that are involved in regulation or modulating the time and / or speed of gene transcription. For example, the promoter may be a cis transcriptional control element, including an enhancer, promoter, transcription terminator, origin of replication, chromosome integration sequence, 5 'and 3' untranslated regions, or intron sequence that are involved in regulating transcription. Cis sequences usually interact with proteins or other biomolecules to carry out (on / off, regulate, modulate, etc.) transcription. "Constitutive" promoters are those that direct expression continuously, under most environmental conditions and developmental stages or cell differentiation. "Induced" or "regulated" promoters direct the expression of the nucleic acid disclosed herein under the influence of environmental or developmental conditions. Examples of conditions environments that may affect transcription through inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.

[0109] "Tissue-specific" promoters are transcriptional control elements that are active only in specific cells or tissues or organs, e.g. in plants or animals. Tissue-specific regulation can be achieved by the action of certain intrinsic factors that ensure expression of the coding genes tissue-specific proteins, known to occur in mammals and plants, allowing the formation of specific tissues.

[0110] The term "plant" includes whole plants, parts of plants (e.g., leaves, stems, flowers, roots, etc.), plant protoplasts, plant seeds and cells and their progeny. A class of plants that can be used in the method provided herein includes in general, a whole class of higher plants subject to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous) as well as gymnosperms. It includes plants with varying levels of ploidy, including polyploids, diploids, haploids, and hemizygous states. The term "transgenic plant" as used herein includes plants or plant cells into which a heterologous nucleic acid sequence has been introduced, e.g., nucleic acids disclosed herein, and various recombinant constructs (e.g., expression cassettes).

[0111] In one aspect, the nucleic acid encoding the polypeptide disclosed herein is assembled in an appropriate phase using a leader sequence capable of regulating the secretion of the polypeptide obtained by translation or a fragment thereof.

[0112] Fusion proteins and nucleic acids encoding them have been disclosed. The polypeptide disclosed herein can be fused to a heterologous peptide or polypeptide, such as N-terminal identifying peptides, conferring desired characteristics, such as increased stability or simplified purification. The peptides and polypeptides disclosed herein may also be synthesized and expressed as fusion proteins with one or more additional domains attached thereto, e.g. in order to produce a peptide with greater immunogenicity, easier isolation of a peptide synthesized by recombination, to identify and isolate antibodies and B cells expressing antibodies, etc. Domains facilitating detection and purification include, e.g. metal chelating peptides, such as polyhistidine sequences and histidine-tryptophan modules, which allow purification of the mobilized metals on them, protein A domains that allow purification of the mobilized immunoglobulin on them, and the domain used by the FLAG epitope affinity purification kit (Immunex Corp, Seattle WA, USA). Incorporation of a linker sequence containing a cleavage site, such as sequences with a factor Xa cleavage site or enterokinase (Invitrogen,

San Diego CA, USA) between the purification domain and the peptide or polypeptide containing the appropriate motif, can facilitate purification. For example, the expression vector may contain a nucleic acid sequence encoding an epitope associated with six histidine residues with downstream thioredoxin and an enterokinase cleavage site (see, e.g., Williams (1995) Biochemistry 34: 1787-1797; Dobeli (1998) Protein Expr. Purif 12: 404-414). Histidine residues facilitate detection and purification, while the enterokinase cleavage site provides agents for purifying the epitope from the remainder of the fusion protein. Techniques for vectors encoding fusion proteins and the use of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12: 441-53.

Transcription and translacyine control sequences [0113] Nucleic acid sequences (e.g., DNA, iRNA) are disclosed operably linked to expression control sequences (e.g., transcriptional or translational), e.g., promoters or enhancers, to direct or modulate RNA synthesis / expression. The expression control sequence may be present in the expression vector. Examples of bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplary eukaryotic promoters include direct-early CMV, HSV thymidine kinase, SV40 early and late, retrovirus LTR sequences, and murine metallothionein.

[0114] Promoters suitable for expression of a polypeptide in bacteria include E. coli lac or trp promoters, lacl promoter, lacZ promoter, T3 promoter, T7 promoter, gpt promoter, lambda PR promoter, lambda PL promoter, promoters from operons encoding glycolytic enzymes, such as phosphoglycerate 3-kinase (PGK) and the acid phosphatase promoter. Eukaryotic promoters include the early-early CMV promoter, the HSV thymidine kinase promoter, heat shock protein promoters, SV40 early and late promoter, LTR sequences from retroviruses and the mouse metallothionein-I promoter. Other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses may also be used.

Tissue-specific plant promoters [0115] Expression cassettes are disclosed that can be expressed in a tissue-specific manner in which, for example, the expression of the hydrolase disclosed herein can be expressed in the tissue-specific disclosed manner. Plants or seeds have been disclosed which express the hydrolases disclosed herein in a tissue-specific manner. Tissue specificity can mean sperm specificity, stem specificity, leaf specificity, root specificity, fruit specificity, etc.

[0116] In one aspect, a constitutive promoter such as the CaMV 35S promoter can be used to express in certain parts of the plant or seed, or throughout the entire plant. For example, to overexpress the hydrolase disclosed herein, a plant promoter fragment may be used that will direct the expression of nucleic acid in some or all of the plant's tissues, e.g., a regenerated plant. Such "constitutive" promoters are active under most environmental conditions and developmental stages or cell differentiation. Examples of constitutive promoters include the transcription initiation region of the cauliflower mosaic virus (CaMV) 35S, the 1'- or 2'-promoter derived from the Agrobacterium tumefaciens T-DNA. and other transcription initiation areas from various plant genes known to those skilled in the art, such as, for example, ACT11 from Arabidopsis (Huang (1996) Plant Mol. Biol. 33: 125-139); Cat3 from Arabidopsis (GenBank No. U43147, Zhong (1996) Mol. Gen. Genet. 251: 196-203); gene coding for stearoyl-ACP desaturase from Brassica napus (Genbank No. Χ74782, Solocombe (1994) Plant Physiol. 104: 1167-1176); Maize GPcl (GenBank No. Χ15596; Martinez (1989) 1 Mol. Biol 208: 551-565); Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol. Biol. 33: 97-112); plant promoters described in U.S. Patent Nos. 4,962,628; 5 633 440.

[0117] Tissue-specific or constitutive promoters derived from viruses have been disclosed, which may include, e.g., the subgenomic tobamovirus promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92: 1679-1683; Bacillus rice tungro virus (RTBV), which replicates only in the bast cells of infected rice plants, together with a promoter that directs the expression of a strong bast-specific reporter gene; CVMV (cassava nerve mosaic virus) promoter, showing the highest activity in vascular elements, in mesophyll leaf cells, and in the root apexes (Verdaguer (1996) Plant Mol. Biol. 31: 1129-1139).

[0118] Alternatively, the plant promoter may direct the expression of a hydrolase expressing nucleic acid in a particular tissue, organ or cell type (i.e., they are tissue specific promoters) or may otherwise be under more stringent environmental or developmental control or under the control of an inducible promoter. Examples of environmental conditions that may affect transcription include anaerobic conditions, elevated temperature, the presence of light, or chemical / hormone spraying. Drought-induced maize promoters have been disclosed (Busk (1997) see above); Potato promoter induced by cold, drought and high salinity (Kirch (1997) Plant Mol. Biol. 33: 897 909).

[0119] Tissue specific promoters can promote transcription only during a certain development period within that tissue. See. e.g. Blazquez (1998) Plant Cell 10: 791-800, where the LEAFY gene promoter in Arabidopsis was characterized. See. also Cardon (1997) Plant J 12: 367-77, which describes the SPL3 transcription factor that recognizes the conserved sequence motif in the promoter region of the A. thaiiana · flower meristem1 gene identity, and Mandel (1995) Plant Molecular Biology, Tom 29, p. 995-1004, where the eIF4 meristem promoter is described. Tissue-specific promoters that are active throughout the entire life cycle of a particular tissue can be used. In one aspect, the nucleic acids disclosed herein are functionally linked to a promoter substantially active only in cotton fibrous cells. In one aspect, the nucleic acids disclosed herein are functionally linked to an active promoter mainly during the stages of cotton fibrous cell elongation, e.g. as described by Rinehart (1996), see above. Nucleic acids can be functionally linked to the Fbl2A gene promoter so that they have their preferential expression in cotton fibrous cells (ibid.). See. also John (1997) Proc. Natl. Acad. Sci. USA 89: 5769-5773; John et al., U.S. Patent Nos. 5,608,148 and 5,602,321, which describe cotton-specific promoters and methods for constructing transgenic cotton plants. Root-specific promoters can also be used to express the nucleic acids disclosed herein. Examples of root-specific promoters include the promoter from the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol. 123: 39-60). Other promoters that can be used to express the nucleic acids disclosed herein include, e.g. ovule-specific, embryo-specific, endosperm-specific, shell-specific, shell-specific promoters, or some combinations thereof; leaf specific promoter (see e.g. Busk (1997) Plant J. 11: 1285 1295, where a maize leaf specific promoter is described); Agrobacterium rhizogenes ORF13 promoter (showing high root activity, see e.g. Hansen (1997) above); maize pollen specific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224: 161 168); an active tomato promoter during fruit ripening, aging and leaf fall, and to a lesser extent, flowers (see, e.g., Blume (1997) Plant J. 12: 731 746) can be used; potato-specific SK2 gene promoter (see, e.g., Ficker (1997) Plant Mol. Biol. 35: 425 431); the Blec4 gene from peas, which is active in the epidermal tissue of the tips of vegetative and flower shoots of transgenic alfalfa, which makes it a useful tool for directing the expression of foreign genes to the epidermis layer of actively growing shoots or fibers; ovule-specific BELI gene (see e.g. Reiser (1995) Cell 83: 735-742, GenBank No. U39944); and / or the promoter described by Klee in U.S. Patent No. 5 589 583, depicting a plant promoter region providing high levels of transcription in creative tissue and / or rapidly dividing cells.

[0120] Alternatively, plant promoters that are induced by exposure to plant hormones such as auxins are used to express the nucleic acids disclosed herein. Promoters are disclosed comprising an El promoter fragment with auxin response elements (AuxREs) in soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115: 397-407); Auxin-responsive GST6 promoter from Arabidopsis (also responding to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); auxin-induced tobacco parC promoter (Sakai (1996) 37: 906-913); plant biotin response element (Streit (1997) Mol. Plant Microbe Interact. 10: 93346

937); and the stress hormone responsive promoter abscinic acid (Sheen (1996) Science 274: 1900-1902).

[0121] Nucleic acids disclosed herein may also be functionally linked to plant promoters that are induced by exposure to plant chemical reagents such as herbicides or antibiotics. For example, the In2-2 maize promoter activated by benzenesulfonamide herbicide detoxifiers can be used, (De Veylder (1997) Plant Cell Physiol. 38: 568-577); the use of various safeners added to the herbicide induces different gene expression patterns, including root, hydatode and apical meristem expression. Coding sequences can be controlled, e.g., by a tetracycline-inducible promoter, e.g., as described for transgenic tobacco plants containing the arginine decarboxylase gene from Avena sativa L. (oats) (Masgrau (1997) Plant 1 11: 465-473); or salicylic acid responder (Stange (1997) Plant J. 11: 1315-1324). By using chemically induced promoters (e.g., hormones or pesticides), i.e. a chemical responsive promoter that can be applied to a transgenic plant in the field, the expression of the polypeptide disclosed herein can be induced at a particular stage of plant development. Transgenic plants containing an induced gene encoding for the polypeptides disclosed herein have been disclosed whose host range is limited to target plant species, such as corn, rice, barley, wheat, potato or other crops, induced at any stage of crop development.

[0122] Tissue specific plant promoters can direct the expression of functionally related sequences in tissues other than the target tissue. Thus, a tissue-specific promoter is one that directs expression preferably in the target tissue or cell type, but can also lead to some expression in other tissues.

[0123] Nucleic acids disclosed herein may also be functionally linked to plant promoters that are induced by exposure to chemical reagents. These reagents include, e.g., herbicides, synthetic auxins or antibiotics, which can be applied, e.g. by spraying, to transgenic plants. The induced expression of the hydrolase producing nucleic acids disclosed herein will allow the grower to select plants with an optimal starch: sugar ratio. Thus, the development of individual plant parts can be controlled.

[0124] Agents facilitating the collection of plants and individual plant parts have been disclosed. For example, in various embodiments, the maize In2-2 promoter activated by a benzenesulfonamide herbicide detoxifier (De Veylder (1997) Plant Cell Physiol. 38: 558-577) is used; the use of various safeners added to the herbicide induces different gene expression patterns, including root, hydatode and apical meristem expression. The coding sequences disclosed herein are also controlled by a tetracycline-inducible promoter, e.g. as described for transgenic tobacco plants containing the arginine decarboxylase gene from Λνε / 73 sativa L. (oats) (Masgrau (1997) Plant J. 11: 465-473); or salicylic acid responder (Stange (1997) Plant J. 11: 1315-1324).

[0125] If adequate expression of the polypeptide is desired, a polyadenylation region at the 3'-end of the coding region should be attached. The polyadenylation region may be derived from a natural gene, from a variety of other plant genes, or from Agrobacterium T-DNA genes.

Expression Vectors and Cloning Carriers [0126] Expression vectors, expression cassettes and cloning carriers containing nucleic acids, e.g., hydrolase and antibody coding sequences, have been disclosed. The expression vectors and cloning carriers disclosed herein may include viral particles, baculoviruses, phages, plasmids, phagemids, cosmids, phosmids, artificial bacterial chromosomes, viral DNA (e.g. bovine pox, adenovirus, avipox virus, false rabies and SV40 derivatives), artificial chromosomes based on PI phage, yeast plasmids, artificial yeast chromosomes and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast). The vectors disclosed herein may include chromosomal, non-chromosomal and synthetic DNA sequences. A large number of suitable vectors are known to those skilled in the art and are commercially available. Examples of vectors include: bacterial: pQE vectors (Qiagen), plasmids pBLUESCRIPT ™, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRlT2T (Phannacia); eukaryotic: pXTl, pXTl, pEg , pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vector can be used as long as it can replicate and remain viable in the host. Vectors with low copy numbers or vectors with high copy numbers can be used.

[0127] The "expression cassette" disclosed herein comprises a nucleotide sequence that is capable of expressing a structural gene (i.e., a protein coding sequence such as the hydrolase disclosed herein) in a host organism compatible with such a sequence. The expression cassettes contain at least a functionally related promoter with a polypeptide coding sequence, and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or useful for expression may also be used, e.g. enhancers. As used herein, the term "operably linked" refers to the combination of a promoter upstream of a DNA sequence in such a way that the promoter mediates transcription of the DNA sequence. Thus, expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant vector of the "naked" DNA "etc. A "vector" contains a nucleic acid that can infect, transfect, transduce a cell in a transient or stable manner. It is understood that the vector may be a naked nucleic acid or a complexed protein or lipid nucleic acid. The vector optionally contains viral or bacterial nucleic acids and / or proteins and / or membranes (e.g., cell membrane, viral lipid envelope, etc.) Vectors include, but are not limited to, replicons (e.g. RNA replicons, bacteriophages) to which DNA fragments can be attached and replicated. Thus, vectors include, but are not limited to, RNA, autonomous selfplacing and staggering circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, e.g., U.S. Patent No. 5,217,879), and includes both expression and non-expression plasmids. Where a recombinant microorganism or cell culture is described as a host for the "expression vector" this includes both extrachromosomal circular and linear DNA as well as DNA that has been introduced into the host chromosome (s). In the event that the vector is retained by the host cell, this vector can either be stably replicated by cells during mitosis as an autonomous structure, or become incorporated into the host genome.

[0128] The expression vector may include a promoter, ribosome binding site for translation initiation, and transcription terminator. The vector may also contain appropriate sequences to increase expression. Mammalian expression vectors may include an origin of replication, any necessary ribosome binding sites, polyadenylation site, donor and acceptor splice sites, transcription termination sequences, and 5 'non-transcribing flanking sequences. In some aspects, DNA sequences derived from SV40 splice and polyadenylation sites can be used to provide the required non-transcribed genetic elements.

[0129] In one aspect, the expression vectors contain one or more selectable marker genes that allow selection of host cells containing the vector. Such selectable markers include genes encoding dihydrofolic acid reductase or genes conferring neomycin resistance in eukaryotic cell cultures, genes conferring tetracycline or ampicillin resistance in E. coli, and the TRP1 gene in S. cerevisiae. Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers.

[0130] Vectors for expressing the polypeptide or fragment thereof in eukaryotic cells may also contain enhancers to increase expression levels. Enhancers (enhancers) are cis-acting ("cis-acting") DNA elements, typically from about 10 to about 300 bp in length, acting on the promoter to increase transcription. Examples include the SV40 enhancer downstream of the bp 100 to 270 replication origin, the cytomegalovirus early promoter enhancer sequence, the polyoma enhancer sequence downstream of the replication origin, and adenovirus enhancer sequences.

[0131] The DNA sequence may be inserted into the vector using a variety of procedures. Generally, the DNA sequence is ligated to the desired position in the vector after digestion of the insert and vector with appropriate restriction endonucleases. Alternatively, the blunt ends of the insert and vector may be ligated. A number of cloning techniques are known in the art, e.g. as described by Ausubel and Sambrook. It can be considered that these and other procedures are within the skill of those skilled in the art.

[0132] The vector may be in the form of a plasmid, viral particle or phage. Other vectors include chromosomal, non-chromosomal and synthetic DNA sequences, SV40 derivatives; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, viral DNA such as bovine pox, adenovirus, bird pox virus and pseudo rabies. It has been described (e.g. by Sambrook) a number of different cloning vectors and expression vectors that are used in prokaryotic and eukaryotic host organisms.

[0133] Specific bacterial vectors that can be used include commercially available plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEMl ™ (Promega Biotec, Madison , Wl, USA) pQE70, pQE60, pQE-9 (Qiagen), pDlO, psiX174 Pbluescript II KS ™, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233R, DR540 ), pKK232-8 and pCM7. Specific eukaryotic vectors include pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any other vector can be used as long as it can replicate and remain viable in the host cell.

[0134] Expression of the nucleic acids disclosed herein may occur in expression cassettes, vectors or viruses, and transiently or stably in plant cells and seeds. One exemplary transient expression system utilizes episomal expression systems, e.g., cauliflower mosaic virus (CaMV) viral RNA produced in the nucleus by transcription of an episomal minichromosome containing super-developed DNA, see e.g. Covey (1990) Proc. Natl. Acad. Sci. USA 87: 16331637. Alternatively, the coding sequences, i.e. all or sub-fragments of the sequences disclosed herein can be introduced into the genome of the plant host cell and become an integral part of the host chromosomal DNA. In this way, sense or antisense transcripts can be expressed. The vector containing these sequences (e.g., promoters or coding regions) from nucleic acids as disclosed herein may contain a marker gene that gives the plant cell or seed a selective phenotype. For example, the marker may encode biocide resistance, in particular antibiotic resistance, e.g. kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as chlorosulfuron or Basta resistance.

[0135] Expression vectors capable of expressing nucleic acids and proteins in plants are well known in the art and may include, e.g., vectors from Agrobacterium spp., Potato virus X (see, e.g., Angell (1997) EMBO J. 16: 3675 -3684), tobacco mosaic virus (see e.g. Casper (1996) Gene 173: 69-73), tomato bushy dwarf virus (see e.g. Hillman (1989) Virology 169: 42-50), tobacco pitting virus ( see e.g. Dolja (1997) Virology 234: 243-252), golden bean mosaic virus (see e.g. Morinaga (1993) Microbiol Immunol. 37: 471-476), cauliflower mosaic virus (see e.g. Cecchini (1997) Mol. Plant Microbe Interact. 10: 1094-1101), Ac / Ds transposons from maize (see e.g. Rubin (1997) Mol. Cell. Biol. 17: 6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204 : 161-194), and a transposon suppressor-mutator (Spm) from maize (see, e.g., Schlappi (1996) Plant Mol. Biol. 32: 717-725); and their derivatives.

[0136] In one aspect, the expression vector may have two replication systems, which allows it to be preserved in two organisms, for example in mammalian, yeast, fungal or insect cells for expression and in a prokaryotic host for cloning and amplification. In addition, to integrate expression vectors, the expression vector may contain at least one sequence homologous to the host cell genome. It may contain two homologous sequences flanking an expression construct. The integration vector can be oriented to a specific iocus in the host cell by selecting the appropriate homologous sequence to be included in the vector. Constructs for integration vectors are well known in the art.

[0137] The expression vectors disclosed herein may also include a selectable marker gene that allows selection of transformed bacterial strains, e.g., genes that confer resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selective markers may also include genes involved in biosynthesis, such as genes involved in histidine biosynthesis pathways, tryptophan and leucine.

Host cells and transformed cells [0138] Transformed cells containing a nucleic acid sequence, e.g., a sequence encoding a hydrolase or antibody, or a vector disclosed herein have been disclosed. The host cell may be any of the host cells known to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells or plant cells.

[0139] The enzymes disclosed herein can be expressed in any host cell, e.g., any bacterial cell, any yeast cell, any Saccharomyces or Schizosaccharomyces spp., Any Pichia spp., E.g. Pichia pastoris, Saccharomyces cerevisiae or Schizosaccharomyces. Examples of bacterial cells include any Streptomyces or Badiius spp., E.g. E. coli, Lactococcus lactis, Bacillus subtilis, Bocillus cereus, Salmonella typhimurium or any species from the genera Bacillus, Streptomyces and Staphylococcus. Examples of insect cells include Drosophila S2 and Spodoptera Sf9. Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. Choosing the right host is within the skill of those skilled in the art. Techniques for transforming various types of higher plant species are well known and have been described in technical and scientific literature. See. e.g. Weising (1988) Ann. Rev. Genet. 22: 421-477, US Patent No. 5,750,870.

[0140] The vector can be introduced into host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene shotguns or gene transfer via Ti plasmid. Specific methods include calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofection or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

[0141] If appropriate, genetically engineered host cells can be cultured in conventional culture media, appropriately modified to activate promoters, select transformants, or amplify the genes disclosed herein. After transformation of the appropriate host strain and growth of the host strain to the appropriate density of the cell suspension, the selected promoter may be induced by appropriate means (e.g. temperature shift or chemical induction), and the cells can be cultured for an additional period of time, allowing them to produce the desired polypeptide or fragment.

[0142] In one aspect, the nucleic acids or vectors disclosed herein are introduced into the cells for screening, so the nucleic acids enter the cells in a manner suitable for further expression of the nucleic acid. The input method is largely dictated by the type of target cell. Examples of methods include precipitation with CaPO₄, fusion to liposomes, lipofection (e.g. LIPOFECTIN ™), electroporaga, viral infection, etc. Candidate nucleic acids can stably integrate into the host cell genome (for example when introduced by retrovirus) or may exist either transiently or stably in cytoplasm (i.e. due to the use of traditional plasmids, the use of standard regulatory sequences, selection markers, etc.). Retroviral vectors capable of transfecting this type of target (e.g., mammalian, human cells) have been disclosed, because e.g. many pharmaceutically important screening procedures require human or model mammalian target cells.

[0143] Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract kept for further purification. Cells of microorganisms used for protein expression can be disrupted by any conventional method, including freezing-thawing, sonication, mechanical disruption, or using cell lysing agents. These types of methods are well known to those skilled in the art. The expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell culture by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose bed chromatography, hydrophobic interaction chromatography, affinity chromatography, on hydroxylapatite and lecithin chromatography. If desired, protein refolding steps can be used to complete the configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be used in the final stages of purification.

[0144] Various mammalian cell culture systems can also be used to express recombinant protein. Examples of mammalian expression systems include COS-7 fibroblast lines derived from monkey kidneys and other cell lines capable of expressing proteins from a compatible vector, such as C127, 3T3, CHO, HeLa and BHK cell lines.

[0145] These constructs in host cells can be used in a traditional manner to obtain the gene product encoded by the recombinant sequence. Depending on the host used in the recombinant production procedure, the polypeptides produced by host cells containing this vector may be glycosylated or may be non-glycosylated. The polypeptides disclosed herein may or may not contain an initial methionine amino acid residue.

[0146] Cell-free translation systems can also be used to obtain the polypeptide disclosed herein. Cell-free translation systems can use mRNA chains transcribed from a DNA construct containing a promoter operably linked to the nucleic acid encoding the polypeptide or fragment thereof. In some aspects, the DNA construct may be linearized prior to performing the in vitro transcription reaction. The transcribed mRNA is then incubated with a suitable cell-free translation extract, such as rabbit reticulocyte extract, to obtain the desired polypeptide or fragment thereof.

[0147] Expression vectors may contain one or more selectable marker genes to provide phenotypic features for the selection of transformed host cells, such as dihydrofolic acid reductase or neomycin resistance in eukaryotic cell culture, or such as tetracycline or ampicillin resistance /and.

Nucleic acid amplification [0148] Nucleic acids encoding polypeptides or modified nucleic acids that can be copied, e.g., by amplification, have been disclosed.

Amplification primer pairs for amplifying nucleic acids encoding hydrolase, e.g., lipase, saturase, palmitase and / or stearatase, have been disclosed, wherein the primer pairs are capable of amplifying the nucleic acid sequences disclosed herein. One skilled in the art can design a pair of amplification primer sequences for any portion or full length of these sequences.

[0149] Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of information contained in a cell sample), nucleic acid labeling (e.g. for use in matrix or blotting techniques), nucleic acid detection, or quantification specific nucleic acid in the sample. As disclosed herein, information isolated from a cDNA cell or library is amplified. One skilled in the art can select and design suitable oligonucleotide primers for amplification. Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, edited by Innis, Academic Press, New York (1990) and PCR STRATEGIES (1995), Innis, Academic Press, Inc., New York, Ligase Chain Reaction (LCR) (see, e.g., Wu (1989) Genomics 4: 560; Landegren (1988) Science 241: 1077; Barringer (1990) Gene 89: 117); transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA 86: 1173); and self-supporting sequence replication (see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87: 1874); amplification with Qbeta replicase (see, e.g., Smith (1997) J. Clin. Microbiol. 35: 1477-1491), an automated amplification method based on Q-beta replicase (see, e.g., Burg (1996) Mol. Cell. Probes 10: 257-271) and other techniques involving RNA polymerase (e.g., NASBA, Cangene, Mississauga, Ontario); see. also Berger (1987) Methods Enzymol. 152: 307-316; Sambrook; Ausubel; U.S. Patent Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13: 563-564.

[0150] Amplification primer pairs containing the sequences disclosed herein have been disclosed, for example wherein the primer pair comprises a first member having a sequence defined by approximately the first (end 5⁷) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more nucleic acid residues disclosed herein, and a second member having the sequence determined by approximately the first (end 59 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more chain residues complementary to the first member.

Determining the Degree of Sequence Identity [0151] Nucleic acids containing at least a nucleic acid, or having complete (100%) sequence identity with the nucleic acid disclosed herein, e.g., having the nucleic acid disclosed herein (e.g., having the sequence given as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID

NO: 22 or SEQ ID NO: 23, or SEQ ID NO: 1 modified to encode one, two, three, four, five, six, seven, eight or more (several) or all policy changes described in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, or their equivalents); and polypeptides showing at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or greater or full (100%) sequence identity with the polypeptide disclosed herein, e.g. an exemplary polypeptide having the sequence given as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or SEQ ID NO: 2 containing one, two, three, four, five, six, seven, eight or more (several) or all amino acid changes described in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, or their equivalents. In alternative aspects, sequence identity may extend over an area of ​​at least about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 3 50, 400, 450, 500, 5 50, 600, 650 , 700, 750, 800, 850, 900, 950, 1000 or more consecutive residues, or the entire length of a nucleic acid or polypeptide. The extent of sequence identity (homology) can be determined using any computer program and related parameters, including those described in the present invention, such as BLAST 2.2.2. or FASTA version 3.0t78, with default parameters. The terms "computer", "computer program" and "processor" as used herein are used in their broadest general context and include all such devices as described in detail below.

[0152] The following table lists selected features of the exemplary nucleic acids and polypeptides disclosed herein, including a comparison of the sequence identities of the exemplary sequences with public databases to determine the activity of the enzymes disclosed herein by homology (sequence identity) analysis. All sequences described in this table (all exemplary sequences disclosed herein) were subjected to BLAST search (as described in detail below) against two database files. The first database collection is made available through the NCBI (National Center for Biotechnology Information). All results of searches against these databases are in the columns titled "NR Description", "NR Accession Code", "NR E Value" or "NR Organism". "NR" refers to non-redundant ( non-redundant) nucleotide database maintained by NCBI. This database is a combination of the GenBank database, GenBank upgrades and EMBL upgrades. Entries in the column "Description NR" refer to the definition line in any given NCBI database record that contains a description of the sequence, such as: source organism, gene name / protein name, or some description of the function of this sequence, thus identifying the activity of the listed examples of the enzymes disclosed herein by homology (sequence identity). Entries in the column "Accession Code NR" refer to the unique identifier given to the record with the sequence. The entries in the column "Value E NR" refer to the expected value (value E; Evalue), showing the probability that the match result as good as the match result between the query sequence (the sequences disclosed here) and the sequence in the database will be found in this the same number of comparisons between random sequences that was with the current BLAST search. The entries in the "Organism NR" column refer to a source organism containing the sequence identified as the nearest BLAST hit (sequence homology). The second database set is collectively referred to as the GENESEQ ™ database, which is provided by Thomson Derwent (Philadelphia, PA, USA). All search results for this database are in the columns titled "GENESEQ ™ - Protein Description", "GENESEQ ™ - Protein Accession Code", "GENESEQ ™ - Protein E Value", "GENESEQ ™ - DNA Description", "GENESEQ ™ - DNA accession code "or" GENESEQ ™ E DNA value ". The information in these columns is comparable to the information in columns NR described above, except that it is from a BLAST search against the GENESEQ ™ database instead of the NCBI database. The columns "Query DNA length" and "Query protein length" refer to the number of nucleotides or the number of amino acids in the sequence disclosed herein, respectively, which was searched or queried against either the NCBI or GENESEQ ™ database. "GENESEQ ™ or NR DNA Length" columns and "GENESEQ ™ or DNA Length

NR "refers to the number of nucleotides or the number of amino acids in the best-fit sequence of the BLAST search, respectively. The results shown in these columns are from that search that returned a lower E value, be it NCBI databases or Geneseq databases. Columns" GENESEQ / NR% Protein ID "and" GENESEQ ™ / NR% ID DNA "refer to the percentage sequence identity between the sequence disclosed herein and the best BLAST alignment sequence. The results shown in these columns are from this search, which returned a lower E value, be it NCBI databases or GENESEQ ™ databases.

<td>Genese q / NO %ID GOUT</td><td></td><td></td>

<td>Value E DNA Genese q</td><td>about</td><td> 00</td>

<td>Code accession GOUT GeneSeq</td><td>AQZ64878</td><td>ACA26233</td>

<td>DNA description GeneSeq</td><td>peptide showing activity SEQ hydrolases ID NO: 2.</td><td>Protein coded by prokaryotic</td>

<td>Value E protein Genese q</td><td>Ι, ΟΟΕ127</td><td>3,00E39</td>

<td>Code accession proteins GeneSeq</td><td>AQZ6487 9</td><td>AQZ64879</td>

<td>Description of the protein GeneSeq</td><td>peptide showing activity SEQ hydrolases ID NO: 2.</td><td>peptide showing activity SEQ hydrolases</td>

<td>Organism NO</td><td>Sphingopyxis aiaskensis RB2256</td><td>Sphingopyxis aiaskensis R2256</td>

<td>'s worth Ć E NR</td><td>7,00E40</td><td>2,00E40</td>

<td>Code accession NO</td><td> 103485 777</td><td> 10348577 7</td>

<td>Description NO</td><td>&lt;u (Λ C ΙΛ Ϊ S - ϊ. £ S. BS 8- «" S 8 f S &amp; V, ~ G cn c «3 o&gt; χΤ cn c vo &lt;u _about oc &lt;u in cn in ni οι n ^c about e 1 r \ i oo m in £ .2 Έ ™ O = c ro -5 mr \ and cn 7? u_ c 8 -sn^ω.9-.5 ro ^ -SIng - cg § .9- .2 # 15 g r η σι ci in ci σ&gt; - &lt;rr ii id a:</td><td>hypothetical protein Sala_0282 [Sphingopyxis</td>

<td>in o- a 2</td><td>f \ l</td><td>NT What</td>

<td></td><td></td>

<td></td><td> 0,53</td>

<td></td><td>AQZ64878</td>

<td>y gen necessary # 30232.</td><td>peptide showing activity SEQ hydrolases ID NO: 2. and</td>

<td></td><td>3,00E39</td>

<td></td><td>AQZ64879</td>

<td>ID NO: 2.</td><td>peptide showing activity SEQ hydrolases ID NO: 2.</td>

<td></td><td>Sphingopyxis alaskensis RB2256</td>

<td></td><td>8,00E42</td>

<td></td><td> 10348577 7</td>

<td>&lt;u ξ .52 S 7 I Ϊ s. (Λ 00 LT) -tt N Q- W (Λ i — i Lf) O&gt; VO tfl r — i C3 LO IX O&gt; &amp; O) C LO o ιλ σι &lt;~ \ and ni &lt;uoc: &lt;u U7 (N 03 lo $ · «.2 Έ &lt;" M wf \ i σι 7? U- c 8 - * · n^ωrc 03 - Π, ω by 8-53 (Λ -53 0Q ro οί σι - &lt;.2 r £) lL ro oi</td><td>ABOUT) In ξ ΙΛ £ 2 - and £ 2 &amp; &amp; 2? ocnc ^&gt; Lno&gt; 2? · cn &lt;uo ca&gt; Lncor \ ini &lt;uoC «£: | ΐ s &lt;: | «S 5 .9 .2 ra £ &lt;2 £ cmo. 8- .53 fi</td>

<td></td><td> 5, 6</td>

<td colspan="2"></td><td colspan="14"></td><td colspan="5"></td>

<td></td><td></td><td>LU</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LLI</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Ο</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>ο</td><td>Μ "</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>C0</td><td>IX</td><td></td><td></td><td></td>

<td></td><td></td><td>τ-1</td><td>ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Τ "Η</td><td>ο</td><td></td><td></td><td></td>

<td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>IX</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>IX</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>KD</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LO</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>(XI</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1X1</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>θ '</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>θ '</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &lt;</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>θ '</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>θ '</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>LU</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LLI</td><td></td>

<td></td><td></td><td></td><td>δστ</td><td>ο</td><td>(Λ δ "</td><td>fN</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>δnr</td><td>: « η</td><td>(Λ δ "</td><td>(Ν</td>

<td></td><td></td><td>ptyd</td><td>σ Ν J2</td><td>C</td><td>03 2 σ</td><td>ό from</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ptyd</td><td>σ Ν 2</td><td>C</td><td>03 2 XI</td><td>ό ζ</td>

<td></td><td></td><td> 0)</td><td></td><td></td><td></td><td>ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ω</td><td></td><td></td><td></td><td>ο</td>

<td></td><td></td><td>CL</td><td> §</td><td>ίϋ</td><td>-C</td><td> 1—1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Ο.</td><td> §</td><td> 03</td><td>-C</td><td> 1—4</td>

<td></td><td></td><td>LLI</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LLI</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>Ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Ο</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>What Γχ</td><td> 44</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ςσ (Ν</td><td> 42</td><td></td><td></td><td></td>

<td></td><td></td><td>σ&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>σ</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>IX</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>IX</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>U0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &lt;£&gt;</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>ιχι</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ΓΧΙ</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>θ '</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>θ '</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &lt;</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>θ '</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>θ '</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>LU</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LLI</td><td></td>

<td></td><td></td><td></td><td>δnr</td><td>: « ο</td><td>ΙΛ δ ·</td><td>ΓΜ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>δ * No.</td><td>: « ο</td><td>ΙΛ δ "</td><td>Ν</td>

<td></td><td></td><td>ptyd</td><td>3 Ν .2</td><td>C</td><td>03 2 Ό</td><td>ό ζ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ptyd</td><td>Ν</td><td>C</td><td>03 2 σ</td><td>ό ζ</td>

<td></td><td></td><td>ω</td><td></td><td></td><td> &gt;~</td><td>ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ω</td><td></td><td></td><td></td><td>Ω</td>

<td></td><td></td><td>ο.</td><td>S</td><td> 03</td><td>_c</td><td>Η-Ι</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ol</td><td></td><td> 03</td><td>-C</td><td>Η-Ι</td>

<td></td><td></td><td>IN</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ω</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> £</td><td rowspan="3">ω 'ω c</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> £</td><td>ιη</td><td></td><td></td><td></td>

<td></td><td></td><td>C..1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Ω_</td><td rowspan="2">σι C</td><td></td><td></td><td></td>

<td></td><td></td><td>ο</td><td>ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ο</td><td>U0</td><td></td><td></td>

<td></td><td></td><td>σι</td><td>&lt;ρ</td><td>LO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>σ&gt;</td><td>Φ</td><td>1_Γί</td><td></td><td></td>

<td></td><td></td><td>C</td><td> 22</td><td>γχι</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>C</td><td></td><td>ΓΜ</td><td></td><td></td>

<td></td><td></td><td>_c</td><td>ω</td><td>(XI</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>_c</td><td>(Λ</td><td>(XI</td><td></td><td></td>

<td></td><td></td><td>spl</td><td>ala</td><td>RB</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>sp</td><td> 03 03</td><td>C0 CC</td><td></td><td></td>

<td></td><td></td><td>LLI</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LLI</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>Ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Ο</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>Ο</td><td>&lt;Χ&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Ο</td><td>Η-τ</td><td></td><td></td><td></td>

<td></td><td></td><td>Η-τ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ΓΟ</td><td>LO</td><td></td><td></td><td></td>

<td></td><td></td><td>IX</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>IX</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>IX</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>IX</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>ιη</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LD</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>'Τ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>m</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ΟΙ</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ο</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 7—|</td><td>IX</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>τ ι-</td><td>IX</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>ω</td><td></td><td></td><td></td><td> —</td><td></td><td></td><td></td><td>ω</td><td></td><td></td><td></td><td></td><td></td><td>ω</td><td></td>

<td></td><td></td><td>AD</td><td></td><td></td><td> £</td><td></td><td>σ_</td><td>-Ω</td><td>τΗ</td><td></td><td> 03</td><td></td><td> £</td><td></td><td></td><td>AD</td><td></td><td></td><td> £</td><td></td>

<td>ω</td><td></td><td>Ν</td><td></td><td>Γ \ Ι</td><td>Ω.</td><td>ΙΛ</td><td></td><td>σι</td><td>ιη</td><td rowspan="2"></td><td>AT</td><td></td><td>Ω.</td><td>ω</td><td></td><td>Ν</td><td></td><td>Γ \ Ι</td><td>Ω.</td><td>ω</td>

<td>ΙΛ</td><td> |—1</td><td>s</td><td></td><td> 00</td><td>Ο</td><td>V)</td><td> |—1</td><td></td><td>ο</td><td> 4-1</td><td></td><td>Ο</td><td>tn</td><td>f-I</td><td>s</td><td></td><td> 00</td><td>Ο</td><td>(Ζ)</td>

<td>C</td><td>ο</td><td>RJ *</td><td></td><td>(Ν</td><td>σ&gt;</td><td>C</td><td>LD</td><td>LO</td><td>ο</td><td>δ</td><td>Φ</td><td></td><td>σι</td><td>C</td><td>ΙΟ</td><td>δ "</td><td></td><td>Γ \ Ι</td><td>σι</td><td>C</td>

<td>ω</td><td>LO</td><td>&lt;υ</td><td>ο</td><td>ο.</td><td>C</td><td>αι</td><td>LD</td><td> 00</td><td>ΓΜ</td><td rowspan="2">Φ (Ζ) C ο</td><td></td><td rowspan="2"> 2</td><td>C</td><td>&lt;υ</td><td>LD</td><td>V</td><td>ο</td><td>ο.</td><td>C</td><td>V</td>

<td>ω _03</td><td>Β22</td><td>4-J Ο Ω.</td><td>12 "ίδ</td><td> 1 25 03</td><td>-C ο. this</td><td>ω 03</td><td>(Ν (Ν C0</td><td>U0 IX σ&gt;</td><td>ιη at_ C0</td><td> &amp;</td><td>-C ο. (Ο</td><td>(Λ 03</td><td>Β22</td><td>4 * Ο Ω.</td><td>12 "ίδ</td><td> 1 25 03</td><td>-C CL this</td><td>ω 03</td>

<td> 03</td><td>cc.</td><td>lc</td><td>jo</td><td>(Ό</td><td>k__l</td><td> 03</td><td>οί</td><td> 00</td><td> &lt;</td><td> 0</td><td>χ:</td><td>Ω.</td><td>ι_ι</td><td> 03</td><td>0C</td><td>ic</td><td>jo</td><td>ΙΛ</td><td></td><td> 03</td>

<td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ο</td><td></td><td></td><td></td>

<td></td><td></td><td>IX</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>σ</td><td>τ-Ι</td><td></td><td></td><td></td>

<td></td><td></td><td></td>

<td></td><td>νο rH</td><td>about</td>

<td></td><td>ACN41328 and</td><td>AOG5399</td>

<td></td><td>Human protein diagnostic and therapeutic SEQ ID NO: 2739.</td><td>Protein</td>

<td></td><td>3,00E42</td><td>Ι, ΟΟΕ-</td>

<td></td><td>AQZ64879</td><td>AOG5399</td>

<td></td><td>peptide showing activity SEQ hydrolases ID NO: 2.</td><td>Protein</td>

<td></td><td>Sphingomonas sp. SKA58</td><td>Plesiocystis</td>

<td></td><td>4,00E46</td><td>3,00E-</td>

<td></td><td> 94497812</td><td> 14992111</td>

<td>&lt;υ ξ (Λ 1 Ξ Η ο £ ro U S- | -¹1 Ο&gt; y Ο (Λ 1 “~¹νο m ο&gt; &amp; cn c &lt;χ&gt; LncorsifljOJocaiLn £ {ίη ιη 8 -g .2 Έ £ m σι m ο .5- .2 (η^- JP co O &lt;00 &lt;-XCC) of L-Lroai</td><td>8 S 8 "S Ϊ "s 2 3 -! S "g" ν ιο E en ιο νο ν about E oo y ή o οι my, -ι om and οι οι and "'£ 1 Ol &lt; (U at 00 C μ- ΙΟ φ at 00 c .9- .2 sts - σι &lt;- .2 (Λ d r Λ 7) id ω oi - ilirrwidw</td><td>hypothetical</td>

<td></td><td>1-T Csl R1 RH</td><td>ro H</td>

<td colspan="12"></td><td colspan="11"></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>WHAT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ABOUT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ABOUT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ABOUT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ABOUT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>PJ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>_l</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>at</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Ρ4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &lt;</td><td>LO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>CT (AT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;1&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td rowspan="2">sequence is</td><td></td><td>(FROM)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ο (Θ ' ΈΓ Ν .5?</td><td>&amp; about c</td><td>£ ίϋ about and- "D</td><td>PJ Q 1-1 θ '</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>I / T 3 _C 4- " c</td><td>LO (N σ&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> &gt;&gt;</td><td>UJ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>a&gt;</td><td></td><td>ίϋ</td><td rowspan="2">p</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>ίϋ</td><td>_c</td><td>LO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LO</td><td>la</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LLI</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ΙΛ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>about about</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ιη</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>and "H</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>τ-1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>m</td><td>PJ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 64</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LO</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>N</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ABOUT¹</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>η</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>θ '</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>&lt;y</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>UJ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LU</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>φ υ στ</td><td>: « about</td><td>cn £</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>er ar</td><td>about</td><td>(Λ</td><td>pj</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>3 Ν .2?</td><td>c</td><td>ίϋ 2 "ABOUT</td><td>Γ-4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ptyd</td><td>N .2</td><td>c</td><td>ίϋ 2 "ABOUT</td><td>about from</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &gt;*</td><td></td><td></td><td>Q</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>φ</td><td></td><td> 15</td><td></td><td>Q</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> *</td><td>ίϋ</td><td>_cz</td><td> 1—1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ABOUT.</td><td> 5</td><td>ίϋ</td><td>JZ</td><td>k-4</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ι-Ι</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>in 0&gt;</td><td>Σ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>cC</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>AT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>L-1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &gt;-</td><td>ΙΛ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>What</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>E</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ίϋ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>about</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>AT &lt;3 Ό</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>CL 2</td><td>E l_ oh</td><td>about What</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ίϋ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4-J</td><td> &gt;</td><td>&lt;X&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>about.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>in</td><td>ίϋ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LLI</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>pd</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>about about</td><td>about about</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>What</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>loam</td><td>loam</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>'Φ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>about</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>about</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>about</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>What</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>What</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>σ&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ΓΜ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>PJ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>σ&gt; ιχ ιχ</td><td></td><td>1 and 1</td><td></td><td> 745</td><td></td><td rowspan="2">Φ c</td><td></td><td> 779</td><td></td><td>I-l tH</td><td></td><td>ω &lt;U</td><td>MA-</td><td></td><td></td><td></td><td>loam</td><td>no</td><td></td><td>ω φ</td><td>MA-</td>

<td></td><td>Tj-</td><td>ω</td><td>Oi I-l</td><td>σ&gt; cn</td><td>X Σ</td><td></td><td></td><td></td><td>IN</td><td>· oL</td><td></td><td> &gt;-</td><td rowspan="2">IN 13</td><td></td><td>sr T "ł</td><td></td><td>RN</td><td rowspan="2">(0 and N ω</td><td></td><td> £</td><td rowspan="2">0Λ 3</td>

<td></td><td>FSJ 1</td><td>ω &amp;</td><td>ΙΛ IN</td><td>ps H 00</td><td>ABOUT UJ</td><td></td><td></td><td></td><td>Ί</td><td>ω £</td><td>IN ίϋ</td><td></td><td>E 2</td><td></td><td>loam IX about</td><td></td><td>IX H loam</td><td></td><td>E 2</td>

<td></td><td>di ►-b</td><td>about 'ω</td><td>0 c</td><td>σ * M "</td><td>XI</td><td></td><td rowspan="2">tt §.</td><td>s</td><td>cn L-1</td><td>in</td><td>at 13</td><td>ίϋ</td><td>CL OJ</td><td>E</td><td>I-l ABOUT</td><td>KLJ σ »</td><td></td><td>IX (J</td><td>ABOUT.</td><td>ίϋ</td><td>CL OJ</td><td>E</td>

<td rowspan="2">BLR</td><td>(Λ ABOUT.</td><td>(AT Q_</td><td>at ro</td><td>vH</td><td>_about</td><td>H</td><td rowspan="2">BLR</td><td>ΙΛ ABOUT.</td><td>Φ Q_</td><td>at ίϋ</td><td>ID CL</td><td>4-&gt; AT)</td><td></td><td>What LO</td><td>PJ</td><td>Έ¹"Q</td><td> £</td><td> £</td><td>&lt;a CL</td><td>4- " (FROM)</td><td> 0) &gt;</td>

<td>Q_</td><td></td><td>cl</td><td>cn</td><td>cn</td><td>What</td><td>ic</td><td>ABOUT.</td><td></td><td>CL</td><td></td><td></td><td>ίϋ</td><td></td><td>cn</td><td></td><td></td><td>CL</td><td></td><td></td><td>ίϋ</td>

<td>ie "</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>lo</td><td>lo</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>loam</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>loam</td><td>loam</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td>

<td></td><td>Ι, ΟΟΕ05</td><td>2,00E04</td>

<td></td><td>AQZ64878</td><td>AQZ64878 and</td>

<td></td><td>peptide showing activity SEQ hydrolases ID NO: 2.</td><td>peptide showing activity SEQ hydrolases ID NO: 2.</td>

<td></td><td>8,00E48</td><td>Ι, ΟΟΕ44</td>

<td></td><td>ABM1591 6 1</td><td>ABM1591 6</td>

<td></td><td>mycobacterial e protein antigenic mycobacterium tuberculosis SEQ ID NO: 5.</td><td>mycobacterial e protein antigenic mycobacterium tuberculosis SEQ ID NO: 5.</td>

<td></td><td>Bradyrhizobiu m japonicum USDA 110</td><td>Bradyrhizobiu m japonicum USDA 110</td>

<td></td><td>l 00E115</td><td>Ι, ΟΟΕ118</td>

<td></td><td> 27377990</td><td> 27377990</td>

<td> 4680]</td><td>Ε by 77 Ε Eo 3 3 TH ΞΙ ΣΙ or \ and Ιο ~ -¹ about . α - ° £ as ϊ £ as &amp; fc-, 2. m οο σι i- .2, --i Oj O -π "&lt;f? Mm" OC zsBEzisSSŚ.sŁEz!</td><td>hypothetical protein blr2879 [Bradyrhizo bium japonicum USDA 110] gi | 27351136 | DBJ IBAC48144. 1 |</td>

<td></td><td>rC oo i-li-l</td><td>cn o ι-H OJ</td>

<td></td>

<td></td>

<td></td>

<td></td>

<td></td>

<td></td>

<td></td>

<td></td>

<td></td>

<td></td>

<td>blr2879 [Bradyrhizo bium japonicum USDA 110]</td>

<td></td>

<td>GeneSeq / NR % DNA ID</td><td></td><td></td><td></td>

<td>GeneSeq / NR % Protein ID</td><td></td><td> 47</td><td> 42</td>

<td>Length of protein GeneSeq / NR</td><td> 227</td><td> 249</td><td> 249</td>

<td>DNA length GeneSeq / NR</td><td> 00 &lt;£&gt;</td><td>about</td><td>about</td>

<td>Length of protein query</td><td> 227</td><td> 210</td><td> 236</td>

<td>DNA length query</td><td>684 and 1 1</td><td> 633</td><td>Ύ — 1 τ — 1</td>

<td>Description NO</td><td>hypothetical protein Sala_0282 [Sphingopyxis alaskensis RB2256] gi | 98975854 | gb | ABF52005.1 | conservative hypothetical protein [Sphingopyxis alaskensis RB22561</td><td>hypothetical protein Sala_0282 [Sphingopyxis alaskensis RB2256] gi 1989758541g b 1ABF52005.11 conservative hypothetical protein [Sphingopyxis alaskensis RB2256]</td><td>hypothetical protein Sala_0282 [Sphingopyxis alaskensis RB2256] gi 198975854 | gb | ABF52005.11 conservative hypothetical</td>

<td>SEQ ID NO:</td><td>r \ and</td><td>cn</td><td> 5, 6</td>

<td></td><td></td><td></td><td></td>

<td></td><td> 46</td><td> 48</td><td> 46</td>

<td></td><td> 249</td><td> 249</td><td> 298</td>

<td></td><td>about</td><td>about</td><td>about</td>

<td></td><td> 222</td><td> 222</td><td>σ&gt; 00 Η-ύ</td>

<td></td><td> 669</td><td> 669</td><td> 570</td>

<td>protein [Sphingopyxis alaskensis RB2256]</td><td>hypothetical protein Sala_0282 [Sphingopyxis alaskensis RB2256] gi | 98975854 | gb | ABF52005.1 | conservative hypothetical protein [Sphingopyxis alaskensis RB2256]</td><td>hypothetical protein Sala_0282 [Sphingopyxis alaskensis RB2256] gi | 98975854 | gb | ABF52005.1 | conservative hypothetical protein [Sphingopyxis alaskensis RB2256]</td><td>hypothetical protein SKA58_17128 [Sphingomonas sp. SKA58] gi 1944227011 gb | EAT07736.11 hypothetical protein SKA58_17128 [Sphingomonas</td>

<td></td><td>8 'L.</td><td> 9, 10</td><td> 11, 12</td>

<td></td><td colspan="7"></td><td colspan="6"></td><td colspan="6"></td><td colspan="2"></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>σ</td><td></td><td></td><td></td><td></td><td></td><td>σ</td><td></td><td></td><td></td><td></td><td></td><td>σ</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ιθ</td><td></td><td></td><td></td><td></td><td></td><td>rx</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td>and ©</td><td></td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td></td><td> 10</td><td></td>

<td></td><td> 10</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td></td><td> &lt;0</td><td></td>

<td></td><td>CM</td><td></td><td></td><td></td><td></td><td></td><td></td><td>r \ l</td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td></td>

<td></td><td>Γν</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td>about</td><td></td><td></td><td></td><td></td><td></td><td>about</td><td></td><td></td><td></td><td></td><td></td><td>about</td><td></td>

<td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LO</td><td></td><td></td><td></td><td></td><td></td><td>in</td><td></td>

<td></td><td> 10</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td></td><td> 10</td><td></td>

<td></td><td>(Ν</td><td></td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td> 00</td><td></td>

<td></td><td>Ο</td><td></td><td></td><td></td><td></td><td></td><td></td><td>about</td><td></td><td></td><td></td><td></td><td></td><td>σ</td><td></td><td></td><td></td><td></td><td></td><td>σ</td><td></td>

<td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 9</td><td>ιη</td><td>Γ “1 ι — Ι</td><td>Η-ι</td><td></td><td>ιη</td><td></td><td>ω</td><td></td><td></td><td>fU N</td><td> &lt;</td><td></td><td>σι hs</td><td>E</td><td>r - and about</td><td></td><td>E</td><td></td><td>cn</td><td>E</td>

<td></td><td>BLR</td><td>ιη £</td><td rowspan="2">□ C L-I (Λ</td><td>00 LO</td><td>"δ Ιο</td><td>ιη £</td><td></td><td>4 ' E</td><td></td><td>i-4 WHAT · r *</td><td>Π3 ABOUT.</td><td>FROM in</td><td></td><td>oo (N</td><td>3 c</td><td>loam loam</td><td>loam</td><td>X5 about</td><td></td><td>Ir28</td><td>3 c</td>

<td></td><td></td><td>'ιη φ</td><td>1? 1? Σ Ο LU</td><td></td><td>ιη Φ</td><td></td><td>(Ό in &lt;υ P</td><td></td><td>τ — 1 and — 1 r *. at s</td><td></td><td>"P E 2</td><td></td><td rowspan="2">protein b</td><td>about about. fO • ri</td><td></td><td>loam 00 HIM 2</td><td>N c "ABOUT 2</td><td>ι-i about loam loam</td><td rowspan="2">protein b</td><td>S · · - »</td>

<td></td><td></td><td></td><td></td><td>Χ5 σ&gt;</td><td></td><td rowspan="2">σ&gt;</td><td></td><td>&gt; · * F</td><td></td><td>x?</td><td rowspan="2">Φ</td><td>(AT</td><td></td><td>E</td><td></td><td>x?</td><td>oo¹</td><td rowspan="2">&lt; about</td><td>E</td>

<td></td><td></td><td>σ</td><td></td><td></td><td></td><td></td><td>about</td><td></td><td>TJ</td><td>in</td><td></td><td></td><td>ZJ</td><td></td><td>Ό</td><td></td><td></td><td> 3</td>

<td></td><td>&lt;υ</td><td>Γ * χ</td><td></td><td>σ</td><td>φ</td><td>Γ s.</td><td></td><td>CL</td><td></td><td>M "</td><td rowspan="3">(Ό and and</td><td> 8</td><td></td><td>φ</td><td>-Ω</td><td></td><td> 10</td><td></td><td>z&gt;</td><td>φ</td><td>XJ</td>

<td>ι-1</td><td>C</td><td></td><td></td><td>σ</td><td>C</td><td></td><td>οί</td><td>Φ</td><td></td><td>ii</td><td> &gt;%</td><td></td><td>c</td><td>ABOUT</td><td></td><td>n</td><td></td><td></td><td>c</td><td>ABOUT</td>

<td>00 ιη &lt;</td><td>and</td><td>ΓΜ 1</td><td>ίϋ</td><td>ι-Ι 00</td><td>and</td><td>(Ν 1</td><td>and SI</td><td>1_ cn</td><td>i-1 about 00</td><td>loam r&gt;. about</td><td>E £</td><td></td><td>and</td><td>N c</td><td></td><td>loam loam LO</td><td>σ</td><td>L · Z3 at</td><td>AND</td><td>N c</td>

<td>Ξ (Λ</td><td>ΰ</td><td>cC</td><td>AT ΙΡ</td><td>cn ^ 3 "</td><td>Β</td><td>0C</td><td>fic</td><td>fr</td><td>&lt;X&gt;</td><td>KO σ »</td><td>about.</td><td>ABOUT. CD</td><td>about</td><td></td><td>ie</td><td> &lt;</td><td>n</td><td>hs 00</td><td>'C</td><td>CD 4&gt; J</td><td>T&gt;</td>

<td>Ω_</td><td> 8.</td><td>ίη CL</td><td>υ ω</td><td>ι-1</td><td>and</td><td>(Λ Ο.</td><td>aci</td><td>Π3 ABOUT-</td><td> &lt;</td><td>CM</td><td> £</td><td>at. 4- " U5</td><td> 68</td><td>and</td><td>2 WHAT</td><td>about ΙΛ</td><td>CM</td><td>CM and_</td><td rowspan="2">and (0 · "</td><td>ABOUT about_</td><td>2 What</td>

<td>ιη</td><td>c</td><td>CL</td><td>CL</td><td>σ&gt;</td><td>ίο</td><td>Ο.</td><td>CL</td><td></td><td>Σ</td><td>σ</td><td>CL</td><td></td><td></td><td>lo</td><td>l__l</td><td>FROM)</td><td>σ</td><td>x&gt;</td><td>c</td><td>l__l</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td></td><td> 00</td><td></td><td></td><td></td><td></td><td></td><td>about</td><td></td>

<td></td><td>Η-ι</td><td></td><td></td><td></td><td></td><td></td><td></td><td>τ-1</td><td></td><td></td><td></td><td></td><td></td><td>τ-1</td><td></td><td></td><td></td><td></td><td></td><td>CM</td><td></td>

<td></td><td>σί</td><td></td><td></td><td></td><td></td><td></td><td></td><td>in</td><td></td><td></td><td></td><td></td><td></td><td>r &lt;</td><td></td><td></td><td></td><td></td><td></td><td>σ</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ιΉ</td><td></td><td></td><td></td><td></td><td></td><td>I-I</td><td></td><td></td><td></td><td></td><td></td><td>loam</td><td></td>

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&lt;υ [0153] Homologous sequences also include RNA sequences in which uridines replace thymines in nucleic acid sequences. Homologous sequences may be obtained using any of the procedures described in the present invention or may result from correction of sequencing error. It should be noted that nucleic acid sequences as defined herein can be presented in a traditional one-character format (see, e.g., Stryer, Lubert. Biochemistry, 3rd edition, W. H Freeman &amp; Co., New York) or in any other format that registers the identity of the nucleotides in the sequence.

