Recombined hexose oxidase, method of obtaining same and application of that enzyme

Field of the Invention

The present invention provides a method of producing hexose oxidase by recombinant DNA technology and such an enzyme produced by this method, and its use in the food industry and other fields.

Technical basis and state of the art

Hexose oxidase (D-hexose: O2-oxidoreductase, EC 1.1.3.5) is an enzyme that in the presence of oxygen is able to oxidize D-glucose and several other reducing sugars including maltose, lactose and cellobiose to their respective lactones, with the following then hydrolysis to the corresponding aldobionic acids. Hexose oxidase differs from another oxidoreductase, glucose oxidase, which can only convert D-glucose that it can use a wider range of substrates. Oxidation catalyzed by hexose oxidase can be exemplified as follows:

D-glucose + O2 -> δ D-gluconolactone + H2O2, or D-galactose + O2 -> γ-D-galactogalactone + H2O2

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To date, hexose oxidase (hereinafter referred to as HOX) has been provided by isolating the enzyme from layman's species of red algae such as Iridophycus flaccidum (Bean and Hassid, 1956) and Chondrus crispus (Sullivan et al., 1973). In addition, the algae species Euthora cristata has been shown to produce glucose oxidase.

It has been reported that hexose oxidase isolated from these natural sources may have potential use in the manufacture of certain food products. Thus, hexose oxidase isolated from Iridophycus flaccidum has been shown to be able to convert lactose in milk to form the corresponding aldobionic acid, and there has been potential interest as a milk acidifying agent, e.g. replacing acidifying microbial cultures (Rand, 1972). To this end, hexose oxidase was mentioned as a more interesting enzyme than glucose oxidase, since the latter can be used in milk or non-glucose foods only after adding glucose or, in the case of dairy products, adding lactose-degrading enzyme, lactase, which breaks down lactose to glucose and galactose. Even if glucose thus becomes an available Substrate for glucose oxidase, it is obvious that only 50% of the lactase products will be used as a substrate by glucose oxidase, and thus it will not be an efficient acidifying agent in natural milk or dairy products.

The ability of oxygen oxidoreductases, including hexose oxidase, to form hydrogen peroxide that exerts an antibacterial effect has been used to improve the storage stability of certain food products, including cheese, butter and fruit juice, as disclosed in JP-B-73/016612. It has also been suggested that oxidoreductases may potentially be useful as oxygen scavengers or antioxidants in food products.

In the bakery and milling industry, the use of oxidizing agents such as iodides, peroxides, ascorbic acid, potassium bromide or azodicarbonamide is known to improve the suitability of baking flour to obtain dough with improved ductility, and thus having the desired strength and stability. The mechanism of oxidizing agents to achieve this effect is that flour proteins, such as e.g. gluten in wheat flour, contain thiol groups, which after oxidation form disulfide bonds, so that the protein forms a more stable matrix resulting in better dough quality and an increase in the volume and structure of baking crumb.

However, the use of some of the currently available oxidizing agents is questioned by consumers or is not allowed by regulatory bodies, so attempts are being made to find alternatives to these conventional dough and flour additives, and prior art suggests the use of glucose oxidase for the above purposes. Thus, US 2,783,150 discloses the addition of glucose oxidase to flour to improve the rheological characteristics of the dough. CA 2,012,723 discloses bread improving agents including cellulolytic enzymes and glucose oxidase, while JP-A-084848 suggests the use of a bread improving composition, including glucose oxidase and lipase.

However, the use of glucose oxidase as a dough and bread improving additive has the limitation that this enzyme requires the presence of glucose as a substrate to be effective in the dough system and the glucose content in cereal flour is generally low. Thus, in wheat flour glucose is present in an amount of 0-0.4% by weight, i.e. the flour may not contain glucose at all. Hence, the absence or low glucose content of the dough will be a factor limiting the use of glucose oxidase as a dough improving agent. In contrast, the maltose content is significantly higher in the freshly prepared dough, after which further maltose is formed due to β-amylase activity present in flour or added.

The current source of hexose oxidase is a crude or partially purified enzyme preparation, isolated by extraction from the abovementioned naturally occurring marine algae species. However, since the amount of hexose oxidase in the algae is small, it is obvious that the production of the enzyme in this way is too burdensome and expensive to ensure economical, commercial production of the enzyme from natural sources. Furthermore, ensuring sufficiently pure enzyme at an economic level is not easy to achieve in this way.

Thus, there is a significant industrial need to provide an alternative and more economical source of this industrially valuable enzyme that would be independent of natural sources as well as providing the enzyme in pure form, i.e. without contaminating enzymatic activities or other contaminating substances, including unwanted algae pigments and environmental contaminants that may occur in marine regions where algae species producing hexose oxidase grow.

In addition, the industrial availability of hexose oxidase with food-grade purity in appropriate quantities and economic prices should undoubtedly open up new applications for this enzyme, not only in the food industry but also in other industries, as will be discussed later. One example of such new uses of recombinant hexose oxidase in the food industry is the use of a dough improving agent, another example is the use of an active hexose oxidase polypeptide or a recombinant polypeptide-producing organism in the production of lactones.

Summary of the Invention

The present invention, through the use of recombinant DNA technology, has for the first time enabled the delivery of active hexose oxidase polypeptide in industrially appropriate amounts and of a quality and purity that makes the active hexose oxidase polypeptide of the present invention extremely useful for important industrial purposes, including for the production of food products and pharmaceuticals.

The invention in a first aspect provides a method of producing a polypeptide with hexose oxidase activity, comprising isolating or synthesizing a DNA fragment encoding the polypeptide, introducing a DNA fragment into a suitable host organism in which the DNA fragment is combined with an appropriate expression signal for the DNA fragment. culturing the host organism under conditions that lead to expression of the active hexose oxidase polypeptide and obtaining the polypeptide from the culture medium or from the host organism.

In a further aspect, the invention relates to an isolated polypeptide having hexose oxidase activity comprising at least one amino acid sequence selected from the group consisting of (i) Tyr-Glu-Pro-Tyr-Gly-Gly-Val-Pro (SEQ ID No. 1) , (ii) Ala-Ile-Ilk-Asn-Val-Thr-Gly-Lku-Val-Glu-Ser-Gly-Tyr-Asp-XXX-Gly-Tyr-X-ValSer-Ser (SEQ ID No. 2) , (iii) Asp-Lku-Pro-Mkt-Ser-Pro-Arg-Gly-Val-Πk-Ala-Ser-Asn-Leu-X-Phe (SEQ ID No. 3), (iv) Asp-Ser-Glu-Gly-Asn-Asp-Gly-Glu-Leu-Phe ^ -Ala-His-Thr (SEQ ID No. 4), (v) Tyr-Tyr-Phe- Lys (SEQ ID No. 5), (vi) Asp-Pro-Gly-Tyr-Ilk-Va [- Ie-Asp-'V-lAsn-Ala-Gly-'ΠlrrX-A_Sp (SEQ ID No. 6), (vii) Lku-Gln-Tyr-Gln-yhr-Tyr-Trp-Gln-Glu-Glu-Asp (SEQ ID No. 7), (viii) X-Ilk-Arg- Asp-Phe-yyr-Glu-Glu-Mkt (SEQ ID NO: 8) where X is an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val and their muteins and varieties.

In a still further aspect, the invention relates to a recombinant DNA molecule, comprising a DNA fragment encoding a polypeptide with hexose oxidase activity and a microbial cell containing said recombinant molecule.

In another aspect, the invention provides the use of a polypeptide with hexose oxidase activity or a microbial cell expressing such a polypeptide in the manufacture of food or animal feed products and in the manufacture of a pharmaceutical product, cosmetic products and dental hygiene.

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In another useful aspect, there is provided a method of reducing the sugar content of food products, comprising adding to the food products such an amount of the polypeptide or microbial cells disclosed herein that is sufficient to remove at least a portion of the sugar initially present in the food product, a method of making a bakery product from dough, comprising adding a polypeptide having hexose oxidase activity or microbial cells expressing said polypeptide to the dough, and a dough improving composition comprising the polypeptide or microbial cell of the present invention and at least one conventional dough component.

In another aspect, the invention relates to the use of a polypeptide or microbial cell of the present invention as an analytical reagent for measuring sugar content.

In an interesting aspect, the invention also provides the use of the polypeptide or microbial cell of the present invention in the production of a lactone in which the polypeptide and / or microbial cell is introduced into a reactor containing a carbohydrate that can be oxidized by the polypeptide and conducting the reaction in the reactor under conditions whose carbohydrate oxidizes.

Detailed disclosure of the invention

Hexose oxidases are produced naturally by some species of marine algae. These species are, among others found in the family Gigartinaceae, which belongs to the order Gigartinales. Examples of algae producing hexose oxidase species belonging to Gigartinaceae are Chondrus crispus and Iridophycus flaccidum. Also, algae species belonging to the order Cryptomeniales, including the species Euthora cristata, are potential sources of the active hexose oxidase polypeptide of the present invention. Thus, these algae species are potentially useful sources of hexose oxidase and DNA encoding active hexose oxidase polypeptides. As used herein, the term "active hexose oxidase polypeptide" means an enzyme that oxidizes at least D-glucose, D-galactose, D-mannose, maltose, lactose and cellobiose.

When using these natural sources for the isolation of native hexose oxidase, as was done in the prior art and in the present invention to identify algae material that could be used as a source of mRNA for use in constructing cDNA and as a starting point for constructing synthetic oligonucleotide DNA primers, the enzyme is usually isolated from the starting algae material by extraction using aqueous extraction media.

As starting material for such extraction, fresh algae can be used, as collected from marine growth areas, or can be used after drying, e.g., with air at ambient temperature or any suitable method of industrial drying, such as drying in circulating heated air or by lyophilization. To facilitate the subsequent extraction step, fresh or dried starting material may be subjected to e.g. milling or mixing.

As an aqueous extraction medium, buffer solutions with a pH in the range of 6 to 8, such as 0.1 M sodium phosphate buffer, 20 mM triethanolamine buffer or 20 mM Tris-HCl buffer are useful. Hexose oxidase is usually extracted from algae material by suspending the starting material in buffer and maintaining the suspension at a temperature in the range of 0-20 ° C, such as 5 ° C for 1 to 10 days, preferably shaking.

The suspended algae material is then separated from the aqueous medium by a suitable separation method, such as filtration, straining or centrifugation, and then hexose oxidase is obtained from the supernatant or filtrate. Optionally, the separated algae material is subjected to one or more extraction steps.

Because some marine algae contain colored pigments, such as phycocyanins, it may be necessary to subject the filtrate or supernatant to a further extraction step during which the pigments are removed. For example, pigments can be removed by treating the filtrate or the supernatant with an organic solvent in which the pigments are soluble, and then separating the solvent containing dissolved pigments from the aqueous medium.

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The extraction of hexose oxidase from the aqueous extraction medium can be carried out by any suitable conventional method allowing the isolation of proteins from the aqueous medium. These methods, examples of which will be described below, include methods such as ion exchange chromatography, optionally followed by a concentration step such as ultrafiltration. It is also possible to obtain the enzyme by adding a substance, such as (NH2SO4), which precipitates the protein, followed by separation of the precipitate and, optionally, subjecting the protein to conditions allowing its dissolution.

For the purposes of the invention, it is desirable to provide the enzyme in substantially pure form, e.g., as a preparation substantially free of other proteins or non-protein impurities, and the relatively crude enzyme preparation resulting from the above extraction and isolation is preferably subjected to further purification, gel filtration or chromatographic focusing steps such as are described, for example, below.

As mentioned above, the active hexose oxidase polypeptide of the present invention is provided by recombinant DNA technology, which enables it to be produced by culturing in a culture medium of a suitable host organism containing a gene encoding hexose oxidase and recovering the enzyme from these cells or from the culture medium.

The method of producing hexose oxidase provided herein includes the first step of isolating or constructing a DNA fragment encoding hexose oxidase. Several strategies are available for delivering such a DNA fragment. Thus, a DNA fragment can be isolated as such from an organism that naturally produces hexose oxidase. In order to identify the location of the DNA coding fragment, it is necessary to have RNA or DNA probes whose sequences under appropriate conditions hybridize with the DNA fragment sought, and then isolate the DNA fragment containing the coding sequence and clone it into the appropriate cloning vector.

Another suitable strategy that is disclosed in detail in the examples below is isolating mRNA from a hexose oxidase producing organism and using such mRNA as a starting point for constructing a cDNA library that can then be used in DNA synthesis in polymerase chain reaction (PCR) using oligonucleotide primers which are synthesized based on the hexose oxidase amino acid sequences. This strategy has been found to be suitable for obtaining a DNA fragment encoding hexose oxidase. For example, this strategy is described in detail below.

Synthetic oligonucleotides were prepared based on HOX-2 and HOX-3 peptide sequences, obtained as described below by digesting endoLys-C with 40,000 hexose oxidase polypeptide extracted from Chondrus crispus. PCR using the first strand cDNA as template and the sense HOX-2 primer and the HOX-3 antisense primer, gave a 407 bp DNA fragment. This fragment was introduced into E. coli vector, pT7 Blue and sequenced. In addition to the HOX-2 and HOX-3 peptide sequences, it was found that this 407 bp fragment also contained an open reading frame containing the HOX-4 and HOX-5 peptides of the above fragment of Chondrus crispus hexose oxidase, size 40000, the isolation of which is described below.

The sense and antisense oligonucleotide was synthesized based on a 407 bp fragment, making it possible for PCR to obtain two fragments, 800 and 1400 bp, using cDNA as template. These two fragments were cloned into pT7 Blue vector and sequenced. The DNA sequence of the 5 'fragment showed an open reading frame containing HOX-6 peptide, which was also isolated from a 40,000 hexose oxidase fragment derived from Chondrus crispus. Similarly, the 3 'fragment showed a reading frame containing the HOK-1 peptide, whose isolation is described below, and HOX-7 and HOX-8, obtained from a 29000 hexose oxidase fragment derived from Chondrus crispus, obtained by digestion with endoLys-C as well described below.

Based on the associated DNA sequences, an oligonucleotide corresponding to the 5 'end of the putative hox gene and an oligonucleotide corresponding to the 3' end of the gene were synthesized. These two oligonucleotides were used in PCR with the first strand cDNA as template, resulting in

187 218 DNA fragment about 1.8 kb in size. This fragment was cloned into E. coli vector and sequenced. The DNA sequence was identical to the combined sequence of the above 5 'end, 407 bp and 3' fragment, and it was concluded that this 1.8 kb sequence encodes both Chondrus crispus, 40,000 and 29,000 hexose oxidase fragments.

As is apparent to those skilled in the art, the above strategy for isolating a DNA fragment encoding an active hexose oxidase polypeptide, including isolating and characterizing hexose oxidase, can be used to construct such fragments encoding hexose oxidase from any source other than Chondrus crispus, including algae species marine species mentioned above, as well as from other plants and microbes.

Alternatively, the DNA sequence of the DNA fragment encoding the active hexose oxidase polypeptide may be constructed synthetically by established standard methods, e.g., the phosphoramidate method described by Beaucage and Caruthers (1981), or the method described by Matthes et al. (1984). Referring to the phosphoamidan method, oligonucleotides are synthesized, e.g. with an automatic DNA synthesizer, purified, aniled, ligated and cloned into a suitable vector.

In addition, the DNA fragment may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin generated by ligating fragments of synthetic, genomic or cDNA origin, fragments corresponding to different parts of the entire DNA fragment, according to standard techniques.

In a next step of the method of the present invention, the isolated or synthesized DNA fragment encoding the active hexose oxidase polypeptide is introduced into a suitable host organism in which the DNA fragment is operably linked to the appropriate expression signal for the DNA fragment. Such introduction may be carried out by methods well known to those skilled in the art, comprising constructing a vector containing the introduced fragment and transforming with this vector the host organism. Suitable vectors include plasmids that are capable of replication in the selected host organism. It is also contemplated that the DNA fragment may be integrated into the chromosome of the host organism e.g. by introducing the fragment into a transposable element, such as a transposon, and subjecting the mixture of a selected host organism and transposon to conditions in which the transposon integrates into the chromosome of the host organism and binds to the appropriate expression signal.

Referring to the invention, a DNA fragment encoding an active hexose oxidase polypeptide comprising a polypeptide gene produced by the methods described above or by any methods known in the art can be expressed in enzymatically active form using an expression vector. The expression vector usually contains components of a typical cloning vector, i.e. element allowing autonomous replication of the vector in the selected host organism and one or more phenotypic markers for selection. The expression vector contains control sequences encoding a promoter, operator, ribosome binding site, translation start signal and optionally a repressor gene or one or more activator genes. To enable secretion of the expressed polypeptide, a signal sequence may be inserted above the gene coding sequence. In the present context, the term "expression signal" includes any of the above control sequences, repressor or activator sequences, and signal sequences. For expression under the direction of control sequences, the gene encoding hexose oxidase is operably linked to the control sequences in a manner appropriate for expression. Promoter sequences that can be included in plasmid vectors and that can assist in transcription of the hexose oxidase gene include, but are not limited to the tac promoter, promoters derived from lambda phage including promoters ^PL^iPR ·

The expression vector carrying the DNA fragment of the present invention can be any vector that is capable of expressing the hexose oxidase gene in the selected host organism, and the choice of vector depends on the host cell into which it will be

187 218 introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector that occurs as an extrachromosomal unit whose replication is independent of chromosome replication, e.g. a plasmid, bacteriophage or extrachromosomal element, a minichromosome or an artificial chromosome. Alternatively, the vector may, when introduced into the host cell, integrate into the host cell genome and replicate with the chromosome.

In the vector, the DNA fragment encoding the active hexose oxidase polypeptide should be operably linked to the appropriate promoter sequence. The promoter can be any DNA sequence that provides transcriptional activity in a selected host organism and can be derived from genes encoding proteins homologous or heterologous to the host organism. Examples of suitable promoters for directing the transcription of DNA fragments of the present invention in the bacterial host are the E. coli lac operon promoter, Streptomyces coelicolor agagase gene dagA promoters, maltogenic amylase (amyM) Bacillus stearothermophilus gene promoters, amylase a (amyac) amylaqu Bacillus subtilis xylA and xy1B gene promoters.

For transcription in fungal species, examples of useful promoters are selected from the genes encoding Pichia pastoris alcohol oxidase, TAKA Aspergillus oryzae amylase, Rhizomucor miehei aspartyl proteinase, Aspergillus niger neutral α amylase, A. A. nigeriase α amylase, glucoamylase , alkaline protease Aspergillus oryzae, isomerase tri-phosphate Aspergillus oryzae or acetamidase Aspergillus nidulans. Examples of suitable promoters for expression in yeast species may be the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae. For expression in bacterial species such as E. coli, a suitable promoter may be selected from bacteriophage promoters, including the T7 promoter or the lambda bacteriophage promoter.

The vector comprising the DNA fragment encoding the active hexose oxidase polypeptide may also contain a selectable tag, e.g. a gene whose product compensates for a host organism defect, such as a mutation providing an auxotrophic phenotype, or the tag may provide antibiotic resistance or resistance to heavy metal ions.

The host organism of the present invention comprising a DNA construct or expression vector as described above can be used as a host cell in the recombinant production of the polypeptide of the present invention. The cell may be transformed with a DNA construct comprising the gene encoding the polypeptide of the present invention or, conveniently by integrating the DNA construct into the host chromosome. Such integration is generally considered beneficial because the DNA fragment is more stable held in the cell. Integration of DNA constructs with the host chromosome can be carried out by conventional methods such as, for example, homologous or heterologous recombination or using a transposable element. Alternatively, the host organism may be transformed with an expression vector as described above.

In accordance with the present invention, the host organism may be a higher organism cell, such as an animal cell, including a mammalian cell, a bird cell or insect cell, or a plant cell. However, in the most preferred embodiments, the host organism is a microbial cell, e.g. a bacterial or fungal cell, including a yeast cell.

Examples of a suitable host organism are Gram-positive bacterial species such as Bacillaceae, including Bacillus subtilis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautusuring Streptomyces such as Streptomyces murinus, lactic acid bacteria such as Lactococcus sp., Such as Lactococcus lactis, Lactobacillus sp., Including Lactobacillus reuteri, Leuconostoc sp. And Streptococcus sp. Alternatively, strains of Gram-negative species belonging to Enterobacteriaceae, including E. coli or Pseudomonaceae may be selected as the host organism.

The yeast host organism may preferably be selected from Saccharomyces species, including Saccharomyces cerevisiae or species belonging to Schizosajjharomyjes. Suitable host organisms among filamentous fungi include Aspergil12 species

187 218 lus, e.g. Aspergillus oryzae, Aspergillus nidulans or Aspergillus niger. Alternatively, strains of Fusarium species, e.g. Fusarium oxysporum, or Rhizomucor species such as Rhizomucor miehei may be used as the host organism. In one preferred embodiment, a strain of the species Pichia pastoris is used as the host organism.

Some of the above useful host organisms, such as fungal species or Gram-positive bacterial species can be transformed by a process that involves protoplast formation and protoplast transformation, followed by cell wall regeneration in a manner known per se.

For the production of the active hexose oxidase polypeptide, the cells of the recombinant host organism as described above are cultured under conditions which lead to expression of the polypeptide in a recoverable form. The medium used to grow the cells may be any conventional medium suitable for growing the host cells in question and obtaining the expression of the polypeptide. Suitable media are available from suppliers or can be prepared according to published recipes.

The resulting polypeptide is usually obtained from the culture medium by conventional procedures involving separating the cells from the medium by centrifugation or filtering, if necessary, after cell destruction, followed by precipitation of the protein components of the supernatant or filtrate, e.g. by adding a salt such as ammonium sulfate, followed by centrifugation. .

An industrially useful aspect of the invention is that microbial cultures, such as e.g. bacterial cultures, which are used to produce food and feed products, can be used as a host organism expressing the gene encoding the active hexose oxidase polypeptide. Thus, lactic acid starter cultures, which are used in the production of dairy products or other food products, such as meat or wine products, and which e.g. include one or more strains of lactic acid bacteria, can be used as host organism in which hexose oxidase can be produced directly in food products to which the initial culture is added.

Similarly, the gene encoding the hexose oxidase of the present invention can be introduced directly into cultures of initial lactic acid bacteria that are used as inoculates added to feed plants such as grass or corn or to waste protein products of animal origin such as fish and slaughterhouse waste material. for the production of silage for feeding animals. In this duty, the expression of hexose oxidase by silage inoculates should cause oxygen initially present in the feed or waste material to be silenced to be deprived of oxygen, which should inhibit the growth of aerobic putrefactive bacteria such as Gram-positive bacteria and yeast.

It is also contemplated that yeast cultures such as baker's yeast or yeast cultures that are used to make alcoholic beverages, including wine and beer, may be used as the host organism for the gene encoding the active hexose oxidase polypeptide of the present invention. For example, for such recombinant baker's yeast strains, the produced hexose oxidase should have a dough improving effect as described below.

From the above, it becomes apparent that the direct addition of recombinant microbial cultures expressing the hexose oxidase of the present invention to food products or any other products where hexose oxidase activity is desired can be used as an alternative to the addition of an isolated enzyme.

In further industrially important embodiments, recombinant microbial cultures expressing the active hexose oxidase polypeptide are used in a bioreactor to produce the enzyme or to produce lactones from the above-mentioned carbohydrates that can be oxidized by the active enzyme - hexose oxidase. For the latter application, the cells of the microbial cultures are preferably immobilized on a solid support such as a polymer material, which is preferably in the form of small particles

187 218 to provide a large surface for cell binding. Alternatively, an isolated enzyme may be used for the above purposes, also preferably associated with the solid support material. In this connection, cell or enzyme binding can be accomplished by any conventional method.

In other preferred embodiments of the invention, the polypeptide with hexose oxidase activity may be a fusion product, i.e. a polypeptide that, in addition to the active amino acid sequences of hexose oxidase, comprises further amino acid sequences having other useful activities. Thus, fusion polypeptides having one or more enzymatic activities in addition to hexose oxidase activity are included. Such additional enzymatic activity can be selected from enzymes capable of breaking down carbohydrates, such as lactase, amylases, including glucoamylases, glucanases, cellulases, hemicellulases, xylanases, lactases or other oxidoreductases such as glucose oxidase, galactose oxidase or pyranose oxidase, as well as proteases and peptidases, lipases or nucleases. The additional sequences selected for integration with the hexose oxidase polypeptide of the present invention depend on the product for which the enzymatically active product is intended. Thus, as an example, it is contemplated that the hexose oxidase fusion polypeptide for use in the preparation of a dairy product preferably includes lactase, protease or peptidase, and the fusion product for dough improvement may contain, as a fusion partner, any of the above carbohydrate-degrading enzymes. It is also evident that the microbial cells of the present invention that express the active hexose oxidase fusion polypeptide having additional enzymatic activity can be used to inoculate other food products and animal feed as described above.

It is also contemplated that a suitable fusion partner may be a sequence that provides altered hexose oxidase characteristics, such as solubility, or a sequence that can serve as a marker group that provides hexose oxidase with the ability to bind more strongly and selectively to a particular solid support for purification or immobilization. hexose oxidase polypeptide. Furthermore, it is within the scope of the present invention to provide a polypeptide as a chimeric product comprising partial sequences of active hexose oxidase polypeptides from various sources and encoded by a DNA fragment that has been constructed by combining DNA sequences encoding the active hexose oxidase polypeptide from these different sources into a DNA fragment encoding the entire chimeric polypeptide .

In one useful application, the method of the present invention is a method wherein the DNA fragment encoding the active hexose oxidase polypeptide comprises at least one DNA sequence encoding an amino acid sequence selected from the group consisting of (i) Tyr-Glu-Pro-Tyr-Gly-Gly-Val- Pro (Sequence Identifier No. 1), (ii) Ala-Ile-Ile-Asn-Val-Thr-Gly-Leu-Val-Glu-Ser-Gly-Tyr-Asp-XXX-Gly-Tyr-X-ValSer- Cheese (Identifier No. 2), (iii) Asp-Leu-Pro-Met-Ser-Pro-Arg-Gly-Val-Ile-Ala-Ser-Asn-Leu-X-Phe (SEQ ID No. 3), (iv) Asp -Ser-Glu-Gly-Asn-Asp-Gly-Glu-Leu-Phe-X-Ala-His-Thr (SEQ ID No. 4), (v) Tyr-Tyr-Phe-Lys (SEQ ID No. 5 ), (vi) Asp-Pro-Gly-Tyr-Ile-Val-Ile-Asp-Val-Asn-Ala-Gly-Thr-X-Asp (SEQ ID

No. 6), (vii) Leu-Gln-Tyr-Gln-Thr-Tyr-Trp-Gln-Glu-Glu-Asp (SEQ ID No. 7), (viii) X-Ile-Arg-Asp-Phe-Tyr -Glu-Glu-Met (SEQ ID NO: 8) where X is an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gin, Głu, Glx, Gly, His, Ile, Leu, Lys, Met , Phe, Pro, Ser, Thr, Trp, Tyr and Val and their muteins and varieties.

In the present context, the term "variation" means any modification of the active hexose oxidase polypeptide sequence that does not completely abolish the activity of hexose oxidase. Modifications may include deletion, residue replacement

187 218 amino acids present in the polypeptide in a form obtained from natural sources or in an already modified polypeptide sequence, or the modification may result in the introduction of additional amino acid residues into such polypeptide. Replacement of one or more amino acid residues can be carried out by modifying or replacing the codon or codons coding for the amino acid or amino acids whose replacement is preferred, e.g. by mutagenesis, in particular site-directed mutagenesis, using methods known per se. Similarly, the removal of one or more amino acids can be done by removing the corresponding codon or codons in the DNA fragment encoding the polypeptide of the present invention.

As mentioned above, the method of the present invention may further comprise purifying the polypeptide preparation initially obtained from the culture medium and / or from microorganisms. The purpose of the further step is to obtain an enzyme preparation in which the hexose oxidase polypeptide is in substantially pure form. The term "substantially pure form" means that the formulation is free of undesirable contaminants derived from the culture medium, host cells, or substances produced by these cells during culture. Thus, it is important for many applications that the polypeptide preparation resulting from the purification step is substantially free of any non-enzymatic activity. hexose oxidase. Purification methods depend on the degree of purity that is desired but are usually selected from conventional protein purification methods such as salting out, affinity or ion exchange chromatography procedures, including hydrophobic interaction chromatography, and gel filtration methods such as the method described in the examples below.

As mentioned above, the invention further relates to a polypeptide in isolated form having hexose oxidase activity, comprising at least one of the above amino acid sequences or a mutein or variant thereof as described above. Preferably, the polypeptide is produced according to the methods described above.

Depending on the method of production, in particular the particular host organism used, the polypeptide of the invention may be glycosylated to varying degrees, or may for certain reasons be preferably expressed in substantially non-glycosylated form.

In the most preferred embodiment of the invention, it is a polypeptide whose functional characteristics are identical to those of hexose oxidase naturally occurring in the Chondrus crispus algae species as described in the prior art. It has been found that such hexose oxidase extracted from algae after subjecting it to SDS-PAGE as described herein may have separate bands of 29,000,40000 and / or 60,000.

In order to achieve a generally economical use of the polypeptide, it is preferred that the enzyme has high specific activity over a wide pH range. Thus, it is preferred that the hexose oxidase of the present invention has at least an enzymatic activity in the pH range from 1 to 9, such as 2-9, including the range 5-9. It is contemplated that the pH range of activity, or the optimum pH of naturally occurring hexose oxidase, could be modified in the desired direction and to the desired extent, by modifying the enzyme as described above, or by random mutagenesis of the replicon or host organism containing DNA encoding hexose oxidase, followed by selection mutants showing the desired altered pH characteristics. Alternatively, such enzyme modifications may be directed to modify the heat tolerance and optimum temperature activity of the active hexose oxidase polypeptide, or change the enzyme's isoelectric point.

In addition, the polypeptide of the present invention is preferably enzymatically active in a wide temperature range from 10-90 ° C, e.g. in the range 15-80 ° C including 2060 ° C. In particular, it may be beneficial for certain purposes that the hexose oxidase polypeptide retains significant residual enzymatic activity at 70 ° C and higher, e.g. when the enzyme is intended for use in dough, where it may be useful to have hexose oxidase activity during the final stage followed by baking.

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The extent of use of hexose oxidase depends on the range of carbohydrates that can be used as substrates. Although hexose oxidase appears to have the highest substrate specificity for hexoses such as glucose, galactose and mannose, it has been found that the range of carbohydrates that can be used as substrates of the polypeptides of the invention is not limited to hexoses. Thus, a preferred polypeptide is one that, in addition to high hexose specificity, also exhibits specificity for other carbohydrates, including disaccharides such as lactose, maltose, and / or cellobiose, and even significant pentose specificity, including, for example, xylose or deoxypentoses or deoxyhexoses such as Rhamnose or Fbkoza. It has significant practical implications that, in addition to high specificity for hexoses and other monosaccharides, hexose oxidase also has significant specificity for disaccharides, in particular lactose present in milk and maltose, which, among others occurs in cereal flour and cakes.

Accordingly, in another preferred embodiment, the polypeptide of the present invention is a polypeptide which, in addition to D-glucose, oxidizes at least one sugar selected from the group consisting of D-galactose, maltose, cellobiose, lactose, D-mannose, D-phicose and D-xylose.

In yet another most preferred embodiment, the active hexose oxidase polypeptide has an isoelectric point in the range of 4-5. Specifically, the polypeptide may preferably have an isoelectric point of 4.3 ± 0.1 or 4.5 ± 0.1.

Generally, the polypeptide of the present invention typically has a molecular weight determined by gel filtration using Sephacryl S-200 Superfine (Pharmacia) in the range of 100,000-150000. The molecular weight determined by this or equivalent methods is also defined as the actual molecular weight. Specifically, the polypeptide may have an actual molecular weight of 110,000 ± 10,000.