[0154] Various sequence comparison programs as defined herein and known to a person skilled in the art are used. Protein and / or nucleic acid sequence identity (homologies) can be evaluated using any of a variety of algorithms and sequence comparison programs known in the art. Such algorithms and programs include, but are not limited to: TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448.1988; Altschul et al., J. Mol. Biol. 215 (3): 403-410, 1990; Thompson et al., Nucleic Acids Res. 22 (2): 4673-4680, 1994; Higgins et al., Methods Enzymol. 266: 383-402, 1996; Altschul et al., J. Mol. Biol.215 (3): 403-410, 1990; Altschul et al., Nature Genetics 3: 266272, 1993).

[0155] Homology or identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package developed by Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705, USA). Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions, and other modifications. The terms "homology" and "identity", in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or contain a certain percentage of the same amino acid residues or nucleotides when compared and adjusted for maximum compatibility within a certain comparison window or designated area, measured using any number of algorithms for sequence comparison or by manual matching and visual checking. When comparing sequences, one sequence can act as a reference sequence (e.g., the exemplary nucleic acid or polypeptide sequence disclosed herein) with which test sequences are compared. Using a sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the coordinates of the subsection are indicated and the program parameters are assigned to the sequence comparison algorithm. Default program parameters can be used, or alternative parameters can be assigned. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences relative to the reference sequence, based on program parameters.

[0156] The term "comparison window" as used herein includes reference to a segment with any number of contiguous residues. For example, in alternative aspects, adjacent residues with a number anywhere within the range of 20 to the full length of an exemplary polypeptide or nucleic acid sequence, are compared with a reference sequence with the same number of adjacent positions after optimal alignment of these two sequences. If the reference sequence has the required sequence identity with an exemplary polypeptide or nucleic acid sequence, e.g. in alternative aspects, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65 %, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or more, or full (100%) sequence identity with the exemplary polypeptide or nucleic acid sequence disclosed herein, then the sequence falls within the scope of the present invention. Subsequence sequences with a residue number in the range of about 20 to 600, about 50 to 200 and about 100 to 150 are compared with a reference sequence with the same number of adjacent positions after optimal alignment of the two sequences. Methods of alignment of sequences for comparative purposes are well known in the art. Optimal alignment of sequences for comparison can be carried out, e.g., using a local homology analysis algorithm by Smith and Waterman, Adv. Appl. Math. 2: 482.1981, homology matching algorithm by Needleman and Wunsch, J. Mol. Biol. 48: 443.1970, by similarity search method by Person &amp; Lipman, Proc. Nat'1. Acad. Sci. USA 85: 2444, 1988, using computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, USA), or by manually adjusting and visual checking. Other algorithms for determining homology or identity include, for example, in addition to the BLAST (Basic Local Alignment Search Tool from National Center for Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET (Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals &amp; Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global Alignment Program) , GENAL, GIBBS, GenQuest, ISSC (Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local Content Program), MACAW (Multiple Alignment Construction 8 and Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN, ΡΙΜΑ (Pattern-Induced Multi-sequence Alignment), SAGA (Sequence Alignment by Genetic Algorithm) and WHAT-IF. Such alignment programs can also be used to screen genomic databases to identify polynucleotide sequences having substantially identical sequences. Many genomic databases are available, for example, a significant portion of the human genome is available as part of the Human Genome Sequencing Project (Gibbs, 1995). Several genomes have been sequenced, e.g. M. genitalium (Fraser et al. 1995), M. jannaschii (Bult et al. 1996), H. influenzae (Fleischmann et al. 1995), E. coli (Blattner et al. 1997) and yeast (5. cerevisiae) (Mewes et al. 1997) and D. melanogaster (Adams et al. 2000). Significant progress has also been made in sequencing the genomes of model organisms such as mouse, C. e / egans and Arabadopsis sp. Different organizations maintain databases containing genomic information, sometimes annotated about the functions of individual genes, and these databases are available online.

[0157] BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used. They are described e.g. in: Altschul (1977) Nuc. Acids Res. 25: 3389-3402; Altschul (1990) J. Mol. Biol. 215: 403-410. BLAST analysis software is publicly available through the National Center for Biotechnology Information. This algorithm first consists in determining pairs of high-scoring sequences (HSPs) by identifying short words of length W in the query sequence that either match or meet a certain threshold criterion with a positive T value when matching a word of the same length in the sequence found in the database. The T value is defined as the threshold score within adjacent words (Altschul (1990), above). These initial hits within adjacent words act as embryos that initiate searches to find longer HSP sequences containing these words. The word hits are expanded in both directions along each sequence as far as the cumulative match score can be increased. Cumulative results are calculated using, for nucleotide sequences, M parameters (reward for a pair of matching residues; always> 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative result. The expansion of word hits in each direction is stopped when: the cumulative match score drops by the X value relative to the maximum value achieved; the cumulative score drops to zero or below, due to the accumulation of one or more negative match matches; or after reaching one of these sequences. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of matching. By default, the BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, BLASTP uses by default, word length 3, expected value (E) 10, and BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) fit (B) 50, expected value (E) 10, M = 5, N = -4 and comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between the two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873). One measure of similarity provided by the BLAST algorithm is the lowest probability sum (P (N)), which indicates the probability at which a match between two nucleotide or amino acid sequences would occur accidentally. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum of probabilities when comparing the analyzed nucleic acid with the reference nucleic acid is less than about 0.2, or alternatively, less than about 0.01, or alternatively less than about 0.001 .

[0158] In one aspect, protein and nucleic acid sequence homologies are determined using the Basic Local Alignment Search Tool ("BLAST"). For example, five specific BLAST programs can be used to accomplish the following task: (1) BLASTP and BLAST3 compare the amino acid sequence of a query with a protein sequence database; (2) BLASTN compares the nucleotide sequence of the query with the nucleotide sequence database; (3) BLASTX compares the conceptual nucleotide sequence translation products of the query (both strands) in six reading frames with a protein sequence database; (4) TBLASTN compares the protein sequence of the query with the translated nucleotide sequence database in all six reading frames (both strands); and (5) TBLASTX compares translations in six nucleotide sequence reading frames from a query with translations in six reading frames from a nucleotide sequence database.

[0159] In one aspect, BLAST programs determine homologous sequences by identifying similar segments, which are referred to herein as "high scoring segment pairs", between the amino acid sequence or the nucleic acid sequence from the query and a test sequence that is obtained alternatively from protein or nucleic acid sequence databases. High-scoring segment pairs can alternatively be identified (i.e. matched) using scoring matrices, many of which are known in the art. In one aspect, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al. Science 256: 1443-1445, 1992; Henikoff and Henikoff, Proteins 17: 49-61, 1993). In one aspect, PAM or PAM250 arrays can also be used (see e.g. Schwartz and Dayhoff, ed., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).

[0160] In one aspect, NCBI BLAST 2.2.2 programs are used to determine whether the nucleic acid has the required sequence identity to be within the scope of the present invention, the default options are blastp. There are about 38 setting options in the BLAST 2.2.2 program. In this example aspect, all default values ​​are used, except the default filtering setting (i.e. all parameters set as default except filtering which is set to OFF); instead, the "-F F" setting is used to disable filtering. Using default filtering often causes violations of Karlin-AItschul statistics due to the short length of the sequence.

[0161] The default values ​​used in this exemplary aspect include:

"Filter for low complexity: ON

Word Size: 3

Matrix: Blosum62

Gap Costs: Existence: 11

Extension: l "

[0162] Other default settings are: filter for low complexity OFF, word size: 3 for protein, BLOSUM62 matrix, gap existence penalty: -11 and gap extension penalty: -1. In one aspect, the "-W" option defaults to 0. This means that if these parameters are not set, word size defaults to 3 for proteins and 11 for nucleotides.

Computer systems and computer program products [0163] In order to determine and identify sequence identity, structural homology, motifs, etc. in siiico, the sequence disclosed herein may be stored, recorded and manipulated on any medium that can be read and available by computer. Computers, computer systems, computer readable media, computer program products, etc., have been disclosed in which the nucleic acid and polypeptide sequences disclosed herein have been contained, stored therein or stored in memory. As used herein, the words "recorded" and "stored" refer to the process of storing information on a computer medium. One skilled in the art can easily adapt any known methods of recording information on a computer readable medium to produce products containing one or more of the nucleic acid and / or polypeptide sequences disclosed herein.

[0164] Disclosed is a computer readable medium on which at least one nucleic acid and / or polypeptide sequence disclosed herein has been recorded. Computer-readable media includes magnetic media, optical media, electronic media, and magnetic / optical media. For example, the computer-readable media may be a hard disk, floppy disk, magnetic tape, CD-ROM, universal video disk (DVD), random access memory (RAM) or read-only memory (ROM), as well as other types of media known to people to those skilled in the art.

[0165] Systems (e.g., Internet systems) have been disclosed, in particular computer systems that store and manipulate the sequences and information about the sequences described in the present invention. One example of the computer system 100 is illustrated in the form of a block diagram in Figure 1. As used herein, the term "computer system" refers to hardware components, software components, and data storage components used to analyze a nucleotide or polypeptide sequence disclosed herein. The computer system 100 may include a processor for processing, accessing, and manipulating sequence data. Processor 105 can be any well-known type of central processing unit, e.g. Pentium III from Intel Corporation or similar processor from Sun, Motorola, Compaq, AMD or International Business Machines. The computer system 100 is a general purpose system comprising a processor 105 and one or more internal data storage components 110 for storing data, and one or more data acquisition devices for retrieving data stored in data storage components. One of skill in the art can easily assess whether any of the computer systems currently available will be suitable for this purpose.

[0166] In one aspect, the computer system 100 includes a processor 105 connected to a bus that is connected to the operating memory 115 (alternatively implemented as RAM) and one or more internal data storage devices 110, such as a hard disk drive and / or other media read by the computer containing data stored on them. The computer system 100 may further include one or more data download devices 118 for reading data stored in the internal data storage devices 110. The data download device 118 may be, for example, a diskette drive, compact disc drive, magnetic tape drive, or modem capable of connecting to a remote data storage system (e.g. via the Internet) etc. Internal data storage device 110 means a removable medium readable by a computer, such as a floppy disk, compact disc, magnetic tape, etc., containing the control logic and / or the data stored on it. The computer system 100 may advantageously include or be programmed by suitable software to read the control logic and / or data from the data storage component after entering the data download device. The computer system 100 includes a display 120 that is used to display output to a computer user. It should also be noted that the computer system 160 may be combined with other 125a-c computer systems in the network or wide area network (WAN) to provide centralized access to the computer system 100. Software for accessing and processing the nucleotide or amino acid sequences disclosed herein may be in operational memory 115 when performing these operations. In some aspects, the computer system 100 may further include a sequence comparison algorithm for comparing the nucleic acid sequence disclosed herein. This algorithm and sequence (s) may be stored on a computer readable medium. "Sequence comparison algorithm" refers to one or more programs that are implemented (locally or remotely) on a computer system 100 to compare nucleotide sequences with other nucleotide sequences and / or compounds stored in data storage means. For example, the sequence comparison algorithm may compare the nucleotide sequences disclosed herein on a computer readable medium with reference sequences stored on a computer readable medium to identify homology or structural motifs.

[0167] The parameters used in the above algorithms can be adjusted depending on the length of the sequence and the degree of homology tested. In some respects, these parameters may be the default parameters used by the algorithms in the absence of user instructions. Figure 2 is a flowchart illustrating one aspect of the process 200 for comparing a new nucleotide or protein sequence with a sequence database to determine the level of homology between the new sequence and the sequences in the database. The sequence database may be a private database stored within the computer system 100, or a public database, such as GENBANK, which is made available via the Internet. The process 200 starts in the initial state 201 and then goes to the state 202 in which the new sequence for comparison is stored in memory in the computer system 100. As discussed above, memory can be any type of memory, including RAM or an internal storage device. This process 200 then proceeds to state 204, in which the sequence database is opened for analysis and comparison. Process 200 then proceeds to state 206 in which the first sequence stored in the database is loaded into the computer's memory. A comparison is then made at state 210 to determine if the first sequence is the same as the second sequence. It should be noted that this step is not limited to making an accurate comparison between the new sequence and the first sequence in the database. Those skilled in the art are familiar with well-known methods for comparing two nucleotide or protein sequences, even if the sequences are not identical. For example, gaps can be introduced into one sequence to increase the level of homology between the two sequences tested. Parameters that control whether gaps or other features are entered into the sequence during the comparison are typically entered by the computer system user. After comparing the two sequences in state 210, it is determined in decision state 210 whether these two sequences are the same. Of course, the concept of "the same" is not limited to sequences that are absolutely identical. Sequences that fall within the range of homology parameters entered by the user will be marked in the process as "the same." If it is determined that the two sequences are the same, then the process 200 goes to state 214 in which the sequence name from the database is displayed to the user. This state notifies the user that the display name sequence meets the introduced homology restrictions. When the name of the stored sequence is displayed to the user, the process 200 goes into decision state 218, in which it is determined whether there are more sequences in the database. If there are no more sequences in the database, then the process 200 ends in the final state 220. However, if there are more sequences in the database, then the process 200 goes to state 224, in which the pointer is moved to the next sequence in the database so that it was to compare with the new sequence. In this way, the new sequence is matched and compared with each sequence in the database. Note that if it is determined in decision state 212 that the sequences are not homologous, then the process 200 will immediately go to decision state 218 to determine if any other sequences are available in the database for comparison purposes. A computer system comprising a processor, a data storage device comprising the nucleic acid sequence disclosed herein and a means of comparing the sequences for performing the comparison is disclosed. The sequence comparison agent may indicate the level of homology between the compared sequences or identify structural motifs, or it may identify structural motifs in the sequences that are compared with those nucleic acid and polypeptide codes. Figure 3 is a flowchart illustrating one embodiment of process 250 on a computer to determine if two sequences are homologous. The process 250 starts in the initial state 252 and then goes to the state 254 in which the first sequence for comparison is stored in memory. The second comparison sequence is then stored in memory at state 256. Process 250 then proceeds to state 260 where the first character in the first sequence is read and then to state 262 where the first character in the second sequence is read. It should be understood that if this sequence is a nucleotide sequence then that character will usually be one of the characters A, T, C, G or U. If the sequence is a protein sequence then it may denote a single letter amino acid code, so that the first and second sequence. It is then determined in decision-making state 264 whether these two characters are the same. If they are the same, then process 250 goes to state 268, in which the next characters of the first and second sequences are read. Then it is determined whether the subsequent characters are the same. If they are, then process 250 stays in this loop until two characters are not the same. If it is determined that the next two characters are not the same, the process 250 goes to decision state 274 to determine if any of the sequences have any characters to read. If there are no more characters to read then process 250 goes to state 276, in which the user is displayed a level of homology between the first and second sequences. The level of homology is determined by calculating the proportion of characters between the sequences that were the same for the total number of characters in the first sequence. Thus, if each character in the first 100 nucleotide sequence is matched to each respective character in the second sequence, the homology level will be 100%.

[0168] Alternatively, the computer program may compare the reference sequence to the sequence disclosed in this specification to determine if the sequences differ at one or more positions. The program may record the length and identity of nucleotides or amino acid residues that have been inserted, deleted or substituted with respect to a reference sequence or disclosed herein. The computer program may be a program determining whether the reference sequence contains a single nucleotide polymorphism (SNP) relative to the sequence disclosed herein, or whether the sequence disclosed herein comprises the SNP of a known sequence. Thus, in some aspects, a computer program is a program that identifies SNP polymorphisms. This method can be implemented in the computer systems described above and by the method illustrated in Fig. 3. This method may involve reading the sequences and reference sequences disclosed herein by using a computer program and identifying differences using a computer program.

[0169] In other aspects, the computer-based system includes an identifier for identifying features within the nucleic acid or polypeptide disclosed herein. The term "identifier" refers to one or more programs that identify certain features within a nucleic acid sequence. For example, the identifier may include a program that identifies an open reading frame (ORF) in a nucleic acid sequence. Figure 4 is a flow chart illustrating one aspect of the identifier process 300 for detecting the presence of a certain feature in a sequence. The process 300 starts in the initial state 302 and then goes to the state 304, in which the first sequence to be checked for certain features is saved in the memory 115 in the computer system 100. The process 300 then goes to the state 306, in which the base is opened sequence characteristics data. Such a database could contain a list of attributes of each feature along with the feature name. For example, the name of the feature could be "initiation codon" and the attribute could be "ATG". Another example would be the feature name "TAATAA cassette" and the attribute attribute would be "TAATAA". An example of such a database was created by Genetics Computer Group of the University of Wisconsin. Alternatively, the features may be structural polypeptide motifs, such as alpha-helixes, beta-sheets, or functional polypeptide motifs, such as enzymatic active sites, helix-twist-helix motifs, or other motifs known to those skilled in the art. After opening the feature database, in state 306, the process 300 goes to state 308, in which the first feature is read from the database. Then, in state 310, a comparison of the attribute of the first feature with the first sequence is performed. Then it is determined in decision state 316 whether the attribute attribute was found in the first sequence. If the attribute was found, then the process 300 goes to state 318, in which the name of the found feature is displayed to the user. The process 300 then proceeds to decision state 320, in which it is determined whether the database has more features. If there are no more features, then the process 300 ends in the final state 324. However, if there are more features in the database, then the process 300 reads the next feature of the sequence, in state 326, and performs a return loop to state 310, in which the attribute of the next feature is compared with the first sequence. If no attribute attribute is found in the first sequence in decision state 316, the process 300 proceeds directly to decision state 320 to determine if there are more features in the database. Thus, in one aspect, a computer program is provided that identifies open reading frames (ORFs).

[0170] The polypeptide or nucleic acid sequence disclosed herein may be stored and manipulated in various data processing programs in a variety of formats. For example, the sequence can be stored as text in a word processing file such as MICROSOFT WORD ™ or WORDPERFECT ™, or as an ASCII file in a series of database management programs known to those of skill in the art, such as DB2, SYBASE or ORACLE ™. Additionally, many computer programs and databases can be used as algorithms to compare the sequence, identifiers or reference sources of nucleotide sequences or polypeptide sequences to compare with the nucleic acid sequences disclosed herein. Programs and databases may include: MACPATTERN ™ (EMBL), DISCOVERYBASE ™ (Molecular Applications Group), GENEMINE ™ (Molecular Applications Group), LOOK ™ (Molecular Applications Group), MACLOOK ™ (Molecular Applications Group), BLAST and BLAST2 (NCBI) ), BLASTN and BLASTX (Altschul et al., J. Mol. Biol. 215: 403, 1990), PASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB ™ ( Brutlag et al. Biosci Comp. 6: 237245, 1990), CATALYST ™ (Molecular Simulations Inc.), CATALYST ™ / SHAPE ™ (Molecular Simulations Inc.), CERIUS2.DBACCESS ™ (Molecular Simulations Inc.), HYPOGEN ™ (Molecular Simulations Inc.), Insight II , (Molecular Simulations Inc.), DISCOVER ™ (Molecular Simulations Inc.), CHARMm ™ (Molecular Simulations Inc.), FELIX ™ (Molecular simulations Inc.), DELPHI ™ s (Molecular Simulations Inc.), QUANTEMM ™, (Molecular Simulations Inc.), HOMOLOGY ™ (Molecular Simulations Inc.), MODELER (Molecular Simulations Inc.), ISIS ™ (Molecular Simulations Inc.), Quanta / Protein Design (Molecular Simulations Inc.), WEBLAB ™ (Molecular Simulations Inc.), WEBLAB ™ Diversity Explorer (Molecular Simulations Inc.), GENE EXPLORER ™ (Molecular Simulations Inc.), SEQFOLD ™ (Molecular Simulations Inc.), MDL Available Chemicals Directory database, MDL Drug Data Report database, Comprehensive Medicinal Chemistry database, Derwent's World Drug Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and databases will be apparent to one of skill in the art in view of this description.

[0171] Motifs that can be detected using the above programs include leucine zipper coding sequences, helix-torsion-helix motifs, glycosylation sites, ubiquitination sites, alpha helices and beta sheets, signal sequences encoding signal peptides that direct the secretion of encoded proteins, sequences involved in transcription regulation, such as homeo cassettes, stretched acid fragments, enzymatic active sites, substrate binding sites and enzymatic cleavage sites.

Nucleic acid hybridization [0172] Isolated, synthetic or recombinant nucleic acids have been disclosed that hybridize under stringent conditions to the nucleic acid disclosed herein, e.g. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 , SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22 or SEQ ID NO: 23, or SEQ ID NO: 1 modified to encode one, two, three, four, five, six, seven, eight or more (several ) or all base changes described in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23, or their equivalents, and their sub-sequences and complementary sequences, or the nucleic acid encoding the polypeptide disclosed herein. Stringent conditions can mean the highly stringent, medium stringent and low stringency conditions, including the high and limited stringency conditions described in the present invention.

[0173] The term "hybridization" refers to the process by which a nucleic acid strand binds to a complementary strand by base pairing. Hybridization reactions can be sensitive and selective, so that a specific sequence of interest can be identified even in samples where it is present at low concentrations. Stringent conditions can, for example, be defined by salt or formamide concentrations in prehybridization and hybridization solutions, or by hybridization temperature, and are well known in the art. For example, stringency may be increased by limiting the salt concentration, increasing the formamide concentration or increasing the hybridization temperature, changing the hybridization time as detailed below. In alternative aspects, the nucleic acids disclosed herein are determined by their ability to hybridize under conditions of different stringency (e.g., high, medium and low) as defined herein.

[0174] Nucleic acids disclosed herein defined by their ability to hybridize under stringent conditions may be between about five residues in length and the full length of the nucleic acid disclosed herein; e.g. they can be at least 5, 10, 15, 20, 25, 30, 35, 40.50, 55, 60, 65, 70.75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residues. This also includes nucleic acids shorter than full length nucleic acids. These nucleic acids may be useful as, e.g., hybridization probes, labeling probes, PCR oligonucleotide probes, iRNAs, antisense strands or antibody-binding peptide coding sequences (epitopes), motifs, active sites and the like.

[0175] In one aspect, the nucleic acids disclosed herein are defined by their ability to hybridize under high stringency conditions, including the use of about 50% formamide at a temperature of about 37 ° C to 42 ° C. In one aspect, the nucleic acids disclosed herein are defined by their ability to hybridize under conditions of limited stringency involving the use of about 35% to 25% formamide at a temperature of about 30 ° C to 35 ° C.

[0176] Alternatively, the nucleic acids disclosed herein are defined by their ability to hybridize under high stringency conditions, including conditions at 42 ° C in 50% formamide, 5X SSPE, 0.3% SDS, and a nucleic acid blocking repetitive sequence like cot-1 or salmon sperm DNA (e.g., 200 ug / ml cut and denatured salmon sperm DNA). In one aspect, the nucleic acids disclosed herein are defined by their ability to hybridize under conditions of limited stringency, including the use of 35% formamide at a reduced temperature of 35 ° C.

[0177] After hybridization, the filter can be washed with 6X SSC, 0.5% SDS at 50 ° C. These conditions are considered "moderate" conditions for formamide levels above 25% and "low stringency" conditions for formamide levels below 25%. A specific example of "moderate" hybridization conditions is when the above hybridization is carried out in 30% formamide. A specific example of "low stringency" hybridization conditions is when the above hybridization is carried out in 10% formamide.

[0178] The temperature range corresponding to a given level of stringency can be further narrowed by calculating the ratio of purines to pyrimidines in the nucleic acid of interest and adjusting the temperature accordingly. Nucleic acids disclosed herein are also defined by their ability to hybridize under high, medium and low stringency conditions such as those reported in Ausubel and Sambrook. Variations of the above ranges and conditions are well known in the art. The hybridization conditions are discussed in more detail below.

[0179] The above procedure can be modified to identify nucleic acids showing a decreasing level of homology with the probe sequence. For example, to obtain nucleic acids with decreasing homology with a detectable probe, less stringent conditions may be used. For example, hybridization temperature may be reduced every 5 ° C from 68 ° C to 42 ° C in Na hybridization buffer⁺about 1 M. After hybridization, the filter can be washed with 2X SSC, 0.5% SDS at the hybridization temperature. These conditions are considered "moderate" conditions above 50 ° C and "low stringency" conditions below 50 ° C. A specific example of "moderate" hybridization conditions is when the above hybridization is carried out at 55 ° C. A specific example of "low stringency" hybridization conditions is when the above hybridization is carried out at 45 ° C.

[0180] Alternatively, hybridization can be carried out in buffers such as 6X SSC, containing formamide, at 42 ° C. In this case, the concentration of formamide in the hybridization buffer can be reduced every 5% from 50% to 0% to identify clones showing a decreasing level of homology with the probe. After hybridization, the filter can be washed with 6X SSC, 0.5% SDS at 50 ° C. These conditions are considered "moderate" conditions with a formamide concentration above 25% and as "low stringency" conditions with a concentration below 25% formamide. A specific example of "moderate" hybridization conditions is when the above hybridization is carried out at 30% formamide. A specific example of "low stringency" hybridization conditions is when the above hybridization is carried out at 10% formamide.

[0181] However, the choice of hybridization format is not critical - it is the stringency of the washing conditions that determine whether a nucleic acid falls within the scope of the invention disclosed herein. Washing conditions used to identify nucleic acids in the present description include, e.g., a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 50 ° C or about 55 ° C to about 60 ° C; or a salt concentration of about 0.15 M NaCl at 72 ° C for about 15 minutes; or a salt concentration of about 0.2X SSC at a temperature of at least about 50 ° C or about 55 ° C to about 60 ° C for about 15 to about 20 minutes; or the hybridization complex is washed twice with a solution of about 2X SSC containing 0.1% SDS salt at room temperature for 15 minutes, and then washed twice with 0.1X SSC containing 0.1% SDS at 68 ° C for 15 minutes; or equivalent conditions apply. See. Sambrook, Tijssen and Ausubel, where SSC buffer description and equivalent conditions are given.

[0182] These methods can be used to isolate the nucleic acids disclosed herein.

Oligonucleotide probes and methods of their use [0183] Nucleic acid probes for recognizing nucleic acids encoding a polypeptide having hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity have been disclosed. In one aspect, the probe contains at least 10 further bases of the nucleic acid disclosed herein. Alternatively, the probe disclosed herein may be at least about 5.6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120 , 130, 150, 160, 170, 180, 190, 200 or more, or about 10 to 50, about 20 to 60 about 30 to 70, further bases with the sequence given as the nucleic acid disclosed herein. These probes recognize nucleic acid through binding and / or hybridization. These probes can be used in the arrays disclosed here, see discussion below, including e.g. in capillary matrices. Probes disclosed herein can also be used to isolate other nucleic acids or polypeptides.

[0184] The probes disclosed herein may be used to determine whether a biological sample, such as a soil sample, contains an organism having the nucleic acid sequence disclosed herein (e.g., a hydrolase encoding nucleic acid) or the organism from which the nucleic acid was obtained. In such procedures, a biological sample is obtained, which is a potential habitat for the organism from which the nucleic acid has been isolated, and nucleic acids are obtained from this sample. Nucleic acids are contacted with the probe under conditions that allow the probe to specifically hybridize with any complementary sequences present in the sample. When necessary, conditions enabling specific probe hybridization with complementary sequences can be determined by placing the probe in contact with the complementary sequences of samples known to contain the complementary sequence, as well as with control sequences that do not contain the complementary sequence. Hybridization conditions, such as salt concentration in hybridization buffer, formamide concentration in hybridization buffer, or hybridization temperature, can be changed to determine conditions that allow for specific hybridization of the probe with complementary nucleic acids (see discussion of specific hybridization conditions).

[0185] If the sample contains the organism from which the nucleic acid has been isolated, then specific hybridization with the probe can be detected. Hybridization can be detected by labeling the probe with a detectable agent such as a radioactive isotope, fluorescent dye or enzyme capable of catalyzing the reaction leading to the detection of a detectable product. Persons skilled in the art know many methods for using labeled probes to detect the presence of complementary nucleic acids in a sample. These include Southern hybridization, northern hybridization, colony hybridization procedures and point hybridization. Reports for each of these procedures are presented in the work of Ausubel and Sambrook.

[0186] Alternatively, more than one probe (of which at least one is capable of specifically hybridizing to any complementary sequences present in the nucleic acid sample) may be used in the amplification reaction to determine whether the sample includes the organism comprising the nucleic acid sequence disclosed herein (e.g. the organism from which this nucleic acid was isolated). In one aspect, the probes contain oligonucleotides. In one aspect, the amplification reaction may include a PCR reaction. PCR reaction protocols were described in Ausubel and Sambrook (see discussion of amplification reactions). In such procedures, the nucleic acids contained in the sample are contacted with the probes, an amplification reaction is performed, and any resulting amplification product is detected. The amplification product can be detected by performing gel electrophoresis of the reaction products and staining the gel with an intercalator such as ethidium bromide. Alternatively, one or more probes can be radiolabelled and the presence of the radioactive amplification product can be detected by autoradiography after gel electrophoresis.

[0187] Probes derived from sequences near the 3 'or 5' end of the nucleic acid sequence disclosed herein can also be used in "chromosome walking" procedures to identify clones containing additional sequences, e.g. genomic sequences. Such methods allow isolation from the host organism of genes that encode additional proteins of interest.

[0188] In one aspect, the nucleic acid sequences disclosed herein are used as probes to identify and isolate related nucleic acid sequences. In some aspects, the nucleic acid sequences thus identified may be cDNA or genomic DNA from organisms other than the one from which the nucleic acid disclosed herein was first isolated. In these types of procedures, the nucleic acid sample is contacted with the probe under conditions that allow the probe to specifically hybridize with the associated sequences. Hybridization of the probe with nucleic acids from the associated organism is then detected using any of the methods described above.

[0189] In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary depending on the nature of the hybridized nucleic acids. For example, when choosing hybridization conditions, the length, degree of complementarity, nucleotide sequence composition (e.g., GC content relative to AT) and type of nucleic acid (e.g. RNA or DNA) of hybridizing nucleic acid regions may be taken into account. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Hybridization can be carried out under low stringency, moderate stringency or high stringency conditions. During an exemplary nucleic acid hybridization, a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45 ° C in a solution consisting of 0.9 M NaCl, 50 mM NaH₂AFTER₄, pH 7.0, 5.0 mM Na₂EDTA, 0.5% SDS, 10Χ Denhardt's solution and 0.5 mg / ml polybyboadenylic acid. An end-labeled oligonucleotide probe is then added to the solution³²P with activity around 2Χ10⁷ cpm (specific activity 4-9 X 10⁸ cpm / pg). After 12-16 hours of incubation, the membrane is washed for 30 minutes at room temperature (RT) in IX SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, ImM Na₂EDTA) containing 0.5% SDS, and then washed for 30 minutes in fresh IX SET at Tm-10 ° C for the oligonucleotide probe. The membrane is then applied to an autoradiographic film to detect hybridization signals. [0190] By changing the stringency of hybridization conditions used to identify nucleic acids, such as cDNA or genomic DNA, which hybridize to a detectable probe, nucleic acids with different levels of probe homology can be identified and isolated. Strictness can be changed by conducting hybridization at various temperatures below the melting point of these probes. The melting point Tm is the temperature (at a given ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly complementary probe. Very stringent conditions are selected to be equal to or about 5 ° C lower than the Tm for a particular probe. The melting point of the probe can be calculated using the following example formulas. For probes containing between 14 and 70 nucleotides, the melting point (Tm) is calculated by the formula: Tm = 81.5 + 16.6 (log (Na +]) + 0.41 (G + C fraction) - (600 / N) where N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting point can be calculated by the equation: Tm = 81.5 + 16.6 (log [Na +]) + 0.41 (fraction G + C ) - (0.63% formamide) - (600 / N), where N is the length of the probe. Prehybridization can be carried out in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, 100 pg DNA from denatured fragmented salmon sperm or 6X SSC, 5X Denhardf's reagent, 0.5% SDS, 100 g denatured, fragmented salmon sperm DNA , 50% formamide. The compositions of SSC and Denhardt and other solutions are listed, e.g., in Sambrook.

[0191] In one aspect, hybridization is performed by adding a detectable probe to the prehybridization solutions listed above. If the probe contains double-stranded DNA, it is denatured prior to addition to the hybridization solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNA or genomic DNA containing sequences complementary or homologous thereto. For probes containing more than 200 nucleotides in length, hybridization can be carried out at 15-25 ° C below Tm. For shorter probes, such as oligonucleotide probes, hybridization can be carried out at a temperature of 5-10 ° C below Tm. In one aspect, hybridization in 6 X SSC is carried out at a temperature of about 68 ° C. In one aspect, hybridization in solutions containing 50% formamide is carried out at a temperature of about 42 ° C. It is believed that all of the above hybridizations are carried out under highly stringent conditions.

[0192] In one aspect, after hybridization, the filter is washed to remove non-specifically bound detectable probe. The stringency used to wash the filters can also be changed depending on the type of hybridized nucleic acids, length of hybridized nucleic acids, degree of complementarity, composition of the nucleotide sequence (e.g. GC content in AT see) and type of nucleic acid (e.g. RNA in see GOUT). Examples of increasingly stringent conditions are: 2 X SSC, 0.1% SDS at room temperature for 15 minutes (low stringency); 0.1 X SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0.1 X SSC, 0.5% SDS for 15 to 30 minutes, at a temperature between hybridization temperature and 68 ° C (high stringency); and 0.15 M NaCl for 15 minutes at 72 ° C (very high stringency). Final washing under low stringency conditions can be carried out even at 0.1 X SSC at room temperature. The above examples are merely illustrative of one set of conditions that can be used to wash filters. The skilled artisan will know that there are many recipes to flush with varying rigor. [0193] Nucleic acids that hybridized to the probe can be identified by autoradiography or other conventional techniques. The above procedure can be modified to identify nucleic acids with decreasing homology to the probe sequence. For example, less stringent conditions can be used to obtain nucleic acids with decreasing homology to a detectable probe. For example, the hybridization temperature can be reduced in 5 ° C increments from 68 ° C to 42 ° C in hybridization buffer containing a Na + concentration of about IM. After hybridization, the filter can be washed with 2X SSC, 0.5% SDS at hybridization temperature. These conditions are considered "moderate" stringency conditions above 50 ° C, and "low" stringency conditions below 50 ° C. An example of "moderate stringency" hybridization conditions is when the above hybridization is carried out at 55 ° C. An example of "low stringency" hybridization conditions is when the above hybridization is carried out at 45 ° C.

[0194] Alternatively, hybridization can be carried out in buffers, such as 6X SSC, containing formamide at 42 ° C. In this case, the concentration of formamide in the hybridization buffer can be reduced using 5% jumps from 50% to 0% to identify clones with reduced level of homology to the probe. After hybridization, the filter can be washed with 6X SSC, 0.5% SDS at 50 ° C. These conditions are considered as "moderate stringency" conditions above 25% formamide and "low stringency" conditions below 25% formamide. A particular example of "moderate stringency" hybridization conditions is when the above hybridization is carried out in 30% formamide. A special example "low stringency" hybridization conditions are those where the above hybridization is carried out in 10% formamide. [0195] Such probes and methods disclosed herein can be used for isolation or identification (e.g. using a matrix), nucleic acids containing a sequence of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% , 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above sequence identity with the nucleic acid sequence disclosed herein, comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more of its subsequent bases and sequences complementary to them. Homology can be measured using a comparison algorithm, as discussed herein. For example, polynucleotides may be homologous to a coding sequence, which is a naturally occurring allelic variant of one of the coding sequences described herein. Such allelic variants may have the substitution, deletion or addition of one or more nucleotides, as compared to the nucleic acid disclosed herein.

[0196] In addition, the probes and methods disclosed herein can be used to isolate or identify (e.g., using an array), nucleic acids encoding polypeptides having at least 50%, 51%, 52%, 53%, 54%, 55 %, 56%, 57%, 58%,

59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72 %, 73%,

74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above sequence identity (homology) with the polypeptide disclosed herein, comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or 150 or more of their subsequent amino acids as determined by a sequence comparison algorithm, e.g. such as the FASTA 3.0t78 algorithm version with default parameters or the BLAST program 2.2.2 with sample settings as described in this description.

Inhibition of Hydrogen Expression of [0197] Nucleic acids complementary to the sequences (e.g., antisense sequences) of the nucleic acid disclosed herein, e.g., hydrolase coding sequences, are disclosed. Antisense sequences are capable of inhibiting the transport, splicing or transcription of genes encoding hydrolases. Inhibition can be accomplished by targeting genomic DNA or RNA. Inhibition can be carried out using DNA, e.g. god-inhibiting or RNA, e.g. double-stranded iRNAs containing the sequence disclosed herein. The transcription or function of the target nucleic acid can be inhibited, e.g., by hybridization and / or cleavage. Disclosed herein are sets of oligonucleotides containing inhibitors capable of binding the gene and / or hydrolase information, in each of these cases, preventing or inhibiting the production or function of the hydrolase. Linking can occur through a specific hybridization sequence. Another useful class of inhibitors contains oligonucleotides that cause inactivation or cleavage of hydrolase information. The oligonucleotide may have an enzyme activity that causes such cleavage, such as ribozymes. The oligonucleotide may be chemically modified, or conjugated to an enzyme or composition capable of cleaving complementary nucleic acid. A pool of many such oligonucleotides can be screened to obtain those with the desired activity.

Antisense Oligonucleotides [0198] Antisense oligonucleotides are disclosed, capable of binding hydrolase information that can inhibit hydrolase activity by targeting mRNA or genomic DNA. Antisense oligonucleotide design strategies are well described in the scientific and patent literature, and one of skill in the art can design such hydrolase oligonucleotides using the new reagents disclosed herein. For example, RNA gene walking / mapping protocols are well known in the art to screen for effective antisense oligonucleotides, see e.g. Ho (2000) Methods Enzymol.314: 168-183, describing an RNA mapping assay that is based on standard molecular techniques to provide an easy and reliable way to select an active antisense sequence. See also Smith (2000) Eur. J. Pharm. Sci.ll: 191-198.

[0199] In one aspect, recombinantly produced or isolated naturally occurring nucleic acids are used as antisense oligonucleotides. Antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40 lengths. The antisense oligonucleotides may be single-stranded or double-stranded RNA or DNA. Optimal length can be determined by routine screening. Antisense oligonucleotides can be present at any concentration. Optimal concentrations can be determined using routine screening. Many different synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known that can address this potential problem. For example, peptide nucleic acids (PNA) containing non-ionic backbones can be used, e.g. N- (2-aminoethyl) glycine units. Antisense oligonucleotides having phosphoorothioate linkages as described in WO 97/03211 may also be used; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144: 186-197; Antisense Therapeutics, Agrawal edition (Humana Press, Totowa, NJ., 1996). Disclosed herein are antisense oligonucleotides having synthetic DNA backbone analogs that may also contain phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphorotriester, sulfamate, 3'-thioacetal, methylene (methylimine), 3'-carboxamate and m-carbamate nucleic as described above.

[0200] Combinatorial chemistry methodology can be used to create a huge number of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificity for any target, such as the sense and antisense hydrolase sequences disclosed herein (see, e.g., Gold (1995) J. of Biol. Chem. 270: 13581-13584).

Inhibiting Ribozymes [0201] Ribozymes capable of binding hydrolase information that are able to inhibit hydrolase activity by targeting mRNA are disclosed. Strategies for ribozyme design and selection of the hydrolase specific antisense sequence for targeting are well described in the scientific and patent literature, and one of skill in the art can design such ribozymes using the new reagents disclosed herein. Ribozymes act by binding to target RNA through a portion of the ribozyme that binds to the target RNA that is kept in close proximity to the enzymatic portion of the RNA that cleaves the target RNA. Thus, ribozyme recognizes and binds to the target RNA through complementary base pairing, and when bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of the target DNA in this way destroys its ability to directly synthesize the encoded protein if cleavage occurs in the coding sequence. After binding and cleavage by ribozyme of its target RNA, it is usually released from this RNA and can repeatedly bind and cleave new targets.

[0202] Under certain circumstances, the enzymatic nature of ribozyme may be advantageous compared to other technologies, such as antisense technology (where the nucleic acid molecule binds easily to the target nucleic acid to block its transcription, translation or binding to another molecule) as that the effective ribozyme concentration necessary to induce therapeutic treatment may be lower than that of an antisense oligonucleotide. This potential benefit reflects the ability of ribozyme to have enzymatic activity. Thus, a single ribozyme molecule is able to split multiple target RNA molecules. In addition, ribozyme is usually a very specific inhibitor with inhibition specificity dependent not only on the mechanism of base pairing binding, but also on the mechanism by which the molecule inhibits the expression of RNA to which it binds. That is, inhibition is caused by target RNA cleavage, so specificity is defined as the ratio of target RNA cleavage rate to non-target RNA cleavage rate. This mechanism of cleavage depends on factors additional to those involved in base pairing. Thus, the specificity of ribozyme action may be greater than when binding an antisense oligonucleotide to the same RNA site.

[0203] The enzyme RNA ribozyme molecule may be formed in the form of a hammer head motif, but may also be formed in form of a hairpin motif, delta hepatitis virus, intron group I or RNase P-like RNA (in combination with a lead sequence RNA). Examples of such hammerhead motifs are described by Rossi (1992) Aids Research and Uman Retroviruses 8: 183; hairpin motifs by Hampel (1989) Biochemistry 28: 4929, and Hampel (1990) Nuc. Acids Res. 18: 299; delta hepatitis virus motif by Perrott (1992) Biochemistry 31:16; RNazyP motif by Guerrier-Takada (1983) Cell 35: 849; and the intron of group I by Cech (US Patent No. 4,987,071). The purpose of these specific motives is not to limit; it will be apparent to those skilled in the art that the enzyme RNA molecule disclosed herein may have a specific substrate binding site complementary to one or more RNA regions of the target genes, and has a nucleotide sequence within or around that substrate binding site that gives the molecule activity RNA cleavage.

RNA interference (RNAi) [0204] Disclosed herein are inhibitory RNA molecules, so-called "RNAi" molecules comprising the sequence of the hydrolase disclosed herein. The RNAi molecule may contain a double-stranded RNA molecule (dsRNA), e.g., siRNA and / or miRNA. RNAi may inhibit the expression of a hydrolase gene or transcript (e.g., lipase, saturase, palmitase and / or stearatase). In one aspect, the RNAi molecule e.g. siRNA and / or miRNA, is a duplex nucleotide about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 in length, 29 or 30 or above. Although the invention is not limited to any particular mechanism of action, RNAi may enter the cell and cause degradation of single-stranded RNA (ssRNA) with similar or identical sequences, including endogenous mRNA molecules. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). The likely underlying mechanism behind RNAi is the cracking of double-stranded RNA (dsRNA), corresponding to a specific gene sequence, into short segments called short interfering RNA that triggers mRNA degradation that matches its sequence.

[0205] In one aspect, the RNAi disclosed herein are used for gene silencing therapies, see, e.g., Shuey (2002) Drug Discov. Today 7: 1040-1046. Methods for selective RNA degradation using RNAi are disclosed. This process can be used in vitro, ex vivo or in vivo. In one aspect, the RNAi molecules disclosed herein can be used to generate loss-of-function mutations in a cell, organ or animal. Methods for producing and using RNAi molecules for the selective degradation of RNA are well known in the art, see, e.g., US Patent No. 6,506,559; US 6,511,824; US 6,515,109; US 6,489,127.

Nucleic Acid Modification [0206] Methods for generating nucleic acid variants are disclosed, e.g., those disclosed herein encoding a hydrolase or antibody. These methods can be repeated or used in various combinations to produce hydroiazides or antibodies having altered or different activity or altered or different stability from the hydrolase or antibody encoded by the template nucleic acid. These methods can also be repeated or used in various combinations, e.g., to generate a change in gene expression / information, information translation or information stability. In another aspect, the genetic composition of the cell is altered by, e.g., altering the ex vivo homologous gene and then reintroducing it into the cell.

[0207] The term "variant" may include polynucleotides or polypeptides disclosed herein modified on one or more base pairs, codons, introns, exons or amino acid residues (as appropriate), while retaining the biological activity of the hydrolase disclosed herein. Variants can be produced using any number of agents, including methods such as, for example, error-prone PCR, shuffling, oligonucleotide-guided mutagenesis, assembly-type PCR, "sexual" PCR mutagenesis, in-vivo mutagenesis, cassette mutagenesis, recursive team mutagenesis , exponential syndrome mutagenesis, site-specific mutagenesis, Gene Reassembly, GSSM^sm and any combinations thereof. Techniques for producing hydrolase variants having activity at a given pH or temperature, for example, which differ from the wild-type hydrolase, are included herein.

[0208] The nucleic acid disclosed herein may be modified in any way. For example, by random or stochastic or non-stochastic or "directed evolution" methods, see, e.g., US Patent No. 6,361,974. Methods for random gene mutation are well known in the art, see, e.g., U.S. Patent No. 5,830,696. For example, a mutagen may be used to mutate the gene at random. Mutagens include, e.g. ultraviolet light or gamma radiation or a chemical mutagen, e.g. mitomycin, nitrous acid, photoactivated psoralens, alone or in combination, to cause DNA breaks that can be repaired by recombination. Other chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Other mutagens are analogs of nucleotide precursors e.g. nitrosoguanidine, 5-bromouracil, 2-aminopurine or acridine. These factors can be added to the PCR reaction instead of the nucleotide precursor thus mutating the sequence. Intercalating agents, such as proflavin, acrylavlavin, quinacrine and the like can also be used.

[0209] Any molecular biology technique can be used, e.g., random PCR mutagenesis, see e.g. Rice (1992) Proc. Natl. Acad. Sci. USA 89: 5467-5471; or multiple combinatorial cassette mutagenesis, see e.g. Crameri (1995) Biotechniques 18: 194-196. Alternatively, nucleic acids, e.g. genes, may be reassembled after random or "stochastic" fragmentation, see, e.g., US Patent Nos. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. In alternative aspects, modifications, additions or deletions are introduced by error-prone PCR, shuffling, oligonucleotide-guided mutagenesis, assembly-type PCR, "sexual PCR" -type mutagenesis, in-vivo mutagenesis, cassette mutagenesis, recursive team mutagenesis, exponential team mutagenesis site-specific saturation (GSSM^sm), synthetic re-assembly with ligation (SLR) synthetic ligation reassembly), recombination, recursive team mutagenesis, phosphothioate-modified DNA mutagenesis, matrix-containing uracil mutagenesis, intermittent duplex mutagenesis, repair mutagenesis, host strain mutagenesis with repair disorder, chemical mutagenesis, radiation mutagenesis, delagen mutagenesis restriction selection, restriction mutagenesis with purification, artificial gene synthesis, team mutagenesis, multimeric formation of the chimeric nucleic acid and / or a combination of these and other methods.

[0210] The following publications describe various procedures and / or methods of recursive recombination that may be included in the methods disclosed herein: Stemmer (1999) "Molecular breeding of viruses for targeting and other clinical properties" Tumor Targeting 4: 1-4; Ness (1999) Nature Biotechnology 17: 893-896; Chang (1999) "Evolution of a cytokine using DNA family shuffling" Nature Biotechnology 17: 793-797; Minshull (1999) "Protein evolution by molecular breeding" Current Opinion in Chemical Biology 3: 284-290; Christians (1999) "Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling" Nature Biotechnology 17: 259-264; Crameri (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature 391: 288-291; Crameri (1997) "Molecular evolution of an arsenate detoxification pathway by DNA shuffling," Nature Biotechnology 15: 436-438; Zhang (1997) "Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening" Proc. Natl. Acad. Sci. USA 94: 45044509; Patten et al. (1997) "Applications of DNA Shuffling to Pharmaceuticals and Vaccines" Current Opinion in Biotechnology 8: 724-733; Crameri et al. (1996) "Construction and evolution of antibody-phage libraries by DNA shuffling" Nature Medicine 2: 100-103; Gates et al. (1996) "Affinity selective isolation of ligands from peptide libraries through display on a lac repressor 'headpiece dimer' 'Journal of Molecular Biology 255: 373-386; Stemmer (1996) "Sexual PCR and Assembly PCR" In: The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457; Crameri and Stemmer (1995) "Combinatorial multiple cassette mutagenesis creates all the permutations of mutant and wildtype cassettes" BioTechniques 18: 194-195; Stemmer et al. (1995) "Single-step assembly of a gene and entire plasmid form large numbers of oligodeoxyribonucleotides" Gene, 164: 49-53; Stemmer (1995) "The Evolution of Molecular Computation" Science 270: 1510; Stemmer (1995) "Searching Sequence Space" Bio / Technology 13: 549-553; Stemmer (1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370: 389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution." Natl. Acad. Sci. USA 91: 10747-10751.

[0211] Mutational methods for generating diversity include, for example, site-directed mutagenesis (Ling et al. (1997) "Approaches to DNA mutagenesis: an overview" Anal Biochem. 254 (2): 157-178; Dale et al. (1996) " Oligonucleotide-directed random mutagenesis using the phosphorothioate method "Methods Mol. Biol. 57: 369-374; Smith (1985)" In vitro mutagenesis "Ann. Rev. Genet. 19: 423-462; Botstein and Shortile (1985) "Strategies and applications of in vitro mutagenesis" Science 229: 1193-1201; Carter (1986) "Site-directed mutagenesis" Biochem. J. 237: 1-7; and Kunkel (1987) "The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids and Molecular Biology (Eckstein, F. and Lilley, DMJ eds.,

Springer Verlag, Berlin)); mutagenesis using matrices containing uracil (Kunkel (1985) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) "Rapid and efficient site -specific mutagenesis without phenotypic selection "Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant Trp repressors with new DNA-binding specificities" Science 242: 240245); Oligonucleotide-guided mutagenesis (Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987); Zoller and Smith (1982) "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment "Nucleic Acids Res. 10: 6487-6500; Zoller and Smith (1983) "Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors" Methods in Enzymol. 100: 468-500; and Zoller and Smith (1987) Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template "Methods in Enzymol. 154: 329-350); phosphorothioate-modified DNA mutagenesis (Taylor et al. (1985) "The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA" Nuci. Acids Res. 13: 8749-8764; Taylor et al. (1985) "The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA" Nuci. Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) "Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis" Nuci. Acids Res. 14: 9679-9698; Sayers et al. (1988) "YT Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis" Nuci. Acids Res. 16: 791-802; and Sayers et al. (1988) "Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide" Nuci. Acids Res. 16: 803-814); mutagenesis using gaps in DNA duplexes (Kramer et al. (1984) "The gapped duplex DNA approach to oligonucleotide-directed mutation construction" Nuci. Acids Res. 12: 9441-9456; Kramer and Fritz (1987) Methods in Enzymol. "Oligonucleotide-directed construction of mutations via gapped duplex DNA" 154: 350-367; Kramer et al. (1988) "Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations" Nuci. Acids Res. 16: 7207; and Fritz et al. (1988) "Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro" Nuci. Acids Res. 16: 6987-6999).

[0212] Additional protocols used in the methods of the invention include spot mismatch repair (Kramer (1984) "Point Mismatch Repair" Cell 38: 879-887), mutagenesis of the host strain with repair disorder (Carter et al. (1985) "Improved oligonucleotide site -directed mutagenesis using M13 vectors "Nuci. Acids Res. 13: 4431-4443; and Carter (1987)" Improved oligonucleotide-directed mutagenesis using M13 vectors "Methods in Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) "Use of oligonucleotides to generate large deletions" Nuci. Acids Res. 14: 5115), restriction-selectivity and restriction-selection and restriction-purification (Wells et al. ( 1986) "Importance of hydrogenbond formation in stabilizing the transition State of subtilisin" Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis using whole gene synthesis (Nambiar et al. (1984) "Total synthesis and cloning of a gene coding for the ribonuclease S protein" Science 223: 12991301; Sakamar and Khorana (1988) 'Total synthesis and expression of a gene for the asubunit of bovine rod outer segment guanine nucleotide-binding protein (transducin) "Nuci. Acids Res. 14: 6361-6372; Wells et al. (1985)" Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites "Gene 34: 315-323; and Grundstrom et al. (1985) "Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis" Nuci. Acids Res. 13: 3305-3316), repair of double-thread break (Mandecki (1986); Arnold (1993) "Protein engineering for unusual environments" Current Opinion in Biotechnology 4: 450-455. "Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis "Proc. Natl. Acad. Sci. USA, 83: 7177-7181). Additional details on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for solving problems with various mutagenesis methods.

[0213] Additional protocols used in the methods disclosed herein include those discussed in US Patent No. 5,605,793 issued to Stemmer (February 25, 1997), "Methods for In Vitro Recombination;" U.S. Patent No. 5,811,238 to Stemmer et al. (September 22, 1998) "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" U.S. Patent No. US 5,830,721 to Stemmer et al. (November 3, 1998), "DNA Mutagenesis by Random Fragmentation and Reassembly;" U.S. Patent No. 5,834,252 to Stemmer, et al. (November 10, 1998) "End-Complementary Polymerase Reaction;" U.S. Patent No. 5,837,458 to Minshull, et al. (November 17, 1998), "Methods and Compositions for Cellular and Metabolic Engineering;" WO 95/22625, Stemmer and Crameri, "Mutagenesis by Random Fragmentation and Reassembly;" WO 96/33207 by Stemmer and Lipschutz "End Complementary Polymerase Chain Reaction;" WO 97/20078 by Stemmer and Crameri "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" WO 97/35966 by Minshull and Stemmer, "Methods and Compositions for Cellular and Metabolic Engineering;" WO 99/41402 filed by Punnonen et al. 'Targeting of Genetic Vaccine Vectors;' WO 99/41383 filed by Punnonen et al. 'Antigen Library Immunization;' WO 99/41369 filed by Punnonen et al. 'Genetic Vaccine Vector Engineering; "WO 99/41368 by Punnonen et al. "Optimization of Immunomodulatory Properties of Genetic Vaccines;" EP 752008 by Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation and Reassembly;" EP 0932670 by Stemmer "Evolving Cellular DNA Uptake by Recursive Sequence Recombination;" WO 99/23107 by Stemmer et al., "Modification of Virus Tropism and Host Rank by Viral Genome Shuffling;" WO 99/21979 filed by Apt et al., "Humań Papillomavirus Vectors;" WO 98/31837 by del Cardayre et al. "Evolution of Whole Cells and Organisms by Recursive Sequence Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and Compositions for Polypeptide Engineering;" WO 98/27230 by Stemmer et al., "Methods for Optimization of Gene Therapy by Recursive Sequence Shuflling and Selection," WO 00/00632, "Methods for Generating Highly Diverse Libraries," WO 00/09679, "Methods for Obtaining in Vitro Recombined Polynucleotide Sequence Banks and Resulting Sequences," WO 98/42832 filed by Arnold et al., "Recombination of Polynucleotide Sequences Using Random or Defined Primers," WO 99/29902 filed by Arnold et al., " Method for Creating Polynucleotide and Polypeptide Sequences, "WO 98/41653 by Vind," An in Vitro Method for Construction of a DNA Library, "WO 98/41622 by Borchert et al., "Method for Constructing a Library Using DNA Shuffling," and WO 98/42727 by Pati and Zarling, "Sequence Alterations using Homologous Recombination."

[0214] Protocols that can be used (providing details about the various diversity generating methods) are described, eg, in US Patent Application Serial No. (USSN) 09 / 407,800, "SHUFFLING OF CODON ALTERED GENES". filed on September 28, 1999 by Patten et al; "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION" filed by del Cardayre et al., US Patent No. 6,379,964; "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" filed by Crameri et al., US Patent Nos. US 6,319,714; US 6,368,861; US 6,376,246; US 6,423,542; US 6,426,224 and PCT / US00 / 01203; "USE OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING", filed by Welch et al., US Patent Nos. 6,436,675; "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES and POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" granted to Selifonov et al., Reported on January 18, 2000, (PCT / US00 / 01202) and, e.g. "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES and POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al., Filed July 18, 2000 (U.S. Application Serial No. 09 / 618,579); "METHODS OF POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer, reported January 18, 2000 (PCT / US00 / 01138); and "SINGLE-STRANDED NUCLEIC ACID TEMPLATEMEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION" by Affholter, filed September 6, 2000 (US Application Serial No. 09 / 656,549); and US patents No. US 6,177,263; US 6,153,410.

[0215] Non-stochastic methods or "site-directed evolution" include, e.g., site-specific saturation mutagenesis ^SM (GSSM^sm), synthetic ligation reassembly (SLR or Gene Reassembly) or combinations thereof are used to modify the nucleic acids disclosed herein to generate hydrolases with new or changed properties (e.g., activity under high acidity or alkalinity, high temperatures and the like). Polypeptides encoded by modified nucleic acids can be screened for activity before testing for proteolytic or other activity. Any testing procedure or protocol can be used, e.g., using a capillary matrix platform. See, e.g., US Patent Nos. 6,361,974; US 6,280,926; US 5,939,250.

Saturation mutagenesis or GSSM technology^sm [0216] In one aspect disclosed herein, non-stochastic gene modification, "directed evolution" is used to generate hydrolases and altered properties. Variations of this method have been termed "site saturation mutagenesis", "site saturation mutagenesis" "mutagenesis saturation "or simply" GSSM^sm". It can be used as disclosed in conjunction with other mutagenesis methods. In one aspect, the present disclosure discloses methods of producing enzymes and antibodies using GSSM technology^sm, e.g., as described herein as well as in US Patent Nos. 6,171,820; US 6,579,258; US 6,238,884.