In a still further aspect, the invention provides a recombinant DNA molecule comprising a DNA fragment encoding a polypeptide having hexose oxidase activity. As described above, such a DNA fragment may be isolated from natural sources or may be constructed e.g. as described in detail in the examples below. In addition, the coding fragment can also be synthesized based on the amino acid sequence of naturally occurring hexose oxidases. The recombinant molecule may be selected from the group of expression vectors of any type described above. In a most preferred embodiment, the recombinant DNA molecule comprises a DNA fragment encoding a hexose oxidase polypeptide that comprises at least one of the sequences (i) to (viii) or a mutein or derivative of such a polypeptide. In a specific embodiment, the recombinant DNA molecule comprises the following DNA sequence (SEQ ID No. 30):

TGAATTCGTG GGTCGAAGAG CCCTTTGCCT CGTCTCTCTG GTACCGTGTA TGTCAAAGGT 60

TCGCTTGCAC ACTGAACTTC ACG ATG GCT ACT CTT CCT CAG AAA GAC CCC 110

<td>GGT</td><td>TAT</td><td>ATT</td><td>GTA</td><td>ATT</td><td>GAT</td><td>GTC</td><td>AAC</td><td>GCG</td><td>GGC</td><td>ACC</td><td>GCG</td><td>GAC</td><td>AAG</td><td>CCG</td><td>GAC</td><td> 158</td>

<td>CCA</td><td>CGT</td><td>CTC</td><td>CCC</td><td>TCC</td><td>ATG</td><td>AAG</td><td>CAG</td><td>GGC</td><td>TTC</td><td>AAC</td><td>CGC</td><td>CGC</td><td>TGG</td><td>ATT</td><td>GGA</td><td> 206</td>

<td>ACT</td><td>AAT</td><td>ATC</td><td>GAT</td><td>TTC</td><td>GTT</td><td>TAT</td><td>GTC</td><td>GTG</td><td>TAC</td><td>ACT</td><td>CCT</td><td>CAA</td><td>GGT</td><td>GCT</td><td>TGT</td><td> 254</td>

<td>ACT</td><td>GCA</td><td>CTT</td><td>GAC</td><td>CGT</td><td>GCT</td><td>ATG</td><td>GAA</td><td>AAG</td><td>TGT</td><td>TCT</td><td>CCC</td><td>GGT</td><td>ACA</td><td>GTC</td><td>AGG</td><td> 302</td>

<td>ATC</td><td>GTC</td><td>TCT</td><td>GGC</td><td>GGC</td><td>CAT</td><td>TGC</td><td>TAC</td><td>GAG</td><td>GAC</td><td>TTC</td><td>GTA</td><td>TTT</td><td>GAC</td><td>GAA</td><td>TGC</td><td> 350</td>

<td>GTC</td><td>AAG</td><td>GCC</td><td>ATC</td><td>ATC</td><td>AAC</td><td>GTC</td><td>ACT</td><td>GGT</td><td>CTC</td><td>GTT</td><td>GAG</td><td>AGT</td><td>GGT</td><td>TAT</td><td>GAC</td><td> 398</td>

<td>GAC</td><td>GAT</td><td>AGG</td><td>GGT</td><td>TAC</td><td>TTC</td><td>GTC</td><td>AGC</td><td>AGT</td><td>GGA</td><td>GAT</td><td>ACA</td><td>AAT</td><td>TGG</td><td>GGC</td><td>TCC</td><td> 446</td>

<td>TTC</td><td>AAG</td><td>ACC</td><td>TTG</td><td>TTC</td><td>AGA</td><td>GAC</td><td>CAC</td><td>GGA</td><td>AGA</td><td>GTT</td><td>CTT</td><td>CCC</td><td>GGG</td><td>GGT</td><td>TCC</td><td> 494</td>

<td>TGC</td><td>TAC</td><td>TCC</td><td>GTC</td><td>GGC</td><td>CTC</td><td>GGT</td><td>GGC</td><td>CAC</td><td>ATT</td><td>GTC</td><td>GGC</td><td>GGA</td><td>GGT</td><td>GAC</td><td>GGC</td><td> 542</td>

187 218

<td>ATT</td><td>TTG</td><td>GCC</td><td>CGC</td><td>TTG</td><td>CAT</td><td>GGC</td><td>CTC</td><td>CCC</td><td>GTC</td><td>GAT</td><td>TGG</td><td>CTC</td><td>AGC</td><td>GGC</td><td>GTG</td><td> 590</td>

<td>GAG</td><td>GTC</td><td>GTC</td><td>GTT</td><td>AAG</td><td>CCA</td><td>GTC</td><td>CTC</td><td>ACC</td><td>GAA</td><td>GAC</td><td>TCG</td><td>GTA</td><td>CTC</td><td>AAG</td><td>TAT</td><td> 638</td>

<td>GTG</td><td>CAC</td><td>AAA</td><td>GAT</td><td>TCC</td><td>GAA</td><td>GGC</td><td>AAC</td><td>GAC</td><td>GGG</td><td>GAG</td><td>CTC</td><td>TTT</td><td>TGG</td><td>GCA</td><td>CAC</td><td> 686</td>

<td>ACA</td><td>GGT</td><td>GGC</td><td>GGT</td><td>GGC</td><td>GGA</td><td>AAC</td><td>TTT</td><td>GGA</td><td>ATC</td><td>ATC</td><td>ACC</td><td>AAA</td><td>TAC</td><td>TAC</td><td>TTC</td><td> 734</td>

<td>AAG</td><td>GAT</td><td>TTG</td><td>CCC</td><td>ATG</td><td>TCT</td><td>CCA</td><td>CGG</td><td>GGC</td><td>GTC</td><td>ATC</td><td>GCA</td><td>TCA</td><td>AAT</td><td>TTA</td><td>CAC</td><td> 782</td>

<td>TTC</td><td>AGC</td><td>TGG</td><td>GAC</td><td>GGT</td><td>TTC</td><td>ACG</td><td>AGA</td><td>GAT</td><td>GCC</td><td>TTG</td><td>CAG</td><td>GAT</td><td>TTG</td><td>TTG</td><td>ACA</td><td> 830</td>

<td>AAG</td><td>TAC</td><td>TTC</td><td>AAA</td><td>CTT</td><td>GCC</td><td>AGA</td><td>TGT</td><td>GAT</td><td>TGG</td><td>AAG</td><td>AAT</td><td>ACG</td><td>GTT</td><td>GGC</td><td>AAG</td><td> 878</td>

<td>TTT</td><td>CAA</td><td>ATC</td><td>TTC</td><td>CAT</td><td>CAG</td><td>GCA</td><td>GCG</td><td>GAA</td><td>GAG</td><td>TTT</td><td>GTC</td><td>ATG</td><td>TAC</td><td>TTG</td><td>TAT</td><td> 926</td>

<td>ACA</td><td>TCC</td><td>TAC</td><td>TCG</td><td>AAC</td><td>GAC</td><td>GCC</td><td>GAG</td><td>CGC</td><td>GAA</td><td>GTT</td><td>GCC</td><td>CAA</td><td>GAC</td><td>CGT</td><td>CAC</td><td> 974</td>

<td>TAT</td><td>CAT</td><td>TTG</td><td>GAG</td><td>GCT</td><td>GAC</td><td>ATA</td><td>GAA</td><td>CAG</td><td>ATC</td><td>TAC</td><td>AAA</td><td>ACA</td><td>TGC</td><td>GAG</td><td>CCC</td><td> 1022</td>

<td>ACC</td><td>AAA</td><td>GCG</td><td>CTT</td><td>GGC</td><td>GGG</td><td>CAT</td><td>GCT</td><td>GGG</td><td>TGG</td><td>GCG</td><td>CCG</td><td>TTC</td><td>CCC</td><td>GTG</td><td>CGG</td><td> 1070</td>

<td>CCG</td><td>CGC</td><td>AAG</td><td>AGG</td><td>CAC</td><td>ACA</td><td>TCC</td><td>AAG</td><td>ACG</td><td>TCG</td><td>TAT</td><td>ATG</td><td>CAT</td><td>GAC</td><td>GAG</td><td>ACG</td><td> 1118</td>

<td>ATG</td><td>GAC</td><td>TAC</td><td>CCC</td><td>TTC</td><td>TAC</td><td>GCG</td><td>CTC</td><td>ACT</td><td>GAG</td><td>ACG</td><td>ATC</td><td>AAC</td><td>GGC</td><td>TCC</td><td>GGG</td><td> 1166</td>

<td>CCG</td><td>AAT</td><td>CAG</td><td>CGC</td><td>GGC</td><td>AAG</td><td>TAC</td><td>AAG</td><td>TCT</td><td>GCG</td><td>TAC</td><td>ATG</td><td>ATC</td><td>AAG</td><td>GAT</td><td>TTC</td><td> 1214</td>

<td>CCG</td><td>GAT</td><td>TTC</td><td>CAG</td><td>ATC</td><td>GAC</td><td>GTG</td><td>ATC</td><td>TGG</td><td>AAA</td><td>TAC</td><td>CTT</td><td>ACG</td><td>GAG</td><td>GTC</td><td>CCG</td><td> 1262</td>

<td>GAC</td><td>GGC</td><td>TTG</td><td>ACT</td><td>AGT</td><td>GCC</td><td>GAA</td><td>ATG</td><td>AAG</td><td>GAT</td><td>GCC</td><td>TTA</td><td>CTC</td><td>CAG</td><td>GTG</td><td>GAC</td><td> 1310</td>

<td>ATG</td><td>TTT</td><td>GGT</td><td>GGT</td><td>GAG</td><td>ATT</td><td>CAC</td><td>AAG</td><td>GTG</td><td>GTC</td><td>TGG</td><td>GAT</td><td>GCG</td><td>ACG</td><td>GCA</td><td>GTC</td><td> 1358</td>

<td>GCG</td><td>CAG</td><td>CGC</td><td>GAG</td><td>TAC</td><td>ATC</td><td>ATC</td><td>AAA</td><td>CTG</td><td>CAG</td><td>TAC</td><td>CAG</td><td>ACA</td><td>TAC</td><td>TGG</td><td>CAG</td><td> 1406</td>

<td>GAA</td><td>GAA</td><td>GAC</td><td>AAG</td><td>GAT</td><td>GCA</td><td>GTG</td><td>AAC</td><td>CTC</td><td>AAG</td><td>TGG</td><td>ATT</td><td>AGA</td><td>GAC</td><td>TTT</td><td>TAC</td><td> 1454</td>

<td>GAG</td><td>GAG</td><td>ATG</td><td>TAT</td><td>GAG</td><td>CCG</td><td>TAT</td><td>GGC</td><td>GGG</td><td>GTT</td><td>CCA</td><td>GAC</td><td>CCC</td><td>AAC</td><td>ACG</td><td>CAG</td><td> 1502</td>

<td>GTG</td><td>GAG</td><td>AGT</td><td>GGT</td><td>AAA</td><td>GGT</td><td>GTG</td><td>TTT</td><td>GAG</td><td>GGA</td><td>TGC</td><td>TAC</td><td>TTC</td><td>AAC</td><td>TAC</td><td>CCG</td><td> 1550</td>

<td>GAT</td><td>GTG</td><td>GAC</td><td>TTG</td><td>AAC</td><td>AAC</td><td>TGG</td><td>AAG</td><td>AAC</td><td>GGC</td><td>AAG</td><td>TAT</td><td>GGT</td><td>GCC</td><td>CTC</td><td>GAA</td><td> 1598</td>

<td>CTT</td><td>TAC</td><td>TTT</td><td>TTG</td><td>GGT</td><td>AAC</td><td>CTG</td><td>AAC</td><td>CGC</td><td>CTC</td><td>ATC</td><td>AAG</td><td>GCC</td><td>AAA</td><td>TGG</td><td>TTG</td><td> 1646</td>

<td>TGG</td><td>GAT</td><td>CCC</td><td>AAC</td><td>GAG</td><td>ATC</td><td>TTC</td><td>ACA</td><td>AAC</td><td>AAA</td><td>CAG</td><td>AGC</td><td>ATC</td><td>CCT</td><td>ACT</td><td>AAA</td><td> 1694</td>

<td>CCT</td><td>CTT</td><td>AAG</td><td>GAG</td><td>CCC</td><td>AAG</td><td>CAG</td><td>ACG</td><td>AAA</td><td colspan="6">TAGTAGGTCA CAATTAGTCA</td><td></td><td> 1741</td>

TCGACTGAAG TGCAGCACTT GTCGGATACG GCGTGATGGT TGCTTTTTAT AAACTTGGTA 1801

187 218

In addition, the invention provides in another aspect a microbial cell that comprises the above recombinant DNA molecule. The above general description of the host organism comprising the DNA fragment encoding the polypeptide of the present invention includes such a microbial cell and the cell can be selected from any of the above-mentioned groups, families, genera and species of microorganisms, i.e. the microbial cell can be selected from bacterial cells, fungi and yeast, including, for example, E. coli cells, lactic acid bacteria cells, Saccharomyces ckrkvisiak cells and Pichia pastoris cells.

The microorganism cell of the present invention, if it is intended to be added directly to the product, if desired has hexose oxidase activity, e.g. during the production process it may be provided in the form of a microorganism culture, preferably in a condensed form. Thus, such a culture may preferably contain the microbial cell of the present invention in a density that is preferably in the range of 10 to 10 ° per gram of culture. Such culture may be in fresh form, i.e., not frozen cell suspension in liquid medium, or it may be in frozen or dried form, e.g., lyophilized culture. A microbial cell may also be immobilized on a solid support for specific purposes.

As mentioned above, the invention further relates to the use of the active hexose oxidase polypeptide of the present invention or a microbial cell expressing said polypeptide in the production of food products. In this context, the term "production" should be understood in the broadest sense in that it includes the addition of hexose oxidase or a microbial cell to the ingredients of a given food product, before, during or after any subsequent stage, during packaging and storage of the final product, until consumption. Food products, when such use is preferred, can be any products in which the final product hexose oxidase provides a beneficial effect of the food product.

Naturally, the desired hexose oxidase activity is only obtained if the enzyme substrate is present in appropriate amounts. Carbohydrate substrates may be naturally present in the food product or its component, or may be added or produced during the manufacturing process. An example of a substrate produced during the manufacturing process is the degradation of di-, oligo- or polysaccharides to lower sugars that are broken down by hexose oxidase, which may occur as a result of enzymatic activity naturally present in the food product or added during manufacture. In addition, a substrate for the active hexose oxidase polypeptide can be produced as a result of the enzymatic activity of the fusion partner as described above.

The desired effects of hexose oxidase activity in a product containing substrates for the enzyme include the production of lactones from the Substrate sugars, which can then be converted to the corresponding acids to produce hydrogen peroxide or using oxygen.

Typical examples of food products in which hexose oxidase activity may be beneficial include dairy products, starch-containing food products and non-dairy drinks. Thus, in the production of a wide range of dairy products, it is desirable to lower the pH. This is conventionally obtained by inoculating milk with lactic acid producing starter cultures. As mentioned above, it is contemplated that hexose oxidase or the body expressing this enzyme may be used as an alternative method for acidifying milk. The same effect may be desired in other food products that are acidified during manufacture, such as some meat and vegetable products that are currently acidified by the addition of starter cultures of lactic acid bacteria.

Oxygen consumption due to hexose oxidase activity has several beneficial effects in the production of food and pharmaceutical products. By reducing the content or removing oxygen in foods or pharmaceuticals that contain lipids that are sensitive to oxidative processes, hexose oxidase can act as

187 218 antioxidant, and in addition, reducing the amount of oxygen can inhibit the growth of putrefactive organisms, which increase is dependent on the presence of oxygen, so that the active hexose oxidase polypeptide can also act as an antibacterial agent.

The latter effect can be used to extend the shelf life of pre-packaged food, where it can be prevented from rotting by incorporating the active hexose oxidase polypeptide of the present invention in the food product itself, or by providing a mixture of hexose oxidase and a suitable substrate in the package, but separate from the content of the food product . In a typical example, such a mixture is attached to the inside of a food container, such as a can or jar. Accordingly, the hexose oxidase of the present invention can be used as an oxygen scavenger in a food container.

It is clear that the above effects of the polypeptide of the present invention in the production of food products will also be applicable to the production of animal feed. In particular, these effects are desirable in the production of silage produced either from fodder crops such as grass or corn or from protein by-products of slaughterhouses or fish processing plants. Such feed products are siloed by the addition of acid or acid producing bacteria, such as lactic acid bacterial inoculates. In order to facilitate the growth of acidifying bacteria and to prevent the growth of aerobic putrefactive organisms such as Gram-negative bacteria and yeast, it is important to obtain low oxygen concentration in the silos. It is therefore contemplated that the hexose oxidase of the present invention is useful as an oxygen removal or acidifying agent in silage feed, optionally in the form of a composition further comprising one or more conventional silage additives, such as lactic acid bacterial inoculates or enzymes that produce low-molecular sugar substances.

A further preferred use of the hexose oxidase polypeptide of the present invention is the use of the enzyme for reducing sugar content in food products, comprising adding to the product some amount of the polypeptide or polypeptide-producing microorganism cell in an amount sufficient to remove at least a portion of the sugar initially present in the food product. Such an application can be e.g. beneficial in the manufacture of products for patients with diabetes, where low sugar content is desired, and the production of wines with reduced alcohol content. In the latter application, hexose oxidase is preferably added to the must before yeast inoculation.

In a further preferred aspect, the invention relates to the use of an active hexose oxidase polypeptide or a microbial cell producing said enzyme according to the present invention in the manufacture of pharmaceutical, cosmetic or oral hygiene products such as toothpastes. The desired effect of hexose oxidase in these products has been described above for food products and animal feeds.

A particularly interesting use of the hexose oxidase of the present invention is its use as a dough improving agent. It has been found that the addition of hexose oxidase to the dough results in increased resistance to baking when the dough is stretched, i.e. the enzyme provides increased dough strength, making it less sensitive to mechanical deformation. This means that based on the known effect of glucose oxidase, it is considered that this effect of adding the hexose oxidase of the present invention to the dough causes crosslinking between thiol groups of sulfur-containing amino acids in flour proteins, which occurs when the hydrogen peroxide produced by the dough enzyme reacts with thiol groups that oxidize in this way.

Accordingly, the invention also provides a method of making bakery dough products, comprising adding to the dough an effective amount of a polypeptide or microorganism of the present invention that is capable of expressing that polypeptide and a dough improving composition comprising a polypeptide or microorganism capable of expressing such a polypeptide in dough, and at least one conventional dough ingredient. In a useful embodiment, such a composition may further comprise at least one enzyme

187 218 a dough improving or bakery product, e.g. selected from cellulase, hemicellulase, pentosanase, lipase, xylase, amylase, glucose oxidase and protease.

In yet a further aspect of the invention, hexose oxidase is used as an analytical reagent in the method of determining the concentration of any sugar in biological and other samples that can be converted by the enzyme. Usually, the sugar content is measured by determining the amount of end product resulting from the enzymatic conversion of the substrate sugar present in the measured sample. In this way, it is contemplated that hexose oxidase may be used directly as a reagent in the in vitro diagnostic test or may be embedded in the sensor.

The invention will now be described by way of example in the following examples and attached drawings, in which:

Figure 1 shows a schematic overview of hexose oxidase (HOX) purification and two strategies used to obtain amino acid sequence information.

Figure 2 shows native, non-dissociative polyacrylamide gel electrophoresis (native PAGE) of hexose oxidase preparations at various purification stages. The samples indicate enzyme preparations obtained after anion exchange chromatography and concentration (lane 1), after gel filtration (lane 2) and either after cation exchange chromatography (lane 3) or isoelectric focusing (lane 4). Phast gel (Pharmacia) was stained with silver. Molecular weights of the protein standard (x 10 '³) are indicated on the left. The band corresponding to hexose oxidase, which is indicated by an arrow, was identified by labeling the enzyme on another gel run in parallel (not shown). Four tracks were run on separate gels.

Figure 3 shows the UV profile obtained during the purification of hexose oxidase by gel filtration on Sephacryl S-200 HR as described in the text. Fractions containing hexose oxidase (HOX) are marked in the filled area.

Figure 4 shows SDS-PAGE of hexose oxidase purified from Chondrus crispus by anion exchange chromatography on DEAE-Sepharose Fast Flow, gel filtration on sephacryl S-200 followed by cation exchange chromatography on S-Sepharose Fast Flow (lane 1) or column chromatographic focusing Mono P (track 2). Molecular weights of the protein standard (x 10 '³) marked on the left. Polypeptides with a molecular weight of 60,000, 40,000 and 29,000 are indicated by arrows. The reduced samples were run on a 12% polyacrylamide gel that was stained with Coomassie R-250 brilliant blue. The last two tracks were run on separate gels.

Figure 5 shows isoelectric focusing (IEF) of hexose oxidase. The gel was stained with either Coomassie Brilliant Blue R-250 (lane 1) or labeled with enzymatic activity as described in the text (lane 2). The location of isoelectric point markers released in parallel is marked on the left. Two tracks were run on separate gels.

Figure 6 shows reverse-phase HPLC separation of peptides generated by digestion of a 40,000 HOX polypeptide with Lys-C endoproteinase. Signals marked 1, 2, 3, 4 and 5 were subjected to amino acid sequencing.

Figure 7 shows reverse phase HPLC separation of peptides produced by digestion of a 40,000 HOX polypeptide with Lys-C endoproteinase. Signals marked 1 and 2 were subjected to amino acid sequencing.

Figure 8 shows a Northern blot test of RNA extracted from Chondrus crispus. The denaturing agarose gel was loaded with 30 pg (lane 1) or 3 pg (lane 2) of total RNA. The left arrow shows a transcript specific for hexose oxidase. The positions of the molecular weight standard in kb are shown on the right.

Figure 9 shows the construction of plasmid pUP0153 which mediates the expression of recombinant hexose oxidase in Pichia pastoris. Small arrows show PCR primers. The gray box shows the hexose oxidase gene.

Figure 10 shows the purification of recombinant hexose oxidase from Pichia pastoris by anion exchange chromatography on a HiTrap-Q column (first step). Aktyw20

187 218 Alcohol oxidase (AOX) (·) and hexose oxidase (HOX) (O) activity in the collected fractions were tested as described in the text.

Figure 11 shows the purification of recombinant hexose oxidase from Pichia pastoris by gel filtration on Sephacryl S-200 HR (second step). Alcohol oxidase (AOX) (·) activity and hexose oxidase (HOX) (O) activity in the collected fractions were tested as described in the text.

Figure 12 shows the construction of plasmid pUP0181 that mediates the expression of recombinant hexose oxidase in E. coli. Small arrows show PGR primers. The gray box shows the hexose oxidase gene.

Figure 13 shows SDS-PAGE of recombinant hexose oxidase produced in E. coli. Crude extracts of lysed cells were analyzed on a 14% denaturing gel. Molecular weights of protein standards (x10 '³) marked on the left. The gel was stained with Coomassie R-250 brilliant blue. Lane 1 shows E. coli extract from pUP0181, lane 2 shows plasmid-free control. The arrow shows the hexose oxidase band.

Figure 14 shows the construction of plasmid pUP0155, which mediates the expression of recombinant hexose oxidase in Saccharomyces cerevisiae. Small arrows show PCR primers. The gray box shows the hexose oxidase gene.

Example 1.

Purification of hexose oxidase with Chondrus crispus

A schematic review of purification and two strategies adapted to obtain the enzyme amino acid sequence is shown in Figure 1.

1.1. Harvesting, drying and grinding Chondrus crispus

The red Chondrus crispus seaweed was collected from April to September on the coast near Grena, Jutland, Denmark at a depth of 2-5 meters. Freshly collected algae thallus was immersed in cold water and stored on ice during transport to the laboratory (&lt;24 hours). The seaweed was dried immediately or stored frozen for further processing. For enzyme isolation, the material was stored at -18 ° C, while the material to isolate mRNA was stored in liquid nitrogen.

Chondrus crispus thaws were thawed at 4 ° C and air dried at room temperature (20-25 ° C) for 2-3 days. The dried material was ground to a fine powder in a Waring Commercial Blender (Model 34BL97, Waring, New Harford, Connecticut, USA).

1.2. Extraction of the enzyme

About 500 g of Chondrus crispus powder was mixed with 2.5 L of 20 mM Tris-Cl, pH 7.0. Water used throughout the extraction and purification procedure was obtained from the Milli-Q UF Plus Laboratory Water Purification System (Millipore). The buffer was previously cooled to 4 ° C. The mixture was kept at 4 ° C for 6-8 days. The extract was collected by filtration through several layers of gauze.

The seaweed material was subjected to subsequent extractions, which were carried out as described above. The material was usually removed after 5-8 extractions when the residual activity decreased to insignificant values.

The filtrate was clarified by centrifugation at 10,000 xg in a Sorvall GSA rotor (Sorvall Instruments). The supernatant was filtered through Whatman chromatography paper (chr 1) and diluted with water to a conductivity of 7-8 mS / cm. The pH was adjusted to 7.5. The extract was ready for anion exchange chromatography as described below.

1.3. Hexose oxidase test

The procedure was essentially identical to that described by Sullivan and Ikawa, 1973. This test is based on the principle that hydrogen peroxide produced during the oxidation of sugar in the presence of peroxidase reacts with the chromogenic substance o-dianisidine, forming a dye with a maximum absorbance at 402 wounds .

The test mixture consisted of 370 µL 0.1 M sodium phosphate buffer, pH 7.0; 462 en 0.1 M D-glucose in 0.1 M sodium phosphate buffer, pH 7.0; 9 pl of horseradish peroxidase, 0.1 mg / ml in water (Sigma Chemicals, cat. No. P-6782 or Boehringer Mannheim, cat. No. 814 393);

187 218 and 9 μΐ o-dianisidine * 2HC1, 3.0 mg / ml in water (3,3'-dimethoxy-benzidine, Sigma Chemicals). After incubation at room temperature for 15-30 minutes, the test was stopped by adding a drop of 37% HCl (Merck, pa). 100 ul samples were transferred from test tubes to the wells of a microtiter plate (NUNC, Denmark) and absorbance was read at 410 nm on a Titertek Multiscan II PLUS plate reader (Labsystems / Flow Laboratories Finland) To ensure that the observed activity was from hexose oxidase, not glucose oxidase. The test was periodically performed with Dgalactose instead of D-glucose.

1.4. Anion exchange chromatography

This step was performed on a BioPilot chromatography system (Pharmacia Biotech, Sweden) connected to a SuperRac fraction collector (LKB-Produkter AB, Sweden).

This and subsequent purification steps were carried out at room temperature (2025 ° C), but the fraction collector was placed in a cold room, so that the collected fractions were stored at 4 ° C until the enzymatic test was performed. Absorbance at 280 nm and conductivity were recorded. The extract was applied to a ΧΚ5Ο / 3Ο column (Pharmacia, 5.0 x 25 cm) with a bed volume of 500 ml, which was DEAE-Sepharose Fast Flow (Pharmacia), equilibrated with buffer A: 20 mM Tris-Cl, pH 7.5. The flow rate was 5 ml / min during sample introduction and 10 ml / min. During the subsequent stages of chromatography. After sample introduction, the column was washed with 1200 ml buffer A. Adsorbed proteins were eluted with 2800 ml gradient from 0 to 100% buffer B: 20 ​​mM Tris-Cl, 500 mM NaCl, pH 7.5. 15 ml fractions were collected.

After each chromatography run, the column was regenerated with 500 ml 0.5 M NaOH, neutralized with 500 ml 1.0 M Tris-Cl pH 7.5 and equilibrated with 1200 ml buffer A. Collected fractions were tested for hexose oxidase activity as described above (40 μΐ sample , 30 minutes incubation). Fractions of hexose oxidase activity were pooled and stored at 4 ° C.

1.5. Concentration of fractions containing hexose oxidase activity

Several pools of fractions from DEAE-Sepharose chromatography were combined and concentrated by ultrafiltration on a Millipore Lab Ultrafiltration Cassette System (catalog number XX420LCS0). The system was equipped with a membrane chamber with a nominal cut-off molecular weight of 30,000 (catalog number PTTKOLCP2) and was driven by a peristaltic pump. After concentration at room temperature to about 50 ml, the enzyme preparation was further concentrated to 10-20 ml by ultrafiltration at 4 ° C in a Centriprep apparatus (Amicon, USA, NMWC = 30,000) according to the manufacturer's instructions. The concentrated enzyme solution was stored at 4 ° C.

1.6. Native protein polyacrylamide gel electrophoresis (PAGE)

The enzyme preparation composition obtained by ion exchange chromatography and ultrafiltration was analyzed by PAGE on a Pharmacia Phast System, see. Figure 2. 8-25% gradient gels were run and silver labeled according to the manufacturer's instructions. A kit containing the following molecular weight standards was also purchased from Pharmacia: thyroglobulin (669,000); ferritin (440000); catalase (232000); lactate dehydrogenase (140,000) and albumin (67,000).

Labeling for hexose oxidase activity was performed as described for glucose oxidase by Sock and Rohringer (1988). The principle of the test was that the redox reaction catalyzed by glucose oxidase or hexose oxidase was coupled with the reduction of the tetrazole salt to the colored, insoluble formazan.

Immediately after electrophoresis, the Phast gel was immersed in 10 ml of a freshly prepared staining solution containing: 0.1 M D-glucose (or D-galactose); 85 mM citric acid buffer / sodium phosphates pH 6.5; 0.2 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole bromide ("thiazolyl blue", MTT, Sigma Chemicals, cat. No. M 2128); and 0.1 mg / ml N-methyl-dibenzopyrazine-methyl sulfate salt ("phenazin methosulphate", PMS, Sigma Chemicals, cat. no. P 9625). The gel was incubated at room temperature in the dark until a clearly visible purple band appeared (usually 5-90 minutes), followed by washing with 10% acetic acid, 5% glucose and air drying.

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Silver-stained gel is shown in Fig. 2, lane 1. As can be seen in Fig. At this stage of purification, numerous proteins were visible. However, only one band marked with an arrow in Fig. 2 was stained by enzymatic labeling (result not shown).

1.7. Gel filtration

This purification step was performed on an FPLC system (Pharmacia) equipped with a XK26 / 70 column (2.6 x 66 cm, Pharmacia) with a bed volume of 350 ml. The column was packed with Sephacryl S-200 HR gel (Pharmacia) according to the manufacturer's instructions. The buffer was 20 mM Tris-Cl, 500 mM NaCl, pH 7.5, and the flow rate was 5 ml / minute. Absorbance at 280 nm was recorded. 2.5 ml fractions (Pharmacia) were collected on the FRaC-100 fraction collector, which was then refrigerated (4 ° C) after FPLC. The concentrated hexose oxidase preparation was centrifuged at 30,000 rpm. In a swinging bucket rotor (Beckman) in an L7 ultracentrifuge (Beckman, for 60 minutes at 4 ° C. 3.0-4.0 ml aliquots of the supernatant were mixed with 5% glycerin (Sigma Chemicals), filtered through disposable filters 0.22 pm pore size (Millipore, catalog number SLGV 025 BS) and introduced into the column using SA-5 sample applicator (Pharmacia) connected to the column inlet. Fractions showing hexose oxidase activity were identified using the test described above (10 µl sample, 15 minutes incubation) and stored separately at -18 ° C for further processing.

The UV profile and elution location of hexose oxidase is shown in Fig. 3. As can be seen in Fig., A major part of the UV absorbing material has been eliminated at this stage. Electrophoretic analysis of native PAGE and silver labeling (Fig. 2, lane 2) showed that only few contaminants remained after this step.