[0217] In one aspect, GSSM technology^sm comprising providing a polynucleotide template and multiple oligonucleotides, each oligonucleotide having a sequence homologous to the template polynucleotide, thereby targeting the specific sequence of the template polynucleotide, and a sequence that is a variant of the homologous gene; generating daughter polynucleotides containing non-stochastic sequence variants by replicating the template polynucleotide using oligonucleotides, thereby generating polynucleotides containing gene homologous sequence variants. [0218] In one aspect, primer codons containing degenerate N, N, G / T sequences are used to introduce point mutations into a polynucleotide, so as to generate a set of daughter polypeptides in which the full range of single amino acid substitutions are represented at each amino acid position, e.g., an amino acid residue at an active enzyme site or ligand binding site that the modification targets. These oligonucleotides may include a first continuous homologous sequence, a degenerate N, N, G / T sequence and, optionally, a second homologous sequence. Follow-up translation descendants using such oligonucleotides contain all possible amino acid changes at each amino acid position along the polypeptide because the degeneracy of the N, N, G / T sequence contains codons for all 20 amino acids. In one aspect, one such degenerate oligonucleotide (consisting of, e.g., one degenerated N, N, G / T cassette) serves to subject each original codon in the parent polynucleotide matrix to a full range of codon substitutions. In another aspect, at least two degenerate cassettes are used in the same oligonucleotide or another to subject at least two primary codons in the parent polynucleotide matrix to a full range of codon substitutions. For example, more than one N, N, G / T sequence may be contained in one oligonucleotide to introduce an amino acid mutation in more than one site. This multiplicity of N, N, G / T sequences can be directly adjacent or separated by one or more additional nucleotide sequences. In another aspect, oligonucleotides useful for introducing insertions and deletions may be used alone or in combination with codons containing the N, N, G / T sequence to introduce any combination or permutation of amino acid insertion, deletion and / or substitution.

[0219] In one aspect, the simultaneous mutagenesis of two or more adjacent amino acid positions takes place with an oligonucleotide that contains adjacent N, N, G / T triplets, i.e., a degenerate (N, N, G / T) n sequence. In another aspect, cassettes with less degeneration than the N, N, G / T sequence are used. For example, in some cases use may be desirable (e.g. e oligonucleotide) of a degenerate triplet sequence consisting of only one N, with the N being in the first, second or third position of the triplet. All other rules, including their combinations and permutations, can be used in the other two positions of the triplet. Alternatively, in some cases it may be desirable to use (e.g. in an oligonucleotide) the degenerate triplet sequence Ν, Ν, Ν.

[0220] In one aspect, the use of degenerate triplets (e.g., N, N, G / T triplets) allows easy generation of the full range of possible natural amino acids (20 amino acids in total) at each individual amino acid position in the polypeptide (in alternative aspects methods include also generating less than all possible substitutions at the amino acid residue or codon position). For example, for a 100 amino acid polypeptide, 2000 different types can be generated (i.e. 20 possible amino acids per position X from 100 amino acid positions). By using an oligonucleotide or a set of oligonucleotides containing a degenerated N, N, G / T triplet, 32 individual sequences can encode all 20 possible natural amino acids. Thus, in a reaction vessel in which the parent polynucleotide sequence is subjected to saturation mutagenesis using at least one such oligonucleotide, 32 different daughter polynucleotides encoding 20 different daughter polypeptides are generated. In contrast, the use of a non-degenerated oligonucleotide in site-directed mutagenesis results in only one progeny polypeptide product per reaction vessel. Non-degenerate oligonucleotides can optionally be used in combination with the disclosed degenerate primers; e.g., non-degenerated oligonucleotides can be used to generate specific point mutations in a processed polynucleotide. This provides a means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that result in the generation of stop codons and the associated expression of polypeptide fragments.

[0221] In one aspect, each reaction vessel in which saturation mutagenesis occurs contains polynucleotides encoding at least 20 daughter polypeptide molecules (e.g., hydrolase, e.g. (lipase, saturase, palmitase and / or stearatase) such that all 20 natural amino acids are represented at one specific amino acid position corresponding to the position of the codon mutagenized in the parent polynucleotide (the other aspects use less than all 20 natural combinations). 32-degenerate daughter polypeptides generated from each reaction vessel in which saturation mutagenesis occurs may be subjected to clonal amplification (e.g. cloning to a suitable host, e.g. an E. coli host, using e.g. expression vector) and screened for expression. When a particular daughter polypeptide is identified by screening as showing a favorable change in properties (compared to the parent polypeptide, such as increased selectivity for palmitate ester hydrolysis compared to oleate ester hydrolysis), it can be sequenced to identify the appropriate beneficial amino acid substitution contained therein.

[0222] In one aspect, after mutagenizing each amino acid position in a parent polypeptide using saturation mutagenesis as disclosed herein, beneficial amino acid changes can be identified in more than one amino acid position. One or more new daughter molecules may be generated that contains a combination of all or part of these preferred amino acid substitutions. For example, if 2 specific beneficial amino acid changes were identified at each of the 3 amino acid positions in a polypeptide, permutation involves 3 possibilities at each position (no change from the original amino acid and each of two preferred changes) and 3 positions. So, there is 3 x 3 x 3 or a total of 27 possibilities, including 7 that have previously been studied - 6 single point mutations (i.e. 2 at each of the three positions) and no change in any position.

[0223] In another aspect, site saturation mutagenesis may be used together with other stochastic or non-stochastic agents for sequence differentiation, e.g., ligation re-assembly synthetic (see below), shuffling, chimerization, recombination and other mutagenic processes and mutagenizing agents. The present specification discloses a mutagenizing method (s), including saturation mutagenesis, used in an iterative manner.

Synthetic Ligatia Reassembly (SLR. Synthetic Ligation

Reassembly) [0224] A non-stochastic gene modification system has been disclosed called "ligation synthetic reassembly," or simply "SLR," also known as "Gene Reassembly" technology, "directed evolution process" to generate polypeptides, e.g. , enzymes (such as hydrolases, e.g., lipases, saturases, palmitases and / or stearatases) or antibodies disclosed herein with new or changed properties. SLR is a method of non-stochastically ligating oligonucleotide fragments with each other. This method differs from stochastic shuffling of oligonucleotides in that nucleic acid building blocks are not shuffled, linked or chimerized randomly, but are rather non-stochastically linked. See, e.g., US Patent Nos. 6,773,900; US 6,740,506; US 6,713,282; US 6,635,449; US 6,605,449; US 6,537,776.

[0225] In one aspect, SLR comprises the following steps: (a) providing a polynucleotide template, said template comprising a polynucleotide sequence encoding a homologous gene; (b) providing multiple polynucleotide building blocks, wherein the building block polynucleotides are intended to be re-assembled with the polynucleotide matrix in a predetermined order and the building block polynucleotide contains a sequence that is a variant of the homologous gene and homologous sequence for the polynucleotide matrix variant sequence; (c) combining the building block polynucleotide with the matrix polynucleotide such that the cross-over assembly of the building block polynucleotide with the matrix polynucleotide to generate polynucleotides containing variants of homologous gene sequences.

[0226] SLR does not depend on the presence of high levels of homology between polynucleotides to be reassembled. Therefore, this method can be used to generate in a non-stochastic manner libraries (or sets) of daughter particles containing more than 10^1θθ different chimeras. SLR can be used to create libraries consisting of over ιο^1θθθ various daughter chimeras. Non-stochastic methods for generating a set of final chimeric nucleic acid molecules with a complete assembly order that is selected by design are disclosed. This method involves the following steps: generating by designing a plurality of specific nucleic acid building blocks having useful mutually compatible ligation ends and assembling these nucleic acid building blocks so that a designed complete assembly order is achieved.

[0227] Those mutually compatible ligationable ends of the nucleic acid building blocks to be assembled are considered "useful" for this type of ordered assembly if they allow the building blocks to connect in a predetermined order. Thus, the complete assembly order in which nucleic acid building blocks can be linked is determined by the design of the ligationable ends. If more than one assembly step is to be used, then the complete assembly order in which the nucleic acid building blocks can be connected is also determined by the sequential order of the assembly step (s). In one aspect, molten building blocks are treated with an enzyme, such as a ligase (e.g., T4 DNA ligase), to achieve covalent binding of building blocks.

[0228] In one aspect, the arrangement of oligonucleotide blocks is obtained by analyzing a set of child nucleic acid sequence templates that serve as the basis for generating a child set of final chimeric polynucleotides. Thus, these parent oligonucleotide matrices serve as a source of sequence information that helps design nucleic acid building blocks to be mutagenized, e.g., chimerized or shuffled. In one aspect of this method, the sequences of multiple parent nucleic acid templates are compared to select one or more borderline points. The demarcation points may be in the area of ​​homology, and consist of one or more nucleotides. These demarcation points are possibly common to at least two child matrices. The demarcation points can thus be used to delineate the building blocks of oligonucleotide blocks to be generated for the reassembly of parent polynucleotides. The demarcation points identified and selected for daughter molecules serve as potential chimerization points in the assembly of final chimeric daughter molecules. The borderline may be an area of ​​homology (consisting of at least one homologous nucleotide base) common to at least two parent polynucleotide sequences. Alternatively, the demarcation point may be a region of homology that is common to at least half of the parent polynucleotide sequences, or, it may be a region of homology that is common to at least two-thirds of the parent polynucleotide sequences. A useful demarcation point is an area of ​​homology that is common to at least three-quarters of the parent polynucleotide sequences, or can be common to almost all parent polynucleotide sequences. In one aspect, the demarcation point is an area of ​​homology that is common to all parent polynucleotide sequences. [0229] In one aspect, the ligation reassembly method is performed exhaustively to generate a library of daughter chimeric polynucleotides. In other words, all possible ordered combinations of nucleic acid building blocks are represented in a set of final chimeric nucleic acid molecules. At the same time, in another aspect, the assembly order (e.g. the order of assembly of each building block in the order of 5 'to 3 of each completed chimeric nucleic acid) in each composition is designed (or non-stochastic) as described above. Non-stochastic methods that reduce the possibility of undesirable by-products are disclosed.

[0230] In another aspect, the ligation reassembly method is performed systematically. For example, the method is carried out to generate a systematically segmented library of child molecules, with segments that can be searched in a systematic manner, e.g., one after the other. The methods disclosed herein involving the selective and intelligent use of specific nucleic acid building blocks, in combination with the selective and intelligent use of sequential assembly reactions, can be arranged in which a specific set of daughter products produced in each of several reaction vessels. This enables systematic testing and screening. Thus, these methods allow the systematic study of potentially very large numbers of daughter molecules in small groups. Because of their ability to chimerize in a manner that is highly flexible, but at the same time exhaustive and systematic, in particular when there is low homology between daughter molecules, these methods provide for the generation of a library (or kit) containing a large number of daughter molecules. Due to the non-stochastic nature of exemplary league reassembly methods, the generated daughter molecules may contain a library of final chimeric nucleic acid molecules with a complete assembly order that has been selected by design. Saturation mutagenesis and optimized directed evolution methods can also be used to generate different types of daughter molecule units.

[0231] In one aspect, the present methods provide freedom of choice and control with respect to the selection of demarcation points, size and number of nucleic acid building blocks, and the size and arrangement of linkers. The requirement for intermolecular homology can be highly stringent. In fact, demarcation points can even be selected in regions with little or no intermolecular homology. For example, due to codon ambiguity, i.e. codon degeneration, nucleotide substitutions can be introduced into building blocks of nucleic acids without changing the originally encoded amino acid in the respective daughter matrix. Alternatively, the codon may be changed such that the coding of the original amino acid is changed. In one aspect, substitutions can be introduced into a nucleic acid building block to increase the frequency of intermolecular homologous separation points and thus allow an increased number of connections between building blocks to be achieved, which in turn enables the generation of more daughter chimeric molecules.

[0232] In another aspect, the synthetic nature of the step at which building blocks are generated enables the design and introduction of nucleotides (e.g., one or more nucleotides, which can be, for example, codons or introns or regulatory sequences), which can be later optionally removed by in vitro (e.g. mutagenesis) or in vivo (e.g. utilizing the ability of the host organism to gene splicing). It should be noted that in many cases the introduction of these nucleotides may be desirable for many reasons besides the potential benefit of creating a useful borderline.

[0233] In one aspect, a nucleic acid building block is used to introduce the intron. Thus, functional introns are introduced into a human-generated gene generated according to the methods described herein. Artificially introduced intron (s) may be functional in host cells for gene splicing in a way that naturally occurring introns functionally function in gene splicing.

Optimized directed evolution system [0234] A non-stochastic gene modification system has been disclosed called an "optimized directed evolution system" to generate hydroiodes and antibodies with new or changed properties. Optimized directed evolution refers to the use of repeated cycles of reductive reshuffling, recombination and selection that allow obtaining directed molecular evolution of nucleic acids by recombination. Optimized directed evolution enables the generation of a large population of altered chimeric sequences, wherein the generated population is significantly enriched with sequences that have a predetermined number of crossover events.

[0235] A crossover event is a point in a chimeric sequence where the sequence shifts from one parent variant to another parent variant. Such a point is usually at the junction at which two parent oligonucleotides were ligated to form a single sequence. This method allows the calculation of appropriate concentrations of oligonucleotide sequences so that the final population of chimeric sequences is enriched with a selected number of crossover events. This provides better control over the selection of chimeric variants with a fixed number of crossover events.

[0236] In addition, this method provides convenient means for testing a huge number of possible protein variants compared to other systems. Previously, for example, if 10 were generated¹³ chimeric molecules during the reaction, it would be very difficult to check for such a large number of chimeric variants relative to a particular activity. Furthermore, a significant proportion of the child population would have a very large number of crossover events that resulted in proteins for which they would be less likely to show elevated levels of activity. Using these methods, the population of chimeric molecules can be enriched in those variants that have a specific number of crossover events. So, although you can still generate 10¹³ chimeric molecules during the reaction, each of these molecules selected for further analysis most likely shows, for example, only three crossover events. Because the resulting child population can be targeted to exhibit a predetermined number of crossover events, the boundaries of functional variation between chimeric molecules have been

100 reduced. This provides a better regulated number of variables when calculating which oligonucleotide from the original parent polynucleotides may be responsible for influencing a particular trait.

[0237] One way to create chimeric daughter polynucleotide sequences is to generate oligonucleotides corresponding to fragments or parts of each parent sequence. Each oligonucleotide has a unique overlapping region, so mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order. Alternatively, protocols for implementing these methods disclosed herein can be found in US Patent Nos. 6,773,900; US 6,740,506; US 6,713,282; US 6,635,449; US 6,605,449; US 6,537,776; US 6,361,974.

[0238] The number of oligonucleotides generated in each parent variant refers to the total number of crossover cases obtained in the chimeric molecule that was eventually formed. For example, three parent variants of nucleotide sequences may be provided to be subjected to ligation to find a chimeric variant exhibiting, for example, higher activity at high temperature. As one example, a set of 50 oligonucleotide sequences can be generated for each fragment of each parent variant, respectively. Accordingly, up to 50 crossover events within each chimeric sequence may occur during the ligation reassembly process. The likelihood that each of the generated chimeric polynucleotides will contain the oligonucleotides of each parent variant in alternating order is very low. If each oligonucleotide fragment is present in the ligation reaction in the same molar amount, it is possible that at some positions oligonucleotides from the same parent polynucleotide will be ligated side by side and thus may not result in a crossover event. If the concentration of each oligonucleotide from each parent molecule is kept constant during any ligation step in this example, there is a 1/3 chance (assuming 3 parent molecules) that the oligonucleotide from the same parent variant will be ligated within the chimeric sequence and will not generate crossover events.

[0239] Accordingly, probability density functions (PDF) can be determined to predict the population of crossover events that may occur during each ligation reaction step, for a given size of a set of parent variants, oligonucleotides corresponding to individual variants, and concentrations of each variant during each reaction step ligation. The statistics and mathematics on which the PDF calculation is based are described below. By using these methods, you can calculate this probability density function and thus enrich the descending chimeric population with a fixed number of crossover events from specific ligation reactions. In addition, the target number

101 crossover events can be predetermined and then programmed to calculate the starting amounts of each parent oligonucleotide at each stage in the ligation reaction to obtain a probability density function that focuses on a predetermined number of crossover events. These methods are directed to the use of repetitive cycles of reductive shuffling, recombination and selection that allow targeted molecular evolution of the nucleic acid encoding the polypeptide as a result of recombination. This system allows the generation of a large population of developed chimeric sequences, the generated population being significantly enriched with sequences that have a predetermined number of crossover events. A crossover event is a point in a chimeric sequence where there is a change in the sequence from one parent variant to another parent variant. Such a point is usually at the junction at which oligonucleotides derived from two parent sequences are ligated together to form a single sequence. This method allows the calculation of correct oligonucleotide sequence concentrations so that the final chimeric sequence population is enriched with a selected number of crossover events. This provides better control over the selection of chimeric variants having a predetermined number of crossover events.

Determination of crossover events [0240] Aspects disclosed herein include a system and software that receive the desired probability density (PDF) function for a crossover event, the number of parent genes to be reassembled, and the number of fragments during the reassembly process as input values. The output value of this program is the "PDF fragment" that can be used to specify the recipe for the production of reassembled genes, and the estimated PDF for crossover for these genes. The processing described in this document is alternatively performed in a programming language and programming environment for calculations MATŁAB® technical (Mathworks, Natick, Massachusetts).

Iteracyine processes [0241] Processes that can be iteratively repeated are disclosed. For example, some nucleic acid (or a given nucleic acid) responsible for the altered hydrolase or antibody phenotype is identified and re-isolated, and then it is modified and re-tested for activity. This process can be iteratively repeated until the desired phenotype has been constructed. For example, the entire biochemical anabolic or catabolic pathway may be constructed in the cell, including proteolytic activity.

[0242] Similarly, if it is determined that a particular oligonucleotide does not affect the desired property in any way (e.g., a new hydrolase phenotype), it can be removed as a variable, by synthesizing larger parent oligonucleotides that include the sequence to be deleted. As the incorporation of sequences within a larger sequence prevents any

102 crossover events, there will no longer be any variation with respect to this sequence in the daughter polynucleotides. This iterative practice for determining which oligonucleotides are most closely associated with a desired trait and which are unrelated enables more efficient exploration of all possible protein variants that could provide a particular trait or activity.

In vivo shuffling [0243] In vivo particle shuffling is used in the methods disclosed herein, which provide variants of the polypeptides disclosed herein, e.g., antibodies, hydrolases, and the like. In vivo shuffling can be carried out using the natural property of cells to recombine multimers. While in vivo recombination provides a major natural pathway to molecular diversity, genetic recombination remains a relatively complex process that involves 1) recognition of homology; 2) thread cleavage, thread invasion and metabolic steps leading to the production of recombinant chiazas; and finally 3) the separation of chiazisms into separate recombinant molecules. Chiazasm formation requires recognition of homologous sequences.

[0244] Methods for producing a hybrid polynucleotide from at least the first polynucleotide and second polynucleotide are disclosed. Disclosed are methods used to generate a hybrid polynucleotide by introducing at least the first polynucleotide and second polynucleotide that exhibit at least one common region with partial sequence homology in a suitable host cell. Regions with partial sequence homology promote processes that result in sequence reorganization to form a hybrid polynucleotide. In one aspect, the term "hybrid polynucleotide" includes any nucleotide sequence that is obtained as a result of the method disclosed herein, and includes a sequence from at least two primary polynucleotide sequences. Such hybrid polynucleotides may be the result of intermolecular recombination events promoting the integration of sequences between DNA molecules. In addition, such hybrid polynucleotides may be the result of reductive intra-molecular reshuffle processes that use repeat sequences to change the nucleotide sequence within a DNA molecule.

Preparation of sequence variants [0245] Methods for producing nucleic acid and hydrolase and antibody sequences disclosed herein, or isolating hydrolases using the nucleic acids and polypeptides disclosed herein are disclosed. Disclosed are variants of the hydrolase gene disclosed herein that can be altered by any means, including, e.g. by random or stochastic or non-stochastic or "directed evolution" methods as described above.

[0246] Methods for generating a nucleic acid variant encoding are disclosed

103 a polypeptide with hydrolase activity, e.g., lipase, saturase, palmitase, and / or stearatase activity, comprising the steps of: (a) providing a nucleic acid matrix comprising the nucleic acid disclosed herein; (b) modifying, removing or adding one or more nucleotides in a template sequence, or a combination thereof, to generate a variant nucleic acid template. In one aspect, the method may further comprise expressing a nucleic acid variant to generate a hydrolase polypeptide variant, e.g., lipase, saturase, palmitase, and / or stearatase. Modifications, additions or deletions can be introduced by error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly-type PCR, "sexual" PCR mutagenesis, in-vivo mutagenesis, cassette mutagenesis, recursive team mutagenesis, exponential team-specific mutagenesis, site-specific mutagenesis , gene reassembly, gene site saturation mutagenesis (GSSM^sm), synthetic reassembly involving ligation (SLR or Gene Reassembly) or a combination thereof. In one aspect, modifications, additions or deletions are introduced by a method including recombination, recursive sequence recombination, phosphorothioate-modified DNA mutagenesis, matrix mutagenesis of uracil, intermittent duplex mutagenesis, point mismatch repair mutagenesis, repair mutagenesis, non-repairable mutagenesis, radiogenic, deletion mutagenesis, restriction-selective mutagenesis, purifying-restriction mutagenesis, synthesis of artificial genes, team mutagenesis, formation of chimeric nucleic acid multimers and combinations thereof.

[0247] In one aspect, the method is iteratively repeated until a hydrolase is produced, e.g., lipase, saturase, palmitase and / or stearatase having altered or different activity or altered or other stability than the peptide encoded by the nucleic acid matrix. In one aspect, the polypeptide variant hydrolase, e.g., lipase, saturase, palmitase and / or stearatase, is thermally tolerant and retains some activity when exposed to elevated temperature. In another aspect, the polypeptide variant hydrolase, e.g., lipase, saturase, palmitase and / or stearatase has increased glycosylation compared to hydrolase, e.g., lipase, saturase, palmitase and / or stearatase encoded by the template nucleic acid. Alternatively, the polypeptide variant hydrolase, e.g. lipase, saturase, palmitase and / or stearatase exhibits hydrolase activity, e.g. lipase, saturase, palmitase and / or stearatase at high temperature, whereas hydrolase, e.g. lipase, saturase, palmitase and / or stearatase encoded by the template nucleic acid is not active at high temperature. In one aspect, the method may be iteratively repeated until a hydrolase coding sequence, e.g., lipase, saturase, palmitase and / or stearatase has been produced having altered codon usage from that of the template nucleic acid. In another aspect, the method may be iteratively repeated until a hydrolase gene has been produced, e.g. lipases, saturases, palmitases

104 and / or a stearatase having a higher or lower level of expression of information or stability than that of the template nucleic acid hydrolase, e.g. lipase, saturase, palmitase

In another aspect, the formulation of the final product and / or stearatase makes it possible to increase or modulate the action of the hydrolase, e.g. lipase, saturase, oalmitase and / or stearatase in the product.

[0248] Isolated variants may be those naturally occurring. The variant can also be made in vitro. Variants can be obtained using site-directed engineering techniques, random chemical mutagenesis, exonuclease III deletion procedures and standard cloning techniques. Alternatively, such variants, fragments, analogs or derivatives can be produced using chemical synthesis or modification procedures. Other methods for making variants are also known to the person skilled in the art. These include procedures in which nucleic acid sequences derived from natural isolates are modified to produce nucleic acids that encode polypeptides having properties that increase their value in industrial or laboratory applications. In such procedures, large numbers of sequence variants having one or more nucleotide differences with respect to sequences obtained from natural isolates are generated and characterized. These nucleotide differences may result in amino acid changes relative to polypeptides encoded by nucleic acids from natural isolates.

[0249] For example, variants can be generated using error-prone PCR. In error-prone PCR, PCR is carried out under conditions where the correctness of DNA polymerase copying is low, so that a high degree of point mutations along the entire length of the PCR product is obtained. Susceptible to PCR errors is described, e.g., in Leung, DW, et al., Technique, 1: 11-15, 1989) and Caldwell, RC and Joyce GF, PCR Methods Applic., 2: 28-33, 1992. Briefly, in such procedures, the nucleic acids to be mutagenized are mixed with PCR primers, reaction buffer, MgCl₂, MnCI₂, Taq polymerase and an appropriate concentration of dNTP to achieve a high degree of point mutations along the entire length of the PCR product. For example, the reaction can be carried out using 20 fmoles of nucleic acid to be mutagenized, 30 pmoles of each PCR primer, reaction buffer containing 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mM MnCI₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and ImM dTTP. PCR can be carried out for 30 cycles at 94 ° C for 1 min, 45 ° C for 1 min, and 72 ° C for 1 min. However, it should be noted that these parameters can be changed accordingly. The mutagenized nucleic acids are cloned into a suitable vector, and the activity of polypeptides encoded by mutagenized nucleic acids is assessed. [0250] Variants can also be obtained using oligonucleotide-driven mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988) Science 241: 53-57. In short, in such procedures

105 a number of double-stranded oligonucleotides carrying one or more mutations to be introduced into cloned DNA are synthesized, and introduced into the cloned DNA to be mutagenized. Clones containing mutagenized DNA are recovered, and the activities of the polypeptides encoded by them evaluated.

[0251] Another method for generating variants is assembly-type PCR. Assembling PCR requires assembly of a PCR product from a mixture of small DNA fragments. There are a large number of different parallel PCRs in the same vial, with the products of one reaction producing the products of the next reaction. PCR-folding type is described, e.g., in US Patent No. 5,965,408.

[0252] Yet another way to generate variants is "sexual" PCR mutagenesis. "Sexual" PCR mutagenesis involves forced homologous recombination between DNA molecules of different but highly related DNA sequences in vitro as a result of random fragmentation of the DNA molecule in based on sequence homology and then to fix the crossover event by primer extension in a PCR reaction. Mutagenesis by "sexual" PCR is described, e.g. in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-1075. Briefly, in such procedures a series of nucleic acids are recombined for recombination, using DNase, to generate fragments having an average size of 50-200 nucleotides. Fragments of the desired average size are purified and resuspended in the PCR mixture. PCR is carried out under conditions that facilitate recombination between nucleic acid fragments. For example, PCR can be carried out by resuspending purified fragments at a concentration of 10-30 ng / l in a solution of 0.2 mM each dNTP, 2.2 mM MgCl2, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1 % Triton Χ-100. 2.5 units of Taq polymerase per 100: 1 reaction mixture are added and PCR is carried out using the following scheme: 94 ° C for 60 seconds, 94 ° C for 30 seconds, 50-55 ° C for 30 seconds, 72 ° C for 30 seconds (30-45 times) and 72 ° C for 5 minutes. However, it should be noted that these parameters can be changed accordingly. In some aspects, oligonucleotides can be included in PCR reactions. In other aspects, the Klenow fragment of DNA polymerase I may be used in the first set of PCR reactions, and Taq polymerase in the subsequent set of PCR reactions. The recombinant sequences are isolated and the activities of the polypeptides they encode are evaluated.

[0253] Variants can also be obtained by in vivo mutagenesis. In some aspects, random mutations are generated in the sequence of interest by amplifying the sequence of interest in a bacterial strain, such as a β coli strain that carries mutations in one or more DNA repair pathways. Such "mutator" strains have a higher rate of random mutation than that of the wild-type parent. DNA propagation in one of these strains will eventually generate random mutations within DNA. Mutant strains suitable for use in in vivo mutagenesis are described, e.g., in PCT Publication No. WO 91/16427.

106 [0254] Variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double-stranded DNA molecule is replaced by a synthetic oligonucleotide "cassette" that differs from the native sequence. The oligonucleotide often contains a fully and / or partially randomized native sequence.

[0255] Recursive team mutagenesis can also be used to generate variants. Recursive team mutagenesis is an algorithm for constructing protein (protein mutagenesis) developed to produce a diverse population of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control subsequent rounds of combinatorial cassette mutagenesis. Recursive team mutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815.

[0256] In some aspects, variants are generated using exponential team mutagenesis. Exponential team mutagenesis is the process of generating combinatorial libraries with a high percentage of unique and functional mutants, with small groups of residues being parallel randomized to identify the amino acids that lead to functional proteins at each changed position. Exponential syndrome mutagenesis is described, e.g., in Delegrave (1993) Biotechnology Res. 11: 1548-1552. Random and site-directed mutagenesis is described, e.g., in Arnold (1993) Current Opinion in Biotechnology 4: 450-455.

[0257] In some aspects, variants are generated using shuffling procedures in which portions of many nucleic acids that encode separate polypeptides are fused together to form chimeric nucleic acid sequences that encode chimeric polypeptides, as described, e.g., in US Patent Nos. US 5,965,408; US 5,939,250. [0258] Variants of polypeptides comprising sequences in which one or more amino acid residues (e.g. for an exemplary polypeptide, e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 8 or SEQ ID NO: 20 or SEQ ID NO: 2, having one, two, three, four, five, six, seven, eight or more (many) or all amino acid variants described in Table 3, Table 4, Table 9, Table 1.0, Table 11, Table 16 and Table 23 or equivalent) are substituted with a conservative or non-conservative amino acid residue (e.g., a conservative amino acid residue) and such substituted amino acid residue may or may not be encoded by the genetic code. Conservative substitutions are those that substitute a given amino acid in a polypeptide with another amino acid with similar properties. Thus, polypeptides herein include those with conservative sequence substitutions, e.g., exemplary sequences disclosed herein (e.g. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20 or SEQ ID NO: 2, having one, two, three, four, five, six, seven, eight or more (many) or

107 all amino acid variants described in Table 3, Table 4, Table 9, Table 1.0 Table 11, Table 16 and Table 23 or their equivalent), including but not limited to the following replacements: replacement of an aliphatic amino acid such as alanine, valine, leucine and isoleucine to another aliphatic amino acid; exchange of serine for threonine or vice versa; exchanging an acid residue such as aspartic acid and glutamic acid for another acid residue; replacing a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; replacing a basic residue such as lysine and arginine with another basic residue; and replacing an aromatic residue such as phenylalanine, tyrosine or tryptophan with another aromatic residue. Other variants are those in which one or more of the amino acid residues of the polypeptides disclosed herein contains a substituent group. [0259] Other variants disclosed herein are those in which the polypeptide is combined with another compound, such as a compound to increase the half life of the polypeptide, for example, polyethylene glycol. Additional variants disclosed herein are those in which additional amino acids are fused to a polypeptide, such as a leader sequence, secretory sequence, protein sequence or sequence that facilitates purification, enrichment or stabilization of the polypeptide. In some aspects, variants, fragments, derivatives and analogs of the polypeptides disclosed herein retain the same biological function or activity as the exemplary polypeptides, e.g., proteolytic activity, as described herein. In other aspects, the variant, fragment, derivative or analog comprises a protein, such that the variant, fragment, derivative or analog can be activated by cleaving a fragment of the protein to form an active polypeptide.

Codon optimization to obtain high levels of protein expression in host cells [0260] Methods of modifying nucleic acids encoding hydrolases to modify codons have been disclosed. Methods are disclosed for modifying nucleic acid codons encoding hydrolase to increase or decrease its expression in a host cell, e.g., a bacterial, insect, mammalian, yeast or plant cell. In addition, the present invention discloses nucleic acids encoding modified hydrolase to increase expression in host cells of such modified hydrolase and methods for producing modified hydrolases. The method includes identifying a "non-preferred" and "less preferred" codon in the nucleic acid encoding hydrolase and replacing one or more of these non-preferred or less preferred codons with a preferred codon encoding the same amino acid as the replaced codon and in which at least one - a preferred or less preferred codon in the nucleic acid is replaced with a preferred codon encoding the same amino acid. The preferred codon is the codon over represented in coding sequences in the genes in the host cell, and not preferred or less

108 preferred codon means a codon under-represented in coding sequences in genes in the host cell.

[0261] Host cells for expressing the nucleic acids, expression cassettes and vectors disclosed herein include bacteria, yeast, fungi, plant cells, insect cells and mammalian cells. Methods for optimizing codon usage in all of these cells, codon-modified nucleic acids and polypeptides derived from modified-codon nucleic acids are disclosed. Exemplary host cells include gram negative bacteria such as Escherichia coli and Pseudomonas fiuorescens, gram positive bacteria such as Lactobaciiius gasseri, Lactococcus iactis, Lactococcus cremoris, Bacillus subtiiis. Exemplary host cells also include eukaryotic organisms, e.g. various yeasts such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and Kiuyveromyces iactis, Hansenuia poiymorpha, Aspergillus niger, and mammalian cells, cell lines and insect cells and cell lines. Other examples of host cells include bacterial cells such as E. coii, Streptomyces, Bacillus subtiiis, Bacillus cereus, Salmonella typhimurium and various species within the genus Pseudomonas, Streptomyces and Staphyiococcus, fungal cells such as Aspergillus, yeast such as any species of Pichia, Saccharomyces, Schizosaccharomyces, cerevisiae or Schizosaccharomyces pombe, insect cells such as Drosophiia S2 and Spodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma and adenoviruses. The choice of a suitable disclosed host is within the capabilities of those skilled in the art. Disclosed herein are nucleic acids and polypeptides optimized for expression in these organisms and species.

[0262] For example, nucleic acid codons encoding hydrolase isolated from a bacterial cell are modified such that the nucleic acid is optimally expressed in a bacterial cell different from the bacterium from which the hydrolase is derived, in a yeast, fungus, plant cell, insect cell or mammalian cell. Methods for codon optimization are well known in the art, see e.g. US Patent No. 5,795,737; Baca (2000) Int. 1 Parasitol. 30: 113-118; Hale (1998) Protein Expr. Purif. 12: 185-188; Narum (2001) Infect. Immun. 69: 7250-7253. See also Narum (2001) Infect. Immun. 69: 7250-7253, describing codon optimization in mouse systems; Outchkourov (2002) Protein Expr. Purif. 24: 1824, describing codon optimization in yeast; Feng (2000) Biochemistry 39: 1539915409, describing codon optimization in E. coli; Humphreys (2000) Protein Expr. Purif. 20: 252-264, describing the optimization of codon usage that affects secretion in E. coii.

Non-human transgenic animals [0263] Non-human transgenic animals containing a nucleic acid, polypeptide (e.g., hydrolase or antibody disclosed herein) disclosed, cassette for

109 expression, vector, transfected or transformed cell disclosed herein. Transgenic, non-human animals may be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice containing the nucleic acids disclosed herein. These animals can be used, e.g., as in vivo models for testing for hydrolase activity, or as models for screening for factors that alter in vivo hydrolase activity. Coding sequences for polypeptides to be expressed in transgenic non-human animals can be designed as constitutive, or under the control of tissue-specific, development-specific or inducible transcription regulatory factors. Transgenic non-human animals can be designed and generated using any method known in the art; see e.g. U.S. Patents No. US 6,211,428; US 6,187,992; US 6,156,952; US 6,118,044; US 6,111,166; US 6,107,541; US 5,959,171;

US 5,922,854; US 5,892,070; US 5,880,327; US 5,891,698; US 5,639,940; US 5,573,933;

US 5,387,742; US 5,087,571, describing the preparation and use of transformed cells and eggs, and transgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g., Pollock (1999) J. Immunol. Methods 231: 147-157, describing the production of recombinant proteins in the milk of transgenic dairy animals; Baguisi (1999) Nat. Biotechnol. 17: 456-461, depicting the production of transgenic goats. U.S. Patent No. 6,211,428, describing the preparation and use of transgenic, non-human mammals that express in their brains a nucleic acid construct comprising a DNA sequence. U.S. Patent No. 5,387,742 describes the injection of cloned recombinant or synthetic DNA sequences into fertilized mouse eggs, implantation of injected eggs in pseudo-pregnant females, and breeding for transgenic mice whose cells express proteins associated with Alzheimer's disease pathology. US Patent No. 6,187,992, describes the preparation and use of a transgenic mouse whose genome contains an interrupted gene encoding an amyloid precursor protein (APP).

[0264] "Knockout animals" may also be used to implement the methods provided herein. For example, the transgenic or modified animals disclosed herein include "knockout animals", e.g., "knockout mice" constructed not to express endogenous a gene that is replaced by a gene expressing hydrolase, or, a fusion protein containing the hydrolase disclosed herein. As mentioned above, functional knockouts can also be generated using the antisense sequences disclosed herein, e.g., double-stranded RNAi molecules.

Transgenic Plants and Seeds [0265] Transgenic plants and seeds comprising a nucleic acid, polypeptide (e.g., hydrolase or antibody disclosed herein), expression cassette, vector or transfected or transformed cell disclosed herein are disclosed. The transgenic plant can be dicot or monocotyledonous

110 monocot). Also disclosed herein are methods for producing and using these transgenic plants and seeds. The transgenic plant or plant cell expressing the polypeptide disclosed herein can be constructed according to any method known in the art. See, for example, US Patent No. 6,309,872.

[0266] The nucleic acids and expression constructs disclosed herein can be introduced into a plant cell by any means. For example, nucleic acids or expression constructs may be introduced into the genome of the desired host plant, or, nucleic acids or expression constructs may be episomes. The introduction of the desired plant into the genome may be such that the production of host hydrolase is regulated by endogenous transcriptional or translational control elements. Disclosed herein are "knockout plants" in which the introduction of a gene sequence, e.g., by homologous recombination, disrupted the expression of an endogenous gene. Methods for generating "knockout" plants are well known in the art, see, e.g., Strepp (1998) Proc Natl . Acad. Sci. USA 95: 4368-4373; Miao (1995) Plant J 7: 359-365. See discussion on transgenic plants below.

[0267] The nucleic acids disclosed herein can be used to impart desirable characteristics to virtually any plant, e.g., oilseed producing plants, including rice bran, rapeseed (canola varieties), sunflower, olive, palm or soybean, and the like, or glucose or starch-producing plants, such as corn, potato, wheat, rice, barley and the like. The nucleic acids disclosed herein can be used to manipulate plant metabolic pathways to optimize or change the expression of a hydrolase or hydrolase substrate or product, e.g., an oil, lipid such as mono-, di- or tri-acylglyceride and the like. It can change the proportion of lipids and lipid conversion and metabolic turnover in the plant. This can facilitate the industrial processing of the plant. Alternatively, the hydrolases disclosed herein can be used in the production of a transgenic plant for the production of a compound that is not naturally produced by that plant. This can reduce production costs or create a new product.

[0268] In one aspect, the first step in producing the transgenic plant comprises forming an expression construct for expression in the plant cell. These techniques are well known in the art. These may include selection and cloning of a promoter, coding sequence to facilitate efficient ribosome binding to mRNA, and selection of appropriate gene terminator sequences. An example of a constitutive promoter is CaMV35S from cauliflower mosaic virus, which usually results in high expression in plants. Other promoters are more specific and react to stimuli in the plant's internal or external environment. An example of a light-inducible promoter is the cab gene promoter encoding the main chlorophyll a / b binding protein.

111 [0269] In one aspect, the nucleic acid is modified to achieve a higher level of expression in the plant cell. For example, the sequence disclosed herein may have a higher percentage of AT nucleotide pairs relative to that observed in the plant, some of which prefer GC nucleotide pairs. Therefore, AT nucleotides in the coding sequence may be substituted with GC nucleotides, without a significant change in the amino acid sequence to increase the production of the gene product in plant cells. [0270] A selectable marker gene can be added to the gene construct to identify plant cells or tissues that have successfully integrated the transgene. This may be necessary because achieving the incorporation and expression of genes in plant cells is a rare event occurring only in a few percent of the target tissues or cells. The selectable marker genes encode proteins that provide resistance to agents that are normally toxic to the plant, such as antibiotics or herbicides. Only plant cells that have integrated the selectable marker gene survive when grown on media containing the appropriate antibiotic or herbicide. As for other introduced genes, marker genes also require promoter sequences and termination for proper functioning.

[0271] In one aspect, the production of transgenic plants or seeds involves incorporating the sequences disclosed herein and, optionally, marker genes into a target expression construct (e.g., plasmid, phage), along with positioning of the promoter and termination. This may involve transfer of the modified gene into the plant using an appropriate method. For example, the construct can be introduced directly into the genomic DNA of a plant cell using techniques such as electroporation and microinjection into plant cell protoplasts, or the constructs can be introduced directly into the plant tissue using ballistic methods such as bombardment with DNA coated particles. For example, see e.g. Christou (1997) Plant Mol. Biol. 35: 197-203; Pawłowski (1996) Mol. Biotechnol. 6: 17-30; Klein (1987) Nature 327: 70-73; Takumi (1997) Genes Genet. Syst. 72: 63-69, discussing the use of particle bombardment to introduce transgenes into wheat; and Adam (1997) above regarding the use of particle bombardment to introduce YACs into plant cells. For example, Rinehart (1997) above, used particle bombardment to generate transgenic cotton plants. A particle acceleration device is described in US Patent No. 5,015,580; and the commercially available BioRad (Biolistics) PDS-2000 device for accelerating particles; see also, John, US Patent No. 5,608,148; and Ellis, US Patent No. US 5,681,730, describing particle-mediated transformation of gymnosperms.

[0272] In one aspect, protoplasts can be immobilized and injected with nucleic acids, for example, an expression construct. Although regeneration of plants from protoplasts is not easy with cereals, regeneration of legume plants is possible with

112 using somatic embryogenesis from callus-derived protoplasts. Organized tissues can be transformed with naked DNA using a gene gun technique in which the DNA is coated on tungsten micro projectiles, a 1/100 shot of the size of cells that carries DNA deep into the cells and organelles. The transformed tissue is then induced for regeneration, usually using somatic embryogenesis. This technique was successfully used in many cereal species as well as corn and rice.

[0273] Nucleic acids, e.g., expression constructs can also be introduced into plant cells using recombinant viruses. Plant cells can be transformed using viral vectors such as, for example, vectors derived from tobacco mosaic virus (Rouwendal (1997) Plant Mol.Biol.33: 989-999), see Porta (1996) Use of viral replicons for the expression of genes in plants, "Mol. Biotechnol. 5: 209-221.

[0274] Alternatively, nucleic acids, e.g. an expression construct, may be combined with the appropriate T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. Agrobacteńum tumefaciens host virulence factors will be directed against the introduction of the construct and the adjacent marker into the plant cell DNA when the cell is infected with bacteria. Transformation techniques with Agrobacterium tumefaciens, including virulence deprivation and the use of binary vectors are well described in the scientific literature. See, e.g., Horsch (1984) Science 233: 496-498; Fraley (1983) Proc. Natl. Acad. Sci. USA 80: 4803 (1983); Gene Transfer to Plants, Potrykus, ed. (Springer-Verlag, Berlin 1995). DNA in the A. tumefaciens cell is found in the bacterial chromosome as well as in another structure known as the Ti plasmid (tumor-inducing). The Ti plasmid contains a stretch of DNA called T-DNA (~ 20 k in length), which is transferred to the plant cell during the infection process, and a number of virulenq'i (vir) genes that direct the infection process. A. tumefaciens can only infect plants through damage: when the root or stem of a plant is damaged, it releases some chemical signals in response to which, vir A. genes tumefaciens become active and direct a series of events necessary to transfer T-DNA from the Ti plasmid to the plant chromosome. T-DNA then enters plant cells through damage. One speculation assumes that T-DNA waits for plant DNA to begin replication or transcription, then inserted into exposed plant DNA. To use A. tumefaciens as a transgenic vector, the T-DNA inducing tumor section must be removed while maintaining the T-DNA border regions and vir genes. The transgene is then inserted between the T-DNA border regions in which it is transferred to the plant cell and integrated into the plant chromosomes.

[0275] Methods of transforming monocotyledonous plants using the nucleic acids disclosed herein, including important cereals, are disclosed, see Hiei (1997) Plant Mol. Biol. 35: 205-218. See also, e.g., Horsch, Science (1984) 233: 496; Fraley (1983) Proc.

113

Natl. Acad. Sci. USA 80: 4803; Thykjaer (1997) above; Park (1996) Plant Mol. Biol. 32: 11351148, discussing the integration of T-DNA into genomic DNA. See also D'Halluin, US Patent No. 5,712,135, describing a process for the stable integration of DNA containing a gene that is functional into a grain cell or other monocotyledonous plant.

[0276] In one aspect, the third step may require the selection and regeneration of entire plants capable of transferring the incorporated target gene to the next generation. Such regeneration techniques are based on the manipulation of certain phytohormones in tissue culture growth medium, typically based on biocide markers and / or herbicides that have been introduced along with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be accomplished via plant callus, explants, organs or parts thereof. Such regeneration techniques are generally described in Klee (1987) Ann. Rev. of Plant Phys. 38: 467-486. To obtain complete plants from transgenic tissues such as immature embryos, they can be grown under controlled environmental conditions in a series of nutrient and hormone containing media, a process known as tissue culture. After regeneration of whole plants and receiving seeds, the progeny assessment begins.

[0277] After stable incorporation of the expression cassette in transgenic plants, it can be introduced into other plants by sexual crossing. Any number of standard breeding techniques can be used, depending on the species to be crossed. As the transgenic expression of the nucleic acids disclosed herein leads to phenotypic changes, the plants containing the recombinant nucleic acids disclosed herein can be sexually crossed with a second plant to obtain the final product. Thus, the seed disclosed herein may be derived from a cross between two transgenic plants disclosed herein, or from a cross between a plant disclosed herein and another plant. The desired effects (e.g., expression of the polypeptides disclosed herein, to produce a plant with altered or elevated and / or reduced lipid or oil content) can be enhanced when both parent plants express the polypeptides disclosed herein. The desired effects can be carried forward to subsequent generations of plants using standard multiplication methods.

[0278] The nucleic acids and polypeptides disclosed herein are expressed in or introduced into any plant or seed. The transgenic plants disclosed herein may be dicotyledonous or monocotyledonous. Examples of monocotyledonous transgenic plants of the invention are grasses such as panicle (panicle, Poa grass), fodder grass such as fescue, ryegrass, temperate climate grasses such as Agrostis,

114 and cereals, e.g. wheat, oats, rye, barley, rice, sorghum and corn. Examples of dicotyledonous transgenic plants disclosed herein are tobacco, papilionaceous plants such as lupine, potato, sugar beet, peas, beans and soybeans, and brassica plants (Brassicaceae family) such as cauliflower, oilseed rape, and the closely related model organism Arabidopsis thaiiana . Thus, the transgenic plants and seeds disclosed herein cover a wide range of plants, including, but not limited to, species of the genus Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citruiius, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Eiaeis, Fragaria, Giycine, Gossypium, Heiianthus, Heteracallis, Hordeum, Hyoscyamus, Lactuca, Linum, Loiium, Lupinus, Lycopersicon, Maius, Manihot, Majorana, Medicago, Nicotiana, Oiea, Oryza, Panieum, Pannisetum, Persea, Phaseoius, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secaie, Senecio, Sinapis, Soianum, Sorghum, Theobromus, Trigoneiia, Triticum, Vicia, Vitis, Vigna, and Zea.

[0279] Nucleic acids disclosed herein can be expressed in plants that contain fiber cells, including, for example, cotton, down pollinia (kapok tree, Ceiba pentandra), desert willow, creosote shrub, Krascheninnikowia, balsa, arm, hemp ketemia, hemp, oxalic hibiscus, jute, agave, abacus and flax. The transgenic plants disclosed herein may belong to the genus Gossypium, including any species of Gossypium, such as G. arboreum, G. herbaceum, G. barbadense and G. hirsutum.

[0280] Transgenic plants of the present description can be used to produce large amounts of the polypeptides (e.g., antibodies, hydrolases) disclosed herein. For example, see Palmgren (1997) Trends Genet. 13: 348; Chong (1997) Transgenic Res. 6: 289-296 (production of human milk beta-casein protein in transgenic potato plants using auxin inducible, bi-directional mannopine synthase promoter (masl ', 2') with Agrobacterium tumefacienś mediated transformation of cotyledons).

[0281] Using known procedures, a skilled person can screen the plants disclosed herein by detecting in transgenic plants the increase or decrease in transgene or protein mRNA level. Methods for detecting and quantifying mRNA or protein levels are well known in the art.

[0282] The present specification discloses fatty acids or fatty acid derivatives from transgenic plants as disclosed herein, e.g., transgenic oil plants. In one aspect, transgenic oil plants containing at least one hydrolase disclosed herein are produced. In one aspect, the transgenic plant contains a hydrolase gene functionally linked to a promoter that allows the expression of the gene in cell, tissue or extracellular compartments other than those in which plant lipids accumulate, or allowing exogenous induction

115 hydrolase. In one aspect, seeds and / or fruit containing lipids of the plant are harvested, seeds and / or fruit ground (if necessary, after treatment to induce a hydrolase gene (e.g., saturase lipase, palmitase and / or stearatase)) to provide for contacting lipids and hydrolase as disclosed herein contained in seeds and / or fruits. The mixture may be allowed to incubate to allow enzymatic hydrolysis of lipids from the particulate material by the catalytic action of the lipase disclosed herein contained in the particulate material. In one aspect, the fatty acids resulting from hydrolysis are extracted and / or converted to obtain the desired fatty acid derivatives.

[0283] This enzymatic hydrolysis process disclosed herein utilizes mild operating conditions and can be used on a small scale and use inexpensive installations. In this aspect, the plant disclosed herein is induced to produce hydrolase to convert plant lipids. Using this strategy, contact of the enzyme with accumulated plant lipids is prevented to avoid any risk of premature hydrolysis ("self-degradation of the plant") prior to harvest. Shredding and incubation units can be light and small-scale; many of them are known in agriculture and can be carried out in places where plants are harvested. [0284] In one aspect, the transgenic plants disclosed herein are produced by transforming natural oilseeds. The genetically transformed plants disclosed herein are then reproduced sexually to produce the transgenic seeds disclosed herein. These seeds can be used to obtain the progeny of a transgenic plant.

[0285] In one aspect, the hydrolase gene is operably linked to an inducible promoter to avoid premature contact of the hydrolase and the lipid plant. This promoter may direct gene expression in compartments other than those in which lipids accumulate, or the promoter may initiate hydrolase expression at a selected time by exogenous induction.

Polypeptides and peptides [0286] Isolated, synthetic or recombinant polypeptides having sequence identity (e.g., at least 50% sequence identity) with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: are disclosed 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20 or SEQ ID NO: 2, having one, two, three, four, five six, seven, eight or more (several) or all amino acid variants described in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23 or their counterparts. Nucleic acids encoding polypeptides having the sequence shown by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 are disclosed. , SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20 or SEQ ID NO: 2, having one, two, three, four, five, six, seven, eight or more (several) or all variants

116 amino acids described in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23 or equivalent.

[0287] Sequence identity may occur along the entire length of the polypeptide, or identity may occur in a region of at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 , 650, 700 or more residues. The polypeptides disclosed in the invention may be shorter than the total length of exemplary polypeptides. Polypeptides containing only a fragment of the sequence from the disclosed sequence are disclosed, exemplary subsequences may contain about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues. In alternative aspects, the polypeptides (peptides, fragments) may range in size between about 5 to full length of the polypeptide, e.g., the enzyme disclosed herein; has an example size consisting of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175 , 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues, e.g., further residues of the exemplary hydrolase disclosed herein. The peptides disclosed herein may be useful as, e.g., labeling probes, antigens, toleragens, motifs, hydrolase active sites.

[0288] The polypeptides disclosed herein also include antibodies capable of binding to the hydrolase disclosed herein.

[0289] The polypeptides disclosed herein also include amino acid sequences that are "substantially identical" to the sequences disclosed herein, including sequences that differ from the reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions or inserts , in particular if such substitution occurs at a site which is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid for another in the same group (e.g. substitution of one hydrophobic amino acid such as isoleucine, valine, leucine or methionine in place of another, or substitution of one polar amino acid in place of another, such as substitution of arginine in place of lysine, glutamic acid in place of aspartic acid or glutamine in place of asparagine). One or more amino acids can be removed, for example, from hydrolase, resulting in a modification of the structure of the polypeptide without a significant change in its biological activity. For example, amino- or carboxyterminal amino acids that are not required for hydrolase activity may be removed.

[0290] An "amino acid" or "amino acid sequence" may include an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof, portion or subunit of each, as well as naturally occurring or synthetic molecules.

117 [0291] The terms "polypeptide" and "protein" may include amino acids linked together via peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than 20 amino acids encoded by the genes. The term "polypeptide" also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides. The peptides and polypeptides disclosed herein also include all "mimetic" and "peptidomimetic" forms as described in more detail below.

[0292] The polypeptides disclosed herein include hydrolases in active or inactive form. For example, the polypeptides disclosed herein include pre-mature protein or pre-pro sequence processing, e.g., processing an enzyme protein such as a convertase protein to generate an "active" mature protein. The polypeptides disclosed herein include hydrolases inactive for other reasons, e.g., prior to "activation" by post-translational processing, e.g. using endo-or exo-peptidase or proteinase activity, a phosphorylation, amidation, glycosylation or sulfation event, dimerization event and the like. Methods for identifying "prepro" domain sequences and signal sequences are well known in the art, see, for example, Van de Ven (1993) Crit. Rew Oncog. 4 (2): 115-136. For example, to identify the prepro sequence, the protein is purified from the extracellular space and the N-terminal protein sequence is determined, and compared to the untreated form.

[0293] The polypeptides disclosed herein include all active forms, including active subsequences, e.g., catalytic domains or active sites, of the enzyme disclosed herein. Catalytic domains and active sites were disclosed as shown below. Peptides or polypeptides containing or consisting of an active site domain are disclosed in the form predicted using a database such as Pfam (which is a large collection of multiple sequence comparisons and hidden Markova models involving multiple shared protein families, Pfam protein family database, A. Bateman, E. Birney, L. Cerruti, R. Durbin, L. Etwiller, SR Eddy, S. Griffiths-Jones, KL Howe, M. Marshall, and ELL Sonnhammer, Nucleic Acids Research, 30 (1): 276-280, 2002) or equivalent.

[0294] Polypeptides with or without signal and / or prepro sequences are disclosed. Polypeptides with heterologous signal sequences and / or prepro sequences are disclosed. The prepro sequence (including the sequence disclosed herein used as the heterologous prepro domain) can be located at the amino or carboxy terminus of the protein. Isolated, synthetic or recombinant signal sequences, prepro sequences and catalytic domains (e.g. "active centers") comprising or consisting of the sequences disclosed herein. The signal sequence, prepro domain and / or catalytic domain disclosed herein may be part of a fusion protein, e.g. as a heterologous domain of a chimeric protein. Acids disclosed

118 nucleic codes encoding these catalytic domains (CDs), prepro domains and signal sequences (SPs, e.g., a peptide having a sequence comprising / consisting of terminal amino acid residues of a polypeptide disclosed herein). Signal sequences have been disclosed, comprising a peptide comprising / consisting of sequences as shown in residues 1 to 12, from 1 to 13, 1 to 14, from 1 to 15, from 1 to 16, 1 to 17, from 1 to 18, from 1 to 19, 1 to 20, from 1 to 21, from 1 to 22, from 1 to 23, from 1 to 24, 1 to 25, from 1 to 26, from 1 to 27, from 1 to 28, from 1 to 28, from 1 to 30, from 1 to 31, from 1 to 32, from 1 to 33, from 1 to 34.1 to 35, from 1 to 36, from 1 to 37, from 1 to 38.1 to 39 , from 1 to 40.1 to 41.1 to 42, from 1 to 43, from 1 to 44, 1 to 45, from 1 to 46, from 1 to 47, from 1 to 48, from 1 to 49 or from 1 to 50, of the polypeptide disclosed herein.

[0295] The polypeptides and peptides disclosed herein may be isolated from natural sources, or synthetic, or may be recombinantly generated polypeptides. Peptides and proteins can be expressed recombinantly in vitro or in vivo. The peptides and polypeptides disclosed herein can be made and isolated by any method known in the art. The polypeptide and peptides disclosed herein can also be synthesized, in whole or in part, by chemical methods well known in the art. See, e.g., Caruthers (1980) Nucleic Acids Res. Symp. Cheese. 215-223; Horn (1980) Nucleic Acids Res. Symp. Cheese. 225-232; Banga, AK, Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA. For example, peptide synthesis can be carried out using various solid phase techniques (see e.g., Roberge (1995) Science 269: 202; Merrifield (1997) Methods Enzymol. 289: 3-13) and automated synthesis, e.g., using the ABI 431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions.

[0296] The peptides and polypeptides disclosed herein may also be glycosylated. Glycosylation can be added post-translational chemically or using cellular biosynthesis mechanisms, the latter introducing the use of known glycosylation motifs that may be native to the sequence or may be added as a peptide or added as a coding nucleic acid sequence. Glycosylation can be O- or N-binding.

[0297] The term "recombinant" polypeptides or proteins refers to polypeptides or proteins produced by recombinant DNA techniques; i.e. produced from cells transformed with an exogenous DNA construct encoding the desired polypeptide or protein. "Synthetic" nucleic acids (including oligonucleotides), polypeptides or proteins disclosed herein include those produced by any chemical synthesis method, e.g. as described below.

[0298] As used herein, "fragments" are parts of a naturally occurring protein that can exist in at least two different conformations. Fragments may have

119 the same or substantially the same amino acid sequence as a naturally occurring protein. As used herein, "enzymatically active fragments" are the part of the amino acid (coding for a protein) sequence that retains at least one functional activity of the protein to which it relates. The term "substantially the same" means that the amino acid sequence is substantially, but not completely, the same, but retains at least one functional activity of the sequence to which it relates. Generally, two amino acid sequences are "substantially the same" or "substantially homologous" if they are at least about 85% identical. Fragments that have different three-dimensional structures from the naturally occurring protein are also included. An example of this is a "pro" molecule such as a low activity protein that can be modified by cleavage to produce a mature enzyme with much higher activity.

[0299] The peptides and polypeptides disclosed herein, as defined above, include all "mimetic" and "peptidomimetic" forms. The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound that has essentially the same structural and / or functional characteristics of the polypeptides disclosed herein. The mimetic may be completely composed of synthetic analogues of non-natural amino acids, or is a chimeric complex molecule partly from natural peptide amino acids and partly from non-natural amino acid analogs. The mimetic may also contain any number of conservative substitutions of natural amino acids, as long as such substitutions also do not substantially change the structure and / or activity of the mimetic. As with the polypeptides disclosed herein, which are conservative variants, routine testing will determine whether the mimetic is within the scope of this disclosure, i.e. that its structure and / or function is not significantly changed. Thus, in one aspect, the mimetic composition is within the scope of the present disclosure if it has hydrolase activity.