1.8. Determination of the molecular weight of native hexose oxidase by analytical gel filtration.

The molecular weight of native hexose oxidase was determined by gel filtration on Sephacryl S-200 Superfine (Pharmacia). Column sizes, buffer, flow rate and collection of fractions looked as described above. Blue dextran for column vacuum determination (vo) and the following molecular weight standards were obtained from Pharmacia: ovalbumin (43000), albumin (67000), catalase (158000) and aldolase (252000). A sample containing hexose oxidase was obtained by chromatography on DEAE-Sepharose as described above. Based on the determination of the elution volume (v_e) protein and hexose oxidase standards, the corresponding Kav values ​​(Ve-Vo / v_tVo). Finally, the Kav values ​​of the protein standards are plotted against the respective logarithm (molecular weight) values. The kav of hexose oxidase corresponded to a native molecular weight of about 110,000. This is consistent with Sullivani and Ikawa (1973) who stated that the molecular weight is 130,000. Kerschensteiner and Klippenstein (1978) report that the molecular weight is 140,000, which was also determined by filtration gel

1.9. Cation exchange chromatography

This step was performed on a SMART Micropurification Chromatography Sys 1 ml system filled with S-Sepharose Fast Flow resin (Pharmacia). The column was equilibrated with buffer A: 50 mM sodium acetate, pH 4.5 (prepared by tuning 50 mM acetic acid to pH 4.5 using NaOH). Buffer B used for gradient elution contained 50 mM sodium acetate, 500 mM NaCl, pH 4.5. The gel filtration fractions were desalted on previously filled disposable Sephadex G-25 columns (PD-10, Pharmacia) which were equilibrated and eluted with 25 mM sodium acetate pH 4.5. 20 ml of salted sample derived from 6 gel filtration fractions with high hexose oxidase activity were loaded onto a 50 ml Superloop column (Pharmacia) at a flow of 250 pl / min. The column was washed with 4 volumes of buffer A at the same flow rate. Bound proteins were eluted with a gradient from buffer A to buffer B in 5 ml. 250 fractions (il were taken during gradient elution and tested for hexose oxidase activity as described above (1pl sample, 15 minutes incubation) and stored at -18 ° C.

The resulting hexose oxidase preparation was analyzed by PAGE and silver staining (Fig. 2, lane 3). The hexose oxidase band was the only significant band, although small amounts of contaminating proteins were observed.

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1.10. Analytical SDS-PAGE

Fractions from S-Sepharose chromatography showing hexose oxidase activity were also tested by SDS-PAGE according to Laemmli (1970). 12.5% ​​acrylamide / bis-acrylamide (37.5: 1 mixture) with a thickness of 0.75 mm was run on a MiniProtean II 9Bio-Rad apparatus). Gels were stained with brilliant blue R-250, 10% acetic acid, 40% ethanol and decolorized with 10% acetic acid, 30% ethanol.

The result of electrophoresis is shown in Fig. 4, lane 1. The purified glucose oxidase preparation showed strong bands at a relative molecular weight of 40,000 and 29,000 and a weak band at 60,000 and 25,000, respectively. In addition, two sharp doublet bands of 55,000 and 57,000 were observed.

1.11. SDS-PAGE followed by blotting and staining of carbohydrates

The presence of carbohydrates in the isolated hexose oxidase molecule was studied with the DIG Glycan Detection Kit (Boehringer Mannheim), which is designed to detect micrograms of sugars in glycoconjugates on membranes. In principle, adjacent hydroxyl groups in carbohydrates are oxidized to aldehydes. Then, digoxygenin is covalently bound to aldehyde groups and detected with an anti-digoxygenin and alkaline phosphatase antibody conjugate.

Purified hexose oxidase from cation exchange chromatography was run on a 12% SDS-PAGE gel as described above, transferred to nitrocellulose according to standard procedures and stained for carbohydrate content using the Glycan Detection Kit, according to the manufacturer's instructions. None of the 60,000, 40,000, 29,000 and 25,000 hexose oxidase bands were stained. Only the sharp doublet of bands at 57,000-55,000 was strongly colored (result not shown). A doublet of 57000-55000 was identified as residual impurity.

Thus, it can be concluded that none of the hexose oxidase components observed on SDS-PAGE were glycosylated.

1.12. Isoelectric focusing

The hexose oxidase fractions from S-Sepharose chromatography were pooled and concentrated by ultrafiltration on a Centricon (Amicon) and analyzed by isoelectric focusing (IEF) on Isogel agarose plates, pH 3-10, according to the manufacturer's instructions 9FMC Bioproducts, Rockland, ME, USA. A mixture of pI standards (FMC Bioproducts) was run in parallel with the hexose oxidase samples. The mixture consisted of cytochrome C (pI = 10.2), myoglobin larger / smaller band (7.4 / 7.0), carbonic anhydrase (6.1), β-lactoglobulin A / B (5.4 / 5, 5), ovalbumin (4.8), glucose oxidase (4.2) and amyloglucosidase (3.6). Gels were stained with Coomassie R-250 brilliant blue. As shown in Fig. 5, lane 1, the purified hexose oxidase preparation consisted of two varieties with a pI of 4.3 and 4.5, respectively. Purified hexose oxidase was also analyzed by isoelectric focusing on previously cast polyacrylamide gels pH 3.5-9.5 (Pharmacia, Ampholine PAGplates) according to the manufacturer's instructions. These gels were also labeled for enzymatic activity, by incubating the labeling mixture as described above for native PAGE. As shown in Fig. 5, lane 2, both pI variants are enzymatically active.

1.13. Chromatographic focusing.

Observation of several bands in SDS-PAGE purified on S-Sepharose hexose oxidase as the last step raised the suspicion that one or more bands could constitute residual contamination. In addition, S-Sepharose chromatography continuously gives a poor yield. Hence, chromatographic focusing was introduced as the last purification step instead of cation exchange chromatography on S-Sepharose. Chromatographic focusing was carried out on a SMART chromatographic system equipped with a Mono P HR 5/5 column (0.5 x 5 cm, Pharmacia), with a bed volume of 1 ml and 50 ml Superloop for sample feeding. The start buffer to be separated in the pH 5.0 and 3.5 range was 25 mM piperazine, adjusted to a pH of 5.5 using HCl. The eluent was Polybuffer 74 (Phar24

187 218 macia) 10 times diluted with water and adjusted to pH 5.5. The column was previously treated and equilibrated with start buffer as recommended by the manufacturer.

Sample preparation was performed as follows: In a typical experiment, the best fractions from two gels (2x4 fractions, 20 ml) were pooled and passed through a Phenyl Sepharose 6 Fast Flow column (high sub, Pharmacia) packed in a disposable Poly-Prep column (Bio-Prep Rad) and equilibrated in the buffer used for gel filtration (20 mM Tris-Cl, 500 mM NaCl, pH 7.5). This treatment almost completely removed the remaining amount of red phycoerythrin protein and other colored substances that adsorbed on the gel matrix at this ionic strength, thereby eliminating impurities that were only partially removed during other stages of the purification process. The Phenyl Sepharose column was destroyed after use. Hexose oxidase activity was quantitatively recovered from the eluent, which was salted out on Sephadex G-25 disposable columns (PD-10, Pharmacia) equilibrated and eluted with starting buffer.

Before the sample was introduced, 1 ml of eluent was pumped into the column. The flow rate was 0.5 ml / min. After introducing the sample, a pH gradient was prepared by pumping 11 ml of eluent through the column. During the elution of the pH gradient, 44 fractions of 250 μί were collected. Fractions containing hexose oxidase were identified by the test method described above (1 μί aliquots, 15 minutes incubation) and stored at -18 ° C until further use.

Hexose oxidase purified by chromatographic focusing was examined by PAGE and silver staining (Fig. 2, lane 4) and by SDS-PAGE and Coomassie blue staining (Fig. 4, lane 2). In native PAGE, the hexose oxidase band was the only significant band and only insignificant amounts of impurities were observed. By SDSPAGE it has been clearly shown that this purification method is able to remove the sharp doublets of the 57000 and 55000 bands. The 25,000 band observed after S-Sepharose chromatography was very weak after chromatographic focusing. In summary, hexose oxidase obtained by DEAE chromatography, gel filtration and chromatographic focusing showed one band in native PAGE. Strong 40,000 and 29,000 bands and a weak 60,000 band were observed in SDS-PAGE.

Because the intensity of the 60,000 component relative to the 29000 and 40,000 components differed between different enzyme preparations, it was suspected that the 29000 and 40,000 polypeptides could be derived from proteolytic cleavage of approximately 60,000 precursors. This could match the idea of ​​the homodimeric structure of the enzyme, with a native molecular weight of 110,000 120,000, as found by gel filtration as described above. In addition, this hypothesis should be consistent with the results obtained by Kirschensteiner and Klippenstein, who found a native molecular weight of 140,000 in gel filtration and a molecular weight of 70,000 in the SDS-PAGE subunit (Kirschensteiner and Klippenstein, 1978).

Example 2.

Generation and analysis of the amino acid sequence of a hexose oxidase peptide fragment

2.1. Digestion of purified hexose oxidase with cyanogen bromide

This procedure was carried out when cation exchange chromatography on S-Sepharose was still used as the last purification step.

The hexose oxidase obtained by purification on DEAE Sepharose, Sephacryl S-200 and S-Sepharose was transferred to an aggressive buffer by buffer exchange on PC3.2 / 10 Fast Desalting Columns containing Sephadex G-25 Superfme (Pharmacia, 0.32 x 10 cm, bed volume 0.8 ml) which was installed in the SMART system. The column was equilibrated and eluted with 200 mM ammonium bicarbonate (BDH, AnalaR). To obtain satisfactory recovery, it was necessary to add 500 mM sodium chloride to the hexose oxidase sample before injection.

Eluted hexose oxidase with said buffer was separated into 1.5 ml tubes and lyophilized on Speedvac (Savant Instruments). Cyanogen bromide (CNBr,

Pierce), 200 μί 10 mg / ml solution in 70% formic acid (Promega). (Reagents obtained from Promega were components of the "Probe Design ™ Peptyde Separation System" cat. No.

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V60300. Tubes were incubated overnight in the dark at room temperature. The solutions were dried in Speedvac, suspended in 50 [mu] l water and dried again.

2.2. Separation of cyanogen bromide fragments by high-resolution SDS-PAGE and electroblotting on polyvinyl diene difluoride (PVDF) membrane.

Peptides produced by cyanogen bromide digestion were separated by high resolution SDS-Page according to Schaegger and von Jagow (1987). This system ensures perfect separation of low molecular weight peptides of 20-250 amino acids). The gel system includes a 16.5% separation gel, 10% separation gel and 4% viscous gel, prepared using a 29: 1 acrylamide / bisacrylamide mixture by Promega).

Minikels with a thickness of 0.75 were run in a Mini Protean II apparatus (Bio-Rad). Ammonium persulfate and N, N, N ', N'-teramethyl-ethylenediamine (TEMED) were from BioRad, SDS was from USB (highest purity). Tris was from Fluka (catalog number 93350). Tricine and sodium thioglycolate were from Promega. Glycine (pa), 2-mercaptoethanol (pa) and bromophenol blue were from Merck, while glycerin from Gibco BRL (highest purity). Sodium thioglycolate, 0.1 mM, was added to the cathode buffer immediately before use to prevent chemical blockage of the amino terminus of the peptides during separation. The gel was sterilized for 60 minutes at 30 V to allow thioglycolate to sweep out any substances that react with amine residues. Sample preparation: dried cyanide bromide cleaved peptide fragments were suspended in 30 µl buffer containing 63 mM Tris-Cl, pH 6.8, 1% SDS, 2.5% 2-mercaptoethanol, 10% glycerin and 0.0012% bromophenol blue. Samples that turned yellow during preparation due to residual formic acid content were neutralized by the addition of 1-3 µL 1.0 M Tris base until they returned to blue. Samples were denatured by heating to 95 ° C for 5 minutes before applying to the gel. A mixture of low-range molecular weight standards (Promega) with molecular weights in the range of 31000 and 2500 were run in parallel with the fragments of hexose oxidase. Electrophoresis was carried out at 150 volts.

Electrophoretic transfer to PVDF membranes was performed on a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) according to the manufacturer's instructions. Three problott membrane sheets (Applied Biosystems) cut to gel size were briefly wetted in methanol (Merck, pa) and then immersed in transfer buffer (25 mM tris, 192 mM glycine, pH 8.5, cooled to 4 ° C) before a combination of layered transfer system. After electrophoresis, the gel was incubated in transfer buffer for 5 minutes at 4 ° C and then incorporated into a layered system with the following layers: Whatman paper sheet (3MM chr), two sheets of Problott membrane, SDS-PAGE gel with separated peptides, a third Problott sheet and Whatman paper layer. The layered system was directed with two layers of Problott towards the positive electrode. The cooling unit was mounted in the buffer chamber before it was filled with cooled transfer buffer, followed by a transfer at room temperature for 60 minutes at a constant voltage of 100 V. During the transfer, the current was increased from about 270 mM to about 400 mA.

After transfer, the membrane was rinsed in water for one minute and then stained for 30-45 seconds with 100 ml freshly prepared staining solution containing 0.1% Coomassie Brilliant Blue R-250 (Bio-Rad), 5% acetic acid (Merck pa) and 45 % methanol (Merck pa). The membrane was then decolorized in 3 lesions with approximately 80 ml of freshly prepared 5% acetic acid, 45% methanol after 30-60 seconds each. Finally, the membranes were washed in 3 water changes to remove residual glycine, and then air dried. Well separated and relatively rare bands with molecular weights of around 2500, 9000 and 16000. Amino acid analysis and sequencing were excised.

2.3. Amino acid analysis and sequencing of the cyanogen bromide cleaved fragment of 9,000 hexose oxidase.

Amino acid analysis was performed by ion exchange chromatography and derivatization by column, using ophthalmialdehyde. Samples were hydrolyzed at 110 ° C for 20 hours in 6M HCl, 0.05% phenol and 0.05% dithiodipropionic acid (Barkholt and Jensen, 1989). Peptides were referenced on an Applied automated protein / peptide sequencer

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Biosystems model 477A, ​​equipped with PTH analyzer connected in series, model 120A and data analysis system. Protein sequencing reagents were obtained from Applied Biosystems. Amino acid and peptide sequence analysis were kindly done by Anie L. Jensen, Department of Protein Chemistry, University of Copenhagen, Denmark.

The peptide sequence identified by analysis of the 9000 fragment is shown in Table 2.1. The initial yield of phenylthiohydantoin-tyrosine (PTH-Tyr) in the first stage was 22 pmole. The amino acid composition of fragment 9000 is shown in Table 2.2.

Table 2.1. Peptide sequence obtained by sequence analysis of the 9000 hexose oxidase fragment after cyanogen bromide peptide sequence start sequence identification amino acid sequence fragment fragment 9000 CMBr peptide HOK-1 YEPYGGYP

Abbreviations: Y = Tyr; E = Glu; P = Pro; G = Gly; V = Val

Table 2.2. Amino acid composition of the 9,000 hexose oxidase fragment after cyanogen bromide

<td>Amino acid</td><td>% molamy</td><td>N</td>

<td>Asx</td><td> 16,4</td><td> 14</td>

<td>Thr</td><td> 4,8</td><td> 4</td>

<td>Cheese</td><td> 4,6</td><td> 4</td>

<td>Glx</td><td> 9,9</td><td> 8</td>

<td>Pro</td><td> 8,1</td><td> 7</td>

<td>Gly</td><td> 11,2</td><td> 9</td>

<td>ala</td><td> 4,3</td><td> 4</td>

<td>Cys</td><td> 0</td><td> 0</td>

<td>val</td><td> 5,2</td><td> 5</td>

<td>net</td><td> 0,2</td><td> 0</td>

<td>How much</td><td> 3,6</td><td> 3</td>

<td>Leu</td><td> 9,3</td><td> 8</td>

<td>Tyr</td><td> 6,1</td><td> 5</td>

<td>phe</td><td> 4,6</td><td> 4</td>

<td>His</td><td> 1,0</td><td> 1</td>

<td>lys</td><td> 8,1</td><td> 7</td>

<td>Arg</td><td> 2,7</td><td> 2</td>

<td>Trp</td><td>ND</td><td> -</td>

<td>in total</td><td> 100,0</td><td> 85</td>

ND - not found

2.4. Preparative SDS-PAGE and electro biot on a PVDF membrane

The following procedure was performed to obtain amino acid sequences that are known to be from 40,000 or 29,000 hexose oxidase preparation polypeptide.

Preparative SDS-PAGE gels were run according to Laemmli (laemmli, UK 1970). Minikels containing 12.5% ​​acrylamide / bisacrylamide (37.5: 1 mixture) with a thickness of 0.75 mm were run on a Mini-Protean II (Bio-Rad) apparatus. A solution of acrylamide (BDH, catalog number 44313) and Ν, Ν'-methylene-bis-acrylamide (BDH, catalog number 44300) was stored above the Bio-Rad mixed ion exchange resin, catalog number 142-6425). The sources of all other reagents are described above.

Sample preparation: Fractions from chromatographic focusing were concentrated by centrifugal ultrafiltration at 4 ° C in Ultrafree-MC units with NMWL 10,000 and a sample capacity of 400 μΐ (Millipore, catalog number UFC3 LGC25). The filtrate was mixed with one volume of 2x gel loading buffer containing 125 mM Tris-Cl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 20% glycerin and 0.0025% bromophenol blue. Samples that turned yellow due to the acidic content of Polybuffer were neutralized by adding 1-3 μΐ 1.0 M Tris base until the blue color was restored. Denat samples were heated by heating to 95 ° C for 5 minutes and applied to the gel in portions of approximately 30 µl per lane.

A mixture of molecular weight standards (Bio-Rad) with molecular weights in the range from 974000 to 14400 were released in parallel with the hexose oxidase fragments. Electrophoresis was carried out at a low 10 mA current to minimize the risk of thermally induced chemical modification of the proteins in the sample.

Electrophoretic transfer to PVDF membranes was carried out as described above, except that one Immobilon P membrane sheet (Millipore, catalog number IPVH 15150) was used instead of three Problott sheets. The layered system was placed with the membrane towards the positive electrode.

After transfer to the Immobilon P membrane, it was washed in water for 10 seconds and stained for 45-60 seconds in 100 ml of a freshly prepared staining solution consisting of 0.025% Coomassie R-250 brilliant blue, 5% acetic acid, 40% methanol. The membrane was decolorized for 2-3 minutes in 250 ml of a freshly prepared solution of 5% acetic acid, 30% ethanol (96% vol. Danisco, Denmark). The membrane was finally air dried and stored at 4 ° C.

The band pattern on the membrane was identical to that observed in analytical SDS-PAGE after final purification by chromatographic focusing. It showed strong 40,000 and 29,000 bands and a weak 60,000 band.

40,000 and 29,000 bands were excised and used for amino acid analysis and for enzymatic digestion of membrane bound polypeptides as described below. The amount of 60,000 was too small to allow further analysis of the polypeptide.

2.5. Amino acid analysis of 40,000 and 29,000 hexose oxidase polypeptides

The amino acid compositions of components 29000 and 40,000 hexose oxidase are shown in Table 2.3.

Table 2.3. Amino acid composition of 40,000 and 29,000 hexose oxidase polypeptides

<td>Amino acid</td><td>40 K.</td><td>molar% 29 K.</td>

<td>Asx</td><td> 11,5</td><td> 12,5</td>

<td>Thr</td><td> 5,9</td><td> 5,2</td>

<td>Cheese</td><td> 6,1</td><td> 4,7</td>

<td>Glx</td><td> 9,7</td><td> 15,1</td>

<td>Pro</td><td> 5,2</td><td> 5,4</td>

<td>Gly</td><td> 13,6</td><td> 9,7</td>

<td>ala</td><td> 6,6</td><td> 6,4</td>

<td>Cys</td><td> 1,1</td><td> 0,9</td>

<td>val</td><td> 7,3</td><td> 5,5</td>

<td>Underworld</td><td> 1,5</td><td> 2,3</td>

<td>How much</td><td> 3,7</td><td> 4,4</td>

<td>Leu</td><td> 8,5</td><td> 8,6</td>

<td>Tyr</td><td> 4,2</td><td> 5,3</td>

<td>phe</td><td> 5,5</td><td> 4,1</td>

<td>His</td><td> 2,2</td><td> 1,4</td>

<td>lys</td><td> 3,9</td><td> 6,1</td>

<td>Arg</td><td> 3,5</td><td> 2,4</td>

<td>Trp</td><td>ND</td><td>ND</td>

<td>in total</td><td> 100,0</td><td> 100,0</td>

ND - not found

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2.6. Enzymatic digestion of hexose oxidase polypeptides bound to PVDF membrane

Digestion of hexose oxidase polypeptides bound to PVDF membrane and extraction of the resulting proteolytic peptides was performed as described in Fernandez et al. (1992) and Femandez et al. (1994).

Digestion of 40,000 hexose oxidase polypeptide: 11 40,000 bands with a total estimated amount of protein of 5 pg (corresponding to 125 pmolom) were excised from Coomassie stained PVDF membranes, decolorized in methanol for 1-2 minutes and washed in water for 2-3 minutes. The membrane bands were cut into 1 x 1 mm pieces and transferred to a microcentrifuge tube.

The empty region of the PVDF membrane served as background control. The cut membrane pieces were soaked in 50 µl digestion buffer containing 1% (v / v) hydrogenated Triton 100-100 (RTK-100), Sigma Chemicals, catalog number X-100R-PC, or Calbiochem, protein purity catalog number 648464), 10% acetonitrile (Merck, Lichrosolv grade to gradients) and 100 mM TrisCl, pH 8.0. The proteolytic enzyme selected for digestion was Lys-C endoproteinase (endoLys-C), which cleaves the peptide chains on the C-side of lysine residues. Portions of 5 pg endoLys-C (Boehringer Mannheim, sequencing purity, catalog number 1047 825) were diluted in 20 pl of water. 2 µl enzyme solution, corresponding to 0.5 pg / ml (enzyme: 1:10 substrate) was added. Digestion was carried out at 37 ° C for 22-24 hours.

After digestion, the samples were sonicated in an ultrasonic tank (Elma Transonic) for 5 minutes and centrifuged at 1700 rpm in a microcentrifuge for 5 minutes, after which the supernatant was transferred to a new tube. Subsequent rinses in 50 µL digestion buffer and 100 µL 0.1% trifluoroacetic acid (TFA, Pierce, Cat. No. 28902) were performed by sonication and centrifugation as described above. All supernatants were pooled, giving a volume of 200 pl. The extracts were kept at -18 ° C until the peptide was purified.

Digestion of the 29000 hexose oxidase polypeptide was performed as described for component 40,000, except that four bands with a total protein content of 2.4 pg (approximately 80 pmol) were used, based on amino acid analysis.

2.7. Purification of peptides produced by endoLys-C treatment.

Peptide fragments obtained by digestion of 29000 and 40,000 hexose oxidase polypeptides were separated on a SMART chromatographic system. The system was equipped with a pPeak variable wavelength monitor and fraction collector for 60 tubes. The reverse phase column used for the separation was silica based pRPC C2 / C18 SC2.1 / 10 small diameter column (Pharmacia, column dimensions 2.1 x 100 mm, particle size 3 (im, average pore size 125 A). Buffers were used: A: 0.1% TFA (Pierce) in Milli-Q water and B: 0.1% TFA in acetonitrile (Merck). The buffers were filtered and degassed by vacuum filtration on a 0.5 pm fluoropore filter (Millipore). The flow rate was 100 (pl / min. UV absorbance of the filtrate was followed at 220 nm 254 nm and 280 nm. The gradient was from 0 to 30% B (0.65 minutes), 30-60% B (65-95 minutes) and 60-80% B (95-105 minutes) Then the column was washed in 80% B for 45 minutes at 200 µl / minute. 50 µl fractions were collected between t = 15 minutes and t = 105 minutes (3x60 fractions) and stored at -18 ° C for amino acid sequencing.

The peptide map obtained after digestion with endoLys-C 4000 polypeptide is shown in Figure 6. As can be seen in this Figure, digestion and HPLC separation resulted in several signals (peaks) with a high signal-to-background ratio. A corresponding chromatogram of the empty digestion mixture (not shown) indicated that the signals before t = 83 were not protein, they came from solvents probably UV-absorbing impurities Triton 100-100 or traces of Coomassie dye. Signals marked 1-5 in Fig. 6, selected for amino acid sequencing based on the following criteria: 1) signal height; 2) actual cleanliness; 3) high ratio of A280: A220 and / or A254: A220, indicating the presence of aromatic residues that are most useful for selecting PCR primer sequences because of their low degeneracy of the genetic code; 4) late elution time, which may indicate a relatively long peptide.

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The chromatogram of peptides derived from 29000 after endoLys-C is shown in Fig. 7. Of course, this component of hexose oxidase only gave few peptide fragments compared to the 40,000 polypeptide in Fig. 6. When comparing the chromatograms, there was no indication that any fragment was present in both polypeptides. This finding indicates that the components of hexose oxidase 29000 and 40,000 do not have common amino acid sequences, which would have happened if the 29000 polypeptide had been produced by the transformation of the 40,000 proteolytic polypeptide. (Compared with 40,000 digestion products, the 29000 digestion products contained only a small amount of contaminating eluting substances later than t = 83 minutes. The reason for this may be that hydrogenated Triton X-100 with Calbiochem was used to digest 29,000, while digestion was carried out using Triton X-100 from Sigma Chemicals).

Fractions corresponding to signals designated 1 and 2 on the 29000 peptide map (Fig. 7) were amino acid-conditioned.

2.8. Amino acid sequence analysis of peptides produced proteolytically from hexose oxidase.

Peptide sequences identified by analysis of the fraction corresponding to signals 1-5 in Fig. 6 (HOX-2, HOX-3, HOX-4, HOX-5 and HOX-6 peptides) and signals 1-2 in Fig. 7 (HOX-peptides 7 and HOX-8) are shown below in Table 2.4. The initial yield of PTH amino acids was from 46 pmoles for PTH-Tyr in the first stage in the HOX-5 peptide to 6 pmoles for PTH-Ile in the second stage in the HOX-8 peptide. As expected from absorbance at 254 nm and 280 nm, respectively, all sequenced peptides contained at least one aromatic amino acid.

Table 2.4. Peptide sequences obtained by sequence analysis of Lys-C endoproteinase peptides obtained from 40,000 and 29,000 hexose oxidase polypeptides.

<td>Origin</td><td>identification</td><td>sequence</td>

<td colspan="2">sequenced sequence</td><td>The amino acid</td>

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

<td>40 K,</td><td>HOX-2 peptide signal</td><td>aiinvtglvesgydxx-</td>

<td> 1</td><td></td><td>XGYXVSS-</td>

<td>40 K, 2</td><td>HOX-3 peptide signal</td><td>dlpmsprgviasnlwf-</td>

<td>4.0 K, α</td><td>HOX-4 peptide signal</td><td>DSEGNDGELFXA- (H) -T-</td>

<td>40 K, 4</td><td>HOX-5 peptide signal</td><td>YYFK-</td>

<td>40 K,</td><td>HOX-6 peptide signal</td><td>dpgyividvnagtpd-</td>

<td>29 K, 1</td><td>HOX -7 peptide signal</td><td>LQYQTYWQ- (E) - (E) - (D) -</td>

<td>29 K,</td><td>HOX-8 peptide signal</td><td>XI (R) -DFYEEM-</td>

Residues identified in a dubious manner are enclosed in parentheses.

1) The rest of No. 15 was identified as Asp or Asn

2) The rest of No. 16 was identified as Asp or Ala

3) The rest of No. 17 was identified as Arg or Trp. HOX-2 peptide = Sequence identifier No. 9

HOX-3 peptide = Sequence identifier No. 10 Peptide HOX-4 = Sequence Identifier No. 11 Peptide HOX-5 = Sequence Identifier No. 12 Peptide HOX-6 = Sequence Identifier No. 13

187 218

HOX-7 peptide = Sequence identifier No. 14 Peptide HOX-8 = Sequence Identifier No. 15

Example 3

Isolation of the hexose oxidase gene from Chondrus crispus

3.1. RNA purification from Chondrus crispus

Freshly harvested Chondrus crispus thins were rinsed with cold water and directly frozen in liquid nitrogen for further use. About 15 grams of Chondrus crispus thallus frozen in liquid nitrogen were homogenized to a fine powder in a mortar. Frozen, homogenised material was transferred to a 50 ml tube (Nunc, cat. No. 339497) containing 15 ml extraction buffer (8 M guanidine hydrochloride; 20 mM 2 (N-morpholino) -ethanesulfonic acid (MES), pH 7.0; 20 mM ethylenediaminetetraacetic acid (EDTA); 50 mM β-mercaptoethanol).

The tube was shaken and kept on ice (0 ° C) during the next steps, unless temperature was indicated. The tube was then centrifuged for 20 minutes at 6000 xg in a Heraeus Omnifuge 2.ORS centrifuge and the supernatant containing RNA (about 15 ml) was collected and transferred to a cooled 50 ml tube. Then, 1.5 ml 2 M sodium acetate, pH 4.25, 15 ml water saturated phenol and 3 ml chloroform: isoamyl alcohol (49: 1) were added to the tube containing the RNA extract.

The tube was shaken vigorously for 1/2 minute then the phases were separated by centrifugation of the tube for 20 minutes in Omnifuge at 6000 x g. The aqueous phase (about 17 ml) was transferred to a 30 ml Corex tube (Sorvall, catalog number 00156) and an equal volume was added (about 17 ml) cold isopropanol. The tube was shaken again and incubated for one hour at -20 ° C. The precipitated RNA was centrifuged for 20 minutes at 10,000 rpm in a Sorvall centrifuge with a cooled SS34 rotor. The supernatant was removed and the RNA pellet was suspended in 4 ml 0.3 M sodium acetate, pH 5.5 and 12 ml 96% ethanol was added.

The Corex tube was shaken and re-incubated for at least one hour at -20 ° C, followed by centrifugation of the RNA again by centrifugation for 20 minutes as described above. The supernatant was gently removed, and the RNA pellet was suspended in 2 ml 0.15 M sodium acetate, pH 5.5. Then 8 ml of 4 M sodium acetate, pH 5.5 were added and RNA precipitated on ice for 30 minutes and centrifuged again as described above. The RNA pellet was washed with 70% ethanol and suspended in 500 µl water. The suspended RNA was transferred to a microcentrifuge tube and stored at -20 ° C.

The purity and concentration of RNA were studied by agarose gel electrophoresis and absorbance measurement at 260 nm and 280 nm, as described in Sambrook et al. (1989).

3.2. Isolation of polyadenylated RNA from Chondrus crispus

Polyadenylated RNA was isolated from total RNA by using magnetic beads coated with oligo dT (Dynabeads® oligo (dT) 25, in the mRNA Purification Kit ™, Dynal). About 100 pg of total RNA was mixed with 1 mg Dynabeads® oligo (dT) and polyadenylated RNA was isolated as described in the mRNA Purification Kit ™ protocol, Dynal. The yield of polyadenylated RNA using Dynabeads® was from 1 to 3%.

Other methods are also used to isolate polyadenylated RNA from Chondrus crispus, including the use of oligo- (dT) -cellulose packed columns (Clontech, catalog number 8832-2) or columns (mRNA Separator Kit ™, Clontech, catalog number K1040- 1) as described in the protocol for this kit. The yield of polyadenylated RNA isolated on oligo (dT) columns is from 0.1 to 1% of the initial amount of RNA. Polyadenylated RNA isolated on oligo (dT) columns was used in cDNA synthesis as described below (3.4.), But the cDNA yield was very low (less than 1%).