[0300] Mimetic polypeptide compositions disclosed herein may contain any combination of unnatural structural components. In an alternative embodiment, the mimetic compositions disclosed herein comprise one or all of the following three structural groups: a) linker residues other than natural amide linkage ("peptide bond"); b) unnatural residues in place of naturally occurring amino acid residues; or c) residues that induce a secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., beta twist conformation, gamma twist, beta sheet, alpha helix and the like. For example, the polypeptide disclosed herein may be characterized as a mimetic if all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds

120 or coupling agents such as e.g. glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N, N'-dicyclohexylcarbodiimide (DCC) or N, N'diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to traditional linkages via amide linkages ("peptide linkage") include, e.g., ketomethylene (e.g. C (= O) -CH₂- to -C (= O) -NH-), aminomethylene (CH₂-NH), ethylene, olefin (CH = CH), ether (CH₂0), thioether (CH₂-S), tetrazole (CN₄-), thiazole, retroamide, thioamide or ester (see, e.g., Spatola (1983), in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, vol. 7, p. 267357, "Peptide Backbone Modifcations," by Marceli Dekker, New York).

[0301] The polypeptide disclosed herein can also be termed mimetic in that it contains all or part of the unnatural residue in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature; several examples of non-natural compositions useful as mimetics of natural amino acid residues and instructions are described below. Aromatic amino acid mimetics can be prepared by substitution, for example, with D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2 thienylalanine; D- or Ll, -2, 3- or 4 pyreneylalanine; D-or L-3 thienylalanine; D- or L- (2-pyridyl) -alanine; D- or L- (3-pyridyl) alanine; D- or L- (2-pyrazinyl) -alanine; D- or L- (4-isopropyl) -phenylglycine; D (trifluoromethyl) -phenylglycine; D- (trifluoromethyl) -phenylalanine; Dp-fluoro-phenylalanine; D or Lp-biphenylphenylalanine; D or Lp-methoxy-biphenylphenylalanine; D- or L-2-indolo (alkyl) alanine; and D- or L-alkylamines, wherein the alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, isopentyl, or in the form of non-acidic amino acids. Aromatic rings of non-natural amino acids include, e.g., aromatic rings of thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl and pyridyl.

[0302] Acid amino acid mimetics can be generated by substitution, e.g., with non-carboxylic amino acids while maintaining a negative charge; (Phosphono) alanine; sulfated threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'-NCN-R '), such as, e.g., 1-cyclohexyl-3- (2-morpholinyl- (4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimetholopentyl) carbodiimide. Aspartyl or glutamyl can also be converted by reaction with ammonium ions to asparaginyl residues and Iowa mine gluta. Basic amino acid mimetics can be generated by substitution, e.g., (in addition to lysine and arginine substitution) with ornithine, citrulline or (guanidino) acetic acid or (guanidino) alkyl acetic acid, wherein alkyl is as defined above. Nitrile derivatives (e.g. containing a CN moiety instead of COOH) may be substituted in place of asparagine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues. Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more

121 conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione or ninhydrin, optionally under basic conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidisole and tetranitromethane can be used to form O-acetyl tyrosyl and 3-nitro derivatives, respectively. Cysteine ​​residue mimetics can be prepared by reacting cysteine ​​residues with, e.g., alphahaloacetates), such as 2-chloroacetic acid or chloroacetamide, and the corresponding amines; to obtain carboxymethyl or carboxamidomethyl derivatives. Mystics of cysteine ​​residues can also be generated by reacting cysteinyl residues with, e.g., bromotrifluoroacetone, alpha-bromo-beta- (5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4-nitrophenol; or, chloro-7-nitrobenzo-2-oxa-1,3-diazole. Lysine mimetics can be generated (and terminal amino residues changed) by reacting lysinyl with succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino acid mimetics can also be generated by reacting imidoesters such as picolinimidazolic acid methyl ester; pyridoxal phosphate, pyridoxal, hydrochloride, trinitrobenzenesulfonic acid, Omethylissourea, 2.4 pentanedione; and transaminase catalyzed reactions with glyoxylate. Methionine mimetics can be generated by reacting with, e.g., methionine sulfoxide. Proline mimetics include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline or 3,3-dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylpyrocarbonate or p-bromophenacyl bromide. Other mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of hydroxyl groups on serine or threonine residues; methylation of lysine, arginine and histidine alpha-amino groups; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.

[0303] A residue, e.g., an amino acid, of a polypeptide disclosed herein may also be replaced by an amino acid (or peptidomimetic residue) of opposite chirality. Thus, any amino acid naturally occurring in the L configuration (which can also be referred to as R or S, depending on the chemical structure of the unit) can be replaced with an amino acid of the same structural chemical type with a peptidomimetic, but with the opposite chirality, referred to as the D-amino acid, which may also be referred to as the R- or S- form.

[0304] Methods of modifying the polypeptides disclosed herein by means of natural processes, such as post-translational processing (e.g., phosphorylation, acylation, etc.) or chemical modification techniques, as well as obtained modified polypeptides are disclosed. Modifications can occur anywhere in the polypeptide, including the skeleton

122 peptide, amino acid side chains and at the amino or carboxy termini. It will be understood that the same type of modification may be present in the same or different degrees at several places in a given polypeptide. In addition, a given polypeptide may have many types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent flavin attachment, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, formation of cyclization linkage covalent, cysteine ​​formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation and addition of amino acids to RNA-mediated proteins such as arginylation. See, e.g., Proteins Structure and Molecular Properties 2nd Edition, WH Freeman and Company New York (1993.); Needs cosylation modification of proteins, BC Johnson, ed. Academic Press, New York, pp. 1-12 (1983).

[0305] Solid phase chemical peptide synthesis methods can also be used to synthesize the polypeptides or fragments thereof disclosed herein. Such methods have been known in the art since the early 1960s (Merrifield, RB, J. Am. Chem.Soc., 85: 21492154,1963) (See also Stewart, JM and Young, JD, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co, Rockford, III, pp. 11-12)) and recently used in commercially available laboratory kits for peptide design and synthesis (Cambridge Research Biochemicals). Such commercially available laboratory kits typically utilize the science of HM Geysen et al., Proc. Natl. Natl. Acad. Sci., USA, 81: 3998 (1984) and provide peptide synthesis on the tips of many "rods" or "pins", all of which are connected to one plate. When such a system is used, the plate with rods or pins is inverted and inserted into the second plate with their corresponding wells or reservoirs that contain solutions for attaching or anchoring the corresponding amino acid to the tip of the pin or rod. By repeating this stage of the process, i.e. by inverting and inserting the tip of the pin or rod into the appropriate solutions, the amino acids build into the desired peptides. In addition, many FMOC based peptide synthesis systems are available. For example, fusion of a polypeptide or fragment can be accomplished on a solid support using the Applied Biosystems, Inc Model 431A ™ automated peptide synthesizer. Such equipment provides easy access to the peptides disclosed herein, by direct synthesis or by the synthesis of a number of fragments that can be combined by other known techniques. enzymes

123 [0306] Hydrolases, e.g. saturase lipases, palmitases and / or stearatases, e.g. proteins, at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58 are disclosed %, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or complete (100%) sequence identity with the exemplary polypeptide disclosed herein (e.g. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 1 4, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20 or SEQ ID NO: 2, having one, two, three, four, five, six, seven, eight or more (several) and all the amino acid variants described in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 or Table 23 or equivalent, antibodies that bind them and methods of making and using them. The polypeptides disclosed herein may have any hydrolase activity, e.g., lipase, saturase, palmitase and / or stearatase activity. In alternative aspects, the activity of the enzyme disclosed herein includes hydrolysis or synthesis of lipids or oils. The hydrolases disclosed herein may modify oils by hydrolysis, acid hydrolysis, alcoholysis, glycerolysis, esterification, transesterification and / or interesterification, including "forced migration" reactions.

[0307] In alternative aspects, the hydrolases disclosed herein may have modified or new activities compared to the exemplary hydrolases or activities described herein. The specification discloses hydrolases with or without a signal sequence as well as signal sequences as such. We disclose herein immobilized hydrolases, anti-hydrolase antibodies and fragments thereof. Disclosed herein are proteins that inhibit hydrolase activity, e.g., antibodies that bind to the hydrolase active site. Disclosed herein are homodimers and heterocomplexes, e.g., fusion proteins, heterodimers, etc., containing the hydrolases disclosed herein. Hydrolases with activity in a wide range of high and low temperatures and pH values ​​(e.g. acidic and basic aqueous conditions) are disclosed herein.

[0308] In one aspect, one or more hydrolases (e.g., lipases, saturases, palmitases and / or stearatases) disclosed herein are used for biocatalytic synthesis of structured lipids, i.e. lipids that contain a specific set of fatty acids distributed in a specific manner on the glycerol skeleton, including the cocoa butter variant, polyunsaturated fatty acids (PUFA), 1,3-diacyl glycerides (DAG), 2-monoacylglycerides (MAG) and triacylglycerides (TAG).

[0309] We disclose herein methods for generating enzymes with altered (higher or lower) Kk_at/ K_m. In one aspect, site-directed mutagenesis is used to create additional hydrolase enzymes with alternative substrate specificities. This can be achieved, for example, by redesigning the prison region

124 or enzyme active site. In one aspect, the hydrolases disclosed herein are more stable at high temperatures, such as 80 ° C to 85 ° C to 90 ° C to 95 ° C, compared to hydrolases from conventional or altered organisms. [0310] The various proteins disclosed herein exhibit hydrolase activity, e.g., lipase, saturase, palmitase, and / or stearatase activity, under various conditions. Methods for producing hydrolases disclosed herein with varying catalytic performance and stability to temperature, oxidizing agents and pH conditions. These methods may utilize, e.g., site-directed mutagenesis and / or random mutagenesis techniques. In one aspect, directed evolution can be used to produce hydrolases with alternative specificities and stability.

[0311] The proteins disclosed herein are used in methods that can identify hydrolase modulators, e.g., activators or inhibitors. Briefly, test samples (e.g., compounds such as peptide or combinatorial library members, broths, extracts and the like) are added to hydrolase tests to determine their ability to modulate, e.g., inhibit or activate, cleave the substrate. In industry and research, these inhibitors can be used to reduce or prevent unwanted isomerization. Modulators identified using the methods disclosed herein can be used to alter (e.g., reduce or increase) the spectrum of hydrolase activity.

[0312] Methods of discovering hydrolases using the nucleic acids, polypeptides and antibodies disclosed herein have been disclosed. In one aspect, lambda phage libraries are screened for the detection of hydrolases based on their expression. Disclosed herein are lambda phage libraries for use in screening to detect toxic clones; improved substrate availability; reduced need to modify the host, bypass the possibility of any error resulting from the library mass cutoff; and, faster growth at low clone densities. Screening of lambda phage libraries can take place in the liquid phase or in the solid phase. The present description discloses liquid phase sieving methods. This can provide greater flexibility under test conditions; additional substrate flexibility; higher sensitivity to weak clones; and the ease of automating screening over solids.

[0313] Screening methods using the proteins and nucleic acids disclosed herein using robotic automation are disclosed. This allows you to perform many thousands of biocatalytic reactions and screening tests in a short period of time, e.g. for a day, and also provides a high level of accuracy and repeatability (see matrix discussion below). As a result, a derivative library can be created within weeks.

125 [0314] Hydrolase enzymes are disclosed that are not naturally occurring hydrolases with different hydrolase activity, stability, substrate specificity, pH profile and / or performance characteristics, compared to non-naturally occurring hydrolase. These hydrolases have an amino acid sequence not found in nature. They can be obtained by substituting many amino acid residues of the precursor hydrolase with different amino acids. The precursor hydrolase may be a naturally occurring hydrolase or recombinant hydrolase. In one aspect, the hydrolase variants involve the substitution of any of the naturally occurring L-amino acids at designated positions of amino acid residues.

Hydrolases, signal sequence, prepro and catalytic domains [0315] Signal sequences (e.g. signal peptides (SP, signal peptides)), prepro catalytic domains and domains (CD catalytic domains) are disclosed. The SP, prepro and / or CD domains disclosed herein may be isolated, synthetic or recombinant peptides, or they may be part of a fusion protein, e.g., as a heterologous domain in a chimeric protein. Nucleic acids encoding these catalytic domains (CDs), prepro domains and signal sequences (SPs, e.g., a peptide having an encapsulating / consisting of terminal amino acid residues of the polypeptide disclosed herein) are disclosed. Signal sequences are disclosed, including a peptide comprising / consisting of sequences as represented by residues 1 to 12, from 1 to 13, 1 to 14, from 1 to 15, from 1 to 16, 1 to 17, from 1 to 18.1 to 19, 1 to 20, from 1 to 21, from 1 to 22, from 1 to 23, from 1 to 24, 1 to 25, from 1 to 26, from 1 to 27, from 1 to 28, from 1 to 28 , from 1 to 30, from 1 to 31, from 1 to 32, from 1 to 33, from 1 to 34, from 1 to 35, from 1 to 36, from 1 to 37, from 1 to 38, from 1 to 39 , from 1 to 40, from 1 to 41, from 1 to 42, from 1 to 43, 1 to 44 (or longer peptide) of the polypeptide disclosed herein. Isolated, synthetic or recombinant signal sequences are disclosed comprising / consisting of the signal sequence disclosed herein derived from another enzyme disclosed herein or another type of enzyme or polypeptide.

[0316] The hydrolase (SP), CD and / or prepro sequences disclosed herein may be isolated peptides, or, sequences linked to another hydrolase or non-hydrolase polypeptide, e.g., as a fusion (chimeric) protein. Polypeptides comprising the hydrolase signal sequences disclosed herein are disclosed. In one aspect, polypeptides comprising the SP, CD and / or prepro hydrolase signal sequences disclosed herein include the heterologous sequences of the hydrolases disclosed herein (e.g., the SP, CD and / or prepro containing fusion protein disclosed herein and the sequences derived from hydrolase or proteins other than hydrolase). Disclosed herein are the hydrolases disclosed herein with heterologous SP, CD and / or prepro sequences, e.g. sequences with a signal sequence

126 yeast. The hydrolase disclosed herein in the vector may contain heterologous SP and / or prepro, e.g. in the vector from the pPIC vector series (Invitrogen, Carlsbad, CA).

[0317] In one aspect, the SP, CD and / or prepro sequences disclosed herein are identified by identifying new hydrolase polypeptides. Paths in which proteins are sorted and transported to the appropriate cellular location are often referred to as protein targeting pathways. One of the most important elements in all these targeting systems is the short amino acid sequence at the amino terminus of the newly synthesized polypeptide called the signal sequence. This signal sequence directs the protein to its proper place in the cell and is removed during transport or when the protein reaches its final destination. Most lysosomal, membrane or secretory proteins have a signal sequence at the amino terminus that indicates their translocation to the lumen of the endoplasmic reticulum. These signal sequences may have a different length from 13 to 45 amino acid residues or more. Various methods of recognizing signal sequences are known to those skilled in the art. For example, in one aspect, new hydrolase signal peptides are identified by a method termed SignalP. Signa lP uses a complex neural network that recognizes both signal peptides and their cleavage sites. (Nielsen, et al. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. "Protein Engineering, vol. 10, No. 1, pp. 1-6 (1997).

[0318] It should be understood that in some aspects, the hydrolases disclosed herein do not have SP and / or prepro and / or catalytic (CD) domain sequences. In one aspect, the specification discloses polypeptides (e.g., hydrolases) lacking all or part of the SP, CD and / or prepro domain. In another aspect, the invention discloses nucleic acids encoding the signal sequence (SP), CD and / or prepro of one hydrolase operably linked to the nucleic acid sequence of another hydrolase or, optionally, the signal sequence (SP) and / or the prepro domain of non-hydrolase protein.

[0319] Disclosed herein are single, synthetic or recombinant polypeptides comprising signal sequences (SP), prepro domain and / or catalytic domains (CD) and heterologous sequences. Heterologous sequences are sequences that are not naturally associated (e.g. to hydrolase) with the SP, prepro domain and / or CD. The sequence to which the SP, prepro domain and / or CD are not naturally associated may be at the amino terminus, carboxy terminus and / or at both ends of the SP, prepro domain, and / or CD. Isolated, synthetic or recombinant polypeptides comprising (or consisting of) a polypeptide having a signal sequence (SP), a prepro domain and / or a catalytic domain (CD) disclosed herein are disclosed with the proviso that they are not associated with any sequence, which are naturally bound (e.g., a hydrolase sequence). Isolated or recombinant nucleic acids encoding these polypeptides are disclosed herein. Thus, in one aspect, an isolated, synthetic or recombinant nucleic acid

127 disclosed herein includes the coding sequence for the signal sequence (SP), the prepro-and / or catalytic domain (CD) disclosed herein, and the heterologous sequence (i.e., a sequence that is not naturally associated with a signal sequence (SP), a domain prepro-and / or catalytic domain (CD) disclosed herein). The heterologous sequence may be at the 3 'end, 5' end and / or at both ends of the SP, prepro domain and / or CD coding sequence.

[0320] The fusion of the N-terminal or C-terminal subsection of the enzymes disclosed herein (e.g., signal sequences, prepro sequences) to other polypeptides, active proteins or protein fragments is disclosed. Production of the enzyme as described herein (e.g. hydrolases, e.g. (lipase, saturase, palmitase and / or stearatase) can also be achieved by expression of the enzyme in the form of an inactive fusion protein, which is then activated by proteolytic cleavage (using endogenous or exogenous protease activity, e.g. trypsin), which results in separation of fusion and mature partners an enzyme, e.g., a hydrolase disclosed herein. In one aspect, the fusion protein disclosed herein is expressed from a hybrid nucleotide construct that encodes a single open reading frame containing the following elements: nucleotide sequence for the fusion protein, a linker sequence (defined as a nucleotide sequence that encodes a flexible amino acid sequence that connects two less flexible protein domain sequences), a cleavage site recognized by the protease and the mature enzyme sequence (e.g., any enzyme disclosed herein, e.g. hydrolase). In an alternative aspect, the fusion protein may comprise a pectin lyase sequence, a xylanase sequence, a phosphatidyl acid phosphatase sequence, or other sequence, e.g., a sequence for which it has previously been shown to be overexpressed in the host system of interest. Any host system (see discussion above) can be used, for example E. coli or Pichia pastoris. The nucleotide sequence assignment of the chimeric nucleotide construct can be determined based on protein expression levels obtained from each fusion construct. Starting from the 5'-end of the nucleotide construct to the main 3'-end of the construct, in one aspect, the nucleotide sequence is organized as follows: signal sequence / fusion protein / linker sequence / cleavage site recognized by protease / mature enzyme (e.g., any enzyme disclosed herein, e.g., hydrolase) or signal sequence / pro sequence / mature enzyme / linker sequence / fusion protein. Expression of the enzyme (e.g. any enzyme disclosed herein, e.g., hydrolases) in the form of an inactive fusion protein may improve the overall expression of the enzyme sequence, may reduce the potential toxicity associated with overproduction of the active enzyme, and / or may increase the shelf life of the enzyme before use because the enzyme will be inactive until it is separated a fusion protein, e.g. an enzyme pectin lyase, e.g. the hydrolase disclosed herein.

128 [0321] Specific preparations for activating the hydrolase disclosed herein expressed as a fusion protein are disclosed. In one aspect, activation of the hydrolase activity pre-expressed as an inactive fusion protein occurs using either proteolytic or potentially proteolytic activity in combination with an amino-terminal or carboxy-terminal peptidase (the peptidase may be an enzyme disclosed herein, or another enzyme). This activation can be carried out in various ways and at various points in the manufacturing / storage process prior to use in oil degumming. Exemplary processes include: cleavage by endogenous activity expressed by the host after secretion of the fusion construct into the fermentation medium; cleavage by endogenous protease activity that is activated or contacted with an intracellularly expressed fusion construct after disruption of host cells; passage of the crude or purified fusion construct through a column with immobilized protease activity to effect cleavage and activation of the enzyme (e.g., hydrolase in the disclosed description, e.g., e.g., lipase, saturase, palmitase and / or stearatase) prior to enzyme formulation; treating the crude or purified fusion construct with a soluble source of proteolytic activity; activation of hydrolase (e.g. hydrolase disclosed herein) for oil purification or using a soluble or insoluble source of proteolytic activity immediately prior to use in the process; and / or, activation of hydrolase (e.g. lipase, saturase, palmitase and / or stearatase disclosed herein) using continuous circulation of the preparation of the fusion construct through a column with immobilized protease activity at reduced temperature (for example, any between about 4 ° C and 20 ° C). This activation event can be carried out prior to delivery to the place of use or can occur at an oil purification site.

Glycosylacia [0322] The peptides and polypeptides disclosed herein (e.g., hydrolases, antibodies) may also be glycosylated, for example, in one aspect, containing at least one glycosylation site, for example N-or O-glycosylation. In one aspect, the polypeptide may be glycosylated after expression in P. pastoris or S. pombe. Glycosylation can be added post-translational, chemically or by cellular biosynthesis mechanisms, the latter including the use of known glycosylation motifs that can be sequence-native or can be added as a peptide or added in the nucleic acid coding sequence.

Hybrid hydrolases and peptide libraries [0323] Hybrid hydrolases (e.g., synthetic proteins) and fusion proteins, including peptide libraries, containing the sequences disclosed herein are disclosed. The peptide libraries disclosed herein can be used to isolate peptide target modulators (e.g., activators and inhibitors). Peptide libraries

129 disclosed herein can be used to identify partners of binding targets, such as ligands, e.g., cytokines, hormones and the like.

[0324] In one aspect, the fusion proteins disclosed herein (e.g., a peptide moiety) are conformationally stabilized (with respect to linear peptides) to allow higher binding affinity for targets. In another aspect, the specification discloses fusions of the hydrolases disclosed herein and other peptides, including known and random peptides. They can be fused in such a way that the structure of the enzyme or antibody (e.g. hydrolases) is not significantly impaired, and the peptide is metabolically or structurally conformationally stabilized. This allows the creation of a peptide library that can easily be monitored for both cell presence and quantity. [0325] The amino acid sequence variants disclosed herein may have a predetermined nature of variation, a feature that distinguishes them from a naturally occurring form, e.g., an allelic or inter-species change of the hydrolase sequence. In one aspect, the variants disclosed herein exhibit the same qualitative biological activity as a naturally occurring analog. Alternatively, variants can be selected for changed characteristics. In one aspect, when the amino acid sequence variation site or region is predetermined, the mutation per se need not be predetermined. For example, to optimize the operation of a mutation at a given site, random mutagenesis can be performed in the target codon or region, and expressed hydrolase variants screened for the optimal combination with the desired activity. Techniques for generating substitution mutations at predetermined locations in DNA of known sequence are well known, as discussed herein, for example, M13 primer mutagenesis and PCR mutagenesis. Mutant screening can be performed by testing for proteolytic activity. In alternative aspects, the amino acid substitutions may relate to single residues; insertions can range from about 1 to 20 amino acids, although much longer insertions can be made. Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70 or more residues. In order to obtain a final derivative with optimal properties, substitutions, deletions, insertions or any combination thereof can be used. Generally, these changes are made to several amino acids to minimize changes in the molecule. However, in some circumstances, larger changes may be tolerated.

[0326] Hydrolases are disclosed in which the polypeptide backbone structure, secondary or tertiary structure, for example alpha-helix or beta-sheet structure has been modified. In one aspect, the charge or hydrophobicity has been modified. In one aspect, the side chain mass has been modified. Significant changes in immunological function or identity are made by selecting substitutions that are less conservative. For example, substitutions can be made that more significantly affect: structure

130 a polypeptide backbone in the lesion area, for example an alpha-helix or beta-card structure; a hydrophobic charge or site of a molecule that may be at an active site; or side chain. Proteins containing substitutions in the sequence disclosed herein are disclosed, e.g., when: (a) a hydrophyte moiety, e.g., seryl or threonyl, is substituted in place of (or through) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) the cysteine ​​or proline is substituted in place of (or through) any other residue; (c) an electropositive side chain residue, e.g., lysyl, arginyl or histidyl, is substituted in place of (or through) an electronegative residue, e.g., glutamyl and aspartyl; or (d) a residue having a large side chain, e.g. phenylalanine, is substituted in place of (or by) having no side chain, e.g. glycine. Variants may have the same biological activity (qualitative, i.e., hydrolase activity) although variants may be selected to modify the properties of the hydrolases as desired.

[0327] In one aspect, the hydrolases disclosed herein include epitopes or markers for purification, signal sequences or other fusion sequences, etc. In one aspect, the hydrolases disclosed herein can be fused to a randomly selected peptide to form a fusion polypeptide. As used herein, "fused" or "operably linked" means that the random peptide and hydrolase are linked together in such a way as to minimize disturbances in the stability of the hydrolase structure, e.g., while maintaining hydrolase activity. The fusion polypeptide (or fusion polynucleotide encoding the fusion polypeptide) may also contain additional components, including multiple peptides in multiple loops.

[0328] In one aspect, the peptides (e.g., hydrolase sub-sequences) and the nucleic acids encoding them are randomized, or completely randomized, or are favored at their randomization, e.g. "Randomized" means that each nucleic acid and peptide, respectively, consists essentially of random nucleotides and amino acids. In one aspect, the nucleic acids that give rise to the peptides can be chemically synthesized and therefore may contain any nucleotide at any position. Thus, when nucleic acids are expressed to produce peptides, any amino acid residue can be introduced at any position. The synthesis process can be designed to generate random nucleic acids to allow the formation of all or most of the possible combinations along the length of the nucleic acid, thereby creating a library of random nucleic acids. The library can provide a sufficiently structurally diverse population of random expression products to be able to affect a likely sufficient range of cellular responses to provide one or more cells showing the desired response. In this description, sufficiently extensive is disclosed

131 interaction libraries, so that at least one of its members will exhibit a structure that confers its affinity to a certain molecule, protein, and other factor.

Screening methodology and active monitoring devices [0329] In carrying out the methods as provided herein, various devices and methodologies may be used in combination with polypeptides and nucleic acids as described herein, for example, to screen polypeptides with hydrolase activity to screening compounds as potential activators or inhibitors of hydrolase activity (e.g. to screen for potential drug), to antibodies that bind to the polypeptide disclosed herein, to nucleic acids that hybridize to the nucleic acid disclosed herein, to screen for cells expressing the polypeptide disclosed herein and the like. See, e.g., US Patent No. 6,337,187.

Capillary Arrays [0330] Capillary arrays such as GIGAMATRIX ™, Diversa Corporation, San Diego, CA may be used in the methods disclosed herein. The nucleic acids or polypeptides disclosed herein can be immobilized or applied to an array, including capillary matrices. Arrays can be used to screen or monitor component libraries (e.g. small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of the nucleic acid or polypeptide disclosed herein. Capillary arrays provide another system for storing and screening samples. For example, the sample screening apparatus may comprise a plurality of capillaries formed into a matrix of adjacent capillaries, each capillary having at least one wall defining a lumen to hold the sample. The device may additionally comprise a separating material disposed between adjacent capillaries in the matrix, and one or more markings formed within the separating material. The sample screening capillaries, wherein the capillary is adapted to remain bound in the capillary matrix, may include a first wall defining a clearance for holding the sample and a second wall made of filter material to filter the excitation energy delivered to the lumen to excite the sample.

[0331] The polypeptide or nucleic acid, e.g., ligand or substrate, may be introduced into the first component into at least one portion of the capillary matrix capillaries. Each capillary matrix of the capillary matrix may include at least one wall defining a clearance for holding the first component. An air bubble may be introduced into the capillary after the first component. A second component may be introduced into the capillary, the second component being separated from the first component by an air bubble. The sample of interest may be introduced into the capillary matrix capillary as the first liquid labeled with a detectable particle, each capillary

132 the capillary matrix comprises at least one wall defining a clearance to retain the first liquid and the detectable particle, and wherein the at least one wall is covered with a binding material to bind the detectable particle to the at least one wall. The method may further include removing the first liquid from the capillary tube, the associated detectable particle being held in the capillary, and introducing the second liquid into the capillary tube.

[0332] The capillary array may include a plurality of individual capillaries comprising at least one wall defining a lumen. The outer wall of the capillary may be in the form of one or more walls joined together. Similarly, the wall may define a lumen that is cylindrical, square, hexagonal or any other geometric shape as long as the walls form a lumen to hold the liquid sample. Capillary matrix capillaries can be kept in close proximity to form a flat structure. The capillaries can be connected to each other side by side by melting (e.g. when the capillaries are made of glass), gluing, joining or fixing. The capillary array can be formed from any number of individual capillaries, for example, in the range from 100 to 4,000,000 capillaries. The capillary array can form a microtiter plate having about 100,000 or more individual capillaries connected together.

Matrices or "Biochips".

[0333] The nucleic acids or polypeptides disclosed herein can be immobilized or applied to an array. Arrays can be used to screen or monitor component libraries (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the nucleic acid or polypeptide activity disclosed herein. For example, in one aspect disclosed herein, the expression parameter of the hydrolase gene transcript is monitored. One or more, or all cell transcripts can be measured by hybridizing a sample containing the cell transcripts, or, their representative nucleic acids or complementary to cell transcripts, by hybridizing to nucleic acids of winters mobilized on the matrix, or "biochip". Using a nucleic acid "matrix" on a microchip, some or all of the cell transcripts can be quantified simultaneously. Alternatively, arrays containing genomic nucleic acid can also be used to determine the genotype of a newly modified strain formed by the methods disclosed herein. The polypeptide arrays can also be used for the simultaneous quantification of many proteins. The present disclosure can be carried out using any known "matrix" also called "microarray" or "nucleic acid matrix" or "polypeptide matrix" or "antibody matrix" or "biochip" or variant thereof. In general, matrices are a series of "points" or "targets", each target having a specific amount of one or more biological molecules,

133 e.g. oligonucleotides, immobilized on a defined area of ​​the substrate surface to specifically bind to the sample of the molecule, e.g. mRNA transcripts.

[0334] The "matrices" or "microarrays" or "biochips" or "chips" disclosed herein may contain a plurality of targets, each target containing a specified amount of one or more polypeptides (including antibodies) and immobilized nucleic acids on specific surface area of ​​the substrate. [0335] In one aspect, the hydrolases are used in a cold-mobilized form. Any immobilization method can be used, e.g. immobilization on an inert medium such as diethylaminoethyl cellulose, porous glass, chitin or cells. Cells that express the hydrolases disclosed herein can be mobilized to the surface of the substrate by crosslinking, e.g. with glutaraldehyde, in winter.

[0336] In the methods disclosed herein, any known matrix and / or method of its preparation and use of the matrix may be included in whole or in part, or variations thereof, as described, for example, in US Patent Nos. US 6,277,628; 6,277,489; US 6,261,776; US 6,258,606; US 6,054,270; US 6,048,695; US 6,045,996; US 6,022,963; US

6,013,440; US 5,965,452; US 5,959,098; US 5,856,174; US 5,830,645; US 5,770,456; US

5,632,957; US 5,556,752; US 5,143,854; US 5,807,522; US 5,800.992; US 5,744,305; US

5,700,637; US 5,556,752; US 5,434,049; see also, e.g., WO 99151773; WO 99/09217; WO

97/46313; WO 96/17958; see also, e.g., Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; Solinas-Toldo (1997) Genes, Chromosomes and Cancer 20: 399-407; Bowtell (1999) Nature Genetics Supp. 21: 25-32. See also published US patent applications No. US 20010018642; US 20010019827; US 20010016322; US 20010014449; US 20010014448; US 20010012537; US 20010008765.

Antibodies and screening methods based on antibodies [0337] Isolated, synthetic or recombinant antibodies have been disclosed that specifically bind to the hydrolase disclosed herein. These antibodies can be used to isolate, identify and quantify the hydrolase disclosed herein or related polypeptides. These antibodies can be used to isolate other polypeptides disclosed herein or other related hydroiazides.

[0338] "Antibodies" disclosed herein may include peptide (s) or polypeptide (s) derived from, modeled for or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see e.g. Fundamental Immunology, third edition, WE Paul edition, Raven Press, New York. (1993); Wilson (1994) J. Immunol. Methods 175: 267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25: 85-97. The term antibody includes antigen binding fragments, i.e., "antigen binding sites" (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain antigen binding capacity,

134 including: (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) the F (ab ') 2 fragment, a two-valued fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; (III) Fd fragment consisting of the VH and CHI domains; (iv) the Fv fragment consisting of the VL and VH domains of a single arm of the antibody, (v) the dAb fragment (Ward et al. (1989) Nature 341: 544-546), which consists of the VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies by reference are also included in the term "antibody". Disclosed herein are antigen binding antibodies, and single chain antibodies that specifically bind to the hydrolase disclosed herein. Polypeptides with hydrolase activity may also be used in the methods disclosed herein.

[0339] These antibodies can be used in immunoprecipitation, staining, immunoaffinity columns and the like. If desired, nucleic acid sequences encoding specific antigens can be generated by immunization followed by isolation of the polypeptide or amplification of the nucleic acid or cloning and immobilization of the polypeptide on an array disclosed herein. Alternatively, the methods disclosed herein can be used to modify the structure of the antibody produced by the cell to be modified, e.g., the affinity of the antibody can be increased or decreased. In addition, the ability to create or modify antibodies can be designed with respect to the cell phenotype using the methods disclosed herein.

[0340] Methods for immunizing, producing and isolating (polyclonal and monoclonal) antibodies are known to those skilled in the art and described in the scientific and patent literature, see e.g. Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY (1991); Stites (edition) BASIC AND CLINICAL IMMUNOLOGY (7th edition) Lange Medical Publications, Los Altos, CA ("Stites"); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd edition) Academic Press, New York, New York (1986); Kohler (1975) Nature 256: 495; Harlow (1988) ANTIBODIES, A LABORATORIES MANUAL, Cold Spring Harbor Publications, New York. Antibodies can also be produced in vitro, e.g., using phage display libraries expressing a recombinant antibody binding site, in combination with traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15: 62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26: 27-45.

[0341] Polypeptides and peptides can be used to generate antibodies that bind specifically to the polypeptides disclosed herein. The resulting antibodies can be used in immunoaffinity chromatography procedures to isolate and purify the polypeptide, or to determine whether a polypeptide is present in a biological sample. In such procedures, a protein preparation, such as an extract, or biological sample

135 contacting an antibody capable of specifically binding to one of the polypeptides disclosed herein.

[0342] In immunoaffinity procedures, the antibody is attached to a solid support, such as beads or other column matrix. The protein preparation is placed in contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides disclosed herein. After washing, specifically bound polypeptides are eluted to remove non-specifically bound proteins.

[0343] The ability of proteins in a biological sample to bind an antibody can be determined using one of many procedures known to those of skill in the art. For example, binding can be determined by labeling the antibody with a detectable label, such as a fluorescent agent, enzyme label, or radioisotope. Alternatively, antibody binding to the sample can be detected using a secondary antibody having such a detectable label on it. Specific tests include ELISA, sandwich tests, radioimmunoassays and Western blots.

[0344] Polyclonal antibodies generated against the polypeptides disclosed herein can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to a non-human animal. The antibody thus obtained will bind the polypeptide as such. In this way, only the coding sequence of the polypeptide fragment can be used to generate antibodies that can bind to the entire native polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing the polypeptide. [0345] Any technique that provides antibodies produced by continuous cell line culture can be used to produce monoclonal antibodies. Examples include the hybridoma technique, the trioma technique, the hybridoma technique of human B cells and the EBV hybridoma technique (see, e.g., Cole (1985), in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0346] The described techniques for the production of single chain antibodies (see, e.g., US Patent No. 4,946,778) can be adopted for the production of single chain antibodies directed against the polypeptides disclosed herein. Alternatively, transgenic mice can be used to express humanized antibodies directed against these polypeptides or fragments thereof.

[0347] Antibodies generated against the polypeptides disclosed herein (including anti-idiotype antibodies) can be used to screen for similar polypeptides from other organisms and samples. In such techniques, polypeptides from the body are contacted with the antibody and those polypeptides that specifically bind the antibody are detected. Any of the procedures described above can be used to detect antibody binding.

Immobilized hydrolases

136 [0348] In one aspect, the hydrolase disclosed herein, e.g., lipases, saturases, palmitases, and / or stearatases, are used in immobilized forms, e.g., for lipid processing, in structural lipid synthesis for protein digestion, and the like. The immobilized lipases disclosed herein can be used, e.g. for the hydrolysis of triacylglycerides, diacylglycerides or esters or the esterification or transesterification of fatty acids and triacylglycerides, diacylglycerides, or, in interesterification of fats. In one aspect, lipase is specific for the esterification of fatty acids with alcohol, 1,3 specific or specific for the hydrolysis of partial glycerides, esters or triacylglycerides. The immobilized lipases disclosed herein can be used in a packed bed for the continuous transesterification of solvent-free fats. See, e.g., US Patent Nos. 4,818,695; 5,569,594.

[0349] Any method of immobilization or substrate form may be used, e.g., matrices, beads, capillary substrates and the like as described above. In one aspect, the hydrolase immobilization can take place on an inert carrier such as diethylaminoethyl cellulose, porous glass, chitin or cells. Cells that express the hydrolases disclosed herein can be immobilized by cross-linking, e.g. with glutaraldehyde, to the surface of the substrate. The immobilized hydrolases disclosed herein may be prepared from hydrolase bound to a dry porous hydrophobic particulate substrate with a surfactant such as fatty acid ester and polyoxyethylene sorbitan or fatty acid ester with polyglycerol. The support may be an aliphatic olefinic polymer such as polyethylene or polypropylene, a styrene homo- or copolymer or a blend thereof or a pre-treated inorganic substrate. These substrates may be selected from aliphatic olefin polymers, oxidative polymers, a blend of these polymers and pretreated inorganic substrates to render these substrates hydrophobic. This pre-treatment may include silanization with an organic silicon compound. The inorganic material may be silica, alumina, glass or ceramics. The substrates can be made of polystyrene, styrene copolymers, polyethylene, polypropylene or co-polymers derived from (meth) acrylates. See, e.g., US Patent No. 5,773,266.

[0350] Hydrolase enzymes, fragments thereof, and nucleic acids that encode these enzymes and fragments thereof can be attached to a solid support. This is often economical and efficient when using hydrolases in industrial processes. For example, a consortium or cocktail of hydrolytic enzymes (or active fragments thereof) that are used in a particular chemical reaction can be attached to a solid support and immersed in a process vat. An enzymatic reaction may occur. Then, the solid support can be removed from the vat, along with the enzymes attached thereto, for multiple use. As disclosed herein, an isolated nucleic acid as disclosed herein,

137 attached to a solid support. As disclosed herein, the solid support is selected from the group of gel, resin, polymer, ceramics, glass, microelectrodes, and any combination thereof. [0351] For example, the solid substrates disclosed herein include gels. Some examples include SEPHAROSE ™ gels (GE Healthcare, Piscataway, NJ), gelatin, glutaraldehyde, chitosan treated glutaraldehyde, albumin-glutaraldehyde, xanthanum chitozanguma, toyopearl gel (polymer gel), alginate, alginate-polylysine, carrageenan, agar glyoxal agarose, magnetic agarose particles, dextran-agarose, poly (carbamoyl sulfonate) hydrogel, BSA-PEG hydrogel, phosphorylated polyvinyl alcohol (PVA), monoaminoethyl-N-aminoethyl (MANA), amine, or any combination thereof.

[0352] Other solid substrates disclosed herein include resins or polymers. Some examples of resins or polymers include cellulose, acrylamide, nylon, rayon, polyester, anion exchange resin, AMBERLTTE ™ XAD-7, AMBERLITE ™ XAD-8, AMBERLITE ™ IRA-94, AMBERLITE ™ IRC-50 (from Rohm and Haas, Philadelphia, PA), polyvinyl, polyacrylic, polymethacrylate, or any combination thereof.

[0353] Another type of solid support disclosed herein is ceramics. Some examples include, non-porous ceramics, porous ceramics, SiO₂, AI₂ABOUT₃. Another type of solid support usable herein is glass. Some examples include non-porous glass, porous glass, aminopropyl glass or any combination thereof. Microelectrodes are another type of solid support that can be used. An example is magnetite coated with polyethyleneimine. Graphite particles can be used as solid support.

[0354] Another type of solid support disclosed herein includes diatomaceous earth products and silicates. Some examples include CELITE®, ΚΕΝΓΓΕ®, DIACTIV®, PRIMISIL®, DIAFIL® diatomites and MICRO-CEL®, CALFLO®, SILASORB ™, and CELKATE® (World Minerals Inc, Santa Barbara, CA), synthetic calcium and magnesium silicates.

[0355] Another example of a solid support is a cell or contains it, such as red blood cells.

Kits [0356] Kits containing compositions, e.g., nucleic acids, expression cassettes, vectors, cells, transgenic seeds, plants or plant parts, polypeptides (e.g., hydrolases) and / or antibodies disclosed herein are disclosed. Kits may also contain instructional material and methodologies, and industrial applications.

Industrial and Medical Applications [0357] The hydrolases (e.g., lipases, saturases, palmitases and / or stearatases) disclosed herein have many industrial and medical applications, and several examples of uses and compositions are described below. Processes as envisaged herein include converting non-hydrated phospholipids into a hydrable form, oil degumming, food processing, oil processing (e.g.

138 production of low saturated oil) from plants, fish, algae and the like, to name a few.

Food and feed processing [0358] Processes for making cheese using hydrolases (e.g. lipases, saturases, palmitases and / or stearatases) disclosed herein are disclosed. Hydrolase-containing cheeses are disclosed. In one aspect, the enzymes disclosed herein (e.g., lipases, saturases, palmitases and / or stearatases or combinations thereof) are used to process cheeses to improve taste, increase yield, and / or "stabilize" cheeses, e.g., by reducing the tendency to "release oil", or, in one aspect, the enzymes disclosed herein are used to make cheese from whey. These processes may include any method or protocol, e.g. as described e.g. in US Patent Nos. US 6,551,635 and US 6,399,121, WO 03/070013, WO 00/054601. For example, in one aspect, the hydrolases (e.g., lipases, saturases, palmitases and / or stearatases) disclosed herein are used to stabilize fat emulsions in milk or milk-containing compositions, e.g., in cream, and are used to stabilize milk compositions, e.g. in the production of creams or cream liqueurs. Processes of enhancing the taste of cheese are disclosed using at least one enzyme disclosed herein, which process comprises incubating protein, fat and protease and lipase (e.g., disclosed herein) in an aqueous medium under conditions that produce an improved cheese flavor ( e.g. reduced bitterness), e.g. as described in WO 99/66805. In one aspect, the lipases disclosed herein are used to improve the aroma of cheese (e.g. clot) by mixing with water, protease, phospholipase at elevated temperature, e.g., between about 75 ° C to 95 ° C, as described, e.g., in the disclosure of US Patent No. 4,752,483. In one aspect, the lipases disclosed herein are used to accelerate cheese aging by adding the enzyme disclosed herein to cheese (e.g., whey), before adding the disclosed coagulant to milk, or adding an enzyme (e.g. lipase) disclosed herein to a salt curd prior to extrusion, e.g. as described e.g. in U.S. Patent No. 4,707,364. In one aspect, the lipases disclosed herein are used to degrade triacylglycerides in milk fat to release free fatty acids, resulting in improved aroma. The enzyme disclosed herein can also be used in any of these methods as provided herein, see e.g. Brindisi (2001) 1 of Food Sci. 66: 1100-1107.

Structured Synthesis and Oil Treatment [0359] In some embodiments, rice structuring methods for the synthesis of oils, lipids and the like are provided using hydrolases (e.g., lipases, saturases, palmitases and / or stearatases). Methods as provided herein include biocatalytic synthesis of structured lipids, i.e., lipids that contain a specific set of fatty acids distributed in a specific manner on the backbone, e.g., on the glycerol backbone. products

139 generated using hydrolases and carrying out the methods provided herein include low saturated oils, e.g., oils from vegetables (e.g., soybean, canola), animals, plants, fish, algae, which oils have been processed or treated with the polypeptide; and food, feed, additives, pharmaceuticals and the like containing low saturated oils prepared by carrying out methods and / or compositions (e.g. enzymes) as provided herein. Products made using hydrolases and carrying out the methods as provided herein also include alternatives to cocoa butter, lipids containing polyunsaturated fatty acids (PUFAs), lipids containing essential fatty acids, lipids containing monounsaturated fatty acids, lipids containing phospho-choline and phosphoserine. lipids containing phytosterols, 1,3-diacyl glycerides (DAG), 2-monoacylglycerides (MAG) and triacylglycerides (TAG).

[0360] The methods provided by the present invention allow the synthesis of lipids or fatty acids with defined regioselectivity and stereoselectivity. This description discloses lipids and the like and oils that can be used in foodstuffs and feed and cooking materials (e.g., cooking oils, frying oils, baking oils, sauces, marinades, food additives, spray oils) , margarines, mayonnaises, spooning dressings and pouring, cocoa butter alternatives, and the like) that have been treated or treated with polypeptides or peptides (e.g., hydrolases such as lipases, saturases, palmitases and / or stearatases). Pharmaceuticals, nutrients and cosmetics disclosing the polypeptides disclosed herein (e.g., hydrolases, such as lipases, saturases, palmitases and / or stearatases or peptides or antibodies).

[0361] In some embodiments, the present invention provides methods for treating (modifying) oils, lipids and the like using hydrolases. These methods can be used to process oils from plants, animals, microorganisms. The methods as provided herein can be used in structured synthesis of oils similar to those found in plants, animals and microorganisms. Lipids and oils can be processed to obtain the desired characteristics. Lipids and oils that can be processed by the methods provided herein (using hydrolases) include alternatives to cocoa butter, lipids containing polyunsaturated fatty acids (PUFAs), lipids containing essential fatty acids, lipids containing monounsaturated fatty acids, phosphololine-containing lipids and phospho-serine, phytosterol-containing lipids, 1,3-diacyl glycerides (DAG), 2-monoacylglycerides (MAG) and triacylglycerides (TAG). In one aspect, processed and synthetic oils and fats (e.g., cocoa butter alternatives and vegetable oils) can be used in many applications, e.g. in food production (e.g., confectionery, cookies) and in pharmaceutical preparations, nutrients and cosmetics. Methods for processing fats and oils, e.g., from oilseeds, from plants, including, e.g.

140 rape, canola, towel, coconut, coriander, corn, cotton beans, hazelnuts, hemp, linseed, meadow cress, olives, palm oil, palm kernels, peanuts, rape, rice bran, saffron, camellia, soybean, sunflower , thallium, tsubaki, varieties of "natural" oils with a changed fatty acid composition through genetically modified organisms (GMOs) or traditional cultivation, such as oils with high oleic acid content, low linolenic acid, or low saturated oil (canola oil with high oleic acid, low linolenic soybean or sunflower with high stearic acid), or a mixture of any of the above using a hydrolase.

[0362] In some embodiments, the present description provides processing methods for processing oils from animals, e.g. fish (olacon, cod liver, orange roughy, sardine, herring, menhaden and the like), mammals (pork, beef, and the like) and poultry (chicken and the like), using hydrolase. In some embodiments, the present description provides methods for structured synthesis of oils similar to those found in animals, for example fish, poultry and mammals and microorganisms, using hydrolases. In one aspect, these synthetic or processed oils are used as feed and food additives, as ingredients in pharmaceutical preparations, nutrients and in cosmetics. For example, in one aspect, hydrolysis is used to dehydrolyze fatty acids from fish oils, so that fatty acids can be recovered and used as feed additives. In one aspect, hydrolases can be used to treat oil waste from restaurant waste and rendered animal fats.

[0363] In other embodiments, the present invention provides methods for processing fats and oils, e.g., algae oil, including, e.g., Neochloris oleoabundans oil, Sceriedesmus dimorphus oil, Euglena grad lis oil, Phaeodactyium tricornutum oil, from Pieurochrysis carterae, oil from Prymnesium parvumo, oil from Tetraseimis Chub, oil from Tetraseimis suedca, oil from Isochrysis gaibana, oil from Nannochioropsis Salina, oil from Botryococcus brauń, oil from Dunaliella tertioiecta, Nannochioris oil, Spiruiina oil, Chiorophycease oil (green algae) and Badiiiarophy oil, or mixtures of any of these fats and oils.

[0364] In one aspect, hydrolases are universal biocatalysts in organic synthesis, e.g., structured synthesis of oils, fats and the like. Enzymes (including, e.g., hydrolases, e.g., lipases, saturases, palmitases and / or stearatases) can accept a wide range of substrates, including secondary and tertiary alcohols, e.g. derived from natural products such as alpha-terpineol, linalool and the like. In some aspects, hydrolases exhibit good to excellent enantiospecificity (e.g., stereospecificity).

141 [0365] A method for converting oil (e.g. vegetable oils, cocoa butter and the like) comprising at least one enzyme (e.g. lipase, saturase, palmitase and / or stearatase) disclosed herein is disclosed. In one aspect, the oil transformation process involves controlled hydrolysis and acylation, e.g., acylation of glycerol, which can result in high purity for a wide range of products. In one aspect, hydrolases (e.g. lipase, saturase, palmitase and / or stearatase) disclosed herein are used for the production of diacylglycerol oils and structured nutritional oils. In some embodiments, the present description provides methods for the esterification of propylene glycol using an enzyme, e.g., a regio-and / or chemo-selective lipase for mono-substituted esterification at the Sn-1 position. The present description provides methods of structured synthesis of oils with target profiles of saturated or unsaturated fatty acids using an enzyme, e.g., in regio-and / or chemo-selective lipase for removal of saturated fatty acid, or for targeted addition of fatty acid to the glycerol chain.

[0366] In one aspect, the methods as provided herein further include methods for the selective removal of fatty acids (e.g., undesirable) fatty acids from oils, e.g., using a separation of saturated and / or unsaturated fatty acids from oils, using a hydrolase (e.g., lipases, saturases, palmitases and / or stearatases). The process as provided herein may separate saturated and / or unsaturated fatty acids from any oil, e.g. soybean oil. The enzyme may be chemoselective and / or enantioselective. In one aspect, these methods generate highly stable fats and oils, e.g. "healthy" frying oils. This exemplary method, as provided herein, can be used to make oils with less sulfur, e.g., using a method comprising removing sulfur from crude oil. Enzymes can also be used in interesterification methods for these and other purposes. [0367] In one aspect, an enzyme is used to produce "non-trans" oil fat. In one aspect, "non-trans" oil is generated from partially hardened olive oil to produce only cis oil. The enzyme may be chemoselective and / or enantioselective.

[0368] In other embodiments, the present description provides methods for modifying cocoa butter using an enzyme. About 80% of cocoa butters contain POP, SOS and POS triacylglycerides (P is palmitic fatty acid, O is oleic fatty acid, S is stearic fatty acid). The structure of saturated-unsaturated-saturated fatty acids of cocoa butter gives its characteristic melting profiles, e.g. in chocolates. In one aspect, the structured and direct synthetic methods provided herein are used for cocoa butter to reduce the variability of cocoa butter or to produce synthetic cocoa butter ("cocoa butter alternatives"). In one aspect, chemoselective and / or enantioselective (e.g.

142 regio-selective) hydrolase (e.g. lipase or esterase) is suitable for the production of an alternative cocoa butter, e.g. a cocoa butter substitute, a cocoa butter substitute and / or cocoa butter equivalent. Cocoa butter alternatives, including cocoa butter substitutes, cocoa butter substitutes and cocoa butter equivalents, and enzyme-containing intermediate products thereof have been provided herein. The process provided herein (using an enzyme) for the production of cocoa butter alternatives may include mixing, e.g. vegetable oil, e.g., palm oil, with shea butter or equivalent, illipe butter or equivalent and Sal stearin or equivalent, and treatment of mixed oils polypeptides. In one aspect, the process provided herein includes the use of interesterification. The process envisaged herein may generate composition or crystalline forms that mimic "natural" cocoa butter.

[0369] In some embodiments, the present invention provides processes (using an enzyme) for producing diacylglycerol (DAG), for example, 1,3 diacylglycerol, using vegetable oil, e.g., inexpensive oil. The enzyme may be chemoselective and / or enantioselective. The process provided herein may result in a DAG containing composition with good stability, long shelf life, and high temperature resistance. [0370] Enzymes (hydrolases, e.g., lipases, palmitase saturases and / or stearatases) as well as the methods provided herein can also be used in the enzymatic treatment of edible oils as described, e.g., in US Patent No. 6,025,171. In this exemplary method, the enzymes are immobilized by preparing an emulsion containing a continuous hydrophobic phase, such as triacylglyceride oil and a dispersed aqueous phase containing an amphiphilic enzyme, such as lipase, and a carrier material that is partially dissolved and partly insoluble in the aqueous phase, and removing water from aqueous layer until the phase changes into solid carrier particles coated with enzyme. The insoluble part of the carrier material can be material that is insoluble in water and oil, or water-soluble material in insoluble form, because the aqueous phase is already saturated with water-soluble material. The aqueous phase may be formed from a raw lipase fermentation liquid containing fermentation residues and biomass that can serve as carriers. Immobilized lipase is useful for rearrangement of the ester and deacidification in oils. After the reaction, the immobilized enzyme can be regenerated in a subsequent reaction by adding water to obtain partial dissolution of the carrier and evaporation of water from the resulting aqueous phase containing the enzyme and the carrier dispersed in the hydrophobic phase to re-form the enzyme-coated carrier particles.

[0371] Enzymes (e.g., lipases, saturases, palmitases and / or stearatases) and methods provided herein may also be used to prepare transesterified oils, as described, e.g., in US Patent No. 5,288,619. In the present description, methods are provided for the enzymatic transesterification of margarine oil characterized by

143 both low trans and low medium chain fatty acids. The method includes the steps of providing a mixture of transesterification reaction containing stearic acid source material and edible liquid vegetable oil, transesterification of stearic acid source material and vegetable oil using a lipase specific for positions 1-, 3-, and finally hydrogenating the mixture of fatty acids to provide recycled source material stearic acid for recycling with vegetable oil. The invention provides a countercurrent process for the production of transesterified oil. The method includes the steps of providing a transesterification reaction zone containing a lipase specific to positions 1-, 3-, introducing vegetable oil into the transesterification reaction zone, introducing a stearic acid source material, conducting a supercritical or subcritical liquid gas in a countercurrent liquid, conducting a triacylglyceride stream transesterification reaction with stearic acid or a stearic acid monoester stream in the reaction zone, withdrawal of the transesterified triacylglyceride stream of margarine oil, withdrawal of the countercurrent liquid phase, hydrogenation of the transesterified ester of stearic acid or stearic acid monoester to provide the hydrogenated recycled stearic acid source material and introduction of the recycled hydrogenated stearic acid source material.

[0372] In one aspect, to allow enzyme action, both phases, the oil phase and the aqueous phase that contains the enzyme must be mixed homogeneously. Only mixing them may not be enough. Good dispersion of the enzyme in the oil is aided if the oil dissolves in a small amount of water, e.g. 0.5-5% by weight (based on oil) and emulsifies in the oil in this form to form droplets with a diameter smaller than 10 microns ( and average weight). Droplets can be smaller than 1 micrometer. Turbulent mixing can be carried out at a circular speed above 100 cm / sec. The oil can also be distributed in the reactor by means of an external rotary pump. The aqueous phase containing the enzyme can also be thoroughly dispersed by ultrasound. A dispersion device can be used.

[0373] In one aspect, the enzymatic reaction provided herein proceeds at the interface between the oil phase and the aqueous phase. The purpose of all these mixing means is to create the largest possible surface area for the aqueous phase containing the enzyme. The addition of surfactants increases the microdispersion of the aqueous phase. Thus, in some cases, surfactants with HLB values ​​above 9, such as sodium dodecyl sulfate, are added to the enzyme solution, as described, e.g., in EP-A 0 513 709. Similarly, the addition of lysolecithin is an effective way to improve emulsification. The added amounts can range from 0.001% to 1% for oil. The temperature during enzyme treatment is not critical. Temperatures between 20 ° C and 80 ° C can be used, but the latter can only be used for a short time. In this case,

144 lipase with good temperature tolerance and / or low pH is used. Operating temperatures between 30 ° C and 50 ° C are optimal. The treatment time depends on the temperature and may be shorter as the temperature increases. Times of 0.1 to 10 hours, or 1 to 5 hours are usually sufficient. The reaction, which can be divided into stages, takes place in the reactor. Therefore, continuous work is possible, along with batch work. The reaction can be carried out at various temperature stages. For example, incubation can be carried out for 3 hours at 40 ° C and then for 1 hour at 60 ° C. If the reaction proceeds in stages, it also opens the possibility of adjusting different pH values ​​in individual stages. For example, in a first step the pH of the solution can be adjusted to 7, for example in a second step to 2.5 by the addition of citric acid or other suitable acids. However, in at least one step, the pH of the enzyme solution must be below 4 or below 3. If the pH is subsequently adjusted below this level, the effect may deteriorate. Thus, citric acid can be added to the enzyme solution before the latter is mixed with oil.

[0374] Enzymes (hydrolases, e.g. lipases, saturases, palmitases and / or stearatases) and methods provided herein may also be used to prepare oils, as described, e.g., in US Patent Application No. 11 / 567,318. Continuous enzymatic treatment of lipids is provided herein. The method relates to a process and apparatus for continuous enzymatic interesterification of lipid containing compositions using a plurality of fixed bed reactors, wherein the flow of lipid containing compositions through the apparatus can remain substantially constant, even when the enzyme activity of the fixed bed decreases over time, and even then, when the fixed bed is in an inactive mode, such as repair, replacement or replenishment.

[0375] In one embodiment, the present description provides a method of hydrolyzing an oil or fat by reacting an oil or fat with a palmitase enzyme. In one embodiment, the hydrolysis is carried out in the presence of an emulsifier with an HLB value higher than 12. In one embodiment, the palmitase enzyme is encoded by a nucleic acid sequence of at least 85%, 90%, 95%, 97%, 99%, 99.5% or 100% sequence identity with SEQ ID NO: 1 and having i) a nucleotide (or equivalent) change coding for an amino acid residue at position 95 (or equivalent) as shown in Table 9, ii) a change of nucleotide (or equivalent) coding for amino acid residues at positions 85 and 172 (or their equivalent) equivalent) as shown in Table 15, iii) a change in the nucleotide (or equivalent) encoding the amino acid residue at position 83 (or equivalent) as shown in Table 16 and iv) the following 35GCT silent mutations, 102GTT, 108AGT, 117CTT, 126AGG, 133TCT, and 188ACG. In one embodiment, the nucleic acid sequence is SEQ ID NO: 1 and exhibits i) a nucleotide change (or equivalent) encoding an amino acid residue at position 95 (or equivalent) as shown in Table 9. ii) a nucleotide change (or its equivalent) equivalent), encoding amino acid residues at positions 85 and 172

145 (or equivalent) as shown in Table 15, iii) a change in the nucleotide (or equivalent) encoding the amino acid residue at position 83 (or equivalent) as shown in Table 16, and iv) the following silent mutations 35GCT, 102GTT, 108AGT , 117CTT, 126AGG, 133TCT and 188ACG. In one embodiment, palmitase is a heat tolerant enzyme depicted as hit 29 in Table 9, and exhibits i) a nucleotide change (or equivalent) encoding an amino acid residue at position 95 (or equivalent) as shown in Table 9, ii) a change a nucleotide (or equivalent) encoding the amino acid residues at positions 85 and 172 (or equivalent) as shown in Table 15, iii) a change in the nucleotide (or equivalent), encoding the amino acid residue at position 83 (or equivalent) as shown in Table 16, and iv) the following silent mutations 35GCT, 102GTT, 108AGT, 117CTT, 126AGG, 133TCT and 188ACG. In one embodiment, the palmitase enzyme used in the methods provided herein is the 29 SM enzyme with the following silent mutations 35GCT, 102GTT, 108AGT, 117CTT, 126AGG, 133TCT and 188ACG as described in Example 12.

[0376] In some embodiments, the emulsifier has an HLB higher than 12, 14, 16, or 18. In some embodiments, the emulsifier is selected from sodium oleate, potassium oleate, sodium linolate, potassium linolate, sodium, sodium linolate, potassium linolate, laurate sodium, potassium laurate, sodium stearate, potassium stearate, sodium palmitate, sodium palmitooleate, potassium palmitooleate, or a combination thereof. In some embodiments, the reaction mixture contains from about 1 to 20% by weight of water based on the total weight of reagents. In one embodiment, the reaction mixture contains about 1, 3, 5, 7, 10, 15, 17 or 20% by weight of water based on the total weight of the reagents.

[0377] In some embodiments, the oil or fat is mixed with the emulsifier before adding the palmitase enzyme. In some embodiments, the oil / fat mixture and emulsifier are homogenized before and / or after adding the palmitase enzyme to provide a uniform emulsion.

[0378] In some embodiments, the reaction is carried out at a temperature of about 20 to 70 ° C. In some embodiments, the reaction is carried out at a temperature of about 20, 30, 40, 50, 60 or 70 ° C. In some embodiments, the palmitase enzyme reduces the oil / fat palmitate content to about 5% or less. In some embodiments, the palmitase enzyme reduces the palmitate oil / fat content to about 5, 4, 3, 2, 1% or less. In some embodiments, a desirable reduction in palmitate content occurs at about or less than about 48 hours, 24 hours, 20 hours, 16 hours, 12 hours, 10 hours, 5 hours or 3 hours. In some embodiments, the method further includes oil / fat pretreatment to remove mucus and the aqueous phase and to reduce free fatty acids. Any pre-treatment methods deemed appropriate by one of skill in the art may be used. In some embodiments, the method further includes adding a base (adding lye) to form soaps.

146 [0379] In some embodiments, the oil used in the reaction is a refined or crude oil. In one embodiment, the reaction further comprises adding a phospholipid. Any phospholipid deemed suitable by one of skill in the art may be used in the reactions. In one embodiment, the phospholipid is lecithin. In one embodiment, the oil used in the reactions provided herein is refined oil and the reaction comprises adding a phospholipid.

Nutrients [0380] In one aspect, the compositions and methods provided herein can be used to prepare nutrients by processing or synthesizing lipids and oils using enzymes, e.g. hydrolases, eg, lipases, saturases, palmitases and / or stearatases . In one aspect, processed or synthesized fats or oils contain polyunsaturated fatty acids (PUFA), diacylglycerides e.g. 1,3-diacyl glycerides (DAG), monoacylglycerides, e.g., 2-monoacylglycerides (MAG) and triacylglycerides (TAG). In one aspect, nutrients are produced by processing diacylglycerides e.g. 1,3-diacyl glycerides (DAG), monoacylglycerides, e.g. 2-monoacylglycerides (MAG) and / or triacylglycerides (TAG) from plant sources (e.g. oil plants) or from animals (e.g. fish oil). Nutrients disclosed (e.g. dietetic compositions) containing polypeptides (e.g., enzymes, peptides, antibodies) disclosed herein.

[0381] In one aspect, the compositions and methods provided herein can be used to enrich dietetic compositions, especially cow's milk based products, eg, cow's milk based infant compositions with bile salt activated hydrolases. The compositions made according to the methods and compositions can be used to feed newborns and premature babies, including administration of bile salt activated hydrolases disclosed herein to increase fat digestion and hence growth rates. In some embodiments, the specification discloses compositions and methods for treating subjects with insufficient production of pancreatic enzymes by administration of bile salt activated hydrolase in combination with fat intake; see also the discussion below.

[0382] Dietary compositions containing hydrolase, e.g. bile salt activated hydrolase, are disclosed. Dietary compositions are disclosed comprising a nutritional base comprising fat and an effective amount of bile salt activated hydrolase. Cow's milk based infant compositions containing hydrolase, e.g., bile salt activated hydrolase are disclosed. In one aspect, the hydrolase disclosed herein is active in the digestion of long chain fatty acids, e.g. C_i2 to C.₂₂, which constitute a very high proportion of most milk types, e.g., 99% from human breast milk. See, e.g., US Patent No. US 5,000,975.

[0383] Dietary compositions comprising vegetable fat in the form of an oil and the hydrolase disclosed herein are disclosed. In other embodiments, the present description provides

147 methods for treating milk-based products and / or compositions containing vegetable oil for the preparation of dietary compositions. In one aspect, the processed compositions comprise lauric acid oil, oleic acid oil, palmitic acid oil and / or linolenic acid oil. In one aspect, rice bran oil, sunflower oleic oil, and / or canola oil can be used as oleic acid oils. In one aspect, fats and oils, e.g., oilseeds, from plants, including, e.g., canola rape, castor oil, coconut, coriander, corn, cotton, hazelnut, hemp, linseed, meadow cress, olive, oil palm, palm kernels, peanuts, oilseed rape, rice bran, saffron, camellia, soybean, sunflower, thallium, tsubaki, varieties of "natural" oils with a fatty acid composition changed by genetically modified organisms (GMOs) or from traditional "crops" such as oils with high oleic acid content, low linolenic acid content or low saturated oil content (high oleic canola rapeseed, soybean with low linolenic acid content or sunflower with high stearic acid content), blends of any of the foregoing for use in nutrient and dietetic compositions are processed using a hydrolase as disclosed herein. See, e.g., US Patent 4,944,944.

[0384] In one aspect, the enzymes disclosed herein are provided in a form that is stable in storage in the composition and / or stomach, but active when the preparation reaches part of the gastrointestinal tract, where the composition would normally be digested. Formulations (e.g., microcapsules) for release in the intestine are well known in the art, e.g. biodegradable polymers such as polylactide and polyglycoide as described e.g. in US Patent Nos. 4,767,628; US 4,897,268; US 4,925,673; US 5,902,617.

Confectionery, Cocoa Butter and Food [0385] In one aspect, the compositions and methods provided herein can be used to treat hard butter such as cocoa butter. In another aspect, the present specification discloses the confectionery, cocoa butter and food containing polypeptides (e.g., enzymes, peptides, antibodies) disclosed herein.

[0386] The compositions and methods provided herein can be used to prepare cocoa butter alternatives using "structured" synthesis techniques using enzymes, e.g., hydrolases, e.g., lipases, saturases, palmitases, and / or stearatases disclosed herein. For example, in one aspect, the methods provided herein process or synthesize triacylglycerides, diacylglycerides and / or monoacylglycerides, for use as, e.g., cocoa butter alternatives. In one aspect, the methods provided herein generate hard butter with a defined "plasticity range" to maintain sufficient hardness below or at room temperature. In one aspect, the processed or synthesized lipid is to have a very narrow "plasticity range", e.g., in one aspect when it melts very quickly at temperature

148 about body temperature. Natural cocoa butter begins to soften at about 30 ° C to 32 ° C and melts completely at about 36 ° C. Natural cacao butter may contain 70% by weight or more of three 1,3-doubled 2-oleyl glycerols, which are 1,3-dipalmitoyl-2-oleoyl glycerol (POP), 1-palmitoyl-2-oleoyl-3-stearoyl glycerol (POST) and 1,3-distearoyl-2-oleoyl glycerol (StOSt). These three glycerols show similar melting properties relative to each other and are responsible for the melting properties of cocoa butter, showing a very narrow plasticity area. Synthetic cocoa butters or processed cocoa butters (synthesized or processed using the hydrolase disclosed herein disclosed, all possible compositions are referred to as cocoa butter alternatives) with varying percentages of 1,3-dipalmitoyl-2-oleoyl glycerol (POP), 1- palmitoyl-2-oleoyl glycerol (POSt) il, 3-distearoyl-2-oleoyl glycerol (StOSt), depending on the desired properties of synthetic cocoa butter and synthetic cocoa butter containing more or less than 70 wt. three, 3-dinasaturated-2-oleoyl glycerols. The synthetic cocoa butter disclosed herein may partially or completely replace natural or unprocessed cocoa butter and may maintain or improve the essential properties of hard butter.

[0387] Synthetic cocoa butters or processed cocoa butters (synthesized or processed using the hydrolase disclosed herein) with desirable properties for use in confectionery, bakery and pharmaceutical products are disclosed. Confectionery, bakery and pharmaceutical products and the like are disclosed, comprising the hydrolase disclosed herein. In one aspect, the methods provided herein produce or process lipid (fat) from confection (e.g., chocolate) or can be used in confection. In one aspect, lipid is produced or processed such that chocolate has fewer fingerprint marks than chocolate made from natural cocoa butter, while still maintaining the stringent melting properties in the mouth. In one aspect, lipid is produced or processed such that confectionery (e.g., chocolate) can be produced at a relatively high ambient temperature, or made using cooling water at a relatively high temperature. In one aspect, lipid is made or processed such that confectionery (e.g., chocolate) can be stored in relatively warm conditions, e.g., tropical or semi-tropical conditions, or in centrally heated buildings. In one aspect, lipids are made or processed such that the formulation (e.g., chocolate) will contain a lipid (fat) of consistent structure and quality. Enzymes can be used to provide a cocoa butter substitute composition that can significantly improve its thermal stability and replace it in a wide range of applications. Production of margarine and food fat [0388] Synthetic or processed fats, e.g., margarine and food fat, synthesized or processed by the hydrolase disclosed herein are disclosed.

149

Synthetic or processed fats, e.g., margarine and food fat, containing polypeptides (e.g., enzymes, peptides, antibodies) disclosed herein are disclosed. [0389] Processed fats containing vegetable oil, such as canola, castor, coconut, coriander, corn, cotton, hazelnut, hemp, linseed, meadow watercress, olives, palm oil, palm kernels, peanuts, rapeseed, are disclosed rice bran, saffron, camellia, sesame, soybean, sunflower, thallium, tsubaki, varieties of "natural" oils with a fatty acid composition changed by genetically modified organisms (GMOs) or from traditional "crops" such as oils with high oleic acid content, low linolenic acid content or low saturated oil content (high oil acid canola rapeseed, soybean with low linolenic acid content or sunflower with high stearic acid content), synthesized or processed using the hydrolase disclosed herein. Synthetic or processed fats, e.g., margarine and edible fat, are designed to give the desired "plasticity". Many plastic fat products, such as margarine and edible fat, are made from hard raw materials and liquid oils as raw materials. For example, liquid oils such as canola, castor oil, coconut, coriander, corn, cotton, hazelnut, hemp, linseed, meadow cress, olives, palm oil, palm kernels, peanuts, oilseed rape, rice bran, saffron, camellia, sesame, soybean, sunflower, thallium, tsubaki, varieties of "natural" oils with a fatty acid composition changed by genetically modified organisms (GMOs) or from traditional "crops" such as oils with high oleic acid content, low linolenic acid content or low saturated oil content (high oil acid canola rapeseed, soybean with low linolenic acid content or sunflower with high stearic acid content), it is mixed with their hardened oils (hard starting materials) and the mixture is adjusted to get the right consistency (plasticity). Plastic fat products such as margarine and food fat produced in this way tend to form relatively thick crystallites because the fats and oils used as raw materials consist of fatty acids with almost the same carbon chain length. In other words, they have a highly unified fatty acid composition. For this reason, the plasticity of these products can only be maintained within a narrow temperature range, so that the liquid oils they contain tend to separate. In the present description, methods are provided for producing and processing fats designed in such a way that they have a diverse (and defined) fatty acid composition. The resulting oil, e.g. margarine or food fat may have a wider range of plasticity.

[0390] In one aspect, the methods and compositions provided herein are used for the production or processing of vegetable oils such as canola rape, castor oil,

150 coconut, coriander, corn, cotton, hazelnut, hemp, linseed, meadow cress, olives, palm oil, palm kernels, peanuts, oilseed rape, rice bran, saffron, camellia, sesame, soy, sunflower, thallium, tsubaki, varieties "natural" oils with a fatty acid composition altered by genetically modified organisms (GMOs) or from varieties of oil from traditional "cultivation" such as oils with high oleic acid content, low linolenic acid content or low saturated oil content (high oil acid canola rape, low linolenic acid soybean or high stearic acid sunflower oil), using hydrolases, including inter-esterification and enzymatic transesterification, see e.g. US Patent No. 5,288,619 and US Patent Application Serial Number 11 / 567,318. The methods and compositions provided herein can be used in place of random inter-esterification as described, for example, US Patent No. 3,949,105. In one aspect, the methods and compositions provided herein are used in enzymatic transesterification to produce an oil, e.g., margarine oil, having both low trans-acid content and low medium-chain fatty acid content.

[0391] In one aspect, the symmetrical structure of the oil, e.g., palm or lauric type oils, is modified, e.g., to a random structure. Thus, the methods provided herein can be used to modify the plastic properties of fat products. In one aspect, oil modification by the methods provided herein can be designed to prevent or slow the gradual curing of oil over time, in particular when products are stored.

[0392] In one aspect, by means of the methods and compositions provided herein in a transesterification reaction mixture containing stearic acid as the source material and edible liquid vegetable oil, the stearic acid source material and vegetable oil are trans-esterified using position-specific lipase 1-, 3-, and then hydrogenating the fatty acid mixture to ensure recycling of the stearic acid source material for the recycling reaction with vegetable oil. See, e.g., U.S. Patent No. 5,288,619.

[0393] In one aspect, the inter-esterification reaction is carried out using lipase. In one aspect, lipase is selective for the 1- and 3- triglyceride positions to slow or inhibit the increase in the amount of tri-saturated triacylglycerides in oil. In this reaction provided herein, the deficiencies of conventional random trans-esterification and the difficulty of inter-esterification using a non-specific lipase can be overcome because inter-esterification is carried out with an enzyme with specificity for 1- and 3-triglyceride positions. In one aspect, the release of liquid oils contained in products is slowed down or prevented by increasing the temperature in the reaction to inhibit the increase in melting point caused by increasing the amount of

151 tri saturated triacylglycerides. This refers to the problem of curing products during prolonged storage.

Pharmaceutical compositions and treatment of hydrolase deficiency [0394] Methods and compositions (enzymes disclosed herein, e.g., esterases, acylases, lipases, phospholipases and proteases disclosed herein) are disclosed that can be used to treat hydrolase deficiency in animals, e.g. mammal such as man. For example, in one aspect, the methods and compositions disclosed herein can be used to treat patients suffering from pancreatic lipase deficiency. In one aspect, lipases are administered orally. The enzyme disclosed herein can be delivered to a site or in a porcine pancreatic enzyme preparation.

[0395] Pharmaceutical compositions comprising polypeptides (e.g., enzymes, peptides, antibodies) disclosed herein are disclosed. These pharmaceutical compositions may be in the form of tablets, pills, gels, capsules, hydrogels, sprays, powders, aerosols, implants, liposomes, ointments, creams, lotions, microspheres, in the form of multiple core particles, emulsions, suspensions, nanostructures and the like. Pharmaceutical compositions containing polypeptides (e.g., enzymes, peptides, antibodies disclosed herein) can be administered in any form, e.g., orally, intradermally, intraperitoneally, intravenously, topically, and the like. In one aspect, the pharmaceutical compositions disclosed herein are formulated for topical, sublingual, oral, intravenous, subcutaneous, intramuscular, transdermal, intraarterial, intra-articular or intradermal administration. [0396] In one aspect, the compositions disclosed herein used for this type of treatment are active under acidic conditions. In one aspect, the compositions disclosed herein are administered orally in formulations (e.g., tablets, pills, gels, capsules, hydrogels, sprays, powders, aerosols) that pass through the acidic regions of the stomach and release the enzyme only in a relatively alkaline environment jejunum. In one aspect, the hydrolase disclosed herein is formulated with a carrier such as lactose, sucrose, sorbitol, mannitol, starch, cellulose derivatives or gelatin or any other excipient. A lubricant such as magnesium stearate, calcium stearate, wax polyethylene glycol may also be added. As a coating, a concentrated sugar solution may be added, which may contain additives such as talc, titanium dioxide, gelatin or acacia. Soft or hard capsules can be used to encapsulate the hydrolase in liquid or solid form. See, e.g., US Patent Nos. 5,691,181; US 5,858,755.

Detergents [0397] Methods and compositions (enzymes, e.g., lipases, saturases, palmitases and / or stearatases disclosed herein) that can be used in the preparation and use of detergents are disclosed. The hydrolase disclosed herein may be added to, for example, mixed with, any known detergent composition, solid or liquid, with or without

152 changes in the composition of the detergent composition. For examples, the hydrolases disclosed herein may be added to any soap, for example, aliphatic sulfates, such as straight or branched alkyl or alkenyl sulfates, amide sulfates, alkenyl sulfates or alkenyl ethers of straight or branched chain or having an alkenyl group to which one or more ethylene oxide, propylene oxide or butylene oxide, aliphatic sulfonates such as alkyl sulfonates, amide sulfonates, dialkyl sulfosuccinates, alpha-olefin sulfonates, vinylidene olefins and internal olefins, aromatic sulfonates such as straight or branched chain alkyl benzene sulfonates, alkyl or alkenyl ether carbonates or amides having a straight or branched chain alkyl group to which they are attached one or more ethylene oxide, propylene oxide and butylene oxide, or amides, salts of alpha-sulfo-fatty acid or esters, amino acid type surfactants, phosphate surfactants such as alkyl or acid alkenyl phosphates, and alkyl or alkenyl phosphates, sulfonic acid amphoteric surfactants, betaine type amphoteric surfactants, alkyl or alkenyl ethers or alcohols containing an alkyl group or straight or branched chain alkenyl to which one or more ethylene oxide is attached, propylene oxide and butylene oxide, polyoxyethylene alkyl phenyl ethers containing a straight or branched chain alkyl or alkenyl group to which one or more ethylene oxide, propylene oxide and butylene oxide is attached, higher fatty acid alkanolamides or their alkylene oxide adducts and sucrose esters fatty acids, glycerol and fatty acid monoesters, alkyl or alkenylamine oxides, cationic tetraammonium surfactants of the tetraalkylammonium salt type or a combination thereof, see, e.g., US Patent No. 5,827,718.

[0398] Detergent compositions comprising one or more polypeptides (hydrolases) disclosed herein are disclosed. Surfactant and / or surface inactive forms may be used. In one aspect, the total amount of hydrolase, surfactant and / or surfactant may be from about 0.0001% to about 1.0%, from about 0.0002% to about 0.5% by weight of the detergent composition In one aspect of the detergent composition, the surfactant hydrolase is from about 5% to about 67% and the inactive surfactant hydrolase is from about 33% to about 95% of the total hydrolase activity in the enzyme blend. In one aspect, the optimum pH of the entire enzyme mixture is from about 5 to about 10.5.

[0399] In one aspect, the detergent compositions disclosed herein include the alkaline hydrolases disclosed herein that operate at alkaline pH values, since the pH of the rinsing solution may be in the range of alkaline pH under normal rinsing conditions. See, e.g., US Patent No. 5,454,971

153 [0400] The polypeptides disclosed herein (enzymes disclosed herein) can be used in any detergent composition that is well known in the art, see, e.g., US Patent Nos. 5,069,810; US 6,322,595; US 6,313,081. For example, in one aspect, a laundry detergent composition is disclosed. It may contain 0.8 ppm to 80 ppm of lipase disclosed herein.

[0401] Any method of making and using detergent compositions may be used using the enzymes disclosed herein, see, e.g., US Patent Nos. 6,413,928; US 6,399,561; US 6,365,561; US 6,380,147. The detergent compositions may be in the form of a one and two component aqueous composition, a liquid non-aqueous composition, a solid cast, granular form, particle form, compressed tablet, gel, powder, gel, hydrogel, liposome, aerosol, paste and / or suspension. The hydrolases disclosed herein can also be used as a detergent additive product in solid or liquid form. Such additive products are intended to supplement or increase the performance of conventional detergent compositions and can be added at any stage of the cleaning process.

[0402] Methods have been disclosed for removing large food contaminants, films from food residues, and other small food compositions using these detergent compositions. The hydrolases disclosed herein may facilitate stain removal by catalytic hydrolysis of lipids, fats and oils. The hydrolases disclosed herein may be used in dishwashing detergents and textile laundry detergents.

[0403] The actual active enzyme content depends on the method of making the detergent composition and is not critical, assuming that the detergent composition has the desired enzymatic activity. In one aspect, the amount of hydrolases present in the final composition ranges from about 0.001 mg to 0.5 mg per gram of detergent composition. The particular enzyme selected for use in the process and the products disclosed herein depend on the conditions of end use, including product form, working pH, working temperature and types of impurities to be degraded or changed. The enzyme can be selected to provide optimal activity and stability for any given set of conditions of use. In one aspect, the hydrolases disclosed herein are active in pH ranges from about 4 to about 12 and in a temperature range from about 20 ° C to about 95 ° C. The detergents disclosed herein may contain cationic, semipolar nonionic or zwitteionic surfactants; or mixtures thereof.

[0404] The disclosed enzymes may be formulated into powdered and liquid detergents with a pH between 4.0 and 12.0 at concentrations of about 0.01 to about 5% (alternatively from 0.1% to 0.5%) by weight. These detergent compositions may also contain other enzymes such as proteases, cellulases, lipases or endoglycosidases, endo-beta.-1,4-glucanase, beta154 glucanase, endo-beta-1,3, 4 (4) glucanase, cutinase, peroxidase, laccase, amylase, glucoamylase, pectinase, reductase, oxidase, phenoloxidase, ligninase, pululanase, arabinanase, hemicellulase, mannanase, xyloglucanase, xylanase, pectinacetyl esterase, acetyl-ramase, ramylactalactalactalacturase, galactanases, pectin lyases, pectin methyl esterases, cellobiohydrolases and / or transglutaminases. These detergent compositions may also contain bulking agents and stabilizers.

[0405] The addition of the hydrolases disclosed herein to conventional cleaning compositions does not pose any special purpose restrictions. In other words, any temperature and pH suitable for the detergent is suitable for the compositions disclosed herein, as long as the enzyme is active or abolishes the pH and / or temperature of the intended use. In addition, the hydrolases disclosed herein can be used in a cleaning composition without detergents, either alone or in combination with fillers and stabilizers.

[0406] Cleaning compositions are disclosed, including hard surface detergent compositions, fabric cleaning detergent compositions, dishwashing detergent compositions, oral cleaning detergent compositions, denture cleaning compositions and contact lens cleaning solutions.

[0407] Methods for washing an object are disclosed comprising contacting the object with the polypeptide disclosed herein under conditions sufficient for washing. The hydrolase disclosed herein may be included as a detergent additive. The detergent composition disclosed herein may, for example, be formulated in a hand or machine wash detergent composition containing the polypeptide disclosed herein. A washing additive suitable for pre-treating dyed fabrics may contain the polypeptide disclosed herein. The fabric softening composition may contain the hydrolase disclosed herein. Alternatively, the hydrolase disclosed herein may be formulated in a detergent composition for use in general hard surface cleaning operations for domestic use. In alternative aspects, the detergent additives and detergent compositions disclosed herein may contain one or more other enzymes such as protease, lipase, cutinase, other protease, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g. ., lactase and / or peroxidase (see also above). The properties of the enzyme (s) disclosed herein are selected to be compatible with the selected detergent (i.e. optimum pH, compatibility with other enzymatic and non-enzymatic components etc.) and enzyme (s) were present in effective amounts. In one aspect, the enzymes disclosed herein are used to remove odorous substances from fabrics. Various detergent compositions and methods for their preparation that can be used are described in, e.g., US Patent Nos

155

US 6,333,301; US 6,329,333; US 6,326,341; US 6,297,038; US 6,309,871; US 6,204,232; US 6,197,070; US 5,856,164.

[0408] Hydrolases disclosed herein, when formulated as compositions suitable for use in a washing machine washing method, may contain both surfactant and bulking compound. They may additionally contain one or more detergent ingredients, e.g., organic polymer compounds, bleaching agents, additional enzymes, soap suds inhibitors, dispersants, lime soap dispersants, anti-suspending and anti-dusting agents and corrosion inhibitors. The laundry compositions disclosed herein may also contain emollients as additional detergent ingredients. The hydrolase-containing compositions disclosed herein, when formulated as laundry detergent compositions, can provide fabric cleaning, stain removal, whiteness maintenance, softening, color appearance, inhibition of dye transfer and decontamination.

[0409] The density of the laundry detergent composition disclosed herein may be from about 200 to about 1500 g / liter, or about 400 to 1200 g / liter, or 500 to 950 g / liter, or 600 to 800 g / liter of the composition ; which can be measured at about 20 ° C.

[0410] The "compact" form of the laundry detergent composition disclosed herein is best reflected by the density and, with respect to the composition, in an amount of inorganic filler salt. Inorganic filler salts are conventional ingredients in the powder detergent composition. In typical detergent compositions, filler salts are present in significant amounts, generally 17% to 35% by weight based on the total weight of the composition. In one aspect of the compact compositions, the filler salt is present in an amount not exceeding 15% of the total weight of the composition, or not exceeding 10%, or not exceeding 5% by weight of the composition. Inorganic filler salts may be selected from sulfate and chloride salts with alkaline earth metals and metals, e.g., sodium sulfate.

[0411] Liquid detergent compositions disclosed herein may also be in "concentrated form". In one aspect, liquid detergent compositions may contain less water compared to conventional liquid detergents. In alternative aspects, the water content of the concentrated liquid detergent is less than 40%, or less than 30%, or less than 20% by weight of the detergent composition. The detergent compounds disclosed herein may contain formulations as described in WO 97/01629.

[0412] The hydrolases disclosed herein may be useful in the preparation of various cleaning compositions. A number of known compounds are suitable surfactants, including anionic, nonionic, cationic or zwitterionic detergents, e.g. as disclosed in US Patent Nos. 4,404,128; US 4,261,868; US 5,204,015. In addition, the enzymes disclosed herein can be used, for example, in the use of bar soaps or liquid soaps, care preparations

156 vessels, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile applications such as fusion cleavage enzymes in protein production and the like. The hydrolases disclosed herein may provide better performance in a detergent composition compared to another detergent protease, i.e., the enzyme group may enhance the cleaning of certain enzyme-sensitive spots, such as grass or blood stains, as determined by standard evaluation after standard washing cycle. The hydrolases disclosed herein may be formulated into known powder and liquid detergents with a pH between 6.5 and 12.0 at concentrations of about 0.01 to about 5% (for example, about 0.1% to 0.5%) by weight. These cleaning detergent compositions may also contain other enzymes, such as other known esterases, phospholipases, proteases, amylases, cellulases, lipases and endoglycosidases, as well as bulking agents and stabilizers.

Food treatment and food processing [0413] The hydrolases disclosed herein can be used to separate material components from plant cells. For example, the hydrolases disclosed herein can be used to separate protein-rich material (e.g., plant cells) into components, e.g., sucrose from sugar beet or starches, or sugars from potatoes, pulp or shell fractions. In one aspect, the hydrolases disclosed herein can be used to separate protein rich or oil rich crops into valuable protein and oil fractions and shells. The separation process can be carried out using methods known in the art.

[0414] The hydrolases disclosed herein can be used to produce fruit or vegetable juices, syrups, extracts and the like to increase yield. The hydrolases disclosed herein may be used in enzymatic treatment (e.g., protein hydrolysis) from cell wall material derived from various plants or waste materials, e.g. from wine or the production of juice or agricultural waste such as plant shells, bean husks, sugar beet pulp, olive pulp, potato pulp and the like. The hydrolases disclosed herein can be used to modify the consistency and appearance of processed fruit or vegetables. The hydrolases disclosed herein can be used to treat plant material to facilitate processing of plant material, including food, to facilitate purification or extraction of plant components. The hydrolases disclosed herein can be used to improve nutritional value, reduce water binding capacity, improve degradability in wastewater treatment plants, and / or improve the conversion of plant material to silage, and the like.

Animal feed and food or feed additives [0415] In some embodiments, the present description provides methods for treating animal feed and food or food or feed additives using hydroiazines,

157 animal, including mammalian (e.g., human), avian, fish and the like. This description discloses animal feeds, food, feed and food additives and the additives containing hydrolases disclosed herein.

[0416] In some embodiments, hydrolases are disclosed for use in modifying feed or animal food, e.g., for processing food or feed in vitro (by modifying feed or food ingredients) or in vivo. In another aspect, the hydrolase disclosed herein can be provided by expressing enzymes directly in transgenic feed crops (e.g., transgenic plants, seeds and the like) such as corn, soybean, rapeseed, lupine and the like. In one aspect, the present disclosure discloses transgenic plants, plant parts, and plant cells comprising a nucleic acid sequence encoding a polypeptide disclosed herein. In one aspect, the nucleic acid is expressed in such a way that the hydrolase disclosed herein is produced in recoverable amounts. Hydrolase can be recovered from any plant or part of the plant. Alternatively, the plant or plant part containing the recombinant polypeptide can be used as such to improve the quality of food and feed, e.g., improve nutritional value, taste and theological properties, or to destroy a factor adversely affecting the nutritional value.

Interesterification [0417] In one aspect, methods and compositions can be used to modify the properties of mixtures of triacylglycerides, and in one aspect, their consistency. In one aspect, the enzyme may be used in the presence of a catalyst such as sodium metal or sodium methoxide to promote acyl migration between glyceride molecules, such that the products consist of glyceride mixtures in which the acyl residue of the fat is randomly distributed between the glyceride molecules.

[0418] In one aspect, the enzymes can be used to produce interesterification products under reaction conditions in which fat hydrolysis is minimized such that lipase catalysed interesterification becomes the dominant reaction. These conditions include, for example, limiting the amount of water in the system.

[0419] In one aspect, enzymes can be used to catalyze interesterification reactions using a mixture of triacylglycerides and free fatty acids, as described, for example, in EP 0 093 602 B2. In these cases, free fatty acids can be exchanged with the acyl groups of triacylglycerides to form new triacylglycerides enriched in added fatty acid. In one aspect, 1,3 specific lipases can be used to limit the reaction to the 1- and 3-glyceride positions, which allows a mixture of triacylglycerides that cannot be obtained by chemical interesterification or reaction with non-specific lipase. In one aspect, non-specific lipases are used to achieve results similar to chemical interesterification.

158 [0420] The ability to form new triglyceride mixtures using position specific lipases is useful in the fat and oil industry because some of these mixtures have valuable properties. One example is interesterfikaq 1,3-specific lipase catalysed 1,3-dipalmitoyl-2-monoleine (POP), which is the main triglyceride of the main palm oil fraction, with stearic acid or tri-stearin to obtain products enriched with valuable 1-palmitoyl -1-3-stearoyl-2-monooleine (POSt) and 1,3-distearoyl-2-monooleine (StOSt). POSt and StOSt are important ingredients in cocoa butter. Thus, in one aspect provided herein, interesterification reactions are provided for the production of cocoa butter equivalents from cheap starting materials.

[0421] In one aspect, the present disclosure discloses methods of producing a hard fat substitute using the 1,3-specific lipases disclosed herein. In one aspect, the hard fat replacement contains a mixture of the main palm oil fraction and StOSt, POSt or StOSt / POSt with a purity of at least 85%.

[0422] The invention will be further described with reference to the following examples; however, it should be understood that the invention is not limited to such examples. EXAMPLES

Example 1: Exemplary lipazv-saturazv assays [0423] The following example describes exemplary hydrolase screening tests, e.g., lipase, saturase, palmitase and / or stearatase. Such tests include the use of pH indicator compounds to detect the cleavage of fatty acids from triacylglycerides, spectrophotometric methods, HPLC, GC, MS, TLC and others. Jaeger (1994) FEMS Microbiol. Rev. 15: 29-63; Ader (1997) Methods Enzymol. 286: 351-386; Vorderwiilbecke (1992) Enzyme Microb. Technol. 14: 631-639; Renard (1987) Lipids 22: 539-541.

Screening for lipazv / esterazv activity [0424] Colonies are taken with sterile toothpicks and used to inoculate with a single colony each well in 96 well microtiter plates. The wells contained 250 pL LB medium with 100 pg / mL ampicillin, 80 pg / mL methicillin and 10% v / v glycerol (LB Amp / Met, glycerol). Cells were grown overnight at 37 ° C without shaking. Each well therefore contained the E coli cell culture starting culture, each containing pBLUESCRIPT ™ with a unique DNA insert.

[0425] 96-well plates were used to propagate the inoculum of a single plate ("high density plate") containing 200 pL LB Amp / Met glycerol in each well. This step was performed using the High Density Replicating Tool (HDRT) from a BIOMEK ™ device (Beckman Coulter, Inc., Fullerton, CA) with 1% oxidant, water, isopropanol, an air-drying sterilization cycle between each inoculation. Each well of an increased density plate thus contained 10 to 12 different pBLUESCRIPT ™ clones from each output library plate. Increased tile

159 densities were grown for 16 hours at 37 ° C and then used to inoculate two 96-well microtiter plate derivatives (Polyfiltronics, Inc., Rockland, MA) containing 250 uL LB Amp / Met (glycerol) in each well. The original plate with increased density was stored at -80 ° C. Two concentrated derivative plates were incubated at 37 ° C for 18 hours.

[0426] A '600 μΜ stock solution of the short chain esterase substrate was prepared as follows: 25 mg of each of the following compounds were dissolved in the appropriate volume of DMSO to give a 25.2 mM solution. The compounds used are 4-methylumbelliferyl propionate, 4-methylumbelliferyl butyrate and 4-methylumbelliferyl heptanoate. Two hundred and fifty microliters of each DMSO solution was added to approximately 9 mL of 50 mM HEPES buffer, pH 7.5, which contained 0.6% Triton 100-100 and 0.6 mg per mL of dodecyl maltoside (Anatrace, Maumee, OH). The volume was transferred to 10.5 mL of the above HEPES buffer to give a slightly cloudy suspension.

[0427] A '600 μΜ stock solution of the long chain substrate was prepared as follows: 25 mg of each of the following compounds were dissolved in DMSO to give a 25.2 mM solution as described above. The compounds used are 4-methylumbelliferyl elaidate, 4-methylumbelliferyl palmitate, 4-methylumbelliferyl oleate, 4-methylumbelliferyl stearate. All required short heating at 70 ° C. Two hundred and fifty microliters of each DMSO solution was added to HEPES buffer and diluted to 10.5 mL as above. All seven umbeliferyl derivatives were obtained from Sigma Chemical Co. (St. Louis, MO).

[0428] Fifty μί of the '600 μΜ stock solution of the long chain or short chain esterase substrate was added to each well of a white plate with increased density using BIOMEK ™, resulting in a final substrate concentration of about 100 μΜ. Fluorescence values ​​were recorded immediately after substrate addition (excitation = 326 nm, emission = 450 nm) using a plate reading fluorimeter. The plate was incubated at 70 ° C for 60 minutes for long chain substrates, and 30 minutes at room temperature for short chain substrates. Fluorescence values ​​were rewritten. The initial and final fluorescence values ​​were compared to determine if active clone was present.

[0429] To isolate a single clone that carries activity, GenBank from source plates was thawed and individual wells were used to inoculate a new LB Amp / Met containing plate individually. As above, the plate is incubated at 37 ° C for cell growth, 50 pL 600 μΜ substrate stock solution added, using BIOMEK ™, and fluorescence determined. After identifying the active well from the source plate, cells from this active well are plated on LB Amp / Met agar and grown overnight at 37 ° C to obtain single colonies. Eight individual colonies were harvested with a sterile toothpick and used to inoculate the wells of a 96 well microtiter plate individually.

160

The wells contained 250 μΙ_ LB Amp / Met. Cells were grown overnight at 37 ° C without shaking. A 200 pL aliquot was taken from each well and tested with appropriate long and short chain substrates as above. The most active clone was identified and the remaining 50 pL cultures were used for inoculation on LB Amp / Met agar plate. Eight individual colonies were harvested, cultured and tested as above. The most active clone was used to inoculate a 3 mL LB / Amp / Met culture that was grown overnight. Plasmid DNA was isolated from the culture and used for sequencing.

Example 2: Example protocols for LCMS determination of the liberated fatty acid profile obtained from enzymatic hydrolysis of vegetable oil [0430] The following example describes exemplary methods (protocols) for carrying out enzymatic hydrolysis of vegetable oil such as soybean oil (used in this example) (including preparation enzyme) using, for example, enzymes. This example also describes exemplary methods (protocols) for detecting and quantifying fatty acids released from oil. This method is described by lipase SEQ ID NO: 2, but applies to other enzymes, e.g., exemplary enzymes having sequences as shown in SEQ ID NO: 2 and having one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more or all modifications of the amino acid residues described in Table 3, Table 4, Table 9, Table 10, Table 11, Table 16 and Table 23.

Protein expression in a 96 well deep plate:

[0431]

1. Grow E. coli lipase clones overnight at 30 ° C in 1 ml TB medium containing carbenicillin (100 pg / mL) in deep 96-well plates. Record the location and identity of the clones.

2. Inoculate into freshly deep 96-well plates containing TB medium (1 mL, 100 pg / mL carbenicillin) with liquid cultures (10 pL / well).

3. Incubate the culture overnight at 30 ° C with shaking at 200 revolutions. for a minute.

4. Induce protein expression by transferring 500 pL of each overnight culture to a fresh 96-well plate containing TB medium (500 pL / well, 100 pg / mL carbenicillin) and anhydrotetracycline (200 ng / mL).

5. Incubate at 30 ° C for 2 hours with shaking at 200 revolutions. for a minute

6. Harvest cells by centrifuging each plate for 10 minutes at 3000 x g. Remove supernatant. Cell pellets can be used directly for oil analysis or stored at 20 ° C for later use.

Enzymatic oil hydrolysis reaction:

[0432]

161

1. Add 100 pL B-PER ™ (Pierce Chemical, Rockford, IL) to each of the cell pellets. If the pellets were stored at -20 ° C, allow thaw for 10 minutes at room temperature before adding B-PER ™.

2. Add 400 pL of soybean oil to each deep well of a 96 well plate.

3. Add a few beads (glass 710-1180 pm) per well. Seal CAPMATS ™ (Whatman, Florham Park, NJ).

4. The cells are lysed and an oil / enzyme / buffer emulsion is generated using a mill mixer (Retsch Inc, Newtown, PA). Place a pair of closed plates in a mixer with a grinder and shake for 30 seconds at a frequency of 30 cycles / second.

5. Replace CAPMATS ™ with gas permeable gaskets.

6. Incubate the plates for 2 hours at 37 ° C while shaking at 200 revolutions. for a minute. Fatty Acid Extraction:

[0433]

1. Add 1 mL of extraction solvent (CHCl₃: MeOH: 4N HCl (2: 1: 0.075)) in each deep well of a 96 well plate.

2. Pipette the mixture up and down several times until it becomes homogeneous.

3. Cover the tiles with an aluminum foil gasket.

4. Centrifuge for 5 minutes at 3000 x g. Cut the seal using a razor blade.

5. Puncture the upper phase with the pipette tip and transfer 5 pL of the lower phase to a new deep 96 well plate containing 995 pL / well of MeOH (i.e. 1/200 dilution in the lower phase). Be careful not to contaminate the upper phase. Keep the extraction mixtures separated at 4 ° C.

6. Transfer 150 pL of 1/200 dilution of all samples to a 96-well polystyrene plate.

7. To prevent evaporation, close the heat seal plates. Make sure the gasket does not come in contact with MeOH, as this will prevent proper adhesion.

8. Analyze LC / MS samples.

LC / MS ANALYSIS:

[0434]

1. Samples delivered in a 96-well plate format are injected with an HTCPAL ™ autosampler (LEAP Technologies, Carrboro, NC) into an isocratic mixture of H₂O / MeCN (10/90, v / v) and 0.1% formic acid, supplied by the LC-10ADVP ™ pump (Shimadzu, Kyoto, Japan) at 1.2 mL / min.

2. Separation is achieved using a SYNERGI MAX-RP ™ column (Phenomenex, Sutter Creek CA) 150 x 2.00 mm and detection. Quantitative evaluation is completed by the API 4000 ™ triple quadrupole mass spectrometer (Applied Biosystems, Foster, CA) using electrospray ionization (ESI) and multiple ion monitoring for masses 277, 279, 281, 255, 283 in negative ionization mode.

162

3. Equipment control and data generation are achieved using ANALYST 1.3 ™ software (Applied Biosystems, Foster, CA).

4. LC / MS was calibrated for each fatty acid in the range from 0.5 to 50 pg using standard samples (Sigma). This range best fits the standard quadratic regression curve that is used to calculate the amount of each fatty acid released in enzyme samples.

Example 3: Exemplary HTP screening protocols of lipase evolutionary libraries for increased selectivity for the hydrolysis of paimitate or stearate esters compared to oleate esters [0435] The following example describes exemplary methods (protocols) for high throughput (HTP) 'lipase evolutionary libraries in terms of the increased selectivity of the hydrolysis of paimitate or stearate esters over oleate esters. This exemplary method (screening protocol / HTP) describes the screening of lipase evolutionary libraries derived from SEQ ID NO: 2, but applies to other enzymes, e.g., exemplary enzymes having sequences as shown in SEQ ID NO: 2 and having one, two , three, four, five, six, seven, eight, nine, ten, eleven or twelve or more or all modifications of the amino acid residues described in Table 3, Table 4; and this example method (protocol) applies to other types of libraries.

[0436] These exemplary HTP screenings are carried out using two fluorogenic substrates: methylumbelliferyl palmitate or stearate esters compared to methylumbelliferyl oleate ester.

HTP screening process:

[0437]

1. Library clones were spread on microtiter plates and tested in first order HTP screening.

2. Clones identified as having improved selectivity were designated as first order hits.

3. First-order hits are redistributed in microtiter plates and tested in second-order HTP screening.

4. Clones confirmed as having improved selectivity were designated as second order hits.

5. Second-order hits were sequenced to identify current mutations and tested in oil (see separate protocol).

HTP test protocol [0438]

163

1. Barcode 384 well black test plates; barcode 384-well black culture plates and fill 30 pL / well LB medium (100 pg / mL carbenicillin).

2. Download clones on growth plates with a needle tool or select the best clones and grow overnight at 30 ° C in a humidified incubator.

3. Induce lipase expression by adding 30 pL / well of LB medium (100 pg / mL carbenicillin) containing 4 pg / ml anhydrotetracycline and incubate 2 hours at 30 ° C.

4. Cell lyse by adding 20 pL / well B-PER ™ (Pierce Chemical, Rockford, IL); and keep at room temperature until placed in the robot.

5. Start the lipase activity test on the robot (see below).

6. Clones identified as showing increased selectivity for MeUMB palmitate or stearate esters compared to oleate esters were designated as hits.

7. Download the best clones into deep 96-well plates containing LB medium (1 mL / well, 100 pg / mL carbenicillin) and grow overnight at 30 ° C.

8. For first row hits, arrange them again on 384-well plates and repeat steps 1-8 in second row screening; mark hit clones as second order hits.

9. For second order hits, after stage 8, submit for sequencing.

Automated sample HTP screening protocol [0439]

1. Apricot: mix and transfer a portion (10 ml) of lysed cells from a "growth plate" (see points 1-4 above) to each of two separate test plates (1 and 2).

2. MULTIDROP ™ (Thermo Electron Corporation, Milford, MA): add 70 pL of Substrate 1 (UMB-16: 0) to Test Plate 1; add 70 pL of substrate 2 (UMB-18: 1) to test plate 2.

3. Incubate the test plates for 20 minutes at 37 ° C.

4. Read on fluorimeter: 360 nm excitation and 465nm emission [0440] Second-order clones, identified as having unique sequences, were distributed and grown in 96 well plates and tested in soybean oil (see below).

Structures of fluorogenic substrates used in HTP screening

164

<img file="PL2329032T3_D0001.tif" />

Me

<img file="PL2329032T3_D0002.tif" />

4-methylumbelliferyl oleate

Example 4: Example evolution to obtain improved hydrolysis of palmitate or stearate esters using GSSM technology^sm [0442] The following example describes and summarizes the results of exemplary "enzyme evolution" and screening protocols by which exemplary enzymes were identified, e.g., enzymes with a sequence as shown in SEQ ID NO: 2, but also showing residue modification as shown in Table 3 or Table 4; or enzymes encoded by a nucleic acid having the sequence shown in SEQ ID NO: 1, but also having residue modification as shown in Table 3 or Table 4. In one aspect, in an exemplary screening assay, soybean oil was used as a substrate to identify these exemplary enzymes, and fatty acids released (hydrolyzed) from soybean oil were characterized, e.g., as linolenic acid, such as linoleic acid, oleic acid, palmitic acid or stearic acid.

[0443] Soybean oil has the following fatty acid distribution: linolenic = 8%; linoleic = 53%; oleic = 23%; palmitic = 12%; stearic acid = 4. Thus, if the percentage of palmitic acid released from soybean oil (hydrolyzed) by an exemplary enzyme is higher than 12%, then the enzyme has a preference for hydrolysis (release) over palmitic acid.

Palmitase screening: generating a "palmitase library"

[0444] The library of SEQ ID NO: 2 palmitase variants was generated by technology

GSSM^sm (Patent No. 6,171,820). Point mutations were introduced using degenerate oligonucleotides for one amino acid position at a time, so that each original codon

165 is substituted with each of 20 naturally encoded amino acids. The mutated variants were transformed into an Escherichia coii TOPIO host (Invitrogen, USA) for expression and screening. This library was constructed in the pASK-5 expression vector that has been modified relative to the pASK-IBA vector (IBA GmbH, Germany). To generate pASK-5, the original cloning linker was replaced with new cloning sites, and specifically the sequence from Xbal to Hindlll from pASK-IBA was replaced by the following sequence:

RBS ArgSerHisHisHisHisHieHie

TCTAGATAACOAOGGCAAAACCATGGGAGOATCCAGATCTCATCACCATCACCATCACTAAGCTT (SEQ ID NO: 21)

Xbai Ncol BaniHl Bglll Hindlll [0445] Expression of GSSM variants^sm induced with anhydrotetracycline after obtaining optimal host cell density.

[0446] Enzymes having amino acid sequences generated using GSSM technology^sm was screened using a high-throughput screening protocol (HTP), e.g., the protocol described in Example 3, which determined which fatty acid in this test was preferentially hydrolyzed from fat - soybean oil. The goal of the evolutionary project was to increase the selectivity of the parent sequence, SEQ ID NO: 2 for palmitate, from oil. The test involves contacting the new / modified enzyme with soybean oil that contains various unsaturated fatty acids, including linolenic acid, linoleic acid, oleic acid, palmitic acid and stearic acid (see% distribution, mentioned above) and measuring the amount of each acid fatty acid hydrolysed by each modified enzyme. A sequence "library" has been identified that allows the enzyme to preferentially hydrolyze palmitic acid (or stearic acid, see below) from soybean oil (the so-called "Palmitate Library"):

• First and second order screening was performed using HTP screening, e.g., as described in Example 3;

• Second-order sequencing in HTP screening identified amino acid mutations that result in improved selectivity for palmitate hydrolysis over oleate compared to, for example, the parent sequence, SEQ ID NO: 2.

• For each codon variant coding for an amino acid mutation, one best clone was selected and placed in 96-well plates for oil testing;

• From oil tests, the selectivity of mutated enzymes to palmitate or stearate or other fatty acids was obtained (Table 3) • The best hit was that palmitate constituted 59% of released fatty acids (FAS) compared to (see) 43% for SEQ ID NO: 2 in the same test; this corresponds to an increase in the selectivity coefficient from 3.6 to 4.9;

• Several clones also showed an increase in stearate selectivity.

166 [0447] Table 1 below summarizes GSSM mutations^sm (see above) selected for inclusion in the "palmitate library" to be combined using GeneReassembly technology^SM (see Example 5). In one example test, fourteen (14) single amino acid mutations were identified that resulted in the largest increase in palmitate hydrolysis in oil tests (see also Tables 1, 3 and 4 below). The residues were determined according to the order of their occurrence in parent SEQ ID NO: 2 (see Fig. 7), among the residues that provide significant increases in palmitate or stearate hydrolysis in oil tests. The "original AA" in SEQ ID NO: 2 and preferred mutations ("New Amino Acids"), i.e., exemplary sequences are given. In one aspect, single mutations to arginine (R) at residue at positions 163 and 164 may be alternatively included such that this exemplary library will contain clones with sequences 163V-164D (SEQ ID NO: 2), 163R-164D, and 163V -164R, but not the 163R-164R sequence.

Table 1

<td>Rest</td><td>Original amino acid</td><td>New Amino Acid</td>

<td> 61</td><td>D</td><td>A, E</td>

<td>⁷²</td><td>R</td><td>E, K</td>

<td> 116</td><td>E</td><td>A, P, R, T, V</td>

<td> 133</td><td>s</td><td>AND</td>

<td> 151</td><td>AND</td><td>G, A</td>

<td> 163</td><td>V</td><td>R ......................'.......................... ..'................'............................. ' ........... '</td>

<td> 164</td><td>D</td><td>R ~ ........................................</td>

[0448] Figure 6a illustrates the effect of exemplary GSSM mutations^sm palmitases for palmitate and stearate hydrolysis relative to parent SEQ ID NO: 2. For each of the fourteen (14) single amino acid mutations selected for inclusion in the GeneReassembly library^SMpalmitases, the percentage change in release of palmitate and stearate relative to parent SEQ ID NO: 2, is shown in the graph. Many of these mutations resulted in a significant increase in palmitate hydrolysis, accompanied by small to significant increases in stearate hydrolysis. However, several mutations cause a slight decrease in stearate hydrolysis. Asterisks indicate mutations identified as carrying increased saturase selectivity.

Stearate screening: generation of ... Stearate (Stearatase) Library "[0449] Stearatase library of SEQ ID NO: 2 variants were generated using GSSM technology^sm (patent number 6,171,824). Point mutations were introduced using degenerate oligonucleotides for one amino acid position at a time, so that each

167 the original codon is substituted with each of 20 naturally encoded amino acids. The mutated variants were transformed into an Escherichia coii TOPIO host (Invitrogen, USA) for expression and screening. This library was constructed in the pASK-5 expression vector (as described above). After obtaining optimal host cell density, expression of GSSM variants^sm induced with anhydrotetracycline.

[0450] Enzymes having amino acid sequences generated using GSSM technology^sm was screened using a high-throughput screening protocol (HTP), e.g., the protocol described in Example 3, which determined which fatty acid in this test was preferentially hydrolysed from fat - soybean oil. The test involves contacting the new / modified enzyme sequence with soybean oil that contains various unsaturated fatty acids, including linolenic acid, linoleic acid, oleic acid, palmitic acid and stearic acid (see% distribution mentioned above) and measuring the amount of each fatty acid hydrolyzed by each modified enzyme. A sequence "library" has been identified that allows the enzyme to preferentially hydrolyze stearic acid (or palmitic acid, see below) from soybean oil (the so-called "Stearate Library"):

• First and second order screening was performed using HTP screening, e.g., as described in Example 3;

• Second-order sequencing in HTP screening identified amino acid mutations that result in improved selectivity for stearate hydrolysis over oleate compared to, for example, the parent sequence, SEQ ID NO: 2.

• For each codon variant encoding the amino acid mutation, one best clone was selected and placed in 96-well oil test plates;

• Mutant enzyme selectivity for palmitate or stearate or other fatty acids was obtained from oil tests (Table 3).

• The best hit resulted in stearate accounting for 22% of released fatty acids (FAS) compared to 9% for SEQ ID NO: 2 in the same test; this corresponds to an increase in the selectivity coefficient from 2.3 to 5.5;

• Several clones also showed an increase in palmitate selectivity. Table 2 below summarizes GSSM mutations^sm (see above) selected for inclusion in the "stearatase library" for combination using GeneReassembly technology^SM. In one example test, twenty-two (22) single amino acid mutations were identified that resulted in the largest increase in stearate hydrolysis in oil tests (see also Tables 2, 3 and 4 below). The residues were determined according to the order of their occurrence in the "parent" SEQ ID NO: 2, among the residues that provide significant increases in palmitate or stearate hydrolysis in oil tests.

168

"Original AA" in SEQ ID NO: 2 and preferred mutations ("New Amino Acids"), i.e., exemplary sequences are given. In one aspect, a single mutation to alanine (A) on the residue at position 223 can be included as a constant mutation such that each clone in this exemplary library will contain this mutation

Table 2

<td>Rest</td><td>Original amino acid</td><td>New Amino Acid</td>

<td> 20</td><td>AND</td><td>L</td>

<td> 62</td><td>V</td><td>S</td>

<td>and L and **</td><td>G</td><td>P</td>

<td> 83</td><td>V</td><td>C</td>

<td> 88</td><td>D</td><td>H</td>

<td> 113</td><td>Y</td><td>G</td>

<td> 116</td><td>E</td><td>G, T</td>

<td>and ! about ί 'Τ ί</td><td>H</td><td>K</td>

<td> 146</td><td>K</td><td>S</td>

<td> 167</td><td>AND</td><td>S</td>

<td>about 00 τ-H</td><td>L</td><td>E</td>

<td> 194</td><td>E</td><td>M</td>

<td> 211</td><td>AND</td><td>Q</td>

<td> 212</td><td>S</td><td>Y</td>

<td> 215</td><td>G</td><td>c, v, w</td>

<td> ^218</td><td>AND</td><td>H, S</td>

<td> ;223</td><td>V</td><td>AND</td>

<td> 225</td><td>AND</td><td>Q, M</td>

Figure 6b (see also above) shows the effect of twelve (12) of the twenty-two (22) leading GSSM mutations^sm stearatase for the hydrolysis of paimitate and stearate relative to parent SEQ ID NO: 2. For each of the twelve (12) single amino acid mutations given in Figure 6b and selected for inclusion in the GeneReassembly library^SM stearatase, the percent change in release of paimitate and stearate relative to parent SEQ ID NO: 2, is shown in the graph. Most of these mutations resulted in significant

169 increases in stearate hydrolysis, but small to significant increases in palmitate hydrolysis. Asterisks indicate mutations identified as carrying increased saturase selectivity, i.e. increased selectivity for palmitate and stearate hydrolysis compared to the hydrolysis of unsaturated fatty acids in oil, e.g., oleate, linolate and linolate.

• Summary • Screening of the GSSM library^sm"(see above, where GSSM technology^sm described in detail) based on the parent SEQ ID NO: 2, resulted in clones of single amino acid mutants showing significant improvement in palmitate and stearate selectivity and saturase selectivity, i.e. selectivity for palmitate and stearate hydrolysis (e.g., selective hydrolysis of palmitate and / or soybean oil);

• Clones were identified with a significant improvement in stearate selectivity (selective hydrolysis of stearic acid over other fatty acids);

• GSSM mutants have been identified^sm with increased selectivity of palmitate (selective hydrolysis of palmitic acid over other fatty acids) in relation to the enzyme of the sequence SEQ ID NO: 2.

[0451] Table 3 and Table 4 below describe (further summarize) the sequences of exemplary hydrolytic enzymes, e.g., exemplary enzymes having the sequence set forth in SEQ ID NO: 2 and exhibiting at least one (one, several or all) amino acid residue change described in tables. Table 3 and Table 4 also summarize data on the activity of selected example enzymes; data containing the matching of individual exemplary enzymes with their positive hydrolase activity including the catalysis of the hydrolysis (release) of palmitate fatty acid or stearate from soybean oil as identified by a highly efficient screening protocol (HTP) as described above.

[0452] In Table 3 and Table 4, the term "Original Amino Acid" means the target amino acid residue (referred to as "Amino Acid residue") in the "parent" SEQ ID NO: 2 of the enzyme ("target" to be changed); and the term "new amino acids" means a newly designed amino acid residue (which replaced the corresponding "target" residue in the "old sequence") in an exemplary (new) enzyme as provided herein. The representation of the "New Amino Acid" residue in the "stearate" column compared to "palmitate" indicates which of the two highly efficient fatty acid (HTP) screenings (i.e., screening of palmitic acid release at one screening, and stearic acid release at a second screening, see Example 3) was used to detect (identify) a particular enzyme with the indicated change in residue (new enzyme sequence, "New Amino Acid" residue).

170 [0453] For example, in the first row of Table 3, amino acid residue 7, tyrosine (or "Y") from the "parent" enzyme of SEQ ID NO: 2 is replaced by the amino acid residue of arginine (or "R"), and this new the enzyme (Y7R) has an activity that differs from that of the parent enzyme (see Table 3); for example, "oil data" summarize the substrate preference (fatty acid) of a new enzyme (e.g. enzyme Y7R) by presenting the released (hydrolyzed) fatty acids generated when the enzyme was exposed to (contacted) soybean oil (tests described above), indicating that the soybean oil substrate has many possible fatty acid components suitable for hydrolysis, including linolenic acid, linoleic acid, oleic acid, palmitic acid, stearic acid.