The reason for the lower yield and worse performance of RNA isolated on oligo (dT) columns compared to Dynabeads® may be the presence of carbohydrates or proteoglycans in the total RNA extract. Carbohydrates contaminating total RNA preparations interfere with the purification of polyadenylated RNAs and inhibit cDNA synthesis has been demonstrated, hence methods for isolating carbohydrate-free RNA have been developed (Groppe et al., 1993; Yeh et al.). However, polyadenylated RNA purified by these methods was not as effective in cDNA synthesis as RNA isolated using Dynabeads®. Accordingly, polyadonylated RNA purified on Dynabeads® was used as a template for the first strand cDNA synthesis reaction (cf. 3.4 below).

3.3. Hexose oxidase specific oligonucleotides

Synthetic oligonucleotides (DNA technology, ApS, Forskerparken,

DK-8000 Aarhus C, Denmark) based on amino acid sequences obtained from HOX-2, HOX-3 and HOX-4 hexose oxidase peptides (Table 2.4). Table 3.1 shows oligonucleotides and their respective amino acid sequences. In table 3.1. the DNA sequence of the primers used in DNA or PCR sequencing is also shown.

Table 3.1 Nucleotide sequences of synthetic oligonucleotide specific for hexose oxidase

Peptide Starter

Hox Hox

<td>Hox-2</td><td>AND</td><td>AND</td><td>AND</td><td>N</td><td>V</td><td>T</td><td>G -</td>

<td></td><td>L</td><td>V</td><td>E</td><td>S</td><td>G</td><td>Y</td><td>DXXXGYXVSS</td>

<td></td><td>Hox2-3 +</td><td>5'YTI gti GAR WSI</td><td>GGN TAYGA3 '</td><td></td>

<td>Hox-3</td><td></td><td>DL PM</td><td>e PR G-</td><td></td>

<td></td><td></td><td>VIA</td><td>e NLW</td><td>F</td>

<td></td><td>Hox3-2-</td><td colspan="2">3CAN TAD CGN AGI TTR RAI ACC</td><td>AA^s</td>

<td>Hox-4</td><td></td><td>DSEGN</td><td>DGE</td><td>LFXAHT</td>

Uox4-1 + ⁵Gar gGI AAY GAY GGI GAR CTN TT³'Hox4-2- 3'cty ccN TTR CTR CCI CTY GAI AA⁵'

Hox5 + 5'att GGG GCT CCT TCA AGA CCT T ^ '

Hox5- 5'tgaTGATTCAGTTTC3 '

Hox6 + 5'ttg GAA GAA TAC GGT TOG ^ '

Hox7- 5'tac TATTIC GTC TGCTTG GG3 '

Hox8- 5 GAA CTC TIC CGT GGTCTC CT 3 '

Hox 10-5'cca CCT GCG TGT TGG GGT CT3 '

Hox11 + ⁵ CAG ATC TAC AAAACA TGC GAG3 '

Hox12- 5'tgtCGCAGACTGTACTTG3 '

Hox13- 5 GAGTGTACACGACATAAA3 '

Hox5'-1 5'atg GCT ACT CTT CCC CAG AAA G3 'where Y is C or T, R is A or G; where W is A or T, S is C or G; where D is A, G or T, N is A, C, G or T, and I is deoxyinosine.

HOX-2

Hox2-3 +

HOX-3

HOX3-2 +

HOX-4

HOX4-1 +

HOX4-2HOX5 +

HOX5HOX6 +

HOX7Iden Identifier S el. No. 9 Sequential Identifier No. 16 Sequential Identifier No. 10 Idenbyikator Seq. No. 17 Ι ^ ο ^^ ηΟ ^ SeOw. No. 11 Ideas of the Seine No. 18

Seq. No. 119 j ^ n ^ fii ^^ tt ^ r Seq. No. 20

Seow. No. 21 Μοπ ^^ Οογ Sequence No. 22 Sequential Identifier No. 13

187 218

HOX8- = Sequential ID No. 24

HOX10- = Sequence Identifier No. 25

HOX11 + = Sequential Identifier No. 26

HOX12- = Sequential ID No. 27

HOX13- = Sequence Identifier No. 28

HOX5'-1 = SekWi Identifier No. 29

3.4. CDNA synthesis and polymerase chain reaction (PCR)

Polyadenylated RNA was used as template for the first strand cDNA synthesis reaction using commercially available kits. About 1 pg of polyadenylated RNA was reverse transcribed as described in the Marathon ™ cDNA Amplification Kit (Clontech) protocol with primers Hox3-2- or Hox4-2-. In the next PCR amplification, the anchor and adapter primers of the kit were used, in addition to the hexose oxidase specific primers Hox3-2- or Hox4-2-, respectively. The buffers and amplification conditions used were as described essentially in the Marathon ™ kit protocol. PCR amplification was performed using AmpliTag (Perkin-Elmer Cetus) and a Perkin-Elmer Thermalcycler 480 ™ device programmed for 30 cycles of 1 minute at 94 ° C, 2 minutes at 55 ° C and 2 minutes at 72 ° C. Electrophoresis of a 5 µl reaction mixture on a 1% agarose gel (SeaPlaque® GTG, FMC) showed DNA fragments of approximately 600 base pairs (bp) with the primer Hox4-2- and 700 bp with the primer Hox3-2-.

These DNA fragments were purified on agarose gel using commercially available kits (QIAEX ™ Gel Extraction Kit, Cat. No. 20020, QUIAGEN) and about 100 ng of the fragment was ligated and with 50 ng of the pT7 Blue plasmid as described in the pT7 Blue TVector Kit protocol (Novagen). E. coli DH5a (Life Technologies) or E. coli NovaBlue (Novagen) was transformed with the ligation mixture and further recombinant white colonies were analyzed.

Plasmid DNA from these colonies was purified using the QUIAGEN Plasmid Midi Kit (QUIAGEN) and subjected to DNA sequence analysis using Sequenase (Sequenase Version 2.0 DNA Sequencing Kit, USB). DNA sequencing reactions were subjected to polyacrylamide gel electrophoresis (Sequencing Gel Mix®6, Life Technologies). Sequence DNA analysis of the 700 bp fragment showed an open reading frame with 234 amino acids encoding capability.

Table 3.2. below shows that all peptide sequences from the 40,000 polypeptide, i.e. HOX-2, HOX-3, HOX-4, HOX-5 and HOX-6, were found in the sequence of 234 amino acids obtained from the open reading frame. Thus, it was concluded that the 700 bp fragment encodes part of the hexose oxidase gene. The DNA sequence of the 600 bp fragment was, as it turned out, identical to the proximal part of the 700 bp fragment (see Table 3.2).

The Hox2-3 + and Hox3-2- primers were used similarly in cDNA synthesis and PCR amplification. About 50 ng of polyadenylated RNA was reverse transcribed using the Hox3-2- primer as described in the 3'-Amplifinder ™ RACE Kit protocol (Clontech). The Hox2-3 + and Hox3-2- primers were used in subsequent PCR amplifications. The buffers and amplification conditions used were essentially as described for AmpliTaq polymerase (Perkin-Elmer Cetus) and the 3'-Amplifindkr ™ RACE Kit protocol. Gel electrophoresis on a 5 µl PCR reaction mixture showed a fragment of about 407 bp in size.

This fragment was purified, introduced into plasmid pT7 Blue and sequenced as described above. The DNA sequence of this fragment is identical to the distal 407 bp fragment of the 700 bp fragment.

The sequence below the 700 and 407 bp fragments was amplified with the 3'Amplifinder ™ RACE Kit (Clontech) using the anchor primers from the kit as primer 3 and the Hox5 + and Hox4 + primers specific for hexose oxidase as the 5 'gene specific primers. Buffers and reaction conditions were as described above. PCR and analysis of the agarose gel reaction mixture showed a fragment approximately 1.3 kb in size. The fragment was isolated and subjected to DNA sequence analysis as described above. The DNA sequence of the 1.3 kb fragment showed an open reading frame of 357 amino acids. The 357 amino acid frame contained the amino acid sequences of HOX-2, HOX-3, HOX-4, HOX-5, HOX-7 and HOX-8 peptides.

187 218

From this it was concluded that the 1.3 kb DNA fragment encoded the 9000 post CNBr fragment, the 29000 polypeptide and the 40,000 hexose oxidase part of the polypeptide.

The primer specific for the 5 'end of hexose oxidase, Hox5'-1 was used together with the oligo (dT) primer to amplify probably the entire hexose oxidase reading frame. The gene was amplified using PCR, introduced into pT7 Blue and sequenced as described above. The DNA sequence of this 1.8 kb DNA fragment was identical to the DNA sequences of the fragments described above, with slight differences. Because these differences could be due to polymerase errors during PCR amplification, the entire hexose oxidase gene was amplified and isolated from three independent PCR amplifications. Thus, the DNA sequence shown below in Table 3.2. it is composed of at least three independently obtained DNA sequences to exclude PCR errors.

The amino acid sequence obtained from the open reading frame of the above 1.8 kb DNA sequence contains all of the above HOX peptides, i.e. from HOX-1 to HOX-8. The 1.8 kb DNA sequence encodes the above 9000, 29000 and 40,000 hexose oxidase fragments derived from Chondrus crispus. The molecular weight of the polypeptide derived from the open reading frame is consistent with the suspicion that the polypeptide is a subunit (probably a monomeric fragment) of a dimeric hexose oxidase molecule.

3.5. Northern blot analysis by RNA Chondrus crispus

Total RNA isolated from Chondrus crispus was subjected to Northern blot analysis. RNA was purified as described above (3.1) and fractionated on a denaturing agarose gel with formalin and transferred to a HybondC filter (Amersham) as described in Sambrook et al., 1989. Using primers Hox2-3 + and Hox3-2- synthesized by PCR a 400 bp DNA fragment as described above. This fragment was purified from a 1.2% agarose gel (SeaPlaque® GTG, FMC) and labeled³²P as described in Sambrook et al., 1989. This radiolabeled hybridization probe was used to probe Northern blot.

Hybridization conditions:

3.5.1. Prehybridization at 65 ° C for two hours in buffer containing 10x Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 2x SSC (IX SSC - 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.1% sodium dodecylate sulfate (SDS) and 50 pg / ml denatured elk sperm DNA.

3.5.2. Hybridization at 65 ° C for at least 14 hours in buffer containing 1x Denhardt's solution, 2x SSC, 0.1% dextran sulfate, 50 pg / ml denatured salmon sperm DNA and labeled probe^MP (about 106 dpm / minute). The filter was washed twice at 65 ° C for 10 minutes in 2x SSC, 0.1% SDS and then twice at 65 ° C for 10 minutes in 1x SSC, 0.1% SDS. After final rinsing for 10 minutes at 65 ° C in 0.2x SSC, 0.1% SDS, the filter was wrapped with Saran Wrap foil and exposed to X-ray film (Kodak XAR2) for 2 days at -80 ° C using Siemens screening screens Titan HS. The resulting autoradiogram (Fig. 8) shows a band about 2 kb in size.

Table 3.2. The nucleotide sequence of the 1.8 kb DNA sequence (SEQ ID No. 30) and the open reading frame of the 546 amino acid hexose oxidase sequence derived from the DNA sequence (SEQ ID No. 31).

TGAATTCGTG GGTCGAAGAG CCCTTTGCCT CGTCTCTCTG GTACCGTGTA TGTCAAAGGT 60

TCGCTTGCAC ACTGAACTTC ACG ATG GCT ACT CTT CCT CAG AAA GAC CCC 110

Met Ala Thr Leu Pro Gln Lys Asp Pro

5

GGT TAT ATT GTA ATT GAT GTC AAC GCG GGC ACC GCG GAC AAG CCG GAC 1161

Gly Tyr Ile Val Ile Asp Val Asn Ala Gly Thr Ala Asp Lys Pro Asp

187 218

<td>CCA</td><td>CGT</td><td>CTC</td><td>CCC</td><td>TCC</td><td>ATG</td><td>(AG</td><td>CAG</td><td>AC</td><td>TTC</td><td>(AC</td><td>CGC</td><td>CGC</td><td>TGG</td><td>ATT¹</td><td>AA</td><td> 206</td>

<td>Pro</td><td>Arg</td><td>Leu</td><td>Pro</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Gln</td><td>Gly</td><td>phe</td><td>own</td><td>Arg</td><td>(Arg</td><td>Tir?</td><td>How much</td><td>gle</td><td></td>

<td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 33</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td>

<td>ACT</td><td>AAT</td><td>ATC</td><td>GAT</td><td>TTC</td><td>GTT</td><td>TAT</td><td>GTC</td><td>GTG</td><td>TAC</td><td>ACT</td><td>CCT</td><td>CAA</td><td>AT</td><td>GAT</td><td>(TA</td><td> 254</td>

<td>Thr</td><td>own</td><td>How much</td><td>Asp</td><td>phe</td><td>val</td><td>GTyr</td><td>val</td><td>val</td><td>Tyr</td><td>Thr</td><td>Pro</td><td>Gln</td><td>Gly</td><td>AAE</td><td>gcs</td><td></td>

50 55

<td colspan="3" rowspan="2">ACT ACA CTT Thr Ala Leu 60</td><td colspan="4">GAC CGT GCT ATG</td><td colspan="2">GAAA (AG</td><td rowspan="2">TGT cys</td><td colspan="5">TCT CCC GGT AGA GAC</td><td rowspan="2">(AND (A r</td><td rowspan="2"> 302</td>

<td>Asp</td><td colspan="2">Arg Ala</td><td>Underworld</td><td>Glu 65</td><td>lys</td><td colspan="2">Cheese Pro</td><td>Gly 70</td><td>Thr</td><td>vaa</td>

<td>ATC</td><td>ATC</td><td>TCT</td><td>AC</td><td>GGC</td><td>CAT</td><td>TGC</td><td>TAC</td><td>GAG</td><td>GAC</td><td>TTC</td><td>GTA</td><td>ttt</td><td>GAC</td><td>GAA</td><td>(AND</td><td> 350</td>

<td>lle</td><td>val</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>His</td><td>cys</td><td>Tyr</td><td>Glu</td><td>Asp</td><td>phe</td><td>val</td><td>phe</td><td>Asp</td><td>dL</td><td>(ys</td><td></td>

<td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td></td>

<td>ATC</td><td>AAA</td><td>GCC</td><td>ATC</td><td>ATC</td><td>AAC</td><td>GTC</td><td>ACT</td><td>GGT</td><td>CTC</td><td>GTT</td><td>GAG</td><td>AGT</td><td>GGT</td><td>TAT</td><td>dA</td><td> 398</td>

<td>val</td><td>lys</td><td>ala</td><td>How much</td><td>How much</td><td>own</td><td>val</td><td>Thr</td><td>Gly</td><td>Leu</td><td>val</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>tyy</td><td>aas</td><td></td>

<td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td>

<td>AAC</td><td>AAT</td><td>AGG</td><td>AT</td><td>TAC</td><td>ttc</td><td>GTC</td><td>AGC</td><td>AGT</td><td>AA</td><td>GAT</td><td>ACA</td><td>AAT</td><td>TA</td><td>AC</td><td>(TC</td><td> 446</td>

<td>Asp</td><td>Asp</td><td>Arg</td><td>Gly</td><td>Tyr</td><td>phe</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Gly</td><td>Asp</td><td>Thr</td><td>own</td><td>Τηρ</td><td>Gal</td><td>See</td><td></td>

<td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 11^^</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td>

<td>TTC</td><td>AAA</td><td>ACC</td><td>TTG</td><td>TTC</td><td>AGA</td><td>GAC</td><td>(CC</td><td>AA</td><td>AGA</td><td>GTT</td><td>CTT</td><td>CCC</td><td>GdA</td><td>Gd</td><td>(TC</td><td> 494</td>

<td>phe</td><td>lys</td><td>Thr</td><td>Leu</td><td>phe</td><td>Arg</td><td>Asp</td><td>His</td><td>Gly</td><td>(Arg</td><td>val</td><td>Leu</td><td>Pro</td><td>Gly</td><td>dL</td><td>(ee</td><td></td>

<td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td>U5</td><td></td><td></td><td></td>

<td>TAC</td><td>TAC</td><td>TCC</td><td>GTC</td><td>GGC</td><td>CTC</td><td>AT</td><td>GGC</td><td>ccc</td><td>ATT</td><td>GTC</td><td>AC</td><td>AA</td><td>AT</td><td>dA</td><td>AA</td><td> 554</td>