[0454] For example, in the first place, for the enzyme Y7R, 8.3% of the released fatty acids (from reacted soybean oil) was linolenic acid, 22.1% of the released fatty acids was linoleic acid; 19.7% of the released fatty acids were oleic acid; 41.5% of the released fatty acids were palmitic acid; 8.4% of the released fatty acids were stearic acid (these four numbers add up to 100%).

[0455] The P + S column sums up both P and S data points to count how many total released fatty acids were palmitic acid and stearic acid (41.5% plus 8.4% = 49.9% of the hydrolyzed fatty acids were palmitic acid and stearic acid, or "P + S").

171

Table 3

<td></td><td></td><td>Upload hits</td><td>HTP</td><td></td>

<td>Rest amino acid</td><td>Original amino acid</td><td>palmitate new amino acid</td><td>stearate new amino acid</td><td>P + S</td>

<td> 7</td><td>Y</td><td>R</td><td colspan="2"> 49.9%</td>

<td> 8</td><td>G</td><td>E.A218R</td><td colspan="2"></td>

<td> 12</td><td>R</td><td></td><td>F</td><td> 47.8%</td>

<td></td><td></td><td></td><td>K</td><td> 54.2%</td>

<td></td><td></td><td></td><td>L</td><td> 45.4%</td>

<td></td><td></td><td></td><td>M</td><td> 43.3%</td>

<td> 16</td><td>D</td><td>M</td><td colspan="2"> 43.2%</td>

<td> 18</td><td>P</td><td></td><td>G</td><td> 41.8%</td>

<td> 20</td><td>AND</td><td></td><td>L</td><td> 50.3%</td>

<td></td><td></td><td></td><td>V</td><td> 44.6%</td>

<td> 22</td><td>T</td><td></td><td>M, G215V</td><td> 52.1%</td>

<td> 27</td><td>G</td><td>Q</td><td></td><td> 57.2%</td>

<td></td><td></td><td>s</td><td></td><td> 43.6%</td>

<td> 29</td><td>AND</td><td></td><td>G</td><td> 51.5%</td>

<td> 32</td><td>G</td><td></td><td>E</td><td>Scala</td>

<td></td><td></td><td></td><td>D, L180E</td><td> 44.6%</td>

<td> 34</td><td>L</td><td></td><td>E</td><td> 45.8%</td>

<td></td><td></td><td></td><td>V</td><td>scale</td>

<td> 36</td><td>D</td><td></td><td>AND</td><td> 51.0%</td>

<td></td><td></td><td></td><td>G</td><td> 50.9%</td>

<td> 40</td><td>V</td><td></td><td>P</td><td> 32.2%</td>

<td> 42</td><td>V</td><td></td><td>AND</td><td> 47.2%</td>

<td></td><td></td><td></td><td>L '</td><td> 47.8%</td>

<td> 43</td><td>L</td><td>V</td><td colspan="2"> 51.5%</td>

<td> 45</td><td>G</td><td>AND</td><td></td><td> 44.4%</td>

<td></td><td></td><td>L</td><td></td><td> 52.7%</td>

<td> 48</td><td>AND</td><td></td><td>G</td><td> 45.4%</td>

<td></td><td></td><td></td><td>V</td><td> 70.1%</td>

172

<td rowspan="2">Rest amino acid</td><td rowspan="2">Original amino acid</td><td colspan="2">HTP screening hits</td><td rowspan="2">P + S</td>

<td>palmitate new amino acid</td><td>stearate new amino acid</td>

<td></td><td></td><td></td><td>V</td><td> 55.7%</td>

<td></td><td></td><td>T</td><td></td><td> 33.60%</td>

<td> 54</td><td>s</td><td>H</td><td colspan="2"> 55.6%</td>

<td> 61</td><td>D</td><td>AND</td><td></td><td> 60.5%</td>

<td></td><td></td><td>E</td><td></td><td> 55.0%</td>

<td></td><td></td><td>S</td><td></td><td> 49.8%</td>

<td> 62</td><td>V</td><td>E</td><td>E</td><td> 53.0%</td>

<td></td><td></td><td>AND</td><td></td><td> 56.6%</td>

<td></td><td></td><td>G</td><td></td><td> 56.5%</td>

<td></td><td></td><td>M</td><td></td><td> 51.9%</td>

<td></td><td></td><td>N</td><td></td><td> 49.7%</td>

<td></td><td></td><td>Q</td><td></td><td> 52.4%</td>

<td></td><td></td><td>s</td><td></td><td> 55.5%</td>

<td></td><td></td><td>T</td><td></td><td> 50.7%</td>

<td></td><td></td><td></td><td>D</td><td> 52.5%</td>

<td></td><td></td><td></td><td>L</td><td></td>

<td></td><td></td><td></td><td>IN</td><td> 50.2%</td>

<td> 66</td><td>AND</td><td></td><td>N</td><td> 54.2%</td>

<td></td><td></td><td></td><td>R</td><td> 52.1%</td>

<td> 72</td><td>R</td><td>E</td><td></td><td> 58.3%</td>

<td></td><td></td><td>κ</td><td></td><td> 61.0%</td>

<td></td><td></td><td>P</td><td></td><td> 27.2%</td>

<td></td><td></td><td>s</td><td></td><td> 55.3%</td>

<td></td><td></td><td>T</td><td></td><td> 55.9%</td>

<td></td><td></td><td>Y</td><td></td><td> 50.1%</td>

<td> 74</td><td>F</td><td>AND</td><td></td><td> 53.8%</td>

<td></td><td></td><td>L</td><td></td><td> 54.8%</td>

<td></td><td></td><td>P</td><td></td><td> 52.3%</td>

<td></td><td></td><td>R</td><td></td><td> 50.5%</td>

173

<td rowspan="2">Rest amino acid</td><td rowspan="2">Original amino acid</td><td colspan="2">HTP screening hits</td><td rowspan="2">P + S</td>

<td>palmitate new amino acid</td><td>stearate new amino acid</td>

<td> 77</td><td>G</td><td>P</td><td colspan="2"> 38.1%</td>

<td> 78</td><td>AND</td><td></td><td>D</td><td> 47.1%</td>

<td></td><td></td><td></td><td>E</td><td> 37.1%</td>

<td></td><td></td><td></td><td>P</td><td> 40.9%</td>

<td> 80</td><td>G</td><td>P</td><td colspan="2"> 51.9%</td>

<td> 82</td><td>L</td><td></td><td>P</td><td> 37.3%</td>

<td> 83</td><td>V</td><td>c</td><td></td><td> 47.7%</td>

<td></td><td></td><td>M</td><td></td><td> 59.3%</td>

<td> 84</td><td>D</td><td>V</td><td colspan="2"> 40.2%</td>

<td> 87</td><td>V</td><td></td><td>AND</td><td> 49.2%</td>

<td></td><td></td><td></td><td>C</td><td> 46.1%</td>

<td></td><td></td><td></td><td>D</td><td> 43.9%</td>

<td></td><td></td><td></td><td>E</td><td> 46.6%</td>

<td></td><td></td><td></td><td>G</td><td></td>

<td></td><td></td><td></td><td>P</td><td> 513%</td>

<td></td><td></td><td></td><td>S</td><td> 45.2%</td>

<td></td><td></td><td></td><td>T</td><td> 42.8%</td>

<td></td><td></td><td></td><td>H</td><td> 52.9%</td>

<td></td><td></td><td></td><td>N</td><td> 50.3%</td>

<td> 88</td><td>D</td><td></td><td>E</td><td> 44.6%</td>

<td></td><td></td><td></td><td>F</td><td> 50.3%</td>

<td></td><td></td><td></td><td>H</td><td> 45.9%</td>

<td></td><td></td><td></td><td>L</td><td> 49.1%</td>

<td></td><td></td><td></td><td>P</td><td> 59.6%</td>

<td></td><td></td><td></td><td>P</td><td> 48.9%</td>

<td></td><td></td><td></td><td>Q</td><td> 47.1%</td>

<td> 89</td><td>R</td><td>S</td><td colspan="2"> 54.5%</td>

<td> 92</td><td>AND</td><td></td><td>D</td><td> 47.3%</td>

<td></td><td></td><td></td><td>E</td><td> 59.3%</td>

174

HTP screening hits

Amino acid residue

The original amino acid

Palmitate a new amino acid

New amino acid stearate

P + S

<td colspan="2"></td><td></td><td>R S T V</td><td> 42.6% 48.7% 52.1% 57.5%</td>

<td> 93</td><td>V</td><td></td><td>M</td><td> 48.2%</td>

<td> 96</td><td>AND</td><td>c</td><td>C</td><td> 51.4%</td>

<td></td><td></td><td>AND</td><td>AND</td><td>Scala</td>

<td></td><td></td><td>s</td><td>S</td><td> 46.8%</td>

<td> 98</td><td>G</td><td>AND</td><td></td><td> 45.0%</td>

<td></td><td></td><td>L</td><td></td><td>scale</td>

<td> 101</td><td>K</td><td></td><td>AND</td><td> 49.8%</td>

<td> 103</td><td>AND</td><td></td><td>L</td><td> 36.8%</td>

<td> 107</td><td>in</td><td></td><td>P</td><td> 46.20%</td>

<td></td><td></td><td></td><td>AND</td><td> 39.5%</td>

<td></td><td></td><td></td><td>C</td><td> 39.4%</td>

<td></td><td></td><td></td><td>G</td><td> 47.5%</td>

<td></td><td></td><td></td><td>H</td><td> 42.0%</td>

<td></td><td></td><td></td><td>R</td><td> 68.0%</td>

<td></td><td></td><td></td><td>S</td><td> 36.8%</td>

<td></td><td></td><td></td><td>L</td><td> 64.8%</td>

<td></td><td></td><td></td><td>P, E217Q</td><td> 46.2%</td>

<td></td><td></td><td></td><td>V</td><td> 37,8%</td>

<td></td><td></td><td></td><td>V E217Q</td><td> 44.80%</td>

<td> 108</td><td>s</td><td></td><td>Stop</td><td> 19.0%</td>

<td></td><td></td><td></td><td>AND</td><td> 43.0%</td>

<td></td><td></td><td></td><td>C</td><td> 26.0%</td>

<td></td><td></td><td></td><td>G</td><td> 47.5%</td>

<td></td><td></td><td></td><td>K</td><td> 57.8%</td>

<td></td><td colspan="2"></td><td>L</td><td> 44.0%</td>

175

HTP screening hits

Rest Original amino acid amino acid

Palmitate a new amino acid

New amino acid stearate

P + S

<td colspan="2"></td><td>T.A218T</td><td>P Q R V E, E217Q</td><td> 56.9% 58.6% 54.7% 53.4% 46.50%</td>

<td> 109</td><td>L</td><td></td><td>M</td><td> 49.0%</td>

<td> 110</td><td>G</td><td></td><td>L</td><td> 54.4%</td>

<td> 113</td><td>Y</td><td></td><td>E</td><td> 35.8%</td>

<td></td><td></td><td></td><td>G</td><td> 39.8%</td>

<td></td><td></td><td>F</td><td></td><td> 36.5%</td>

<td> 116</td><td>E</td><td>AND</td><td></td><td> 66.6%</td>

<td></td><td></td><td>F</td><td></td><td> 54.7%</td>

<td></td><td></td><td>G</td><td></td><td> 53.8%</td>

<td></td><td></td><td>H</td><td></td><td> 57.9%</td>

<td></td><td></td><td>L</td><td></td><td> 58.5%</td>

<td></td><td></td><td>L</td><td></td><td> 55.1%</td>

<td></td><td></td><td>P</td><td></td><td> 58.0%</td>

<td></td><td></td><td>Q</td><td></td><td> 59.6%</td>

<td></td><td></td><td>Q</td><td></td><td> 60.5%</td>

<td></td><td></td><td>R, H140R</td><td></td><td> 60.6%</td>

<td></td><td></td><td>R</td><td></td><td> 61.8%</td>

<td></td><td></td><td>S</td><td></td><td> 58.6%</td>

<td></td><td></td><td>S</td><td></td><td> 59.7%</td>

<td></td><td></td><td>T</td><td></td><td> 67.6%</td>

<td></td><td></td><td>V</td><td></td><td> 67.8%</td>

<td></td><td></td><td>R, H140R</td><td></td><td></td>

<td> 117</td><td>L</td><td>R, I161L</td><td></td><td> 54.1%</td>

<td></td><td></td><td></td><td>R</td><td> 51.6%</td>

176

<td></td><td></td><td>The hits will change</td><td>possibly HTP</td><td></td>

<td>Rest amino acid</td><td>Original amino acid</td><td>palmitate new amino acid</td><td>stearate new amino acid</td><td>P + S</td>

<td> 120</td><td>K</td><td>AND</td><td></td><td> 46.7%</td>

<td></td><td></td><td>L</td><td>L</td><td> 60.8%</td>

<td></td><td></td><td>F</td><td></td><td> 52.6%</td>

<td></td><td></td><td>M</td><td></td><td> 49.9%</td>

<td></td><td></td><td>S</td><td>S</td><td> 53.3%</td>

<td> 132</td><td>G</td><td></td><td>D, S212A</td><td> 56.2%</td>

<td> 133</td><td>S</td><td></td><td>AND</td><td> 53.2%</td>

<td></td><td></td><td></td><td>. AND</td><td> 55.8%</td>

<td></td><td></td><td></td><td>G</td><td> 45.6%</td>

<td></td><td></td><td></td><td>P</td><td> 56.0%</td>

<td></td><td></td><td></td><td>R</td><td> 51.7%</td>

<td></td><td></td><td></td><td>T</td><td> 54.9%</td>

<td></td><td></td><td></td><td>V, L139.H</td><td> 53.2%</td>

<td> 134</td><td>P</td><td></td><td>G</td><td> 7.2%</td>

<td></td><td></td><td></td><td>R</td><td></td>

<td> 135</td><td>F</td><td></td><td>K</td><td> 51.8%</td>

<td> 139</td><td>L</td><td></td><td>H, S133V</td><td> 53.2%</td>

<td> 140</td><td>H</td><td>R.E116R</td><td>K</td><td> 45.5%</td>

<td> 141</td><td>AND</td><td></td><td>R</td><td> 40.2%</td>

<td></td><td></td><td></td><td>T</td><td> 43.3%</td>

<td> 142</td><td>N</td><td></td><td>M</td><td> 46.1%</td>

<td></td><td></td><td></td><td>R</td><td> 53.8%</td>

<td></td><td></td><td></td><td>S</td><td> 43.2%</td>

<td></td><td></td><td></td><td>T</td><td> 64.3%</td>

<td> 144</td><td>AND</td><td></td><td>T N142K</td><td> 33.9%</td>

<td> 146</td><td>K</td><td></td><td>S</td><td> 50.2%</td>

<td></td><td></td><td></td><td>G</td><td> 49.4%</td>

<td></td><td></td><td></td><td>L</td><td> 51.6%</td>

190

Fatty acids released from oil by the enzyme

Rest

<td>amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>p + s</td>

<td></td><td> 13.0%</td><td> 38.4%</td><td> 37.5%</td><td>scale</td><td> 11.2%</td><td>scale</td>

<td></td><td> 9.6%</td><td> 29.5%</td><td> 21.1%</td><td> 35.1%</td><td> 4.7%</td><td> 39.8%</td>

<td></td><td> 17.8%</td><td> 12.3%</td><td> 20.5%</td><td> 38.7%</td><td> 10.7%</td><td> 49.4%</td>

<td></td><td> 23.3%</td><td> 23.1%</td><td> 39.1%</td><td>scale</td><td> 14.5%</td><td>scale</td>

<td></td><td> 6.5%</td><td> 15.2%</td><td> 17.1%</td><td> 58.0%</td><td> 3.3%</td><td> 613%</td>

<td></td><td> 9.1%</td><td> 23.1%</td><td> 19.8%</td><td> 40.1%</td><td> 7.8%</td><td> 47.9%</td>

<td></td><td> 9.2%</td><td> 20.1%</td><td> 17.7%</td><td> 41.2%</td><td> 11.8%</td><td> 53.0%</td>

<td></td><td> 15.6%</td><td> 17.7%</td><td> 24.4%</td><td> 29.9%</td><td> 12.4%</td><td> 42.3%</td>

<td></td><td> 15.9%</td><td> 37.0%</td><td> 35.5%</td><td>scale</td><td> 11.7%</td><td>scale</td>

<td> 166</td><td> 5.5%</td><td> 21.8%</td><td> 22.8%</td><td> 44.5%</td><td> 5.4%</td><td> 49,9%</td>

<td></td><td> 14.6%</td><td> 22.3%</td><td> 21.8%</td><td> 33.2%</td><td> 8.1%</td><td> 41.3%</td>

<td></td><td> 22.3%</td><td> 33.3%</td><td> 36.8%</td><td>scale</td><td> 7.6%</td><td>scale</td>

<td> 167</td><td> 7.2%</td><td> 19.4%</td><td> 20.1%</td><td> 44.8%</td><td> 8.4%</td><td> 53.3%</td>

<td></td><td> 10.0%</td><td> 21.9%</td><td> 20.7%</td><td> 363%</td><td> 11.0%</td><td> 47.3%</td>

<td> 170</td><td> 12,5%</td><td> 12.4%</td><td> 29.4%</td><td> 37.8%</td><td> 7.8%</td><td> 45.6%</td>

<td></td><td> 8.5%</td><td> 18.2%</td><td> 20.8%</td><td> 43.0%</td><td> 9.5%</td><td> 52.5%</td>

<td></td><td> 3.5%</td><td> 22.0%</td><td> 39.8%</td><td> 8.9%</td><td> 25.9%</td><td> 34.7%</td>

<td> 171</td><td> 8.0%</td><td> 22.4%</td><td> 35.5%</td><td> 33.4%</td><td> 0.7%</td><td> 34,1%</td>

<td> 172</td><td> 8.0%</td><td> 18.8%</td><td> 21.4%</td><td> 43.6%</td><td> 8.1%</td><td> 51.7%</td>

<td></td><td> 7.4%</td><td> 19.1%</td><td> 18.6%</td><td> 45.1%</td><td> 9.8%</td><td> 54.9%</td>

<td></td><td> 22.5%</td><td> 0.0%</td><td> 37.3%</td><td> 40.2%</td><td> 0.0%</td><td> 40.2%</td>

<td> 178</td><td> 14.5%</td><td> 12.9%</td><td> 22.0%</td><td> 32.3%</td><td> 18.3%</td><td> 50.6%</td>

<td> 180</td><td> 8.6%</td><td> 19.0%</td><td> 18.5%</td><td> 42.1%</td><td> 11.8%</td><td> 54.0%</td>

<td></td><td> 11.8%</td><td> 14.2%</td><td> 29.3%</td><td> 32.1%</td><td> 12.5%</td><td> 44.6%</td>

<td></td><td> 11.5%</td><td> 40.0%</td><td> 36.3%</td><td>scale</td><td> 12.2%</td><td>scale</td>

<td></td><td> 14.6%</td><td> 12.1%</td><td> 28.7%</td><td> 36.6%</td><td> 7.9%</td><td> 44.6%</td>

<td> 183</td><td> 10.6%</td><td> 35.4%</td><td> 40.7%</td><td>scale</td><td> 13.3%</td><td>scale</td>

<td> 193</td><td> 3.0% '</td><td> 32.4%</td><td> 15.2%</td><td> 49.4%</td><td> 0.0%</td><td> 49.4%</td>

<td> 194</td><td> 10.9%</td><td> 38.7%</td><td> 42.0%</td><td>scale</td><td> 8.4%</td><td>scale</td>

191

Fatty acids released from oil by the enzyme

<td>Rest amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td></td><td> 9.6%</td><td> 21.8%</td><td> 20.7%</td><td> 34.6%</td><td> 13.2%</td><td> 47.9%</td>

<td></td><td> 12.6%</td><td> 31.0%</td><td> 37.8%</td><td>scale</td><td> 18.6%</td><td>scale</td>

<td></td><td> 3.0%</td><td> 32.4%</td><td> 15.2%</td><td> 49.4%</td><td> 0.0%</td><td> 49.4%</td>

<td> 197</td><td> 9.8%</td><td> 0.0%</td><td> 50.9%</td><td> 39.4%</td><td> 0.0%</td><td> 39.4%</td>

<td> 198</td><td> 7.7%</td><td> 19.7%</td><td> 16.6%</td><td> 46.8%</td><td> 9.3%</td><td> 56.1%</td>

<td> 200</td><td> 8.5%</td><td> 16.8%</td><td> 19.3%</td><td> 48.7%</td><td> 6.7%</td><td> 55.3%</td>

<td> 204</td><td> 13.7%</td><td> 12.8%</td><td> 27.6%</td><td> 32.2%</td><td> 13.7%</td><td> 45.9%</td>

<td></td><td> 9.9%</td><td> 14.0%</td><td> 30.5%</td><td> 23.2%</td><td> 22.5%</td><td> 45.7%</td>

<td> 210</td><td> 7.2%</td><td> 22.0%</td><td> 20.7%</td><td> 39.0%</td><td> 11.2%</td><td> 50.2%</td>

<td> 211</td><td> 9.0%</td><td> 16.2%</td><td> 39.4%</td><td> 24.0%</td><td> 11-2%</td><td> 35.3%</td>

<td></td><td> 10.2%</td><td> 17.0%</td><td> 24.7%</td><td> 35.7%</td><td> 12.4%</td><td> 48.1%</td>

<td></td><td> 13.8%</td><td> 10.4%</td><td> 36.5%</td><td> 24.1%</td><td> 15.3%</td><td> 39.4%</td>

<td></td><td> 6.5%</td><td> 12.2%</td><td> 36.3%</td><td> 30.5%</td><td> 14.5%</td><td> 45.0%</td>

<td></td><td> 6.9%</td><td> 26.6%</td><td> 16.1%</td><td> 32.7%</td><td> 17.7%</td><td> 50.3%</td>

<td></td><td> 3.4%</td><td> 36.2%</td><td> 27.7%</td><td> 32.6%</td><td> 0.0%</td><td> 32.6%</td>

<td></td><td> 6.9%</td><td> 26.5%</td><td> 20.5%</td><td> 28.9%</td><td> 17.3%</td><td> 46.1%</td>

<td></td><td> 0.0%</td><td> 35.1%</td><td> 15.6%</td><td> 39.6%</td><td> 9.7%</td><td> 49.2%</td>

<td></td><td> 0.0%</td><td> 25.3%</td><td> 19.5%</td><td> 46.8%</td><td> 8.4%</td><td> 55.2%</td>

<td></td><td> 6.6%</td><td> 19.7%</td><td> 25.9%</td><td> 37.2%</td><td> 10.6%</td><td> 47.8%</td>

<td></td><td> 7.7%</td><td> 22.8%</td><td> 20.7%</td><td> 36.6%</td><td> 12.3%</td><td> 48.9%</td>

<td></td><td> 16.3%</td><td> 4.6%</td><td> 28.2%</td><td> 49.1%</td><td> 1.7%</td><td> 50.8%</td>

<td></td><td> 8.0%</td><td> 22.1%</td><td> 17.2%</td><td> 41.2%</td><td> 11.5%</td><td> 52.7%</td>

<td></td><td> 8.0%</td><td> 22.1%</td><td> 17.2%</td><td> 41.2%</td><td> 11.5%</td><td> 52.7%</td>

<td></td><td> 18.2%</td><td> 3.2%</td><td> 28.7%</td><td> 42.4%</td><td> 7.5%</td><td> 49.8%</td>

<td></td><td> 11.9%</td><td> 10.1%</td><td> 31.8%</td><td> 30.5%</td><td> 15.7%</td><td> 46.2%</td>

<td> 212</td><td> 7.5%</td><td> 25.6%</td><td> 17.6%</td><td> 36.5%</td><td> 12.8%</td><td> 49.3%</td>

<td></td><td> 19.1%</td><td> 0.9%</td><td> 29.7%</td><td> 46.8%</td><td> 3.5%</td><td> 50.3%</td>

<td></td><td> 8.8%</td><td> 28.4%</td><td> 9.6%</td><td> 33.7%</td><td> 19.5%</td><td> 53.2%</td>

<td></td><td> 19.4%</td><td> 24.8%</td><td> 18.9%</td><td> 33.5%</td><td> 3.3%</td><td> 36.8%</td>

192

Fatty acids released from oil by the enzyme

<td>Rest amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td></td><td> 19.1%</td><td> 26.9%</td><td> 17.5%</td><td> 31.6%</td><td> 4.9%</td><td> 36.6%</td>

<td></td><td> 5.5%</td><td> 42.3%</td><td> 7.9%</td><td> 30.8%</td><td> 13.5%</td><td> 443%</td>

<td></td><td> 4.6%</td><td> 23.9%</td><td> 25.1%</td><td> 35.5%</td><td> 11.0%</td><td> 46.5%</td>

<td></td><td> 8.8%</td><td> 28.4%</td><td> 9.6%</td><td> 33.7%</td><td> 19.5%</td><td> 53.2%</td>

<td></td><td> 0.0%</td><td> 65.4%</td><td> 22.4%</td><td> 10.5%</td><td> 1.7%</td><td> 12.2%</td>

<td></td><td> 3.3%</td><td> 14.2%</td><td> 40.6%</td><td> 30.7%</td><td> 11.1%</td><td> 41,8%</td>

<td></td><td> 11.2%</td><td> 13.6%</td><td> 25.0%</td><td> 40.3%</td><td> 9.9%</td><td> 50.2%</td>

<td></td><td> 10.6%</td><td> 16.6%</td><td> 19.1%</td><td> 42.7%</td><td> 11.0%</td><td> 53.7%</td>

<td></td><td> 21.1%</td><td> 22.7%</td><td> 17.4%</td><td> 17.5%</td><td> 21.2%</td><td> 38.7%</td>

<td></td><td> 7.6%</td><td> 24.0%</td><td> 20.0%</td><td> 38.9%</td><td> 9.5%</td><td> 48.4%</td>

<td></td><td> 103%</td><td> 20.4%</td><td> 22.2%</td><td> 33.7%</td><td> 13.4%</td><td> 47.1%</td>

<td></td><td> 3.5%</td><td> 22.0%</td><td> 39.8%</td><td> 8.9%</td><td> 25.9%</td><td> 34,7%</td>

<td> 213</td><td> 7.6%</td><td> 28.2%</td><td> 16.3%</td><td> 30.4%</td><td> 17.5%</td><td> 47.9%</td>

<td></td><td> 5.3%</td><td> 18.8%</td><td> 18.1%</td><td> 41.3%</td><td> 16.4%</td><td> 57.7%</td>

<td></td><td> 7.5%</td><td> 21.0%</td><td> 14.8%</td><td> 48.5%</td><td> 8.2%</td><td> 56.7%</td>

<td></td><td> 8.3%</td><td> 17.8%</td><td> 183%</td><td> 44.6%</td><td> 10.9%</td><td> 55.5%</td>

<td> 214</td><td> 9.1%</td><td> 20.2%</td><td> 19.1%</td><td> 47.0%</td><td> 4.5%</td><td> 51.6%</td>

<td></td><td> 8.3%</td><td> 19.8%</td><td> 18.9%</td><td> 44.6%</td><td> 8.4%</td><td> 53.0%</td>

<td></td><td> 7.7%</td><td> 20.5%</td><td> 19.6%</td><td> 45.3%</td><td> 7.0%</td><td> 52.2%</td>

<td></td><td> 7.1%</td><td> 21.5%</td><td> 16.9%</td><td> 42.4%</td><td> 12.1%</td><td> 54.5%</td>

<td></td><td> 7.0%</td><td> 25.9%</td><td> 15.1%</td><td> 39.9%</td><td> 12.0%</td><td> 51.9%</td>

<td></td><td> 6.9%</td><td> 18.0%</td><td> 18.3%</td><td> 48.5%</td><td> 8.4%</td><td> 56.9%</td>

<td></td><td> 7.4%</td><td> 19.2%</td><td> 18.2%</td><td> 45.6%</td><td> 9.5%</td><td> 55.1%</td>

<td></td><td> 5.3%</td><td> 21.1%</td><td> 10.9%</td><td> 47.3%</td><td> 15.4%</td><td> 62.7%</td>

<td></td><td> 5.3%</td><td> 21.1%</td><td> 10.9%</td><td> 47.3%</td><td> 15.4%</td><td> 62.7%</td>

<td> 215</td><td> 7.8%</td><td> 19.8%</td><td> 15.8%</td><td> 46.4%</td><td> 10.2%</td><td> 56.6%</td>

<td></td><td> 7.9%</td><td> 20.2%</td><td> 17.7%</td><td> 40.6%</td><td> 13.6%</td><td> 54.1%</td>

<td></td><td> 20.0%</td><td> 24.8%</td><td> 25.4%</td><td> 25.7%</td><td> 4.1%</td><td> 29.9%</td>

193

Fatty acids released from oil by the enzyme

Rest

<td>amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td></td><td> 4.4%</td><td> 26.2%</td><td> 19.2%</td><td> 45.1%</td><td> 5.1%</td><td> 50.2%</td>

<td></td><td> 8.1%</td><td> 19.6%</td><td> 20.1%</td><td> 42.7%</td><td> 9.4%</td><td> 52.1%</td>

<td></td><td> 2.3%</td><td> 30.1%</td><td> 19.7%</td><td> 31.8%</td><td> 16.1%</td><td> 47.9%</td>

<td></td><td> 5.9%</td><td> 23.7%</td><td> 14.8%</td><td> 39.3%</td><td> 16.3%</td><td> 55.6%</td>

<td></td><td> 9.6%</td><td> 26.0%</td><td> 17.0%</td><td> 36.5%</td><td> 10.9%</td><td> 47.3%</td>

<td></td><td> 4.7%</td><td> 20.8%</td><td> 14.1%</td><td> 42.2%</td><td> 18.2%</td><td> 60.4%</td>

<td></td><td> 4.2%</td><td> 31.0%</td><td> 12.0%</td><td> 40.7%</td><td> 12.1%</td><td> 52.8%</td>

<td></td><td> 6.7%</td><td> 21.3%</td><td> 18.1%</td><td> 41.7%</td><td> 12.3%</td><td> 53.9%</td>

<td></td><td> 8.0%</td><td> 19.5%</td><td> 20.4%</td><td> 47.1%</td><td> 5.0%</td><td> 52.1%</td>

<td> 216</td><td> 8.3%</td><td> 21.9%</td><td> 18.9%</td><td> 40.9%</td><td> 10.0%</td><td> 50.9%</td>

<td></td><td> 0.0%</td><td> 28.0%</td><td> 30.1%</td><td> 22.8%</td><td> . 19.1%</td><td> 41.9%</td>

<td></td><td> 34.6%</td><td> 0.0%</td><td> 30.6%</td><td> 33.7%</td><td> 1.1%</td><td> 34.8%</td>

<td></td><td> 7.3%</td><td> 17.8%</td><td> 17.9%</td><td> 47.0%</td><td> 10.0%</td><td> 56.9%</td>

<td></td><td> 6.6%</td><td> 16.6%</td><td> 17.2%</td><td> 50.0%</td><td> 9.7%</td><td> 59.7%</td>

<td></td><td> 7.7%</td><td> 18.1%</td><td> 19.2%</td><td> 44.5%</td><td> 10.5%</td><td> 55.0%</td>

<td></td><td> 7.5%</td><td> 20.3%</td><td> 16.5%</td><td> 45.0%</td><td> 10.6%</td><td> 55.6%</td>

<td> 217</td><td> 0.0%</td><td> 42.3%</td><td> 21.0%</td><td> 24.3%</td><td> 12.3%</td><td> 36.6%</td>

<td></td><td> 6.8%</td><td> 16.7%</td><td> 17.1%</td><td> 50.1%</td><td> 9.3%</td><td> 59.4%</td>

<td></td><td> 7.4%</td><td> 20.5%</td><td> 18.7%</td><td> 44.1%</td><td> 9.4%</td><td> 53.5%</td>

<td></td><td> 11.9%</td><td> 10.1%</td><td> 31.8%</td><td> 30.5%</td><td> 15.7%</td><td> 46.2%</td>

<td></td><td> 13.2%</td><td> 21.6%</td><td> 20.4%</td><td> 31.3%</td><td> 13.5%</td><td> 44.8%</td>

<td></td><td> 12.1%</td><td> 13.9%</td><td> 27.6%</td><td> 33.8%</td><td> 12.7%</td><td> 46.2%</td>

<td> 218</td><td> 0.7%</td><td> 39.0%</td><td> 17.8%</td><td> 30.3%</td><td> 12.1%</td><td> 42.5%</td>

<td></td><td> 4.7%</td><td> 26.8%</td><td> 19.4%</td><td> 30.5%</td><td> 18.7%</td><td> 49.1%</td>

<td></td><td> 7.1%</td><td> 22.8%</td><td> 22.4%</td><td> 38.3%</td><td> 9.4%</td><td> 47.7%</td>

<td></td><td> 7.2%</td><td> 19.9%</td><td> 19.6%</td><td> 44.1%</td><td> 9.2%</td><td> 53.4%</td>

<td></td><td> 8.5%</td><td> 19.7%</td><td> 19.9%</td><td> 42.2%</td><td> 9.7%</td><td> 51.9%</td>

<td></td><td> 7.2%</td><td> 25.9%</td><td> 15.8%</td><td> 37.6%</td><td> 13.5%</td><td> 51.1%</td>

<td></td><td> 8.0%</td><td> 21.1%</td><td> 20.9%</td><td> 41.9%</td><td> 8.2%</td><td> 50.0%</td>

194

Fatty acids released from oil by the enzyme

<td>Rest amino acid</td><td>Linoienowy</td><td>linoleic</td><td>oleic</td><td>Palmitic</td><td>Stearic 1</td><td>P + S</td>

<td></td><td> 8.7%</td><td> 19.9%</td><td> 19.0%</td><td> 42.9%</td><td> 9.4%</td><td> 52.4%</td>

<td> 223</td><td> 4.5%</td><td> 29.5%</td><td> 17.2%</td><td> 15.8%</td><td> 33.0%</td><td> 48.8%</td>

<td></td><td> 0.0%</td><td> 38.4%</td><td> 30.1%</td><td> 31.6%</td><td> 0.0%</td><td> 31.6%</td>

<td></td><td> 20.2%</td><td> 22.8%</td><td> 33.6%</td><td> 17.3%</td><td> 6.0%</td><td> 23.4%</td>

<td></td><td> 19.0%</td><td> 37.0%</td><td> 34.9%</td><td>scale</td><td> 9.1%</td><td>scale</td>

<td> 224</td><td> 8.0%</td><td> 20.5%</td><td> 22.1%</td><td> 41.0%</td><td> 8.4%</td><td> 49.5%</td>

<td></td><td> 6.6%</td><td> 18.2%</td><td> 17.1%</td><td> 51.4%</td><td> 6.8%</td><td> 58.2%</td>

<td></td><td> 7.9%</td><td> 22.1%</td><td> 21.6%</td><td> 37.0%</td><td> 11.4%</td><td> 48.4%</td>

<td></td><td> 14.4%</td><td> 19.0%</td><td> 24.9%</td><td> 33.1%</td><td> 8.5%</td><td> 41.7%</td>

<td> -</td><td> 3.1%</td><td> 26.3%</td><td> 24.2%</td><td> 40.8%</td><td> 5.6%</td><td> 46.4%</td>

<td></td><td> 10,3%</td><td> 20.1%</td><td> 25.8%</td><td> 38.1%</td><td> 5.7%</td><td> 43.7%</td>

<td> 225</td><td> 9.7%</td><td> 22.2%</td><td> 18.8%</td><td> 41.8%</td><td> 7.5%</td><td> 49.3%</td>

<td></td><td> 4.3%</td><td> 23.5%</td><td> 17.8%</td><td> 47.9%</td><td> 6.4%</td><td> 54.3%</td>

<td></td><td> 12.0%</td><td> 21.9%</td><td> 17.1%</td><td> 39.1%</td><td> 9.9%</td><td> 49.0%</td>

<td></td><td> 12.9%</td><td> 23.8%</td><td> 17.5%</td><td> 34.1%</td><td> 11.7%</td><td> 45.8%</td>

<td></td><td> 15.9%</td><td> 22.6%</td><td> 18.3%</td><td> 38.0%</td><td> 5.2%</td><td> 43.2%</td>

<td> 226</td><td> 4.9%</td><td> 24.9%</td><td> 21.9%</td><td> 45.8%</td><td> 2.5%</td><td> 48.3%</td>

<td></td><td> 6.5%</td><td> 29.5%</td><td> 22.8%</td><td> 32.4%</td><td> 8.8%</td><td> 41.2%</td>

<td> 227</td><td> 13.6%</td><td> 23.1%</td><td> 21.9%</td><td> 38.3%</td><td> 3.2%</td><td> 41.4%</td>

Table 4 is a summary or further compilation of the data presented in Table 3 (above). For example, the term "position" indicates the position of the amino acid residue in SEQ ID NO: 2; the term "original amino acid" as in Table 3 indicates an unchanged "parent" residue while the term "New Amino Acid" as in Table 3 indicates an altered (new) amino acid residue at this position. The terms 'Type Wild_P' and 'Type Wild's_S' indicate

195 substrate preference (fatty acid release) of the "parent" enzyme, eg SEQ ID NO: 2 relative to a particular substrate (fatty acid) by indicating the amount of fatty acid released (hydrolyzed) from soybean oil (as in Table 3), where "P" is palmitic acid and "S" is stearic acid.

[0456] The columns "palmitate" and "stearate" mean the amount of palmitic acid and stearic acid (released by enzymatic hydrolysis) from soybean oil, which contains linolenic acid, linoleic acid, oleic acid, palmitic acid, stearic acid, as discussed above. "P + S" indicates the total amount of hydrolyzed fatty acids that were palmitic acid and stearic acid, or "P + S".

The terms "delta_P" and "delta_S" indicate a change in the preference of an exemplary enzyme (e.g., first order D61 A) to hydrolyze palmitic acid and stearic acid, respectively, compared to the corresponding activity of SEQ ID NO: 2. The term "P + S delta" means a total or total change in preference of an exemplary enzyme (e.g., first order D61 A) to hydrolyze palmitic acid and stearic acid compared to the corresponding activity of SEQ ID NO: 2. The "Palmate Mutations" section summarizes exemplary enzymes showing a preference for activity (fatty acid hydrolysis) toward release of palmitic acid compared to other fatty acids. The "mutations towards stearate" section summarizes exemplary enzymes showing a preference for activity (fatty acid hydrolysis) towards stearic acid release compared to other fatty acids (from soybean oil, the test is described above).

[0457]

TABLE 4

Sample mutations towards palmitate

<td>Position</td><td>Original amino acid</td><td>New amino acid</td><td>Type dziki_P</td><td>Type dziki_S</td><td>palmitate</td><td>stearate</td>

<td> 61</td><td>D</td><td>AND</td><td> 45%</td><td> 6%</td><td> 54%</td><td> 7%</td>

<td> 61</td><td>D</td><td>E</td><td> 45%</td><td> 6%</td><td> 47%</td><td> 8%</td>

<td> 72</td><td>R</td><td>E</td><td> 45%</td><td> 6%</td><td> 54%</td><td> 4%</td>

<td> 72</td><td>R</td><td>κ .........................</td><td> 45%</td><td> 6%</td><td> 52%</td><td> 9%</td>

<td> 116</td><td>E</td><td>AND</td><td> 45%</td><td> 6%</td><td> 56%</td><td> 11%</td>

<td> 116</td><td>E</td><td>Q</td><td> 45%</td><td> 6%</td><td> 50%</td><td> 9%</td>

<td> 116</td><td>E</td><td>R</td><td> 45%</td><td> 6%</td><td> 52%</td><td> 9%</td>

<td> 116</td><td>E</td><td>T</td><td> 45%</td><td> 6%</td><td> 50%</td><td> 17%</td>

196

Sample mutations towards palmitate

<td>Position</td><td>Original amino acid</td><td>New amino acid</td><td>Type dziki_P</td><td></td><td>Type dziki_S</td><td>palmitate</td><td>stearate</td>

<td> 116</td><td>E</td><td>V</td><td> 45%</td><td></td><td> 6%</td><td> 59%</td><td> 9%</td>

<td> ^33</td><td>S</td><td>AND</td><td> 45%</td><td></td><td> 6%</td><td> 45%</td><td> 11%</td>

<td> 151</td><td>AND</td><td>G</td><td> 45%</td><td></td><td> 6%</td><td> 49%</td><td> 4%</td>

<td> 151</td><td>AND</td><td>AND</td><td> 45%</td><td></td><td> 6%</td><td> 46%</td><td> 2%</td>

<td> 163</td><td>V</td><td>R *</td><td> 45%</td><td></td><td> 6%</td><td> 61%</td><td> 2%</td>

<td> 164</td><td>θ</td><td>R *</td><td> 45%</td><td></td><td colspan="2">6% 58% and</td><td> 3%</td>

<td colspan="3">mutations towards stearate</td><td></td><td></td><td></td><td></td><td></td>

<td>Position</td><td>Original amino acid</td><td>New amino acid</td><td>Type dziki_P</td><td></td><td>Type dziki_S</td><td>palmitate</td><td>stearate</td>

<td> 20</td><td>AND</td><td>L</td><td> 45%</td><td></td><td> 6%</td><td> 39%</td><td> 12%</td>

<td> 62</td><td>V</td><td>S</td><td> 45%</td><td></td><td> 6%</td><td> 37%</td><td> 18%</td>

<td> 77</td><td>G</td><td>P</td><td> 45%</td><td></td><td> 6%</td><td> 18%</td><td> 20%</td>

<td> 83</td><td>V</td><td>C</td><td> 45%</td><td></td><td> 6%</td><td> 31%</td><td> 17%</td>

<td> 88</td><td>D</td><td>H</td><td> 45%</td><td></td><td> 6%</td><td> 33%</td><td> 13%</td>

<td> 113</td><td>Y</td><td>G</td><td> 45%</td><td></td><td> 6%</td><td> 15%</td><td> 25%</td>

<td> 116</td><td>E</td><td>T</td><td> 45%</td><td></td><td> 6%</td><td> 50%</td><td> 17%</td>

<td> 116</td><td>E</td><td>G</td><td> 45%</td><td></td><td> 6%</td><td> 33%</td><td> 21%</td>

<td> 140</td><td>H</td><td>K</td><td> 45%</td><td></td><td> 6%</td><td> 33%</td><td> 13%</td>

<td> 146</td><td>K</td><td>S</td><td> 45%</td><td></td><td> 6%</td><td> 31%</td><td> 19%</td>

<td> 167</td><td>AND</td><td>S</td><td> 45%</td><td></td><td> 6%</td><td> 36%</td><td> 11%</td>

<td> 180</td><td>L</td><td>E</td><td> 45%</td><td></td><td> 6%</td><td> 42%</td><td> 12%</td>

<td> 194</td><td>E</td><td>M</td><td> 45%</td><td></td><td> 6%</td><td> 35%</td><td> 13%</td>

<td> 211 </td><td>AND</td><td>Q</td><td> 45%</td><td></td><td> 6%</td><td> 33%</td><td> 18%</td>

<td> 212</td><td>S</td><td>Y</td><td> 45%</td><td>f</td><td> 6%</td><td> 34%</td><td> 13%</td>

<td> 215</td><td>G</td><td>C</td><td> 45%</td><td></td><td> 6%</td><td> 42%</td><td> 18%</td>

<td> 215</td><td>G</td><td>V</td><td> 45%</td><td></td><td> 6%</td><td> 39%</td><td> 16%</td>

and

197

Sample mutaqe towards palmitate

<td>Position</td><td>Original amino acid</td><td>New amino acid</td><td>Type dziki_P</td><td>Type dziki_S</td><td>palmitate</td><td>stearate</td>

<td> 215</td><td>G</td><td>IN</td><td> 45%</td><td> 6%</td><td> 41%</td><td> 12%</td>

<td> 218</td><td>AND</td><td>H</td><td> 45%</td><td> 6%</td><td> 30%</td><td> 19%</td>

<td> 218</td><td>AND</td><td>S</td><td> 45%</td><td> 6%</td><td> 38%</td><td> 14%</td>

<td> 223</td><td>V</td><td>AND</td><td> 45%</td><td> 6%</td><td> 16%</td><td> 33%</td>

<td> 225</td><td>AND</td><td>M</td><td> 45%</td><td> 6%</td><td> 39%</td><td> 10%</td>

<td>P ................... ....... 1</td><td></td><td>Q</td><td> 45%</td><td> 6%</td><td> 34%</td><td> 12%</td>

TABLE 4 (continued) r -------------------------------------------- Mutations towards palmitate

<td>Position</td><td>Original amino acid</td><td>New amino acid</td><td>P + S</td><td>delta_P</td><td>delta_S</td><td>delta_P + S</td>

<td> 61</td><td>D</td><td>AND</td><td> 60%</td><td> 9%</td><td> 1%</td><td> 9%</td>

<td> 61</td><td>D</td><td>E</td><td> 55%</td><td> 2%</td><td> 2%</td><td> 4%</td>

<td> 72</td><td>R</td><td>E</td><td> 58%</td><td> 9%</td><td> -2%</td><td> 7%</td>

<td> 72</td><td>R</td><td>K</td><td> 61%</td><td> 7%</td><td> 3%</td><td> 10%</td>

<td> 116</td><td>E</td><td>AND</td><td> 67%</td><td> 11%</td><td> 5%</td><td> 16%</td>

<td> 116</td><td>E</td><td>Q</td><td> 60%</td><td> 5%</td><td> 3%</td><td> 9%</td>

<td> |116</td><td>E</td><td>R</td><td> 61%</td><td> 7%</td><td> 3%</td><td> 10%</td>

<td> 116</td><td>E</td><td>T</td><td> 68%</td><td> 5%</td><td> 11%</td><td> 17%</td>

<td> 116</td><td>E</td><td>V</td><td> 68%</td><td> 14%</td><td> 3%</td><td> 17%</td>

<td> 133</td><td>S</td><td>AND</td><td> 56%</td><td> 0%</td><td> 5%</td><td> 5%</td>

<td> 151</td><td>¹................................................................................</td><td>G</td><td> 53%</td><td> 4%</td><td> -2%</td><td> 2%</td>

<td> 151</td><td>AND</td><td>AND</td><td> 49%</td><td> 1%</td><td> -4%</td><td> -2%</td>

<td> 163</td><td>V</td><td>R *</td><td> 64%</td><td> 16%</td><td> -4%</td><td> 13%</td>

<td> 164</td><td>D</td><td>R *</td><td> 61%</td><td> 13%</td><td> -3%</td><td> 10%</td>

TABLE 4 (continuation)

Mutations towards stearate

198

<td>Position</td><td>Original amino acid</td><td>New amino acid</td><td>P + s</td><td>delta_P</td><td>delta_S</td><td>delta_P + S</td>

<td> 20</td><td>AND</td><td>L</td><td> 51%</td><td> -6%</td><td> 6%</td><td> 0%</td>

<td> 62</td><td>V</td><td>S</td><td> 55%</td><td> -8%</td><td> 12%</td><td> 4%</td>

<td> 77</td><td>G</td><td>P</td><td> 38%</td><td> -27%</td><td> 14%</td><td> -13%</td>

<td> 83</td><td>V</td><td>c</td><td> 48%</td><td> -14%</td><td> 11%</td><td> -3%</td>

<td> 88</td><td>D</td><td>H</td><td> 46%</td><td> -12%</td><td> 7%</td><td> -5%</td>

<td> 113</td><td>Y</td><td>G</td><td> 40%</td><td> -30%</td><td> 19%</td><td> -11%</td>

<td> 116</td><td>E</td><td>T</td><td> 68%</td><td> 5%</td><td> 11%</td><td> 17%</td>

<td> 116</td><td>E</td><td>G</td><td> 54%</td><td> -12%</td><td> 15%</td><td> 3%</td>

<td> 140</td><td>H</td><td>K</td><td> 46%</td><td> -12%</td><td> 7%</td><td> -5%</td>

<td> 146</td><td>K</td><td>S</td><td> 50%</td><td> -14%</td><td> 13%</td><td> -1%</td>

<td> 167</td><td>AND</td><td>S</td><td> 47%</td><td> -9%</td><td> 5%</td><td> -4%</td>

<td> 180</td><td>L</td><td>E</td><td> 54%</td><td> -3%</td><td> 6%</td><td> 3%</td>

<td> 194</td><td>E</td><td>M</td><td> 48%</td><td> -10%</td><td> 7%</td><td> -3%</td>

<td> 211</td><td>AND</td><td>Q</td><td> 50%</td><td> -12%</td><td> 12%</td><td> -1%</td>

<td> 212</td><td>S</td><td>γ</td><td> 47%</td><td> -11%</td><td> 7%</td><td> -4%</td>

<td> 215</td><td>G</td><td>C</td><td> 60%</td><td> -3%</td><td> 12%</td><td> 9%</td>

<td>²¹⁵</td><td>G</td><td>V</td><td> 56%</td><td> -6%</td><td> 10%</td><td> 5%</td>

<td> 215</td><td>G</td><td>IN</td><td> 53%</td><td> -4%</td><td> 6%</td><td> 2%</td>

<td> 218</td><td>AND</td><td>H</td><td> 49%</td><td> -15%</td><td> 13%</td><td> -2%</td>

<td> 218</td><td>AND</td><td>S</td><td> 51%</td><td> -7%</td><td> 8%</td><td> 0%</td>

<td> 223</td><td>V</td><td>AND</td><td> 49%</td><td> -29%</td><td> 27%</td><td> -2%</td>

<td> 225</td><td>AND</td><td>M</td><td> 49%</td><td> -6%</td><td> 4%</td><td> -2%</td>

<td></td><td></td><td>Q</td><td> 46%</td><td> -11%</td><td> 6%</td><td> -5%</td>

Example 5: Example evolution to obtain improved palmitate hydrolysis using GeneReassemblv technology^SM [0458] Fourteen (14) single amino acid mutations identified by GeneReassembly screening^SMwhich comprise seven (7) amino acid positions have been combined using GeneReassemblySM technology (US Patent

199 No. 6,605,449). Full-length nucleic acid sequences generated from the GeneReassembly phase were cloned into the pASK-5 expression vector (see description above) for expression in an Escherichia coli HMS175 host (Novagen, USA). After obtaining optimal host cell densities, expression of GeneReassembly variants was induced by hydrotetracycline. These 14 mutations that gave the largest increase in palmitate hydrolysis identified in Table 2 were selected for inclusion in the palmitase GeneReassembly library generated by the methods described above. Initial clones screened for activity against umbeliferyl paiminitate yielded about 145 clones with specific sequence that were tested for soybean oil activity as described above.

[0460] Figure 8 shows first and second order screening data for soybean oil tests on selected clones from the palmitase library. Clones that resulted in obtaining more than 70% of the hydrolyzed FA in a basic test (under standard conditions of the ratio of the initial test method) were selected to be repeated in soybean oil test. For each soybean oil test, the extracted FA was diluted 50-fold and 100-fold for LCMS or GC analysis. Additional non-targeted mutations are also indicated: the detected FA hydrolysis ratios and the amount of each FA detected are shown. In the figure, "high" and "low" indicate values ​​outside the range of the calibration curve. Rows are sorted by order of percentage of paimitate released in the second-order test, and then relative to total released paimitate. Numerous clones showed significantly increased selectivity for paimetitate (up to 100%) compared to parent SEQ ID NO: 2 (61.2%) [0461] The top 25 palmitase hits selected based on the second-order test described above were subcloned into Pseudomonas systems (Dow Global Technologies Inc., U.S. Patent Application Publication No. 20050130160 and Dow Global Technologies Inc., U.S. Patent Application Publication No. 20050186666). The nucleic acid sequence encoding the enzyme or polypeptide was introduced into the pMYC vector (Dow Global Technologies Inc., US Patent Application Publication No. 20050130160) or into the vector pDOW1169 ((Dow Global Technologies Inc., US Patent Application Publication No. 20080058262), and then Pseudomonos fluorescens was introduced into the host by electroporation. Transformed cells were selected for growth either in minimal medium for the pDOW1169 constructs or in rich media with the addition of tetracycline for the pMYC constructs. After reaching optimal host cell densities, the expression of the enzyme or polypeptide was induced with IPTG [0462] Table 5 shows data from replicate soybean oil tests from the top 25 hits expressed in Pseudomonas systems. 4 hits constructed in the pDOW1169 vector are listed in bold underlined, all other hits constructed in the pMYC vector. The enzyme is added to 5 g crude

200 oil, resulting in a 20% final water content. The mixture was then homogenized with a 7 mm probe and incubated for 40 hours at 25 ° C with stirring. Samples were removed and analyzed for FA by converting FA to FAMA and quantifying FAMA by GC as described in Example 8. 25 enzymes were loaded into 5 g soybean oil based on equal units of UMB-palmitate activity. In these reactions, the amount of palmitate in oil was significantly reduced from 11% in untreated oil to 5% or below in enzyme-treated oils indicating an increased preference for palmitate hydrolysis compared to the parent enzyme of SEQ ID NO: 2.

177

<td rowspan="2">Rest amino acid</td><td rowspan="2">Original amino acid</td><td colspan="2">HTP screening hits</td><td rowspan="2">P + S</td>

<td>palmitate new amino acid</td><td>stearate new amino acid</td>

<td colspan="2"></td><td>AND</td><td colspan="2"> 52.2%</td>

<td> 147</td><td>AND</td><td>F</td><td> •</td><td> 56.5%</td>

<td></td><td></td><td>F</td><td></td><td> 50.5%</td>

<td></td><td></td><td>L</td><td></td><td> 52.2%</td>

<td> 150</td><td>AND</td><td></td><td>L</td><td> 59.7%</td>

<td></td><td></td><td></td><td>L</td><td> 53.3%</td>

<td> 151</td><td>and</td><td>AND</td><td></td><td> 48.6%</td>

<td></td><td></td><td>G</td><td></td><td> 53.0%</td>

<td></td><td></td><td>H</td><td></td><td> 60.0%</td>

<td></td><td></td><td>P</td><td></td><td> 33.7%</td>

<td></td><td></td><td>S</td><td></td><td> 52.2%</td>

<td></td><td></td><td>T</td><td></td><td> 49.2%</td>

<td> 152</td><td>N</td><td></td><td>E</td><td> 28.0%</td>

<td></td><td></td><td></td><td>G</td><td> 53.0%</td>

<td></td><td></td><td></td><td>H</td><td> 46.7%</td>

<td></td><td></td><td></td><td>M</td><td> 35.7%</td>

<td></td><td></td><td></td><td>R</td><td> 21.1%</td>

<td> 155</td><td>T</td><td>c</td><td colspan="2"> 51.1%</td>

<td> 157</td><td>D</td><td>s</td><td></td><td> 50.4%</td>

<td></td><td></td><td>G</td><td></td><td> 48.7%</td>

<td></td><td></td><td>T</td><td></td><td> 54.7%</td>

<td> 158</td><td>N</td><td>AND</td><td colspan="2"> 51.2%</td>

<td> 159</td><td>L</td><td>M</td><td colspan="2"> 51.5%</td>

<td> 160</td><td>P</td><td>T</td><td colspan="2"> 52.8%</td>

<td> 161</td><td>AND</td><td></td><td>L, L117R</td><td> 54.1%</td>

<td></td><td></td><td></td><td>L</td><td> 51.6%</td>

<td> 162</td><td>P</td><td></td><td>K</td><td>scale</td>

<td></td><td></td><td></td><td>R</td><td>scale</td>

<td> 163</td><td>V</td><td></td><td>E</td><td> 55.7%</td>

178

<td>Rest Original amino acid acid</td><td>The hits will change palmitate new amino acid</td><td>possibly HTP stearate new amino acid</td><td>P + S</td>

<td></td><td></td><td colspan="2">R 63.9% T 49.7%</td>

<td>164 D.</td><td></td><td colspan="2">A 42.1% £ scale H 39.8% K 49.4% L scale R 61.3% S 47.9% T 53.0% V 42.3% In scale</td>

<td>166 Q</td><td></td><td colspan="2">G 49.9% N 41.3% R scale</td>

<td>167 I</td><td>R S</td><td colspan="2">R 53.3% S 47.3%</td>

<td>170 P.</td><td></td><td colspan="2">Q 45.6% A 52.5% A, S212H 34.7%</td>

<td>171 V.</td><td></td><td colspan="2">K 34.1%</td>

<td>172 R</td><td>P Q s</td><td colspan="2"> 51.7% 54.9% 40.2%</td>

<td>178 S.</td><td></td><td colspan="2">K 50.6%</td>

<td>180 L.</td><td></td><td colspan="2">E 54.0% H 44.6% q scale F, G32D 44.6%</td>

179

<td>Rest amino acid</td><td>Original amino acid</td><td>The hits will change palmitate new amino acid</td><td>possibly HTP stearate new amino acid</td><td>P + S</td>

<td> 183</td><td>V</td><td></td><td>AND</td><td>scale</td>

<td> 193</td><td>P</td><td></td><td colspan="2"> 49.4%</td>

<td> 194</td><td>E</td><td></td><td>AND</td><td>scale</td>

<td></td><td></td><td> -</td><td>M</td><td> 47.9%</td>

<td></td><td></td><td></td><td>Q</td><td>Scala</td>

<td></td><td></td><td></td><td>D.P193S</td><td> 49.4%</td>

<td> 197</td><td>D</td><td>K</td><td colspan="2"> 39.4%</td>

<td> 198</td><td>E</td><td>Stop</td><td colspan="2"> 56.1%</td>

<td> 200</td><td>L</td><td></td><td>V</td><td> 55.3%</td>

<td> 204</td><td>V</td><td></td><td>L</td><td> 45.9%</td>

<td></td><td></td><td></td><td>R</td><td> 45.7%</td>

<td> 210</td><td>AND</td><td>V</td><td colspan="2"> 50.2%</td>

<td> 211</td><td>AND</td><td></td><td>E</td><td> 35.3%</td>

<td></td><td></td><td></td><td>H</td><td> 48.1%</td>

<td></td><td></td><td></td><td>K</td><td> 39.4%</td>

<td></td><td></td><td></td><td>L</td><td> 45.0%</td>

<td></td><td></td><td></td><td>Q</td><td> 50.3%</td>

<td></td><td></td><td></td><td>F</td><td> 32.6%</td>

<td></td><td></td><td></td><td>N</td><td> 46.1%</td>

<td></td><td></td><td></td><td>P</td><td> 49.2%</td>

<td></td><td></td><td></td><td>R</td><td> 55.2%</td>

<td></td><td></td><td></td><td>in</td><td> 47.8%</td>

<td></td><td></td><td></td><td>Y</td><td> 48.9%</td>

<td></td><td></td><td>T</td><td></td><td> 50.8%</td>

<td></td><td></td><td>s</td><td></td><td> 52.7%</td>

<td></td><td></td><td></td><td>s</td><td> 52.7%</td>

<td></td><td></td><td>AND</td><td>AND</td><td> 49.8%</td>

<td></td><td></td><td>T.E217A</td><td></td><td> 46.2%</td>

<td> 212</td><td>S</td><td>C</td><td colspan="2"> 49.3%</td>

180

HTP screening hits

Rest Original amino acid amino acid

Palmitate a new amino acid

New amino acid stearate

P + S

<td colspan="2"></td><td>R</td><td colspan="2"> 50.3%</td>

<td></td><td></td><td></td><td>A, G132D</td><td> 53.2%</td>

<td></td><td></td><td>AND</td><td></td><td> 36.8%</td>

<td></td><td></td><td>E</td><td></td><td> 36.6%</td>

<td></td><td></td><td>G</td><td></td><td> 44.3%</td>

<td></td><td></td><td>H</td><td></td><td> 46.5%</td>

<td></td><td></td><td>L</td><td></td><td> 53.2%</td>

<td></td><td></td><td>P</td><td></td><td> 12.2%</td>

<td></td><td></td><td>Q</td><td></td><td> 41.8%</td>

<td></td><td></td><td>R</td><td></td><td> 50.2%</td>

<td></td><td></td><td>T</td><td></td><td> 53.7%</td>

<td></td><td></td><td>V</td><td></td><td> 38.7%</td>

<td></td><td></td><td>IN</td><td></td><td> 48.4%</td>

<td></td><td></td><td>Y</td><td></td><td> 47.1%</td>

<td></td><td></td><td></td><td>Η, P170A</td><td> 34.7%</td>

<td> 213</td><td>K</td><td>AND</td><td></td><td> 47.9%</td>

<td></td><td></td><td>G</td><td></td><td> 57.7%</td>

<td></td><td></td><td>T</td><td></td><td> 56.7%</td>

<td></td><td></td><td>T</td><td></td><td> 55.5%</td>

<td></td><td></td><td>Stop</td><td></td><td></td>

<td> 214</td><td>T</td><td></td><td>C</td><td> 51.6%</td>

<td></td><td></td><td></td><td>G</td><td> 53.0%</td>

<td></td><td></td><td>V</td><td>V</td><td> 52.2%</td>

<td></td><td></td><td>V</td><td></td><td> 54.5%</td>

<td></td><td></td><td>P</td><td></td><td> 51.9%</td>

<td></td><td></td><td>N</td><td></td><td> 56.9%</td>

<td></td><td></td><td>R</td><td></td><td> 55.1%</td>

<td></td><td></td><td>Y</td><td></td><td> 62.7%</td>

<td></td><td></td><td>Y</td><td></td><td> 62.7%</td>

181

HTP screening hits

Amino acid residue

The original amino acid

Palmitate a new amino acid

New amino acid stearate

P + S

<td> 215</td><td>G</td><td>AND</td><td>AND</td><td> 56.6%</td>

<td></td><td></td><td></td><td>AND</td><td> 54.1%</td>

<td></td><td></td><td></td><td>L</td><td> 29.9%</td>

<td></td><td></td><td>H</td><td></td><td> 50.2%</td>

<td></td><td></td><td>S</td><td></td><td> 52.1%</td>

<td></td><td></td><td>M</td><td></td><td> 47.9%</td>

<td></td><td></td><td>V</td><td></td><td> 55.6%</td>

<td></td><td></td><td>P</td><td></td><td> 47.3%</td>

<td></td><td></td><td>C</td><td></td><td> 60.4%</td>

<td></td><td></td><td>.in</td><td></td><td> 52.8%</td>

<td></td><td></td><td>Stop</td><td></td><td> 53.9%</td>

<td></td><td></td><td></td><td>V, T22M</td><td> 52.1%</td>

<td> 216</td><td>AND</td><td>T</td><td>T</td><td> 50.9%</td>

<td></td><td></td><td>R</td><td></td><td> 41.9%</td>

<td></td><td></td><td>Y</td><td></td><td> 34.8%</td>

<td></td><td></td><td>V</td><td>V</td><td> 56.9%</td>

<td></td><td></td><td>c</td><td></td><td> 59.7%</td>

<td></td><td></td><td>s</td><td>S</td><td> 55.0%</td>

<td></td><td></td><td>L</td><td></td><td> 55.6%</td>

<td> 217</td><td>E</td><td>Q</td><td></td><td> 36.6%</td>

<td></td><td></td><td>R</td><td></td><td> 59.4%</td>

<td></td><td></td><td>S</td><td></td><td> 53.5%</td>

<td></td><td></td><td>AND</td><td></td><td> 46.2%</td>

<td></td><td></td><td>G</td><td></td><td> 44.8%</td>

<td></td><td></td><td>P</td><td></td><td> 46.2%</td>

<td> 218</td><td>AND</td><td>M</td><td></td><td> 42.5%</td>

<td></td><td></td><td>H</td><td>H</td><td> 49.1%</td>

<td></td><td></td><td>Q</td><td>Q</td><td> 47.7%</td>

<td></td><td></td><td>R</td><td></td><td> 53.4%</td>

182

<td colspan="2"></td><td colspan="3">HTP screening hits</td>

<td>Rest amino acid</td><td>Original amino acid</td><td>palmitate new amino acid</td><td>stearate new amino acid</td><td>P + S</td>

<td> 1</td><td></td><td>IN</td><td></td><td> 51.9%</td>

<td></td><td></td><td>s</td><td></td><td> 51.1%</td>

<td></td><td></td><td>T</td><td></td><td> 50.0%</td>

<td></td><td></td><td>K</td><td></td><td> 52.4%</td>

<td></td><td></td><td>R.G8E</td><td></td><td></td>

<td></td><td></td><td>R, 228K</td><td></td><td></td>

<td> 223</td><td>V</td><td></td><td>AND</td><td> 48.8%</td>

<td></td><td></td><td></td><td>M</td><td> 31.6%</td>

<td></td><td></td><td></td><td>R</td><td> 23.4%</td>

<td></td><td></td><td></td><td>T</td><td>scale</td>

<td> 224</td><td>AND</td><td></td><td>F</td><td> 49.5%</td>

<td></td><td></td><td></td><td>G</td><td> 58.2%</td>

<td></td><td></td><td></td><td>G</td><td> 48.4%</td>

<td></td><td></td><td></td><td>AND</td><td> 41.7%</td>

<td></td><td></td><td></td><td>Q</td><td> 46.4%</td>

<td></td><td></td><td></td><td>Y</td><td> 43,7%</td>

<td> 225</td><td>AND</td><td></td><td>G</td><td> 49.3%</td>

<td></td><td></td><td></td><td>L</td><td> 54.3%</td>

<td></td><td></td><td></td><td>M</td><td> 49.0%</td>

<td></td><td></td><td></td><td>Q</td><td> 45.8%</td>

<td></td><td></td><td></td><td>T</td><td> 43.2%</td>

<td> 226</td><td>R</td><td></td><td>H</td><td> 48.3%</td>

<td></td><td></td><td></td><td>T</td><td> 41.2%</td>

<td> 227</td><td>L</td><td></td><td>R</td><td> 41.4%</td>

Table 3 (continued)

Fatty acids released from oil by the enzyme

183

Rest

<td>amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td> 7</td><td> 8.3%</td><td> 22.1%</td><td> 19.7%</td><td> 41.5%</td><td> 8.4%</td><td> 49.9%</td>

<td> 8</td><td colspan="5"></td><td></td>

<td> 12</td><td> 11.5%</td><td> 13.0%</td><td> 27.7%</td><td> 38.5%</td><td> 9.3%</td><td> 47.8%</td>

<td></td><td> 5.2%</td><td> 22.1%</td><td> 18.5%</td><td> 47.2%</td><td> 7.1%</td><td> 54.2%</td>

<td></td><td> 14.7%</td><td> 13.9%</td><td> 26.0%</td><td> 34.4%</td><td> 11.0%</td><td> 45.4%</td>

<td></td><td> 10.3%</td><td> 12.8%</td><td> 33.6%</td><td> 34.0%</td><td> 9.4%</td><td> 43.3%</td>

<td> 16</td><td> 7,2%</td><td> 25.1%</td><td> 24.6%</td><td> 36.1%</td><td> 7.1%</td><td> 43.2%</td>

<td> 18</td><td> 12.5%</td><td> 20.0%</td><td> 25.7%</td><td> 36.2%</td><td> 5.6%</td><td> 41.8%</td>

<td> 20</td><td> . 8.2%</td><td> 21.2%</td><td> 20.3%</td><td> 38.5%</td><td> 11.7%</td><td> 50.3%</td>

<td></td><td> 12.2%</td><td> 23.2%</td><td> 20.0%</td><td> 40.1%</td><td> 4.5%</td><td> 44.6%</td>

<td> 22</td><td> 8.0%</td><td> 19.5%</td><td> 20.4%</td><td> 47.1%</td><td> 5.0%</td><td> 52.1%</td>

<td> 27</td><td> 7.5%</td><td> 17.6%</td><td> 17.6%</td><td> 47.4%</td><td> 9.8%</td><td> 57.2%</td>

<td></td><td> 9.1%</td><td> 23.2%</td><td> 24.0%</td><td> 35.8%</td><td> 7.8%</td><td> 43.6%</td>

<td> 29</td><td> 9.0%</td><td> 19.9%</td><td> 19.6%</td><td> 40.9%</td><td> 10.7%</td><td> 51.5%</td>

<td> 32</td><td> 19.8%</td><td> 29.1%</td><td> 34.0%</td><td>scale</td><td> 17.1%</td><td>scale</td>

<td></td><td> 14.6%</td><td> 12.1%</td><td> 28.7%</td><td> 36.6%</td><td> 7.9%</td><td> 44.6%</td>

<td> 34</td><td> 5.6%</td><td> 31.0%</td><td> . 17.5%</td><td> 40.9%</td><td> 4.9%</td><td> 45.8%</td>

<td></td><td> 21.1%</td><td> 35.3%</td><td> 37.1%</td><td>scale</td><td> 6.5%</td><td>scale</td>

<td> 36</td><td> 7.1%</td><td> 22.1%</td><td> 19.9%</td><td> 43.8%</td><td> 7.1%</td><td> 51.0%</td>

<td></td><td> 8.7%</td><td> 22.9%</td><td> 17.6%</td><td> 48.2%</td><td> 2.7%</td><td> 50.9%</td>

<td> 40</td><td> 0.0%</td><td> 51.4%</td><td> 16.4%</td><td> 22.4%</td><td> 9.7%</td><td> 32.2%</td>

<td> 42</td><td> 14.8%</td><td> 12.1%</td><td> 25.8%</td><td> 34.6%</td><td> 12.6%</td><td> 47.2%</td>

<td></td><td> 7.7%</td><td> 13.9%</td><td> 30.7%</td><td> 34.8%</td><td> 13.0%</td><td> 47.8%</td>

<td> 43</td><td> 8.9%</td><td> 19.9%</td><td> 19.7%</td><td> 44.4%</td><td> 7.1%</td><td> 51.5%</td>

<td> 45</td><td> 10.3%</td><td> 23.8%</td><td> 21.5%</td><td> 38.5%</td><td> 5.9%</td><td> 44.4%</td>

<td></td><td> 5.9%</td><td> 22.3%</td><td> 19.1%</td><td> 49.7%</td><td> 3.0%</td><td> 52.7%</td>

<td> 48</td><td> 15.0%</td><td> 18.0%</td><td> 21.7%</td><td> 38.1%</td><td> 7.2%</td><td> 45.4%</td>

<td></td><td> 4.3%</td><td> 11.5%</td><td> 14.2%</td><td> 61.0%</td><td> 9.1%</td><td> 70.1%</td>

<td></td><td> 7.6%</td><td> 17.3%</td><td> 19.4%</td><td> 43.8%</td><td> 12.0%</td><td> 55.7%</td>

<td></td><td> 23.6%</td><td> 13.4%</td><td> 29.3%</td><td> 22.5%</td><td> 11.1%</td><td> 33.60%</td>

<td> 54</td><td> 8.1%</td><td> 19.3%</td><td> 17.0%</td><td> 48.4%</td><td> 7.3%</td><td> 55.6%</td>

184

Fatty acids released from oil by the enzyme

<td>Rest amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td> 61</td><td> 5.6%</td><td> 19.8%</td><td> 14.1%</td><td> 53.9%</td><td> 6.6%</td><td> 60.5%</td>

<td></td><td> 6.4%</td><td> 20.1%</td><td> 18.5%</td><td> 47.3%</td><td> 7.7%</td><td> 55.0%</td>

<td></td><td> 7.7%</td><td> 19.9%</td><td> 22.6%</td><td> 41.5%</td><td> 8.3%</td><td> 49.8%</td>

<td> 62</td><td> 7.6%</td><td> 18.8%</td><td> 20.7%</td><td> 44.6%</td><td> 8.3%</td><td> 53.0%</td>

<td></td><td> 9.2%</td><td> 17.6%</td><td> 16.6%</td><td> 45.6%</td><td> 11.0%</td><td> 56.6%</td>

<td></td><td> 6.7%</td><td> 20.3%</td><td> 16.5%</td><td> 47.6%</td><td> 8.8%</td><td> 56.5%</td>

<td></td><td> 7.7%</td><td> 20.9%</td><td> 19.5%</td><td> 44.9%</td><td> 6.9%</td><td> 51.9%</td>

<td></td><td> 7.9%</td><td> 21.7%</td><td> 20.7%</td><td> 40.7%</td><td> 9.0%</td><td> 49.7%</td>

<td></td><td> 8.5%</td><td> 20.8%</td><td> 18.4%</td><td> 42.6%</td><td> 9.8%</td><td> 52.4%</td>

<td></td><td> 5.4%</td><td> 26.0%</td><td> 13.1%</td><td> 37.0%</td><td> 18.5%</td><td> 55.5%</td>

<td></td><td> 10.0%</td><td> 21.9%</td><td> 17.5%</td><td> 40.2%</td><td> 10.5%</td><td> 50.7%</td>

<td></td><td> 6.1%</td><td> 23.2%</td><td> 18.2%</td><td> 47.1%</td><td> 5.4%</td><td> 52.5%</td>

<td></td><td> 9.9%</td><td> 21.3%</td><td> 18.6%</td><td> 46.7%</td><td> 3.6%</td><td> 50.2%</td>

<td> 66</td><td> 7.5%</td><td> 16.8%</td><td> 21.5%</td><td> 48.0%</td><td> 6.2%</td><td> 54.2%</td>

<td></td><td> 11.4%</td><td> 18.0%</td><td> 18.5%</td><td> 47.2%</td><td> 4.8%</td><td> 52.1%</td>

<td> 72</td><td> 7.9%</td><td> 16.5%</td><td> 17.3%</td><td> 54.2%</td><td> 4.1%</td><td> 58.3%</td>

<td></td><td> 4.4%</td><td> 20.7%</td><td> 13.9%</td><td> 52.2%</td><td> 8.7%</td><td> 61.0%</td>

<td></td><td> 6.8%</td><td> 44.6%</td><td> 21.4%</td><td> 20.2%</td><td> 7.0%</td><td> 27.2%</td>

<td></td><td> 8.6%</td><td> 17.3%</td><td> 18.8%</td><td> 45.7%</td><td> 9.6%</td><td> 55.3%</td>

<td></td><td> 7.5%</td><td> 17.4%</td><td> 19.3%</td><td> 45.1%</td><td> 10.7%</td><td> 55.9%</td>

<td></td><td> 6.7%</td><td> 23.1%</td><td> 20.1%</td><td> 40.2%</td><td> 9.9%</td><td> 50.1%</td>

<td> 74</td><td> 7.4%</td><td> 19.6%</td><td> 19.3%</td><td> 45.4%</td><td> 8.4%</td><td> 53.8%</td>

<td></td><td> 8.0%</td><td> 19.3%</td><td> 18.0%</td><td> 44.8%</td><td> 10.0%</td><td> 54.8%</td>

<td></td><td> 8.7%</td><td> 20.5%</td><td> 18.6%</td><td> 42.2%</td><td> 10.1%</td><td> 52.3%</td>

<td></td><td> 7.1%</td><td> 21.7%</td><td> 20.7%</td><td> 41.1%</td><td> 9.4%</td><td> 50.5%</td>

<td> 77</td><td> 10.3%</td><td> 41.0%</td><td> 10.6%</td><td> 17.8%</td><td> 20.4%</td><td> 38.1%</td>

<td> 78</td><td> 9.8%</td><td> 22.5%</td><td> 20.6%</td><td> 43.8%</td><td> 3.4%</td><td> 47.1%</td>

<td></td><td> 26.2%</td><td> 23.0%</td><td> 13.8%</td><td> 15.4%</td><td> 21.7%</td><td> 37.1%</td>

185

Fatty acids released from oil by the enzyme

<td>Rest amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td></td><td> 14.4%</td><td> 13.2%</td><td> 31.4%</td><td> 32.3%</td><td> 8.6%</td><td> 40.9%</td>

<td> 80</td><td> 7.4%</td><td> 21.0%</td><td> 19.7%</td><td> 42.9%</td><td> 9.0%</td><td> 51.9%</td>

<td> 82</td><td> 13.0%</td><td> 28.3%</td><td> 21.4%</td><td> 33.3%</td><td> 4.0%</td><td> 37.3%</td>

<td> 83</td><td> 7.5%</td><td> 20.0%</td><td> 24.7%</td><td> 31.1%</td><td> 16.6%</td><td> 47.7%</td>

<td></td><td> 6.8%</td><td> 18.6%</td><td> 15.3%</td><td> 51.5%</td><td> 7.8%</td><td> 59.3%</td>

<td> 84</td><td> 0.0%</td><td> 32.4%</td><td> 27.4%</td><td> 21.0%</td><td> 19.2%</td><td> 40.2%</td>

<td> 87</td><td> 12,7%</td><td> 11.9%</td><td> 26.2%</td><td> 39.7%</td><td> 9.5%</td><td> 49.2%</td>

<td></td><td> 14.5%</td><td> 11.8%</td><td> 27.6%</td><td> 33.1%</td><td> 13.0%</td><td> 46.1%</td>

<td></td><td> 9.3%</td><td> 12.3%</td><td> 34.5%</td><td> 32.7%</td><td> 11.2%</td><td> 43.9%</td>

<td></td><td> 12.2%</td><td> 10.5%</td><td> 30.8%</td><td> 33.6%</td><td> 13.0%</td><td> 46.6%</td>

<td></td><td> 10.6%</td><td> 9.9%</td><td> 26.2%</td><td> 40.6%</td><td> 12.7%</td><td> 53.3%</td>

<td></td><td> 14.4%</td><td> 12.4%</td><td> 27.9%</td><td> 36.0%</td><td> 9.2%</td><td> 45.2%</td>

<td></td><td> 6.7%</td><td> 25.8%</td><td> 24.7%</td><td> 39.5%</td><td> 3.3%</td><td> 42.8%</td>

<td></td><td> 4.4%</td><td> 23.2%</td><td> 19.4%</td><td> 48.5%</td><td> 4.5%</td><td> 52.9%</td>

<td></td><td> 11.7%</td><td> 11.3%</td><td> 26.7%</td><td> 31.5%</td><td> 18.8%</td><td> 50.3%</td>

<td> 88</td><td> 14.1%</td><td> 13.4%</td><td> 27.9%</td><td> 34.7%</td><td> 9.9%</td><td> 44.6%</td>

<td></td><td> 13.4%</td><td> 15.2%</td><td> 21.0%</td><td> 36.7%</td><td> 13.6%</td><td> 50.3%</td>

<td></td><td> 13.3%</td><td> 12.5%</td><td> 28.3%</td><td> 32.5%</td><td> 13.4%</td><td> 45.9%</td>

<td></td><td> 13.0%</td><td> 9.2%</td><td> 28.7%</td><td> 40.3%</td><td> 8.8%</td><td> 49.1%</td>

<td></td><td> 2.9%</td><td> 22.5%</td><td> 15.0%</td><td> 59.0%</td><td> 0.7%</td><td> 59.6%</td>

<td></td><td> 4.2%</td><td> 35.5%</td><td> 11.4%</td><td> 35.6%</td><td> 13.3%</td><td> 48.9%</td>

<td></td><td> 14.7%</td><td> 9.7%</td><td> 28.5%</td><td> 34.9%</td><td> 12.2%</td><td> 47.1%</td>

<td> 89</td><td> 0.0%</td><td> 35.4%</td><td> 10.1%</td><td> 39.0%</td><td> 15.5%</td><td> 54.5%</td>

<td> 92</td><td> 13.1%</td><td> 16.1%</td><td> 23.5%</td><td> 36.4%</td><td> 10.9%</td><td> 47.3%</td>

<td></td><td> 12.0%</td><td> 10.4%</td><td> 18.2%</td><td> 48.0%</td><td> 11.4%</td><td> 59.3%</td>

<td></td><td> 13.5%</td><td> 11.3%</td><td> 32.6%</td><td> 34.8%</td><td> 7.7%</td><td> 42.6%</td>

<td></td><td> 8.3%</td><td> 25.1%</td><td> 17.9%</td><td> 46.1%</td><td> 2.6%</td><td> 48.7%</td>

<td></td><td> 13.7%</td><td> 9.8%</td><td> 24.4%</td><td> 39.6%</td><td> 12.5%</td><td> 52.1%</td>

186

Fatty acids released from oil by the enzyme

<td>Rest amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td></td><td> 4.9%</td><td> 20.0%</td><td> 17.5%</td><td> 51.7%</td><td> 5.8%</td><td> 57.5%</td>

<td> 93</td><td> 11.7%</td><td> 9.3%</td><td> 30.8%</td><td> 40.8%</td><td> 7.4%</td><td> 48.2%</td>

<td> 96</td><td> 10.3%</td><td> 12.4%</td><td> 25.9%</td><td> 37.8%</td><td> 13.6%</td><td> 51.4%</td>

<td></td><td> 17.5%</td><td> 35.4%</td><td> 35.5%</td><td>scale</td><td> 11.6%</td><td>scale</td>

<td></td><td> 12.5%</td><td> 12.0%</td><td> 28.7%</td><td> 33.3%</td><td> 13.6%</td><td> 46.8%</td>

<td> 98</td><td> 13.4%</td><td> 19.9%</td><td> 21.7%</td><td> 39.7%</td><td> 5.3%</td><td> 45.0%</td>

<td></td><td> 18.5%</td><td> 30.8%</td><td> 36.8%</td><td>scale</td><td> 13.9%</td><td>scale</td>

<td> 101</td><td> 9.8%</td><td> 12.5%</td><td> 27.9%</td><td> 39.7%</td><td> 10.1%</td><td> 49.8%</td>

<td> 103</td><td> 9.1%</td><td> 36.6%</td><td> 17.5%</td><td> 26.0%</td><td> 10.8%</td><td> 36.8%</td>

<td> 107</td><td> 11.9%</td><td> 10.1%</td><td> 31.8%</td><td> 30.5%</td><td> 15.7%</td><td> 46.20%</td>

<td></td><td> 0.0%</td><td> 20.4%</td><td> 40.1%</td><td> 12.1%</td><td> 27.4%</td><td> 39.5%</td>

<td></td><td> 0.0%</td><td> 29.6%</td><td> 30.9%</td><td> 6.8%</td><td> 32.6%</td><td> 39.4%</td>

<td></td><td> 0.0%</td><td> 29.6%</td><td> 22.9%</td><td> 9.5%</td><td> 38.0%</td><td> 47.5%</td>

<td></td><td> 2.2%</td><td> 12.0%</td><td> 43.9%</td><td> 22.0%</td><td> 19.9%</td><td> 42.0%</td>

<td></td><td> 30.4%</td><td> 12.5%</td><td> 46.2%</td><td> 10.9%</td><td> 57.1%</td><td> 68.0%</td>

<td></td><td> 12.0%</td><td> 20.5%</td><td> 30.7%</td><td> 5.2%</td><td> 31.6%</td><td> 36.8%</td>

<td></td><td> 5.0%</td><td> 16.0%</td><td> 14.2%</td><td> 62.2%</td><td> 2.6%</td><td> 64.8%</td>

<td></td><td> 11.9%</td><td> 10.1%</td><td> 31.8%</td><td> 30.5%</td><td> 15.7%</td><td> 46.2%</td>

<td></td><td> 0.0%</td><td> 15.6%</td><td> 46.5%</td><td> 10.2%</td><td> 27.6%</td><td> 37.8%</td>

<td></td><td> 13.2%</td><td> 21.6%</td><td> 20.4%</td><td> 31.3%</td><td> 13.5%</td><td> 44.80%</td>

<td> 108</td><td> 9.0%</td><td> 49.0%</td><td> 23.0%</td><td> 12.3%</td><td> 6.7%</td><td> 19.0%</td>

<td></td><td> 0.0%</td><td> 51.0%</td><td> 6.1%</td><td> 33.1%</td><td> 9.9%</td><td> 43.0%</td>

<td></td><td> 11.0%</td><td> 18.4%</td><td> 44.6%</td><td> 4.1%</td><td> 21.9%</td><td> 26.0%</td>

<td></td><td> 0.0%</td><td> 29.6%</td><td> 22.9%</td><td> 9.5%</td><td> 38.0%</td><td> 47.5%</td>

<td></td><td> 0.0%</td><td> 32.0%</td><td> 10.2%</td><td> 53.5%</td><td> 4.3%</td><td> 57.8%</td>

<td></td><td> 0.0%</td><td> 45.6%</td><td> 10.4%</td><td> 38.2%</td><td> 5.8%</td><td> 44.0%</td>

<td></td><td> 0.0%</td><td> 28.4%</td><td> 14.7%</td><td> 51.2%</td><td> 5.7%</td><td> 56.9%</td>

<td></td><td> 5.4%</td><td> 18.9%</td><td> 17.2%</td><td> 52.8%</td><td> 5.8%</td><td> 58.6%</td>

<td></td><td> 0.0%</td><td> 10.6%</td><td> 34.7%</td><td> 5.9%</td><td> 48.8%</td><td> 54.7%</td>

187

Fatty acids released from oil by the enzyme

<td>Rest amino acid</td><td>Linoienowy</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td></td><td> 0.0%</td><td> 21.9%</td><td> 24.7%</td><td> 32.7%</td><td> 20.8%</td><td> 53.4%</td>

<td></td><td> 12.1%</td><td> 13.9%</td><td> 27.6%</td><td> 33.8%</td><td> 12.7%</td><td> 46.50%</td>

<td> 109</td><td> 10.9%</td><td> 8.8%</td><td> 31.3%</td><td> 37.7%</td><td> 11.3%</td><td> 49.0%</td>

<td> 110</td><td> 0.4%</td><td> 21.4%</td><td> 23.9%</td><td> 54.4%</td><td> 0.0%</td><td> 54.4%</td>

<td> 113</td><td> 5.0%</td><td> 44.1%</td><td> 15.1%</td><td> 21.0%</td><td> 14.8%</td><td> 35.8%</td>

<td></td><td> 13.6%</td><td> 14.6%</td><td> 32.0%</td><td> 15.2%</td><td> 24.6%</td><td> 39.8%</td>

<td></td><td> 13.9%</td><td> 25.9%</td><td> 23.7%</td><td> 36.5%</td><td> 0.0%</td><td> 36.5%</td>

<td> 116</td><td> 4.8%</td><td> 17.4%</td><td> 11.2%</td><td> 55.5%</td><td> 11.1%</td><td> 66.6%</td>

<td></td><td> 7.8%</td><td> 17.7%</td><td> 19.8%</td><td> 47.0%</td><td> 7.7%</td><td> 54.7%</td>

<td></td><td> 3.3%</td><td> 26.7%</td><td> 16.1%</td><td> 33.1%</td><td> 20.7%</td><td> 53.8%</td>

<td></td><td> 7.3%</td><td> 18.3%</td><td> 16.5%</td><td> 47.8%</td><td> 10.1%</td><td> 57.9%</td>

<td></td><td> 4.3%</td><td> 22.9%</td><td> 14.2%</td><td> 54.3%</td><td> 4.2%</td><td> 58.5%</td>

<td></td><td> 4.6%</td><td> 26.8%</td><td> 13.5%</td><td> 41.6%</td><td> 13.5%</td><td> 55.1%</td>

<td></td><td> 0.0%</td><td> 32.4%</td><td> 9.6%</td><td> 38.3%</td><td> 19.8%</td><td> 58.0%</td>

<td></td><td> 8.1%</td><td> 16.1%</td><td> 16.2%</td><td> 50.4%</td><td> 9.3%</td><td> 59.6%</td>

<td></td><td> 7.3%</td><td> 20.5%</td><td> 11.7%</td><td> 49.2%</td><td> 11.2%</td><td> 60.5%</td>

<td></td><td> 6.9%</td><td> 19.6%</td><td> 12.9%</td><td> 52.1%</td><td> 8.5%</td><td> 60.6%</td>

<td></td><td> 5.4%</td><td> 17.4%</td><td> 15.4%</td><td> 50.8%</td><td> 11.0%</td><td> 61.8%</td>

<td></td><td> 8.7%</td><td> 18.7%</td><td> 13.9%</td><td> 49.1%</td><td> 9.5%</td><td> 58.6%</td>

<td></td><td> 6.7%</td><td> 22.8%</td><td> 10.8%</td><td> 46.3%</td><td> 13.4%</td><td> 59.7%</td>

<td></td><td> 6.4%</td><td> 17.2%</td><td> 8.8%</td><td> 503%</td><td> 17.2%</td><td> 67.6%</td>

<td></td><td> 5.6%</td><td> 17.6%</td><td> 9.0%</td><td> 59.0%</td><td> 8.8%</td><td> 67.8%</td>

<td> 117</td><td> 6.2%</td><td> 21.4%</td><td> 18.3%</td><td> 46.0%</td><td> 8.0%</td><td> 54.1%</td>

<td></td><td> 8.9%</td><td> 21.8%</td><td> 17.7%</td><td> 40.6%</td><td> 11.0%</td><td> 51.6%</td>

<td> 15.3%</td><td> 17.9%</td><td> 20.1%</td><td> 44.4%</td><td> 2.3%</td><td> 46.7%</td>

<td> 7.5%</td><td> 15.4%</td><td> 16.3%</td><td> 51.3%</td><td> 9.5%</td><td> 60.8%</td>

<td> 17.3%</td><td> 4.4%</td><td> 25.7%</td><td> 44.0%</td><td> 8.6%</td><td> 52.6%</td>

188

Fatty acids released from oil by the enzyme

<td>Rest amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td></td><td> 4.1%</td><td> 25.5%</td><td> 20.5%</td><td> 36.9%</td><td> 13.0%</td><td> 49.9%</td>

<td></td><td> 15.7%</td><td> 10.1%</td><td> 20.9%</td><td> 36.8%</td><td> 16.5%</td><td> 53.3%</td>

<td> 132</td><td> 0.0%</td><td> 32.8%</td><td> 10.9%</td><td> 56.2%</td><td> 0.0%</td><td> 56.2%</td>

<td> 133</td><td> 6.6%</td><td> 20.7%</td><td> 19.5%</td><td> 49.9%</td><td> 3.3%</td><td> 53.2%</td>

<td></td><td> 9.3%</td><td> 18.3%</td><td> 16.5%</td><td> 45.1%</td><td> 10.8%</td><td> 55.8%</td>

<td></td><td> 3.3%</td><td> 30.4%</td><td> 20.7%</td><td> 45.6%</td><td> 0.0%</td><td> 45.6%</td>

<td></td><td> 0.0%</td><td> 34.3%</td><td> 9.6%</td><td> 56.0%</td><td> 0.0%</td><td> 56.0%</td>

<td></td><td> 13.2%</td><td> 12.9%</td><td> 22.2%</td><td> 42.7%</td><td> 9.0%</td><td> 51.7%</td>

<td></td><td> 10.1%</td><td> 11.6%</td><td> 23.3%</td><td> 46.5%</td><td> 8.4%</td><td> 54.9%</td>

<td></td><td> 0.0%</td><td> 35.9%</td><td> 10.9%</td><td> 46.9%</td><td> 6.3%</td><td> 53.2%</td>

<td> 134</td><td> 0.0%</td><td> 56.2%</td><td> 36.6%</td><td> 7.2%</td><td> 0.0%</td><td> 7.2%</td>

<td> 135</td><td> 0.0%</td><td> 41.1%</td><td> 7.2%</td><td> 51.8%</td><td> 0.0%</td><td> 51.8%</td>

<td> 139</td><td> 0.0%</td><td> 35.9%</td><td> 10.9%</td><td> 46.9%</td><td> 6.3%</td><td> 53.2%</td>

<td> 140</td><td> 9.7%</td><td> 23.4%</td><td> 21.4%</td><td> 32.7%</td><td> 12.8%</td><td> 45.5%</td>

<td> 141</td><td> 11.2%</td><td> 12.1%</td><td> 36.5%</td><td> 28.2%</td><td> 12.1%</td><td> 40.2%</td>

<td></td><td> 14.7%</td><td> 13.9%</td><td> 28.1%</td><td> 38.3%</td><td> 5.0%</td><td> 43.3%</td>

<td> 142</td><td> 16.3%</td><td> 18.8%</td><td> 18.8%</td><td> 10.3%</td><td> 35.7%</td><td> 46.1%</td>

<td></td><td> 0.0%</td><td> 34.5%</td><td> 11.7%</td><td> 43.4%</td><td> 10.5%</td><td> 53.8%</td>

<td></td><td> 8.6%</td><td> 15.8%</td><td> 32.5%</td><td> 22.8%</td><td> 20.4%</td><td> 43.2%</td>

<td></td><td> 2.4%</td><td> 9.6%</td><td> 23.7%</td><td> 47.7%</td><td> 16.7%</td><td> 64.3%</td>

<td> 144</td><td> 0.0%</td><td> 14.9%</td><td> 51.2%</td><td> 13.4%</td><td> 20.4%</td><td> 33.9%</td>

<td> 146</td><td> 13.6%</td><td> 10.3%</td><td> 26.0%</td><td> 31.4%</td><td> 18.7%</td><td> 50.2%</td>

<td></td><td> 12.6%</td><td> 12.4%</td><td> 25.5%</td><td> 36.5%</td><td> 12.9%</td><td> 49.4%</td>

<td></td><td> 6.6%</td><td> 22.4%</td><td> 19.5%</td><td> 48.2%</td><td> 3.4%</td><td> 51.6%</td>

<td></td><td> 9.0%</td><td> 19.7%</td><td> 19.0%</td><td> 41.7%</td><td> 10.5%</td><td> 52.2%</td>

<td> 147</td><td> 8.0%</td><td> 17.9%</td><td> 17.6%</td><td> 48.8%</td><td> 7.7%</td><td> 56.5%</td>

<td></td><td> 7.2%</td><td> 24.5%</td><td> 17.9%</td><td> 33.0%</td><td> 17.4%</td><td> 50.5%</td>

189

Fatty acids released from oil by the enzyme

Rest

<td>amino acid</td><td>linolenic</td><td>linoleic</td><td>oleic</td><td colspan="2">Palmitic Stearin</td><td>P + S</td>

<td></td><td> 9.5%</td><td> 20.6%</td><td> 17.7%</td><td> 42.2%</td><td> 9.9%</td><td> 52.2%</td>

<td> 150</td><td> 7.7%</td><td> 15.1%</td><td> 17.5%</td><td> 50.4%</td><td> 9.2%</td><td> 59.7%</td>

<td></td><td> 7.5%</td><td> 20.6%</td><td> 18.6%</td><td> 41.3%</td><td> 12.0%</td><td> 53.3%</td>

<td> 151</td><td> 7.8%</td><td> 26.1%</td><td> 17.5%</td><td> 46.4%</td><td> 2.2%</td><td> 48.6%</td>

<td></td><td> 5.0%</td><td> 29.6%</td><td> 12.4%</td><td> 48.9%</td><td> 4.1%</td><td> 53.0%</td>

<td></td><td> 0.0%</td><td> 25.5%</td><td> 14.5%</td><td> 55.5%</td><td> 4.5%</td><td> 60.0%</td>

<td></td><td> 0.0%</td><td> 14.2%</td><td> 52.1%</td><td> 20.0%</td><td> 13.7%</td><td> 33.7%</td>

<td></td><td> 0.0%</td><td> 17.3%</td><td> 30.5%</td><td> 43.3%</td><td> 8.9%</td><td> 52.2%</td>

<td></td><td> 8.0%</td><td> 22.7%</td><td> 20.2%</td><td> 44.0%</td><td> 5.1%</td><td> 49.2%</td>

<td> 152</td><td> 0.0%</td><td> 56.3%</td><td> 15.7%</td><td> 23.6%</td><td> 4.4%</td><td> 28.0%</td>

<td></td><td> 8.0%</td><td> 12.7%</td><td> 26.3%</td><td> 22.5%</td><td> 30.5%</td><td> 53.0%</td>

<td></td><td> 0.0%</td><td> 27.1%</td><td> 26.2%</td><td> 26.2%</td><td> 20.5%</td><td> 46.7%</td>

<td></td><td> 0.0%</td><td> 20.1%</td><td> 44.2%</td><td> 24.8%</td><td> 10.8%</td><td> 35.7%</td>

<td></td><td> 9.5%</td><td> 31.2%</td><td> 38.3%</td><td> 2.2%</td><td> 18.9%</td><td> 21.1%</td>

<td> 155</td><td> 18.4%</td><td> 4.9%</td><td> 25.6%</td><td> 41.5%</td><td> 9.6%</td><td> 51.1%</td>

<td> 157</td><td> 7.9%</td><td> 19.5%</td><td> 22.2%</td><td> 41.2%</td><td> 9.2%</td><td> 50.4%</td>

<td></td><td> 9.6%</td><td> 21.7%</td><td> 20.1%</td><td> 39.1%</td><td> 9.6%</td><td> 48.7%</td>

<td></td><td> 73%</td><td> 25.2%</td><td> 13.0%</td><td> 34.9%</td><td> 19.8%</td><td> 54.7%</td>

<td> 158</td><td> 14.0%</td><td> 1.2%</td><td> 33.5%</td><td> 42.8%</td><td> 8.5%</td><td> 51.2%</td>

<td> 159</td><td> 63%</td><td> 28.4%</td><td> 13.8%</td><td> 36.7%</td><td> 14.8%</td><td> 51.5%</td>

<td> 160</td><td> 5.6%</td><td> 20.8%</td><td> 20.8%</td><td> 46.6%</td><td> 6.2%</td><td> 52.8%</td>

<td> 161</td><td> 6.2%</td><td> 21.4%</td><td> 18.3%</td><td> 46.0%</td><td> 8.0%</td><td> 54.1%</td>

<td></td><td> 8.9%</td><td> 21.8%</td><td> 17.7%</td><td> 40.6%</td><td> 11.0%</td><td> 51.6%</td>

<td> 162</td><td> 10.2%</td><td> 45.6%</td><td> 38.4%</td><td>scale</td><td> 5.7%</td><td>scale</td>

<td></td><td> 22.1%</td><td> 39.2%</td><td> 32.7%</td><td>scale</td><td> 6.0%</td><td>scale</td>

<td> 163</td><td> 5.9%</td><td> 22.9%</td><td> 15.5%</td><td> 47.4%</td><td> 8.3%</td><td> 55.7%</td>

<td></td><td> 8.6%</td><td> 17.1%</td><td> 10.4%</td><td> 61.4%</td><td> 2.5%</td><td> 63.9%</td>

<td></td><td> 6.4%</td><td> 23.5%</td><td> 20.4%</td><td> 45.7%</td><td> 4.0%</td><td> 49.7%</td>

<td> 164</td><td> 8.4%</td><td> 26.9%</td><td> 22.6%</td><td> 39.2%</td><td> 2.9%</td><td> 42.1%</td>

can

ΓΜ and Amino acid position and current amino acid

<img file="PL2329032T3_D0003.tif" />

5.4% 4.0% 23.9% and 58.6% 8.0%

Amino acid position and current amino acid

<td colspan="2"> 164</td><td></td><td></td><td></td><td></td><td></td><td colspan="3">ί: ί ι</td>

<td rowspan="2">M LO tH</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>and:</td><td> .................</td><td></td><td></td><td> ........„.....</td><td>□ ί</td><td> (±</td><td>Οί</td><td></td>

<td>ABOUT about</td><td></td><td></td><td></td><td></td><td></td><td></td><td colspan="2"></td><td></td>

<td>ii IA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> .....................</td><td></td>

<td> 133</td><td> &lt;</td><td> &lt;</td><td> &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>about ΓΊ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>10 rd</td><td> &lt;</td><td></td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td> &gt; _</td><td> &gt;......</td><td></td><td></td>

<td> 72</td><td>LU</td><td></td><td></td><td>LU</td><td>LUI</td><td>LU</td><td>LU</td><td></td><td></td>

<td>tH 10</td><td>LU</td><td>LU</td><td>LU</td><td> &lt;</td><td> &lt;</td><td> &lt;</td><td> &lt;</td><td>LU</td><td></td>

<td>m in</td><td> -..............</td><td> ---------------</td><td></td><td></td><td> _____________</td><td> . -........ ..</td><td></td><td></td><td></td>

<td>c ro</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>c V about c</td><td>θ ' ABOUT</td><td>θ ' THIS</td><td>σ ' 00</td><td>ι-4</td><td>sp θ '* ι-</td><td>ο</td><td>'ζ σ ' Ο</td><td>'ζ σ ' THIS</td><td></td>

<td></td><td> 00</td><td>r &lt;</td><td>Γ '.'</td><td> 00</td><td> 00</td><td>ζ</td><td> 00</td><td>Γν</td><td></td>

<td>and Linolan</td><td>θ ' rx Ltd. " m</td><td>SP σ ' φ What LD</td><td> 58,8%</td><td>% ρ σ ' Μ ° C LD</td><td>ί 57.6%</td><td>ο ζ</td><td>SP ο ** ι-4 οο " LD</td><td>'ζ σ ' THIS οο " LT)</td><td></td>

<td rowspan="2">Oleate stearate</td><td>σ ' ABOUT Φ CM</td><td>£ M ΓΜ</td><td>θ ' ι-Ι WHAT ΓΜ</td><td>sp ΓΩ Φ * ΓΜ</td><td>SP θ '* ΓΩ Φ * ΓΜ</td><td>ο ζ</td><td>χΡ ο · ^ &lt;Ο Φ ΓΜ</td><td>χ ° θ '* * £ Γ Φ ΓΜ</td><td></td>

<td>sp o * t — 1 φ "</td><td>£ cn ΓΌ</td><td>χΡ σ ' ° λ ΓΩ</td><td>sP θ '* ΓΩ Φ *</td><td>sP θ '* ΓΩ φ "</td><td>ο ζ</td><td>χΡ ο ** CD φ "</td><td>χΡ θ '* ΓΜ Φ</td><td></td>

<td>c ro</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>c £</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Ζ-X ο</td>

<td>Ê ro 0.</td><td>n. in</td><td>'ζ θ ' ΊΟ Ίθ "</td><td>&gt; Ρ ο * Φ Ο</td><td>'ξ σ ' Ο ο "</td><td>sP θ ' LO</td><td>ο ζ</td><td>* 5 σ ' Ο Ί&gt; "</td><td>χΡ θ '* &lt; » φ "</td><td>ο α (0 C Ν ο</td>

<td>ε</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Ο</td>

<td>δ * c here</td><td>ΓΜΙ nI</td><td>ml nI</td><td>ΜΙ Ν |</td><td>Φ ΓΜ</td><td>Φ ΓΜ</td><td>LD ΓΜ</td><td>LD ΓΜ</td><td>ΊΟ ΓΜ</td><td>S / ο Ζ</td>

205 [0463] Table 6 below presents the data for thermal stability of the top 25 palmitase hits selected based on the second-order test described above. These data were obtained using hits expressed in an E. coli HMS174 host. The clones were distributed in 96-well plates and incubated for 10 minutes at room temperature (RT), 45, 50 or 55 ° C, after which they were tested at RT against MeUMB palmitate. Percent residual activity was determined by dividing the activity after incubation at each temperature by the activity after incubation at RT. The current mutations, and examples of palmitate selectivity and soybean oil activity are also shown for each palmitase. SEQ ID NO: 2 retained approximately 15% activity after incubation for 10 min. at 50 ° C but had no activity after incubation at 55 ° C.

Table 6

<td></td><td colspan="3">% Stability</td><td colspan="7">Amino acid position and current amino acid</td>

<td>Enzyme</td><td>55C</td><td>50C</td><td>45C</td><td> 61</td><td> 72</td><td> 116</td><td> 133</td><td> 151</td><td> 163</td><td>164 different</td>

<td> 27</td><td></td><td> 23,0%</td><td> 62,7%</td><td>E</td><td>K</td><td>V</td><td colspan="2"> .....~...............1....................... 1</td><td></td><td>1 ΐ</td>

<td> 28</td><td></td><td> 22,8%</td><td> 62,1%</td><td>................ - E</td><td>K</td><td>V</td><td></td><td></td><td>R</td><td> )</td>

<td> 29</td><td></td><td></td><td> 55,9%</td><td>E</td><td>K</td><td></td><td></td><td></td><td>R</td><td>ί and</td>

<td> 30</td><td> 24,1%</td><td> 68,8%</td><td> 75,6%</td><td>E</td><td>E</td><td>V</td><td>AND</td><td></td><td></td><td>and</td>

<td> 31</td><td> 22,0%</td><td> 68,1%</td><td> 82,5%</td><td>E</td><td>E</td><td>V</td><td>AND</td><td></td><td>R</td><td></td>

<td> 32</td><td></td><td> 27,1%</td><td> 58,4%</td><td>E</td><td>E</td><td>V</td><td></td><td></td><td>R</td><td></td>

<td> 33</td><td></td><td> 26,1%</td><td> 56,6%</td><td>E</td><td>E</td><td>V</td><td></td><td>AND</td><td>R</td><td></td>

<td> 34</td><td></td><td> 24,7%</td><td> 54,0%</td><td>E</td><td>E</td><td>V</td><td>AND</td><td></td><td></td><td>R i</td>

<td> 35</td><td> 8,1%</td><td> 64,1%,</td><td> 67,6%</td><td>E</td><td>E</td><td>T</td><td></td><td></td><td>R</td><td></td>

<td> 36</td><td> 10,3%</td><td> 53,8%</td><td> 75,7%</td><td>E</td><td>E</td><td>AND</td><td>AND</td><td></td><td>R</td><td></td>

<td> 37</td><td> 9,2%</td><td> 54,8%</td><td> 61,5%</td><td>E</td><td>E</td><td>AND</td><td></td><td></td><td>R</td><td>and</td>

<td> 38</td><td></td><td> 45,4%</td><td> 68,4%</td><td>E</td><td>E</td><td>AND</td><td>AND</td><td>AND</td><td></td><td></td>

<td> 39</td><td></td><td> 22,9%</td><td> 61,9%</td><td>E</td><td>E</td><td>AND</td><td>AND</td><td></td><td></td><td>R</td>

<td> 40</td><td></td><td> 35,3%</td><td> 77,1%</td><td>E</td><td></td><td>V</td><td colspan="2">A |</td><td>R</td><td></td>

<td> 41</td><td></td><td> 30,6%</td><td> 70,2%</td><td>E</td><td></td><td>V</td><td>AND</td><td></td><td></td><td></td>

<td> 42</td><td> 20,3%</td><td> 71,8%</td><td> 79,3%</td><td>E</td><td></td><td>AND</td><td>AND</td><td>G</td><td>R</td><td></td>

<td> 43</td><td></td><td></td><td> 64,2%</td><td>E</td><td></td><td>AND</td><td>AND</td><td></td><td></td><td></td>

206 % Stability, amino acid position and current amino acid and _

<td>Enzyme</td><td>55C</td><td>50C</td><td>45C</td><td> 61</td><td> 72</td><td> 116</td><td> 133</td><td> 151</td><td> 163</td><td> 164</td><td>different</td>

<td> 44</td><td></td><td></td><td> 63,2%</td><td>AND</td><td>K</td><td>V</td><td></td><td></td><td></td><td></td><td></td>

<td> 45</td><td></td><td></td><td> 56,0%</td><td>AND</td><td>K</td><td>V</td><td>AND</td><td>AND</td><td>R</td><td></td><td></td>

<td> 46</td><td></td><td></td><td> 80,7%</td><td>^AND</td><td>K</td><td>AND</td><td></td><td></td><td>R</td><td></td><td></td>

<td> 47</td><td> 22,2%</td><td> 71,8%</td><td> 88,1%</td><td>AND</td><td>E</td><td>V</td><td>AND</td><td></td><td>R</td><td></td><td></td>

<td> 48</td><td></td><td> 60,6%</td><td> 83,2%</td><td>AND</td><td>E</td><td>V</td><td></td><td>AND</td><td>R</td><td></td><td></td>

<td> 49</td><td></td><td> 50,7%</td><td> 68,0%</td><td>AND</td><td>E</td><td>V</td><td></td><td></td><td></td><td></td><td></td>

<td> 50</td><td></td><td> 50,2%</td><td> 77,2%</td><td>AND</td><td>E</td><td>V</td><td></td><td></td><td>R</td><td></td><td></td>

<td> 51</td><td></td><td> 21,1%</td><td> 53,6%</td><td>AND</td><td>E</td><td>V</td><td></td><td>AND</td><td></td><td>R</td><td></td>

<td> 52</td><td></td><td></td><td> 56,5%</td><td>AND</td><td>E</td><td>Q</td><td>AND</td><td>AND</td><td>R</td><td></td><td></td>

<td> 53</td><td></td><td> 73,6%</td><td> 118,3%</td><td>AND</td><td>E</td><td>AND</td><td>AND</td><td></td><td> . -</td><td></td><td></td>

<td> 54</td><td></td><td> 69,2%</td><td> 110,7%</td><td>AND</td><td>E</td><td>AND</td><td>AND</td><td></td><td>R</td><td></td><td></td>

<td> 55</td><td></td><td></td><td> 82,2%</td><td>AND</td><td></td><td>r .... ....... V</td><td></td><td></td><td></td><td></td><td></td>

<td> 56</td><td></td><td></td><td> 51,4%</td><td>AND</td><td></td><td>AND</td><td>AND</td><td></td><td></td><td></td><td></td>

<td> 57</td><td></td><td></td><td> 82,8%</td><td></td><td>κ ¹</td><td>V</td><td>AND</td><td>G</td><td></td><td></td><td></td>

<td> 58</td><td></td><td></td><td> 60,1%</td><td></td><td>K</td><td>V</td><td></td><td></td><td>R</td><td></td><td></td>

<td> 59</td><td></td><td></td><td> 58,3%</td><td></td><td>K</td><td>V</td><td>AND</td><td></td><td></td><td></td><td>P162S</td>

<td> 60</td><td></td><td></td><td> 57,9%</td><td></td><td>K</td><td>V</td><td>AND</td><td></td><td>R</td><td></td><td>V62F</td>

<td> 61</td><td></td><td></td><td> 56,3%</td><td></td><td>K</td><td>V</td><td>AND</td><td colspan="3">^R</td><td></td>

<td> 62</td><td></td><td></td><td> 51,9%</td><td></td><td>K</td><td>Q</td><td>AND</td><td></td><td>R</td><td></td><td></td>

<td> 63</td><td></td><td></td><td> 74,8%</td><td></td><td>K</td><td>AND</td><td></td><td></td><td></td><td></td><td></td>

<td> 64</td><td></td><td></td><td> 58,8%</td><td></td><td>K</td><td>AND</td><td>AND</td><td></td><td></td><td></td><td></td>

<td> 65</td><td></td><td> 49,6%</td><td> 72,0%</td><td></td><td>E</td><td>V</td><td></td><td></td><td>R</td><td></td><td></td>

<td> 66</td><td></td><td> 46,3%</td><td> 66,0%</td><td></td><td>E</td><td>V</td><td></td><td></td><td></td><td></td><td></td>

<td> 67</td><td></td><td></td><td> 55,1%</td><td></td><td>E</td><td>V</td><td>AND</td><td></td><td>R</td><td></td><td></td>

<td> 68</td><td></td><td></td><td> 51,7%</td><td></td><td>E</td><td>V</td><td>AND</td><td>AND</td><td></td><td></td><td></td>

<td> 69</td><td></td><td> 23,6%</td><td> 54,6%</td><td></td><td>E</td><td>AND</td><td></td><td></td><td></td><td></td><td></td>

<td> 70</td><td></td><td></td><td> 76,2%</td><td></td><td>E</td><td>AND</td><td></td><td></td><td></td><td>R</td><td></td>

207 and% Stability

Amino acid position and current amino acid

<td>55C enzyme</td><td>50C</td><td>45C</td><td> 61</td><td>72 Jll6</td><td> 133</td><td> 151</td><td> 163</td><td> 164</td><td>different</td>

<td>71 ................. Γ ...................... and</td><td></td><td> 59,2%</td><td></td><td>V and</td><td>AND</td><td></td><td></td><td></td><td></td>

<td>⁷²</td><td></td><td> 51,8%</td><td></td><td>V</td><td>AND</td><td></td><td>R</td><td></td><td></td>

Example 6: Laboratory protocol for the evaluation of palmitase, stearatase or saturase enzyme candidates [0464] Exemplary enzymes and polypeptides were expressed in a Pseudomonas system (Dow Global

Technologies Inc, pub. PCT Int. U.S. Patent No. 20050130160). The nucleic acid encoding the enzyme or polypeptide was introduced into the pMYC vector (Dow Global Technologies Inc., US Patent Application Publication No. 20050130160) and then introduced into the auxotrophic host Pseudomonas fiuorescens by electroporation. Transformed cells were selected for growth in minimal medium. After reaching optimal host cell density, expression of the enzyme or polypeptide was induced with IPTG [0465] The following procedure is used to assess the ability of the enzyme or other polypeptide to hydrolyze the oil sample. The palmitase enzyme was added to 1 kg of crude oil, resulting in a 20% final water content. The mixture was then homogenized in a lid mixer and incubated at room temperature using a paddle mixer. Portions (0.5 mL) were taken after Oh, 21h, 43h, 65h and 72h and treated for conversion to FAMA and analyzed by GC as described in Example 8.

[0466] The above procedure was used with SEQ ID NO: 2, the sample was crude soybean oil. After 72 hours, samples of both the untreated oil and the enzyme treated oil gave the results presented in Table 7.

Table 7

<td>Fatty acid composition</td><td>Untreated oil (%)</td><td>Oil treated with enzyme (%)</td>

<td>C16: 0</td><td> 11,1</td><td> 3,7</td>

<td>C18: 0</td><td> 4,1</td><td> 4,2</td>

<td>C18: l</td><td> 22,1</td><td> 24,3</td>

<td>C18: 2</td><td> 54,5</td><td> 59,5</td>

<td>C18: 3</td><td> 8,2</td><td> 8,3</td>

[0467] These results indicate a significant reduction in the amount of palmitic acid (C16: 0), such a reduction is considered desirable.

Example 7: Evaluation of lipases, saturases or palmitases showing homoloaie sequences to an exemplary polypeptide with SEO ID NO: 2

208 [0468] Several homologous lipase sequences were subcloned into the pMAL-c2x vector (New England Biolabs, USA) using the xi cloning method (Genlantis, USA). Constructs containing SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14 or SEQ ID NO: 16 were transformed into an Escherichia coli ArcticExpress RP host (Stratagene, USA) for expression. Expression of lipases is under the control of a promoter that is induced by IPTG after achieving optimal density of host cells. Recombinant enzymes were tested in soybean oil for FA selectivity (Table 8). Lipases containing SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 14 or SEQ ID NO: 16 were expressed and cleaved from the MBP fusion tag using standard conditions. Media was inoculated with a single colony

LB containing 20 pg / ml gentamicin and shaken at 200 rpm per minute overnight at 30 ° C. This overnight culture was inoculated with fresh LB medium containing 20 pg / ml gentamicin with an OD600 reading of 0.05. This culture was shaken at 200 rev. per minute and 30 ° C until an OD600 reading of 0.5 is obtained. The cultures were transferred to 12 ° C with shaking at 200 rpm. per minute and allowed to equilibrate to a lower temperature before inducing lipase expression by adding 0.5 mM IPTG followed by a further increase for 24 hours. Cells were harvested by centrifugation, resuspended in pH 8 Tris buffer containing NaCl, CaCL, DNase and Iizozym, and then lysed by sonication. Cell lysates were clarified by centrifugation. Enzymes were cleaved from MBP by incubating lipase-MBP fusion with factor Xa for 6 hours at room temperature, followed by an additional 18 hours at 12 ° C. All clarified lysates with intact, active recombinant enzymes when tested in soybean oil showed strong and similar preferences for paimitate hydrolysis over other FAs (Table 8).

Table 8

Similarity to SEQ id] (%) of hydrolyzed NO: 2 fatty acids

<td>Enzyme</td><td>Identity</td><td>Similarity</td><td colspan="2">Palmitate j Stearate</td><td>oleate</td><td>linoleate</td><td>linoleate</td>

<td>Oil</td><td>ON</td><td>ON</td><td></td><td></td><td></td><td></td><td></td>

<td>soybean</td><td></td><td></td><td> 11,0%</td><td> 4,3%</td><td> 24,9%</td><td> 59,7%</td><td> 5,1%</td>

<td>SEQ ID</td><td> 100%</td><td> 100</td><td></td><td></td><td></td><td></td><td></td>

<td>NO: 2</td><td></td><td></td><td> 50,9%</td><td> 5,1%</td><td> 16,9%</td><td> 18,1%</td><td> 9,0%</td>

<td>SEQ ID</td><td> 27%</td><td> 42%</td><td colspan="2">................ on ................................</td><td></td><td></td><td></td>

<td>NO: 14</td><td></td><td></td><td> 45,8%</td><td> 2,0</td><td> 14,2%</td><td> 37,9%</td><td> 0,0%</td>

<td>SEQ ID</td><td> 47%</td><td> 62%</td><td></td><td colspan="2">j</td><td></td><td></td>

<td>NO: 12</td><td></td><td colspan="2"> 50,4%</td><td> 4,1%</td><td> 16,1%</td><td colspan="2">23.4% and 6.0%</td>

<td>SEQ ID</td><td> 41%</td><td> 56%</td><td></td><td></td><td></td><td></td><td></td>

<td>NO: 6</td><td></td><td></td><td> 37,0 %</td><td> 6,2%</td><td> 28,5%</td><td> 20,7%</td><td> 7,6%</td>

209

Example 8: Method for converting free fatty acids or triglycerides to fatty acid methyl esters (FAMA) and quantifying FAMA using the oasis chromatography method [0469] Fatty acids released from lipids, triglycerides, fats or oils as a result of lipases, e.g. saturases, palmitases and / or stearatase can be quantified directly by LCMS using the method described in Example 2. Alternatively, these hydrolyzed fatty acids can be converted to fatty acid methyl esters (FAMA) by acid-catalyzed methanolysis and then quantified by Gas Chromatography (GC). In this example:

• The oil after reaction with lipases, eg, saturases, palmitases and / or stearatases, is treated by adding 1 mL of extraction solvent (CHCl₃: MeOH: 4N HCl (2: 1: 0.075)) per 0.5 mL reaction volume.

• 45 pL portion extracted with oil transferred to a 4 mL screw top vial. A small stir bar was inserted into each vial, followed by the addition of 2 mL hexane and 400 pL 20% (v / v) MeOH in HCl.

• The vials were then sealed and heated with stirring for 15 minutes. The vials were then removed from the heat and allowed to cool, after which 800 pL H was added₂ABOUT.

• The mixture was then vortexed and a sample (500 pL) from the top layer of hexane containing FAMA was transferred to the autosampler vial for GC. FAMA was added to each 500 pL sample with 0.5 mg / mL C15: 0 as an internal standard.

[0470] FAMA synthesized using this method is then analyzed by gas chromatography using the following operating parameters:

• Equipment is Hewlett Packard 6890 Series GC with autosampler • The column used is a Supelco SP-2380 Fused Si capillary column 30 mx 0.25 mm and film thickness 0.2 pm • The injector and detector are set to 260 ° C; helium stream as carrier gas was set at 0.6mL / min; the oven was set to an initial temperature of 150 ° C.

• Samples (1 mL) were injected at a 10: 1 injection split. The GC method used includes:

• Ramp 1: 4C / min for 10 minutes = 190 ° C • Ramp 2: 15C / min for 4 min = 250 ° C • Maintenance: 250 ° C for 2 min [0471] Triglyceride FA can also be analyzed by converting to FAMA, even in the presence of hydrolysed fatty acids. Using a combination of the above method and the following method, it can be used to determine lipase selectivity for fatty acid, e.g., saturase, palmitase and / or stearatase, and the effect of the enzyme on oil.

210

The method of FA analysis bound to glycerol (or other alcohols) uses base-catalyzed methanolysis:

• oil after reaction with lipases, for example saturases, palmitases and / or stearatases, is treated by adding 1 mL of extraction solvent (CHCl₃: MeOH: 4N HCl (2: 1: 0.075)) per 0.5 mL reaction volume.

• 45 pL portion extracted with oil transferred to a microtube centrifuge. 500 pL of heptane are added followed by 50 pL of a 2N methanolic KOH solution.

• The mixture is vortexed for 30 seconds and centrifuged.

• A portion (50 pL) from the top layer of heptane containing FAMA is transferred to the autosampler vial and combined with 450 pL hexane containing the internal standard C15: 0.

• FAMA analysis by GC is as specified above.

Example 9: Example evolution to improve palmitase thermal tolerance using GSSM technology^sm.

[0472] An exemplary palmitase of the invention that contained the D61E, R72K, and V163R mutations in SEQ ID NO: 2 (Enzyme 17, Table 5, above) was selected as the leading selectivity mutant from previous evolution rounds (Examples 4 and 5, above). Further evolution to improve the thermal tolerance of the leading selective enzyme (SEQ ID NO: 2 with mutations D61E, R72K, and V163R) was performed using GSSM technology (see, e.g., US Patent No. 6,171,820).

[0473] Briefly, GSSM evolution was carried out by introducing point mutations using degenerate oligonucleotides for one amino acid position at a time, so that each original codon can be substituted with any of 20 naturally encoded amino acids. The library was constructed in a pDOW-Kan vector, analyzed by agarose gel, then treated with DPNI and transformed into competent £ cells. // which XLlBlue. Colonies were grown, collected and sequenced. Colonies were combined and DNA was isolated using the Qiagen mini-prep kit (catalog number 27106, Qiagen, Valencia, CA). Pseudomonas fluorescens competent cells were transformed with DNA. The Pseudomonas fluorescens host was obtained from Dow Global Technologies Inc. (US Patent Application Publication No. 20050130160, US Patent Application Publication No. US 20050186666 and US Patent Application Publication No. 20060110747). The pDOW-Kan vector was constructed by adding a kanamycin resistance tag to pDOW1169 (Dow Global Technologies Inc., US Patent Application Publication No. 20080058262). Cells were grown in M9 medium (Dow Global Technologies Inc., US Patent Application Publication No. 20050186666) with the addition of kanamycin and uracil. All of the following examples, which describe the use of pDOW-kan vector, Pseudomonas fluorescens and M9 medium, all refer to the same pDOW-kan vector, Pseudomonas fluorescens and M9 medium described above.

211 [0474] The GSSM library was screened in a 384-well system. First and second order HTP screening was performed using UMB palmitate (as described above in Example 3). To identify mutations that retain activity while the enzyme is at elevated temperature, a test was performed by incubating the fluorogenic substrate with the entire cell lysate at 54 ° C for 30 minutes, preceded by a 30-minute incubation of the entire cell lysate with buffer for 30 minutes at 54 ° C to ensure that the enzyme reached 54 ° C before substrate loading. The fluorescence of each mutant (see column 2, Table 9) and each mutant with a fluorescence reading above 2 standard deviations above the control was measured as "hit" - that is, a mutant with sufficiently increased thermal tolerance. As a result of screening with palmitate-UMB, 117 hits with unique amino acid changes were identified (see Table 9 below). Amino acid changes in 117 hits with a single mutation occurred for 50 residues; 22% of proteins showed amino acid changes. 117 hits were further evaluated for performance in standard 5g crude oil tests (see Example 5) at 25 ° C and 45 ° C.

The results are shown in Table 10 below.

212

Table 9:

<td>Enzyme *</td><td>Palmita ynianUMB</td><td>orig inaln s kodo n</td><td>New kodo n</td><td>ORIGINAL Stop Limit Current amino acid</td><td>Well you ami nok mustache</td><td>Poz.r eszty amines OK aso input</td><td>Additional mutations</td>

<td>TT1</td><td> 43670</td><td>TAC</td><td>CTT</td><td>Ϋ</td><td>L</td><td> 7</td><td></td>

<td>ΓΓ2</td><td> 40432</td><td>GCC</td><td>CTG</td><td>AND</td><td>L</td><td> 15</td><td></td>

<td>TT3</td><td> 21247</td><td>GCC</td><td>ATG</td><td>AND</td><td>M</td><td> 15</td><td></td>

<td>TT4</td><td> 65535</td><td>GAT</td><td>TGG</td><td>D</td><td>IN</td><td> 16</td><td></td>

<td>TT5</td><td> 23779</td><td>ATG</td><td>ATT</td><td>M</td><td> 1</td><td> 31</td><td></td>

<td>TT6</td><td> 65535</td><td>GGC</td><td>GAG</td><td>G</td><td>E</td><td> 32</td><td></td>

<td>TT7</td><td>6553S</td><td>GGC</td><td>CCT</td><td>G</td><td>P</td><td> 32</td><td></td>

<td>TT8</td><td> 26222</td><td>CTG</td><td>ATG</td><td>L</td><td>M</td><td> 34</td><td></td>

<td>TT9</td><td>G0900</td><td>CTG</td><td>ATT</td><td>L</td><td> 1</td><td> 43</td><td></td>

<td>TT10</td><td> 28008</td><td>TTC</td><td>TTT</td><td>F</td><td>F</td><td> 46</td><td></td>

<td>TT11</td><td> 65535</td><td>GCC</td><td>TGT</td><td>AND</td><td>C</td><td> 48</td><td></td>

<td>TT12</td><td> 55409</td><td>GCC</td><td>ATG</td><td>AND</td><td>M</td><td> 48</td><td></td>

<td>TT13</td><td> 65535</td><td>GCC</td><td>ACT</td><td>AND</td><td>T</td><td> 48</td><td></td>

<td>TT14</td><td> 65535</td><td>GAC</td><td>AAT</td><td>D</td><td>N</td><td> 49</td><td></td>

<td>TT15</td><td> 57979</td><td>GAC</td><td>CGT</td><td> 0</td><td>R</td><td> 49</td><td></td>

<td>TT16</td><td> 65535</td><td>GAC</td><td>TCT</td><td>D</td><td>S</td><td> 49</td><td></td>

<td>ΤΥ17</td><td> 23108</td><td>GCC</td><td>ATG</td><td>AND</td><td>M</td><td> 52</td><td></td>

<td>TT18</td><td> 65535</td><td>TCG</td><td>TTT</td><td>S</td><td>F</td><td> 68</td><td></td>

<td>TT19</td><td> 65535</td><td>TCG</td><td>TAT</td><td>S</td><td>Y</td><td> 68</td><td></td>

<td>TT20</td><td> 24003</td><td>CGG</td><td>GCT</td><td>R</td><td>AND</td><td> 85</td><td></td>

<td>TT21</td><td> 37005</td><td>CGG</td><td>GAT</td><td>R</td><td> 0</td><td> 85</td><td></td>

<td>TT22</td><td> 65535</td><td>CGG</td><td>CAG</td><td>R</td><td>Q</td><td> 85</td><td></td>

<td>TT23</td><td> 39478</td><td>CGG</td><td>TCT</td><td>R</td><td>S</td><td> 85</td><td></td>

<td>TT24</td><td> 65535</td><td>CGG</td><td>ACG</td><td>R</td><td>T</td><td> 65</td><td></td>

<td>TT25</td><td> 65535</td><td>CGG</td><td>TAT</td><td>R</td><td>Y</td><td> 85</td><td></td>

<td>TT26</td><td> 52743</td><td>GAG</td><td>AAG</td><td>E</td><td>K</td><td> 95</td><td>(GCG) 92 (GCT)</td>

<td>TT27</td><td> 30520</td><td>GCG</td><td>GTT</td><td>AND</td><td>V</td><td> 92</td><td></td>

<td>TT28</td><td> 18239</td><td>GCG</td><td>GAG</td><td>AND</td><td>E</td><td> 92</td><td></td>

<td>TT29</td><td> 25819</td><td>GAG</td><td>GAT</td><td>E ·</td><td> 0</td><td> 95</td><td></td>

<td>TT30</td><td> 21805</td><td>GAG</td><td>GCT</td><td>E</td><td>AND</td><td> 95</td><td></td>

<td>TT31</td><td> 42531</td><td>GCG</td><td>AAG</td><td>AND</td><td>K</td><td> 96</td><td></td>

<td>TT32 I</td><td> 23046</td><td>GCG</td><td>AGG</td><td>AND</td><td>R</td><td> 96</td><td></td>

213

<td>ΤΤ33</td><td> 19889</td><td>GCC</td><td>TCG</td><td>AND</td><td>s</td><td> 97</td><td></td>

<td>ΤΤ34</td><td> .17199</td><td>AAG</td><td>CGT</td><td>K</td><td>R</td><td> 101</td><td></td>

<td>ΤΤ35</td><td> 33316</td><td>GTG</td><td>TTG</td><td>V</td><td>L</td><td> 104</td><td></td>

<td>TT36</td><td> 45591</td><td>TAT</td><td>CTT</td><td>Y</td><td>L</td><td>1Ϊ3</td><td></td>

<td>ΤΤ37</td><td> 31618</td><td>GAG</td><td>GCG</td><td>E</td><td>AND</td><td> 116</td><td></td>

<td>ΤΤ38</td><td> 65535</td><td>GAG</td><td>TGT</td><td>E</td><td>C</td><td> 116</td><td> •</td>

<td>ΤΤ39</td><td> 65535</td><td>GAG</td><td>GAT</td><td>E</td><td>p</td><td> 116</td><td></td>

<td>ΤΤ40</td><td> 36485</td><td>GAG</td><td>TTT</td><td>E</td><td>F</td><td> 116</td><td></td>

<td>ΤΤ41</td><td> 65535</td><td>GAG</td><td>ATT</td><td>E</td><td> 1</td><td> 116</td><td>&lt;TTC) 135 (TTT)</td>

<td>ΤΤ42</td><td> 48338</td><td>GAG</td><td>ATT</td><td>E</td><td> 1</td><td> 116</td><td></td>

<td>ΤΤ43</td><td> 38696</td><td>GAG</td><td>CTT</td><td>E</td><td>L</td><td> 116</td><td></td>

<td>ΤΤ44</td><td> 58069</td><td>GAG</td><td>AAT</td><td>E</td><td>N</td><td> 116</td><td></td>

<td>ΤΤ45</td><td> 65535</td><td>GAG</td><td>CAG</td><td>E</td><td>Q</td><td> 116</td><td></td>

<td>ΤΤ46</td><td> 42681</td><td>GAG</td><td>AGT</td><td>E</td><td>S</td><td> 116</td><td></td>

<td>ΤΤ47</td><td> 65535</td><td>GAG</td><td>ACT</td><td>E</td><td>T</td><td> 116</td><td></td>

<td>ΤΤ48</td><td> 65535</td><td>GAG</td><td>GTT</td><td>E</td><td>V</td><td> 116</td><td></td>

<td>ΤΤ49</td><td> 60385</td><td>GAG</td><td>TGG</td><td>E</td><td>in</td><td> 116</td><td></td>

<td>ΤΤ50</td><td> 42924</td><td>GAG</td><td>TAT</td><td>E</td><td>Y</td><td> 116</td><td></td>

<td>ΤΤ51</td><td> 18591</td><td>CTG</td><td>ATG</td><td>L</td><td>M</td><td> 117</td><td></td>

<td>ΤΤ52</td><td> 65535</td><td>AAG</td><td>AGG</td><td>K</td><td>R</td><td> 120</td><td></td>

<td>ΤΤ53</td><td> 50984</td><td>AGT</td><td>GCT</td><td>S</td><td>AND</td><td> 133</td><td></td>

<td>ΤΤ54</td><td> 65535</td><td>GCG</td><td>TCG</td><td>AND</td><td>S</td><td> 136</td><td></td>

<td>ΊΓΓ55</td><td> 32933</td><td>GGC</td><td>TTT</td><td>G</td><td>F</td><td> 137</td><td></td>

<td>ΤΤ56</td><td> 65535</td><td>CTC</td><td>ATG</td><td>L</td><td>M</td><td> 139</td><td></td>

<td>ΤΤ57</td><td> 54461</td><td>CAC</td><td>AGG</td><td>H</td><td>R</td><td> 140’</td><td></td>

<td>ΤΤ58</td><td> 20741</td><td>AAC</td><td>TGG</td><td>N</td><td>IN</td><td> 142</td><td></td>

<td>TT 59</td><td> 25491</td><td>GCG</td><td>ATT</td><td>AND</td><td> 1</td><td> 144</td><td></td>

<td>ΤΤ60</td><td> 19150</td><td>GCG</td><td>TTG</td><td>AND</td><td>L</td><td> 144</td><td></td>

<td>ΤΤ61</td><td> 54979</td><td>GCG</td><td>ATG</td><td>AND</td><td>M</td><td> 144</td><td></td>

<td>ΤΤ62</td><td> 33234</td><td>GCG</td><td>GTG</td><td>AND</td><td>V</td><td> 144</td><td></td>

<td>ΤΤ63</td><td> 51208</td><td>GAG</td><td>CAT</td><td>E</td><td>H</td><td> 149</td><td></td>

<td>ΤΤ64</td><td> 21503</td><td>GCG</td><td>ATT</td><td>AND</td><td> 1</td><td> 150</td><td></td>

<td>ΤΤ65</td><td> 51405</td><td>GCG</td><td>ATG</td><td>AND</td><td>M</td><td> 150</td><td></td>

<td>ΤΤ66</td><td> 65535</td><td>GCG</td><td>TGG</td><td>AND</td><td>IN</td><td> 150</td><td></td>

<td>ΤΤ67</td><td> 25795</td><td>AGC</td><td>AAT</td><td>• s</td><td>N</td><td> 153</td><td></td>

<td>ΤΤ68</td><td>Ι8Ι56</td><td>AGC</td><td>GGT</td><td>S</td><td>G</td><td> 153</td><td></td>

<td>ΤΤ69</td><td> 65535</td><td>AAC</td><td>GAC</td><td>N</td><td>P</td><td> 158</td><td></td>

<td>ΊΓΤ70</td><td> 65535</td><td>CCG</td><td>GGT</td><td>P</td><td>G</td><td> 162</td><td></td>

<td>ΤΤ71</td><td> 18622</td><td>CCG</td><td>AAG</td><td>^p</td><td>K</td><td> 162</td><td></td>

214

<td>ΤΤ72</td><td> 55639</td><td>CCG</td><td>TCG</td><td>P</td><td>S</td><td> 162</td><td>R163F</td>

<td>ΤΤ73</td><td> 28167</td><td>CCG</td><td>TCG</td><td>P</td><td>S</td><td> 162</td><td>I167L</td>

<td>ΤΤ74</td><td> 20774</td><td>CCG</td><td>TCG</td><td>P</td><td>s</td><td> 162</td><td></td>

<td>ΤΤ75</td><td> 65535</td><td>GTG</td><td>ATT</td><td>V</td><td>l</td><td> 183</td><td></td>

<td>ΤΤ76</td><td> 20029</td><td>CAG</td><td>GCG</td><td>Q</td><td>AND</td><td> 186</td><td></td>

<td>ΤΤ77</td><td> 21399</td><td>CAG</td><td>GAG</td><td> 0</td><td>E</td><td> 166</td><td></td>

<td>ΤΤ78</td><td> 32863</td><td>CAG</td><td>ACG</td><td>Q</td><td>T</td><td> 166</td><td></td>

<td>ΤΤ79</td><td> 25576</td><td>ATT</td><td>TTT</td><td>AND</td><td>F</td><td> 167</td><td></td>

<td>ΤΤ80</td><td> 16567</td><td>ATT</td><td>AAG</td><td>AND</td><td>K</td><td> 167</td><td></td>

<td>ΤΤ81</td><td> 26239</td><td>ATT</td><td>CTG</td><td>AND</td><td>L</td><td>¹⁶⁷</td><td></td>

<td>ΤΤ82</td><td> 19330</td><td>ATT</td><td>CGT</td><td>AND</td><td>R</td><td> 167</td><td></td>

<td>ΤΤ83</td><td> 31994</td><td>ATT</td><td>TAT</td><td>AND</td><td>Y</td><td> 167</td><td></td>

<td>πβ4</td><td> €5535</td><td>CGC</td><td>CAT</td><td>R</td><td>H</td><td> 172</td><td></td>

<td>ΤΤ85</td><td> 38603</td><td>CGC</td><td>AAG</td><td>R</td><td>K</td><td> 172</td><td></td>

<td>ΤΤ86</td><td> 24428</td><td>CGC</td><td>CTT</td><td>R</td><td>L</td><td> 172</td><td></td>

<td>ΤΤ87</td><td> 37581</td><td>CGC</td><td>TAT</td><td>R</td><td>Y</td><td> 172</td><td></td>

<td>ττββ</td><td> 30655</td><td>CTC</td><td>AAG</td><td>L</td><td>K</td><td> 180</td><td></td>

<td>ΤΤ89</td><td> 65535</td><td>CTC</td><td>AGG</td><td>L</td><td>R</td><td> 180</td><td></td>

<td>TT90</td><td> 64023</td><td>GCG</td><td>TGT</td><td>AND</td><td>C</td><td> 185</td><td></td>

<td>ΤΤ91</td><td> 41225</td><td>GCG</td><td>AAT</td><td>AND</td><td>N</td><td> 185</td><td></td>

<td>ΤΤ92</td><td> 65535</td><td>GAA</td><td>GCG</td><td>E</td><td>AND</td><td>Ϊ90</td><td></td>

<td>ΤΓ93</td><td> 65535</td><td>GAA '</td><td>AAG</td><td>E</td><td>K</td><td> 190</td><td></td>

<td>ΤΤ94</td><td> 65535</td><td>GAA</td><td>ATG</td><td>E</td><td>M</td><td> 190</td><td></td>

<td>TT9S</td><td> 65535</td><td>GAA</td><td>CAG</td><td>E</td><td>Q</td><td> 190</td><td></td>

<td>ΤΤ96</td><td> 65535</td><td>GAA</td><td>AGG</td><td>E</td><td>R</td><td> 190</td><td></td>

<td>π97</td><td> 17914</td><td>CTA</td><td>ATT</td><td>L</td><td> 1</td><td> 200</td><td></td>

<td>ΤΤ98</td><td> 18910</td><td>CTA</td><td>GTA</td><td>L</td><td>V</td><td> 200</td><td>E201Y</td>

<td>ΤΤ99</td><td> 35222</td><td>CTA</td><td>GTT</td><td>L</td><td>V</td><td> 200</td><td> *</td>

<td>ττιοο</td><td> 38817</td><td>GAG</td><td>TAT</td><td>E</td><td>Ϋ</td><td> 201</td><td></td>

<td>ΤΤ101</td><td> 65535</td><td>GCG</td><td>CAT</td><td>AND'</td><td>H</td><td> 203</td><td></td>

<td>ΤΤ102</td><td> 65535</td><td>GCG</td><td>CCG</td><td>AND</td><td>P</td><td> 203</td><td></td>

<td>ΤΤ103</td><td> 65535</td><td>GCG</td><td>AGG</td><td>AND</td><td>R</td><td> 203</td><td></td>

<td>ΤΤ104</td><td> 47048</td><td>ATG</td><td>CTT</td><td>M</td><td>L</td><td> 207</td><td></td>

<td>ΤΤ105</td><td> 65535</td><td>ACC</td><td>CAT</td><td>T</td><td>H</td><td> 214</td><td></td>

<td>ΤΤ106</td><td> 65535</td><td>ACC</td><td>AAG</td><td>T</td><td>K</td><td> 214</td><td></td>

<td>ΤΤ107</td><td> 48095</td><td>ACC</td><td>AGG</td><td>T</td><td>R</td><td> 214</td><td></td>

<td>ΤΤ108</td><td> 35774</td><td>ACC</td><td>TCG</td><td>T</td><td>S</td><td> 214</td><td></td>

<td>ΤΤ109</td><td> 65535</td><td>ACC</td><td>GTT</td><td>T</td><td>V</td><td> 214</td><td></td>

<td>ΤΤ110</td><td> 53546</td><td>GGG</td><td>GCG</td><td>G</td><td>AND</td><td> 215</td><td></td>

215

<td>ΤΤ111</td><td> 65535</td><td>CTG</td><td>ATT</td><td>L</td><td> 1</td><td> 222</td><td></td>

<td>TM 12</td><td> 24987</td><td>GCG</td><td>TCT</td><td>AND</td><td>S</td><td> 225</td><td></td>

<td>TT113</td><td> 26618</td><td>CGG</td><td>TAT</td><td>R</td><td>Y</td><td> 163</td><td></td>

<td>τη 14</td><td> 22246</td><td>CGG</td><td>ATG</td><td>R</td><td>M</td><td> 163</td><td></td>

<td>TT115</td><td> 42199</td><td>CGG</td><td>ACG</td><td>R</td><td>T</td><td> 163</td><td></td>

<td>τη ie</td><td> 42127</td><td>CGG</td><td>TTG</td><td>R</td><td>L</td><td> 163</td><td></td>

<td>ΤΤ117</td><td> 33933</td><td>CGG</td><td>TGT</td><td>R</td><td>C</td><td> 163</td><td></td>

216

Table 10

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217

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218

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219

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<td></td><td></td><td>£ «r-</td><td>ϊ £ 9&gt; K</td><td>§ this 00</td><td>g 00</td><td></td><td></td><td></td><td>£ td</td><td></td><td></td><td></td>

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220

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221

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222

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224 [0475] A codon change of a given residue that does not cause an amino acid change is referred to as a silent mutation. Silent mutations are obtained as hits due to increased expression relative to the parent enzyme and were observed at 37 points in the GSSM screening (Table 11, below).

Table 11

<td>Original codon</td><td>new codon</td><td>amino acid</td><td>amino acid</td><td>Place amino acid</td>

<td>GCG</td><td>GCT</td><td>AND</td><td>AND</td><td> 35</td>

<td>GGC</td><td>GGT</td><td>G</td><td>G</td><td> 37</td>

<td>CTG</td><td>CTT</td><td>L</td><td>L</td><td> 41</td>

<td>GGC</td><td>GGA</td><td>G</td><td>G</td><td> 45</td>

<td>GCC</td><td>GCT</td><td>AND</td><td>AND</td><td> 52</td>

<td>CGG</td><td>CGA</td><td>R</td><td>R</td><td> 89</td>

<td>GCC</td><td>GCT</td><td>AND</td><td>AND</td><td> 97</td>

<td>GTG</td><td>GTT</td><td>V</td><td>V</td><td> 102</td>

<td>AGC</td><td>AGT</td><td>S</td><td>S</td><td> 108</td>

<td>CTC</td><td>TTG</td><td>L</td><td>L</td><td> 109</td>

<td>GCG</td><td>GCT</td><td>AND</td><td>AND</td><td>¹¹⁴</td>

<td>CGC</td><td>CGG</td><td>R</td><td>R</td><td> 115</td>

<td>CTG</td><td>CTT</td><td>L</td><td>L</td><td> 117</td>

<td>CTG</td><td>TTG</td><td>L</td><td>L</td><td> 124</td>

<td>CGG</td><td>AGG</td><td>R</td><td>R</td><td> 126</td>

<td>GTC</td><td>GTG</td><td>V</td><td>V</td><td> 128</td>

<td>GTC</td><td>GTG</td><td>V</td><td>V</td><td> 129</td>

<td>AGT</td><td>TCT</td><td>s</td><td>S '</td><td> 133</td>

<td>GGC</td><td>GGT</td><td>G</td><td>G</td><td> 137</td>

<td>GAC</td><td>GAT</td><td>D</td><td>D</td><td> 138</td>

<td>CTC</td><td>CTT</td><td>L '</td><td>L</td><td> 139</td>

<td>AAC</td><td>AAT </td><td>Ν ' </td><td>N</td><td> 142</td>

<td>CGC</td><td>AGG</td><td>R</td><td>R</td><td> 172</td>

<td>GTG</td><td>GTT</td><td>V</td><td>V</td><td> 183</td>

<td>ACC</td><td>ACG</td><td>T</td><td>T</td><td> 188</td>

<td>TCG</td><td>AGT</td><td>s</td><td>s</td><td> 192</td>

<td>CCC</td><td>CCT</td><td>P</td><td>P</td><td> 193</td>

225

<td>CTG</td><td>CTT</td><td>L</td><td>L</td><td> 202</td>

<td>GCG</td><td>GCT</td><td>AND</td><td>AND</td><td> 203</td>

<td>ACC</td><td>ACT</td><td>T</td><td>T</td><td> 205</td>

<td>CAC</td><td>CAT</td><td>H</td><td>H</td><td> 206</td>

<td>GGC</td><td>GGT</td><td>G</td><td>G</td><td> 208</td>

<td>TCG .....</td><td>TCT</td><td>S</td><td>S</td><td> 212</td>

<td>CTG</td><td>CTT</td><td>L</td><td>L</td><td> 222</td>

<td>GTC</td><td>GTG</td><td>V</td><td>V</td><td> 223</td>

<td>CGG</td><td>AGG</td><td>R</td><td>R</td><td> 226</td>

<td>CTC</td><td>TTG</td><td>L</td><td>L</td><td> 227</td>

TMCA^sm [0476] The most effective mutations identified during GSSM evolution (Example 9, above) were assessed in first and second order oil tests against inclusion in TMCA evolution. Tests with 5g of crude oil were carried out as described in Example 5, but with a 10% water content, using 117 unique hits at 25 ° C and 45 ° C. The oil profile was evaluated after 24 and 48 hours of reaction. The primary hits were subjected to a second oil test at 25 ° C, 45 ° C and 55 ° C. Samples were taken after 3, 24 and 48 hours to assess oil profiles. The most effective mutants are given in Table 12 below, with the palmitate remaining in the oil presented as a percentage of total bound fatty acids. To assess the effect of indirect purification using lye, tests at 45 ° C were refined with caustic refining (CR) by adding 11% NaOH (estimated at 7% FFA) and heated under constant stirring at 60 ° C. Fresh enzyme was added to the refined oil at 20% water content, homogenized and an oil reaction was carried out with stirring for an additional 24 hours at 45 ° C. The final palmitate content of the lye-refined samples is shown in the last column, "CR 24h". [0477] From the most effective mutations shown in Table 12, 15 mutants (in bold italics in Table 12) were selected for combination using TMCA technology. The TMCA library was constructed as described in PCT publication number WO 2009/018449 and as further described below. This library consists of 9216 unique variants.

226

Table 12:

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228 [0478] Tailored Multi-Site Combinatorial Assembly (TMCA) technology, TMCA technology (cf. PCT Publication No. WO 09/018449), includes a method of producing multiple daughter polynucleotides containing different combinations of different mutations, in many places. This method can be carried out, in part, by combining at least one or more of the following steps:

Obtaining sequence information for a ("first" or "template") polynucleotide. For example, the first or template sequence may be wild type (e.g., SEQ ID NO: 2 with mutations D61E, R72K, and V163R) or a mutated sequence. Sequence information may refer to the complete polynucleotide (e.g., gene or open reading frame) or partial regions of interest, such as the sequence encoding the binding site, binding specificity, catalysis or substrate specificity.

Identifying three or more mutations of interest along the first or template polynucleotide sequence. For example, the mutations can be at 3, 4, 5, 6, 8, 10, 12, 20 or more in the first or template sequence. Positions can be predetermined by absolute position or by the context of surrounding residues or homology. For example, for TMCA purposes of palmitase polypeptides, the best amino acid changes with respect to thermal tolerance that resulted in increased enzyme efficiency were included as mutations of interest. Sequences flanking the position of the mutation on both sides may be known. Each mutation position may contain two or more mutations, such as in relation to different amino acids. Such mutations can be identified using Gene Site Saturation Mutagenesis technology^SM (GSSM^sm) as described herein and e.g. in US Patent Nos. 6,171,820; 6562594; and 6764835. Delivery of primers (e.g., synthetic oligonucleotides) containing mutations of interest. A primer is provided for each mutation of interest. Thus, a first or template polynucleotide having 3 mutations of interest can use 3 primers at this position. The primer can also be provided as a set of primers containing a degenerate position such that the mutation of interest is within the range of each naturally occurring nucleotide or amino acid or a subset of that range. For example, a primer collection can be provided that promotes mutations to aliphatic amino acids. Primers can be prepared as forward or reverse primers, or the primers can be prepared as at least one forward primer and at least one reverse primer. When mutations are located

229 close together, it may be beneficial to use primers containing mutations for more than one position or a combination of different mutations at many positions. Delivery of a polynucleotide containing a template sequence. The first or template polynucleotide may be circular or may be in a supercoiled conformation, such as a plasmid or vector for cloning, sequencing or expressing. The polynucleotide may be single-stranded ("ssDNA") or may be double-stranded ("dsDNA"). For example, in the TCMA method, the superdrive matrix ("sc") dsDNA is subjected to a heating step at 95 ° C for 1 min (see Levy, Nucleic Acid Res, 28 (12). E57 e57 (i-vii) (2000) ).

Addition of primers to the matrix polynucleotide in the reaction mixture.

The primers and template polynucleotide are combined under conditions that allow the primers to fuse with the template polynucleotide. In the TMCA protocol, primers are added to the polynucleotide in a single reaction mixture, but can be added in multiple reactions.

Carrying out the elongation reaction using a polymerase. Extension products (e.g., as "daughter" or "modified extended polynucleotide") can be amplified by conventional methods. Products can be analyzed for length, sequence, desired properties of nucleic acids, or expressed as polypeptides. Other methods of analysis include in-situ hybridization, sequence screening or expression screening. The analysis may include one or more rounds of screening and selection of the desired property.

The products can also be transformed into a cell or other expression system, such as a cell-free system. The cell-free system may contain enzymes associated with DNA replication, repair, recombination, transcription or translaq. Exemplary hosts include bacterial, yeast, plant and animal cells and cell lines and include E. coli, Pseudomonas fiuorescens, Pichia pastoris and Aspergillus niger. For example, strains E can be used as hosts. every // XLl-Blue or Stbl2.

[0479] This method can be used with the same or different primers under different reaction conditions to promote products having different combinations or numbers of mutations.

[0480] By performing the above-described exemplary method, this protocol also provides one or more polynucleotides produced by the TMCA evolution method, which can then be screened or selected for the desired property. One or more daughter polynucleotides can be expressed as polypeptides and optionally screened or selected for the desired property. Thus, this implementation of the TMCA evolution protocol provides polynucleotides and encoded polypeptides, as well as libraries of such polynucleotides encoding such polypeptides. This implementation of the TMCA evolution protocol also provides a screening agent

230 libraries by screening or selecting the library to obtain one or more polynucleotides encoding one or more polypeptides with the desired activity.

[0481] The TMCA evolution protocol described in PCT Publication No. WO 2009/018449 includes a method of producing a number of modified polynucleotides. These methods typically include (a) adding at least three primers to a double-stranded polynucleotide matrix in a single reaction mixture, wherein the at least three primers do not overlap, and wherein each of the at least three primers contains at least one mutation, different from other primers, with at least one primer being a forward primer, which can fuse with a negative template strand and at least one primer is a reverse primer that can fuse with a positive template strand, and (b) subjecting the reaction mixture to an elongation reaction using a polymerase to produce multiple elongated modified polynucleotides from these at least three primers .

[0482] The TMCA evolution protocol described in PCT Publication No. WO 2009/018449 includes a method in which the cell is transformed with a series of elongated products that have not been treated with ligase. Many elongated modified polynucleotides are recovered from the cell. The recovered series of modified polynucleotides are analyzed, for example, by expressing at least one of many elongated modified polynucleotides and analyzing the polypeptide expressed therefrom. Many elongated modified polynucleotides containing mutations of interest are selected. In the TMCA evolution protocol, information is obtained regarding the template polynucleotide sequence and three or more mutations of interest can be identified along the length of the template polynucleotide. Products obtained as a result of elongation using a polymerase can be analyzed before transformation of many elongated modified products into a cell.

[0483] In the TMCA evolution protocol, products obtained as a result of polymerase elongation are treated with an enzyme, e.g., a restriction enzyme, such as a DpnI restriction enzyme, thereby destroying the polynucleotide matrix sequence. The treated products can be transformed into cells, e.g. E.coli cells. In the TMCA evolution protocol, at least two, or at least three, or at least four, or at least five or at least six, or at least seven, or at least eight, or at least nine, or at least ten or every at least eleven or at least twelve or more primers. Each primer contains one point mutation. Two forward or two reverse primers contain different changes at the same position of the polynucleotide template. At least one primer includes at least two changes at different positions of the polynucleotide template. In yet another embodiment, at least one starter

231 it contains at least two changes in different positions, and at least two forward or reverse primers contain different changes at the same position of the polynucleotide template. [0484] In the TMCA evolution protocol, the forward primers are grouped into the forward primers group, and the reverse primers are grouped into the reverse primers group, and the primers in the forward primers group and the primers in the reverse primer group, are independently normalized to have the same concentration in the appropriate group, regardless of position in the polynucleotide matrix, and wherein after normalization the same number of forward and reverse primers are added to the reaction mixture. In this normalization method, the combination of certain items may be biased. The bias may be caused, for example, by a relatively low concentration of primers in one position containing a single primer relative to a position containing multiple primers. "Position bias" refers to the resulting polynucleotides that have a strong preference for incorporating primers in a single position over other positions within its forward and reverse primer group. This results in a combination of modified polynucleotides that have a high percentage of mutations within a single primer position, but a low percentage of mutations at a different position within its forward and reverse primer groups. This bias is unfavorable when the goal of TMCA is to generate daughter polynucleotides containing all possible combinations of matrix changes. The bias may be corrected, for example, by normalizing the primers as a set in each position so that they are equal.

[0485] In the TMCA evolution protocol, primer normalization is performed by organizing primers into multiple groups depending on their location on the polynucleotide template, where primers covering the same selected region on the template are in one group; normalizing the grouped primers in each group so that they have equal concentrations; combining forward primers within one group with a forward primer group and normalizing the concentration between each forward primer group so that they are equal; combining reverse primers within one group with a group of reverse primers and normalizing the concentration between each group of reverse primers so that they are equal; and adding an equal amount of combined forward and reverse primers to the reaction. No bias was observed in relation to the combination of items. In the TMCA evolution protocol, a set of degenerate primers is provided, each containing a degenerate position, wherein the mutation of interest is a set of different nucleotides at the degenerate position. A set of degenerate primers comprising at least one degenerate codon corresponding to at least one codon of a polynucleotide template and at least one adjacent sequence that is homologous to the codon adjacent sequence of the polynucleotide template is provided. The degenerate codon is Ν, Ν, Ν and codes each of 20 naturally

232 occurring amino acids. The degenerate codon codes for less than 20 naturally occurring amino acids.

[0486] The TMCA evolution protocol described in PCT Publication No. WO 2009/018449 includes a method of producing a number of mutated modified polynucleotides of interest. These methods typically include (a) adding at least two primers to a double-stranded polynucleotide matrix in a single reaction mixture, wherein the at least two primers do not overlap, and each of the at least two primers contains at least one mutation, different from other primers (primer), where at least one primer is a forward primer, which can fuse with a negative template strand and at least one primer is a reverse primer that can fuse with a positive template strand, and (b) subjecting the reaction mixture to an elongation reaction using a polymerase to produce multiple elongated modified polynucleotides from these at least two primers , (c) treating many extended modified polynucleotides with an enzyme, thereby destroying the polynucleotide matrix, (d) transforming treated elongated modified polynucleotides that were not ligated into the cell, (e) recovering multiple elongated modified polynucleotides from the cell, and (f) selecting multiple elongated modified polynucleotides containing mutations of interest.

[0487] In this example, 15 mutations selected for inclusion in the TMCA thermal tolerance library are shown in Table 13, below. Six DNA templates were used: SEQ ID NO: 2 with mutations D61E, R72K and V163R (parent), parent with E95K mutation, parent with P162G mutation, parent with P162K mutation, parent with E95K and P162G mutation and parent with E95K and P162K mutations. Parental plasmids with the E95K mutation, parent with the P162G mutation, parent with the P162K mutation were passaged by E ci9 // XLlBlue competent cells for DNA methylation. Six separate TMCA evolutions were performed, one for each of the templates in combination with the oligonucleotides shown in Table 14 below.

Table 13

MUTATION

NEW CODON

A48C

D49R

R85Y

E95K

El 161

TGT

CGT

TAT

AAG

ATT

E116L

CTT

233

<td colspan="3">MUTATION</td><td>NEW CODON</td>

<td>E116N</td><td></td><td></td><td>AAT</td>

<td>A144I</td><td></td><td></td><td>ATT</td>

<td>E149H</td><td></td><td></td><td>CAT</td>

<td>A150I</td><td></td><td></td><td>ATT</td>

<td>P162G</td><td></td><td></td><td>GGT</td>

<td>P162K</td><td></td><td></td><td>AAG</td>

<td>R172H</td><td></td><td></td><td>CAT</td>

<td>R172L</td><td></td><td></td><td>CTT</td>

<td colspan="3">A225S</td><td>TCT</td>

234

Table 14:

<img file="PL2329032T3_D0004.tif" />

235 [0488] Library members were amplified in a pDOW-kan vector, analyzed on an agarose gel, and treated with DpnI. Samples were then transformed into competent coliform XL1 Blue cells. Colonies were picked, cultured and sequenced. Sequencing results indicate that the E116L mutation and amino acid 144, 149 and 150 mutations were under-represented compared to the theoretical maximum. Accordingly, another TMCA library was generated using a standardized mixture of all six matrices. TMCA reactions were repeated using all oligonucleotides listed in Table 14, except for those for the E116I and E116N mutations. The number of primers used for the E116L mutation and for region 4 (amino acids 144, 149 and 150) has been doubled. Samples were amplified in pDOW-kan vector, analyzed by agarose gel and treated with DpnI. Samples were then transformed into competent E cells. coii XL1 Blue. Colonies were picked, cultured and sequenced. The introduction of the amino acid 144, 149, and 150 mutation was improved, but E116L was still under-represented compared to the theoretical maximum. DNA from both libraries was transformed into competent E. co / XLlBlue cells. Colonies were combined and DNA was isolated using the Qiagen mini-Prep kit. DNA was then used to transform competent Pseudomonas fiuorescens cells. Cells were cultured in LB medium with the addition of uracil (750 pg / ml) and kanamycin (50 pg / ml). A sufficient number of colonies was obtained for 10 times overloading the library. The library was reloaded ten times to offset the under-representation of E116L. [0489] The resulting thermally tolerant TMCA library was cultured in M9 minimal medium with the addition of uracil (750 [pg / ml) and kanamycin (50 pg / ml) and screened using the HTP screening described in Example 3, above. The library was incubated at 60 ° C for 30 minutes before and after the addition of UMB palmitate. Then, 384-well plates were read at excitation at 365 nm and emission at 460 nm. Each plate was cooled rapidly by centrifugation and the spectrophotometer was pre-heated to minimize artifacts in reading. The fluorescence values ​​of each of 384 wells are displayed. Hits were identified as showing> 2 standard deviations above the positive control (E116L single mutation). Second-order HTP screening tested the hits obtained from first-order HTP screening at two temperatures of 60 ° C and 63 ° C. Second order screening hits were defined as> 4 standard deviations above the mean for the positive control on the plate at 63 ° C and retaining> 50% activity between 60 ° C and 63 ° C. 318 unique single HTP hit sequences have been identified. These hits contained between 2 and 9 mutations. Standard tests with 5g crude oil (as described in Example 5) were performed with clarified lysates 318 hits. Of 318 hits from second order HTP, those that were more effective at lowering palmitate levels in an oil test at elevated temperatures (45 ° C or 60 ° C) than at 25 ° C were chosen as "oil hits" with heat tolerance. 57 oil hits showing heat tolerance were identified and characterized using additional oil tests (see Tables 15 and 16 below).

236

Further tests were carried out using 5g of oil and 9 oil hits were identified, showing, in addition to different levels of total activity, the preferred temperature performance profiles (see Table 17 below).

237

Table 15:

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<td rowspan="3">Palimitate 24 hours J</td><td>about 3</td><td>£ (0 AND</td><td>£ about</td><td>£ about</td><td>£ ω</td><td>£ AND</td><td>«0 WHAT</td><td>£ | x WHAT</td><td>£ WHAT</td><td>£ © τ-</td><td>£ «ο d Τ-</td><td>s? «9 ot</td><td>£ OT 0 ' T</td><td>£ | x</td><td>£ What</td><td>£ | X</td><td>£ 00 about TF</td><td>£ OT ABOUT T</td><td>ss about AND</td><td>s? T. about T</td><td>&lt; What</td>

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<td> §</td><td> § &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 8 &lt;</td><td></td><td></td><td></td><td></td><td></td><td>5 OT &lt;</td><td></td><td></td>

<td> 2</td><td>X ? UJ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 2</td><td>ί &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> $ &lt;</td><td></td><td></td><td></td>

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<td>VI AXIS</td><td></td><td></td><td></td><td></td><td>tt tf) ABOUT) UJ</td><td>tt §</td><td>tt 8 UJ</td><td></td><td></td><td></td><td>£ S</td><td>tt 8 UJ</td><td></td><td>tt OT AND UJ</td><td></td><td>tt tf) about UJ</td><td></td><td></td><td></td><td>tt 8 UJ</td>

<td> 90</td><td></td><td></td><td></td><td>X</td><td>8 x</td><td>and</td><td>X</td><td>ξ χ</td><td>fc δ</td><td></td><td>fc 00 GC</td><td></td><td>8 X</td><td></td><td>$ x</td><td>X</td><td>§ X</td><td>§ oe</td><td></td><td>§ X</td>

<td> 3</td><td>about 5 &lt;</td><td>at &lt;</td><td>about</td><td></td><td>about AND</td><td>υ 3</td><td>about 3</td><td>ο 3</td><td></td><td></td><td></td><td>about. 3</td><td> 8 3</td><td></td><td></td><td>at 3</td><td> 3 &lt;</td><td> 8 2</td><td>about</td><td> !</td>

<td>THIS 5 y oc - it's at C i_ * »= b ω □ Φ about £ 1—4 · -C</td><td>of l "</td><td>&lt;0 TF</td><td>about.</td><td>a&gt; of</td><td>What &lt;0</td><td>about tf</td><td>4 T " tf</td><td> 8</td><td> 3</td><td> 8</td><td>4 s</td><td>Ο » about·</td><td> «0</td><td> «0 00</td><td> 3</td><td> 3</td><td>3 τ-</td><td> 8</td><td>What about</td><td>a&gt; about</td>

238

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<td> &amp;</td><td>g</td><td></td><td> 2?</td><td> 28</td><td> 28</td><td> £</td><td rowspan="2">£ tt about</td><td> 28</td><td> 28</td><td> £</td><td> £</td><td> £</td><td> £</td><td> 3!</td><td>SP</td><td></td><td> 28</td><td> 2?</td><td> 2?</td><td> £</td><td> £</td><td> £</td><td></td><td> £</td><td> £</td><td></td><td> £</td>

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<td></td><td> £</td><td> £</td><td>s?</td><td> 38</td><td> £</td><td> 28</td><td> £</td><td> 28</td><td> £</td><td> £</td><td>£ tt</td><td> £</td><td> £</td><td> £</td><td> £</td><td> £</td><td> £</td><td> £</td><td></td><td> £</td><td> £</td><td> £</td><td> 2?</td><td>They are 8</td><td>r ·</td><td> 26</td><td>£ tt</td>

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<td> *</td><td> 2®</td><td> £</td><td> £</td><td>.beta.</td><td> £</td><td> £</td><td> ?</td><td> £</td><td> 28</td><td> £</td><td> &lt;5 *5</td><td> £</td><td>28 T ·</td><td>£ tt</td><td> £</td><td> £</td><td> £</td><td> £</td><td>£ &lt;L *</td><td></td><td>tt</td><td>ξ</td><td> 28</td><td></td><td> £</td><td>* 3 tt</td><td>£ • π</td>

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<td> 28</td><td> 28</td><td></td><td></td><td> 28</td><td> 28</td><td> £</td><td>g φ</td><td> 28</td><td> £</td><td> 28</td><td>t</td><td></td><td> 38</td><td> £</td><td>S</td><td> £</td><td> £</td><td> £</td><td> £</td><td> £</td><td> £</td><td></td><td> £</td><td> £</td><td> 28</td><td> £</td><td> £</td>

<td></td><td>r *</td><td></td><td>about</td><td></td><td> &lt;0</td><td> «</td><td>about'</td><td></td><td>IN</td><td>tt</td><td>Q</td><td>&lt;a</td><td>tt</td><td>about</td><td>fs</td><td>tt</td><td>tt</td><td></td><td>tt</td><td>tt</td><td>F-</td><td>tt</td><td></td><td></td><td></td><td>tt</td><td>M-</td>

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<td>m</td><td>r *</td><td>about</td><td>tt</td><td></td><td> &lt;4</td><td> &amp;</td><td>about</td><td> »</td><td></td><td>What</td><td>ABOUT</td><td>c *</td><td>ABOUT</td><td>ABOUT</td><td> ©</td><td> 3</td><td>about</td><td></td><td>«Μ</td><td>these</td><td>these</td><td>about</td><td>tt</td><td>AND**</td><td>about</td><td>tt</td><td>d</td>

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<td></td><td></td><td>in</td><td></td><td></td><td>tn</td><td></td><td></td><td></td><td></td><td></td><td>FS</td><td></td><td> 0</td><td></td><td></td><td>tn</td><td></td><td> 0</td><td></td><td></td><td></td><td> 0</td><td></td><td></td><td></td><td> 0</td><td></td>

<td>and</td><td></td><td>tt §</td><td></td><td></td><td>tt 3</td><td></td><td></td><td></td><td></td><td></td><td>and</td><td>and</td><td> 8 3</td><td></td><td></td><td>tt s</td><td></td><td> 9</td><td>ϋ</td><td></td><td></td><td>and</td><td></td><td></td><td></td><td> §</td><td></td>

<td>X</td><td>X</td><td>J.</td><td>X</td><td>X</td><td>X</td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>-AND</td><td>X</td><td>_J</td><td>_J</td><td></td><td>X</td><td></td><td> —1</td><td></td><td></td><td>X</td><td></td><td>X</td><td>X</td><td></td><td>-J</td><td>mJ</td>

<td>Pi</td><td>R</td><td>s</td><td>CM r *</td><td>R</td><td>CM N.</td><td></td><td></td><td>CM · r *</td><td>CM Γ * »</td><td>R</td><td>R</td><td>Pi</td><td>ri</td><td>R</td><td></td><td>B</td><td></td><td>B</td><td></td><td></td><td>B</td><td></td><td>B</td><td>R.</td><td></td><td>CM r *.</td><td>CM</td>

<td>oc</td><td>X</td><td>and</td><td>x</td><td>fc</td><td>x</td><td></td><td></td><td>fc</td><td>ά</td><td>X</td><td> &amp;</td><td>fc</td><td>ε</td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td></td><td>X</td><td></td><td>X</td><td>X</td><td></td><td>X</td><td>X</td>

<td>about</td><td>and?</td><td> 0</td><td rowspan="2">s tt</td><td>X</td><td>ll of</td><td></td><td></td><td>about</td><td>ABOUT</td><td>about</td><td>Ό</td><td>about</td><td> 0</td><td>ABOUT</td><td> 0</td><td> 2</td><td>g</td><td> 0</td><td>s</td><td> 0</td><td> 53</td><td> 0</td><td> 52</td><td> 0</td><td></td><td></td><td> 0</td>

<td> 3</td><td>s</td><td>CM tt</td><td> 8</td><td>CM WHAT</td><td></td><td></td><td> 8</td><td> 8</td><td> 8</td><td> 8</td><td> 8</td><td>s</td><td> 8</td><td>CM tt</td><td> 8</td><td> 8</td><td> 8</td><td> 8</td><td> 8</td><td> 8</td><td> 8</td><td> 8</td><td> 8</td><td></td><td></td><td>s</td>

<td>CL</td><td> 0-</td><td>fc</td><td>fc</td><td>fc</td><td>about.</td><td></td><td></td><td>fc</td><td>fc</td><td>α</td><td>fc</td><td>and.</td><td>ε</td><td>χ</td><td>fc</td><td>fc</td><td>fc</td><td>E</td><td> £</td><td> £</td><td>fc</td><td>at.</td><td>about.</td><td>about.</td><td></td><td>fc</td><td>about.</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ABOUT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>tt</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td> 3</td><td> 3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td> &lt;</td><td> &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td>2 tt</td><td>FROM tt</td><td> §</td><td> ®</td><td> 8</td><td> &lt;5</td><td></td><td></td><td> 3</td><td></td><td>tt</td><td></td><td>d</td><td> 25</td><td></td><td>tt</td><td>s</td><td></td><td></td><td></td><td> «5</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td> ▼“</td><td></td><td> **</td><td></td><td></td><td>r *</td><td></td><td></td><td>T</td><td></td><td>T</td><td></td><td></td><td> *»</td><td></td><td></td><td>T ·</td><td></td><td></td><td></td><td>J7</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td>S</td><td>ul</td><td>UJ</td><td>LU</td><td>LU</td><td>ul</td><td></td><td></td><td>ω</td><td></td><td>UJ</td><td></td><td>Ε</td><td>LU</td><td></td><td>E</td><td>E</td><td></td><td></td><td></td><td>UJ</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td>Y</td><td>Y</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>se</td><td> *</td><td>X</td><td></td><td></td><td></td><td>and £</td>

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239

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• hits 29, 40, 41 and 74 showing thermal tolerance also contain additional mutations (see additional mutations shown in Table 16 below),

240

Table 16: Additional mutations also present in hits showing heat tolerance 29, 40, 41 and 74.

<td colspan="2">Thermal tolerance Hit No.</td><td>Old codon</td><td>New codon</td><td>Old amino acid</td><td>New amino acid</td><td colspan="2">Position amino acid</td>

<td> 29</td><td></td><td>GTG</td><td>ATG</td><td>V</td><td>M</td><td> 83</td><td></td>

<td colspan="2"> 40</td><td>GCG</td><td>GCT</td><td>AND</td><td>AND</td><td> 92</td><td></td>

<td> 41</td><td></td><td>GCG</td><td>GCT</td><td>AND</td><td>AND</td><td> 92</td><td></td>

<td> 74</td><td></td><td>GCG</td><td>GCT</td><td>AND</td><td>AND</td><td> 92</td><td></td>

Table 17:

<td rowspan="2">Enzyme</td><td rowspan="2">25 ° C</td><td rowspan="2">palmitate an 3 hours 45 ° C</td><td></td><td></td><td rowspan="2">palmitate an 24 hours 45 ° C</td><td rowspan="2">60 ° C</td><td rowspan="2">25C</td><td rowspan="2">palmitate an 48 hours 45C</td><td rowspan="2">60C</td>

<td>60 ° C</td><td>25 ° C</td>

<td>r nEGATIVE and</td><td> 10,7 %</td><td> 10,6%</td><td> 10,7 %</td><td> 10,6 %</td><td> 10,5%</td><td> 10,6 %</td><td> 10,6 %</td><td> 10,5%</td><td> 10,6 %</td>

<td>macierzys this</td><td> 7,8%</td><td> 7,3%</td><td> 10,3 %</td><td> 5,4%</td><td> 6,2%</td><td> 10,3 %</td><td></td><td> 5,9%</td><td> 10,3 %</td>

<td> 29</td><td> 8,1%</td><td> 6,5%</td><td> 9,1%</td><td> 4,9%</td><td> 5,7%</td><td> 8,8%</td><td> 4,2%</td><td> 5,9%</td><td> 8,9%</td>

<td> 40</td><td> 10,5 %</td><td> 10,3%</td><td> 10,0 %</td><td> 10,0 %</td><td> 9,1%</td><td> 8,8%</td><td> 9,7%</td><td> 8,4%</td><td> 9,3%</td>

<td> 41</td><td> 10,5 %</td><td> 10,3%</td><td> 9,9%</td><td> 10,1 %</td><td> 8,4%</td><td> 8,7%</td><td> 9,8%</td><td> 8,8%</td><td> 8,6%</td>

<td> 74</td><td> 9,2%</td><td> 8,0%</td><td> 9,9%</td><td> 6,2%</td><td> 6,1%</td><td> 9,8%</td><td> 5,1%</td><td> 6,1%</td><td> 9,8%</td>

<td> 81</td><td> 10,3 %</td><td> 9,8%</td><td> 9,3%</td><td> 9,5%</td><td> 8,1%</td><td> 8,0%</td><td> 8,2%</td><td> 7,4%</td><td> 7,9%</td>

<td> 202</td><td> 10,2 %</td><td> 9,4%</td><td> 10,5 %</td><td> 9,3%</td><td> 7,4%</td><td> 10,4 %</td><td> 8,5%</td><td> 7,2%</td><td> 10,4 %</td>

<td> 204</td><td> 10,5 %</td><td> 10,1%</td><td> 9,7%</td><td> 10,0 %</td><td> 9,0%</td><td> 8,7%</td><td> 9,4%</td><td> 8,6%</td><td> 8,5%</td>

<td> 238</td><td> 10,4</td><td> 9,8%</td><td> 9,5%</td><td> 9,8%</td><td> 7,7%</td><td> 9,0%</td><td> 8,5%</td><td> 7,6%</td><td> 8,9%</td>

241

<td></td><td rowspan="2">25 ° C %</td><td>palmitate an 3 hours</td><td></td><td></td><td rowspan="2">palmitate an 24 hours 45 ° C</td><td rowspan="2">60 ° C</td><td></td><td rowspan="2">palmitate an 48 hours 45C</td><td rowspan="2">60C</td>

<td>Enzyme</td><td>45 ° C</td><td>60 ° C</td><td>25 ° C</td><td>25C</td>

<td> 244</td><td> 10,3</td><td> 9,6%</td><td> 10,4</td><td> 9,5%</td><td> 8,0%</td><td> 10,2</td><td> 8,3%</td><td> 7,5%</td><td> 10,2</td>

<td></td><td> %</td><td></td><td> %</td><td></td><td></td><td> %</td><td></td><td></td><td> %</td>

Example 11: Example evolution to obtain increased palmitase expression using TMCA technology^sm [0490] 37 single silent mutations identified during GSSM screening (see

Table 11, Example 9, above) were evaluated for expression (see Table 18, below). 50mL cultures were expressed in M9 medium, supplemented with uracil and kanamycin, in 250mL flasks. Hits were cultured at 30 ° C for 16 to 20 hours, before and after induction of 0.3 mM IPTG. Cells were pelleted by centrifugation and lysed using B-PER ™ (catalog number 78248, Pierce Protein Research Products, Rockford, IL). Part of the entire lysate was centrifuged for clarification. Both the total cell lysate and clarified lysates were tested for total protein concentration using the Bio-Rad Protein Assay Bio-Rad test, Hercules, CA, catalog number 500-0006) based on the Bradford method, relative to the activity of the fluorogenic palmitate-UMB substrate and by SDS -PAGE. The values ​​were normalized to the parent value set as 100%. 16 hits showing increased activity relative to the parent were identified).

Table 18:

<td></td><td colspan="2">% of parent activity</td><td colspan="2">% soluble</td>

<td>Place aa</td><td>all</td><td>cleaned</td><td>Activity</td><td>Protein</td>