<td>Cys</td><td>Tyr</td><td>Cheese</td><td>val</td><td>Gly</td><td>Leu</td><td>Gly</td><td>Gly</td><td>hii</td><td>Ill</td><td>val</td><td>Gly</td><td>Gly</td><td>Gly</td><td>AAP</td><td>dL</td><td></td>

140

145

110

187 218

<td colspan="5">ATT TTG GCC CGC ^ TTG</td><td rowspan="2">CAT His</td><td rowspan="2">mc Gly 160</td><td colspan="9">CCC CCC GGT GAT TTG} CTC AGC GAC GGT</td><td rowspan="2"> 555</td>

<td>How much</td><td colspan="3">Leu Ala Arg 155</td><td>Leu</td><td>Leu</td><td>Pro</td><td>VVL</td><td>goat</td><td>TT 110</td><td>Glu</td><td>ter</td><td>Gly</td><td>val</td>

<td>GAG</td><td>GTC</td><td>GTC</td><td>GTT</td><td>AAG</td><td>CCA</td><td>GTC</td><td>CTC</td><td>ACC</td><td>GAA</td><td>GAT</td><td>TCG</td><td>GTA</td><td>CTC</td><td>ATT</td><td>TGT</td><td> 603</td>

<td>Glu</td><td>val</td><td>val</td><td>val</td><td>lys</td><td>Pro</td><td>val</td><td>Leu</td><td>Thr</td><td>G1u</td><td>Cajj</td><td>Tsr</td><td>val</td><td>Leu</td><td>lys</td><td>iyy</td><td></td>

<td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 110</td><td></td>

<td>GTG</td><td>CAC</td><td>AAA</td><td>GAT</td><td>TCC</td><td>GAA</td><td>GIC</td><td>AAC</td><td>CAAC</td><td>taiga</td><td>CAA</td><td>CTC</td><td>gcc</td><td>TCGl</td><td>GCA</td><td>CAT</td><td> 600</td>

<td>val</td><td>His</td><td>lys</td><td>Asp</td><td>Cheese</td><td>Glu</td><td>Gly</td><td>own</td><td>Asp</td><td>Gly</td><td>G1u</td><td>Leu</td><td>phe</td><td>Tc?</td><td>ala</td><td>hii</td><td></td>

<td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td>

<td>ACA</td><td>GGT</td><td>GGC</td><td>CALt</td><td>CGAC</td><td>GGA</td><td>AAC</td><td>TCGł</td><td>DAD</td><td>ATC</td><td>TTC</td><td>ACC</td><td>AAA</td><td>TAC</td><td>TAC</td><td>TTT</td><td> 774</td>

<td>Thr</td><td colspan="2">Gly gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>own</td><td>phe</td><td>Gly</td><td>IIl</td><td>Bullfinch</td><td>Thr</td><td>lys</td><td>Tyr</td><td>GT so ^</td><td>PPe</td><td></td>

<td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td>

<td>AAG</td><td>GAT</td><td>TTG</td><td>CCC</td><td>ATG</td><td>TCT</td><td>C (CA</td><td>CCTG</td><td>CATC</td><td>GTC</td><td>ATC</td><td>GCA</td><td>TCCA</td><td>AAT</td><td>ΤΓΑ</td><td>CCA</td><td> 770</td>

<td>lys</td><td>Asp</td><td>Leu</td><td>Pro</td><td>Underworld</td><td>Cheese</td><td>Pro</td><td>Arg</td><td>Gly</td><td>VVL</td><td>tIL</td><td>ala</td><td>Cheese</td><td>own</td><td>Leu</td><td>hii</td><td></td>

<td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td>

<td>TTC</td><td>AGC</td><td>TGG</td><td>&lt;AAC</td><td>GIT</td><td>TTC</td><td>ACG</td><td>AGA</td><td>GAT</td><td>GCC</td><td>GTG</td><td>GAT</td><td>GAT</td><td>TTG</td><td>TTG</td><td>AAC</td><td> 800</td>

<td>phe</td><td>Cheese</td><td>Trp</td><td>Asp</td><td>Gly</td><td>phe</td><td>Thr</td><td>Arg</td><td>Asp</td><td>ala</td><td>Leu</td><td>Gln</td><td>Asp</td><td>Leu</td><td>Leu</td><td>TCe</td><td></td>

<td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td><td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td></td>

<td>AAG</td><td>TAC</td><td>TTC</td><td>CAAA</td><td>CTT</td><td>GCC</td><td>AGA</td><td>TGT</td><td>GAT</td><td>TGG</td><td>AAG</td><td>AAT</td><td>ACG</td><td>GTT</td><td>GGC</td><td>AAG</td><td> 808</td>

<td>lys</td><td>Tyr</td><td>phe</td><td>lys</td><td>Leu</td><td>ala</td><td>Arg</td><td>C "S</td><td>Asp</td><td>TL?</td><td>lys</td><td>own</td><td>Thr</td><td>val</td><td>Gly</td><td>ly ^^</td><td></td>

<td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 265</td><td></td>

<td>TTT</td><td>CAA</td><td>ATC</td><td>nc</td><td>CAT</td><td>&lt;CAG</td><td>GGA</td><td>GCG</td><td>GAA</td><td>GAG</td><td>ICC</td><td>GTC</td><td>ATG</td><td>TAC</td><td>TTG</td><td>TGT</td><td> 926</td>

<td>phe</td><td>LLN</td><td>How much</td><td>phe</td><td>His</td><td>Gln</td><td>ala</td><td>ala</td><td>Glu</td><td>Glu</td><td>ple</td><td>val</td><td>Underworld</td><td>trr</td><td>Leu</td><td>tcr</td><td></td>

270

5

80

187 218

974

<td colspan="4">ACA TCC TATA TTG</td><td colspan="10">AAC CAC GCC GAG CGC GAG GCT CCC CAA GAC</td><td rowspan="2">CAT ACG</td><td rowspan="2">CAC Cia</td>

<td>Thr</td><td>Cheese</td><td colspan="2">Tyr See 285</td><td>Asa</td><td>acp</td><td>Asa</td><td colspan="2">Gic Acg 2 ^ 0</td><td>Glu</td><td>VGG</td><td>ala</td><td>Glu</td><td>asv 22 ^ 5</td>

<td>TAT</td><td>CAT</td><td>TTG</td><td>GAG</td><td>GCT</td><td>GGC</td><td>GTA</td><td>GAG</td><td>CGG</td><td>ATC</td><td>TAC</td><td>ACC</td><td>CCC</td><td>TGC</td><td>GGG</td><td>CCC</td>

<td>Tyr</td><td>His</td><td>Leu</td><td>Glu</td><td>Gla</td><td>gsp</td><td>How much</td><td>Glu</td><td>Gln</td><td>How much</td><td>Tyr</td><td>lys</td><td>Thr</td><td>Cys</td><td>Glu</td><td>Pro</td>

<td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 331</td><td></td><td></td><td></td>

<td>ACC</td><td>AAA</td><td>GCG</td><td>CTT</td><td>GGC</td><td>GGG</td><td>CGT</td><td>GCT</td><td>CTT</td><td>TGG</td><td>GCG</td><td>CCG</td><td>TTC</td><td>CCC</td><td>GTG</td><td>CGG</td>

<td>Thr</td><td>lys</td><td>ala</td><td>Leu</td><td>Gly</td><td>Gly</td><td>His</td><td>Cla</td><td>Gly</td><td>Trp</td><td>Gla</td><td>Pro</td><td>phe</td><td>Pro</td><td>val</td><td>CRG</td>

<td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 332</td><td></td><td></td><td></td><td></td><td> 332</td><td></td><td></td><td></td><td></td>

<td>CCG</td><td>CGC</td><td>AAG</td><td>AGG</td><td>CGC</td><td>GCG</td><td>TCC</td><td>AGG</td><td>ACG</td><td>TCG</td><td>TGT</td><td>GTG</td><td>CGT</td><td>GCC</td><td>GCG</td><td>GCG</td>

<td>Pro</td><td>Arg</td><td>lys</td><td>Arg</td><td>His</td><td>Thr</td><td>Cheese</td><td>lys</td><td>Thr</td><td>Cheese</td><td>Tyr</td><td>Underworld</td><td>His</td><td>Csp</td><td>Glu</td><td>Thr</td>

<td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 334</td><td></td><td></td><td></td><td></td><td> 334</td>

<td>ATG</td><td>GAC</td><td>TAC</td><td>CCC</td><td>TTC</td><td>TGC</td><td>GCG</td><td>CTC</td><td>CCT</td><td>GCG</td><td>GCG</td><td>GTC</td><td>AAC</td><td>GGC</td><td>TCC</td><td>CTT</td>

<td>Underworld</td><td>Asp</td><td>Tyr</td><td>Pro</td><td>phe</td><td>Tyr</td><td>ala</td><td>Leu</td><td>Thr</td><td>Glu</td><td>Thr</td><td>How much</td><td>own</td><td>Gly</td><td>Cheese</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td>

<td>CCG</td><td>AAT</td><td>CAG</td><td>CGC</td><td>GGC</td><td>CGG</td><td>TGC</td><td>AGG</td><td>TCT</td><td>GCG</td><td>TAC</td><td>CTG</td><td>CTC</td><td>AAG</td><td>GCT</td><td>TTC</td>

<td>Pro</td><td>own</td><td>Gln</td><td>Arg</td><td>Gly</td><td>lys</td><td>Tyr</td><td>lys</td><td>Cheese</td><td>ala</td><td>Tyr</td><td>Underworld</td><td>How much</td><td>lys</td><td>gsp</td><td>phe</td>

<td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td><td></td><td> 3-70</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td>

<td>CCG</td><td>GAT</td><td>TTC</td><td>CAG</td><td>GTC</td><td>GAC</td><td>GTG</td><td>CTC</td><td>TGG</td><td>CCG</td><td>TCC</td><td>CTT</td><td>GCG</td><td>GGG</td><td>GTC</td><td>CCG</td>

<td>Pro</td><td>Asp</td><td>phe</td><td>Gln</td><td>How much</td><td>gsp</td><td>val</td><td>How much</td><td>Trp</td><td>lys</td><td>Tyr</td><td>Leu</td><td>Thr</td><td>Glu</td><td>val</td><td>Pro</td>

<td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 339)</td><td></td><td></td><td></td>

<td>GAC</td><td>GGC</td><td>TTG</td><td>ACT</td><td>GGT</td><td>GCC</td><td>GCA</td><td>GTG</td><td>CCG</td><td>GGT</td><td>GCC</td><td>TTC</td><td>CTC</td><td>CGG</td><td>GTG</td><td>GCC</td>

<td>Asp</td><td>Gly</td><td>Leu</td><td>Thr</td><td>Cheese</td><td>ala</td><td>Glu</td><td>Underworld</td><td>lys</td><td>gsp</td><td>Gla</td><td>Leu</td><td>Leu</td><td>Gln</td><td>val</td><td>Asp</td>

<td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td>

<td>ATG</td><td>TTT</td><td>GGT</td><td>GGT</td><td>GAG</td><td>GTT</td><td>CCC</td><td>AAG</td><td>GTG</td><td>GTC</td><td>TGG</td><td>GCT</td><td>GCG</td><td>GCG</td><td>GCA</td><td>GTC</td>

<td>Underworld</td><td>phe</td><td>Gly</td><td>Gly</td><td>Glu</td><td>How much</td><td>His</td><td>lys</td><td>val</td><td>vai</td><td>Trp</td><td>gsp</td><td>Cla</td><td>Thr</td><td>Cla</td><td>val</td>

1122

1107

1111

1116

1214

1262

1310

410

415

442

420

1358

187 218

<td rowspan="2">GCG ala</td><td colspan="2">CAG CGC</td><td colspan="3">GAG TAC ATT</td><td rowspan="2">ATC AAA But Lys</td><td rowspan="2">CTG Leu</td><td colspan="4">CAG TAC CAG ACA ACA GGA AGA</td><td rowspan="2"> 1106</td>

<td>Gln</td><td>Ar ej</td><td>Glu</td><td>thousand 400</td><td>But</td><td>Gln 405</td><td>Tyr Gln</td><td>Thr</td><td>Tyr Trp Gln 440</td>

<td>GAA</td><td>GAA</td><td>GAC</td><td>CAGA</td><td>AGA</td><td>AGC</td><td>GTG AA ^ CC</td><td>CTC</td><td>GGG</td><td>TCG3 AT</td><td>AGA</td><td>GAC TT TAC</td><td> 11^4</td>

<td>Glu</td><td>Glu</td><td>Asp</td><td>lys</td><td>aAN</td><td>Aasia</td><td>Val Asn</td><td>Leu</td><td>lys</td><td>TTp He</td><td>AAog</td><td>Asp Phe Tyo</td><td></td>

<td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td><td> 450</td><td></td><td></td><td></td><td> 455</td><td></td>

<td>GAG</td><td>GAG</td><td>ATG</td><td>TM?</td><td>AGA</td><td>ACG</td><td>AAT tAAC</td><td>GGA</td><td>GTT</td><td>CCA GAC</td><td>CCC</td><td>AAAC GGA CAG</td><td> 1102</td>

<td>Glu</td><td>Glu</td><td>Underworld</td><td>ITs</td><td>AGU</td><td>Apo</td><td colspan="2">Tyr Gly Aly</td><td>val</td><td>Pro AAsp</td><td>Pro</td><td>AAsn Thr Gln</td><td></td>

<td></td><td></td><td> 460</td><td></td><td></td><td></td><td> 465</td><td></td><td></td><td></td><td> 470</td><td></td><td></td>

<td>GTG</td><td>GAG</td><td>AGT</td><td>(AGG</td><td>AAA</td><td>AGT</td><td>GTG</td><td>GAG</td><td>GGA</td><td>TGC TAC</td><td>TTC</td><td>AAAC TAC CCG</td><td> 1550</td>

<td>val</td><td>Glu</td><td>Cheese</td><td>GGU</td><td>thousand</td><td>Gly</td><td>Val Phe</td><td>Glu</td><td>Gly</td><td>Tys</td><td>phe</td><td>AAsn Tyo · Pro</td><td></td>

<td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td><td></td><td></td><td> 485</td><td></td><td></td><td></td>

<td>GAT</td><td>GTG</td><td>GAC</td><td>TTT</td><td>AAkA</td><td>? AA</td><td>TGG AAG</td><td>AA ^ C</td><td>GGC</td><td>AA ^ G TAT</td><td>GTT</td><td>CGCC CTC GGG</td><td> 1598</td>

<td>Asp</td><td>val</td><td>Asp</td><td>Lye</td><td>aAN</td><td>aAN</td><td>Tto&gt; Lys</td><td>own</td><td>Gly</td><td>Lys Ayr</td><td>Gly</td><td>Ala Leu Glu</td><td></td>

<td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td><td></td><td></td><td> 500</td><td></td><td> 505</td><td></td>

<td>CTT</td><td>TAC</td><td>TTT</td><td>TTT</td><td>AGG</td><td>0AAC</td><td>ACTG AAAC</td><td>CGC</td><td>CTC</td><td>ATC AAAG</td><td>GCC</td><td>AAAAA TCG TTG</td><td> 1646</td>

<td>Leu</td><td>Tyr</td><td>phe</td><td>Alu</td><td>Gly</td><td>own</td><td>Leu Asn</td><td>Arg</td><td>Leu</td><td>How many bald</td><td>ala</td><td>Lys Trp Leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 510</td><td></td><td></td><td></td><td> 515</td><td></td><td></td><td> 220</td><td></td>

<td>TGG</td><td>GAT</td><td>CCC</td><td>aaa</td><td>AGA</td><td>ATC</td><td>TTC ACA</td><td>CGG</td><td>CGG</td><td>CAG AGC</td><td>ATC</td><td>CCT ACT AAA</td><td> 1694</td>

<td>Trp</td><td>Asp</td><td>Pro</td><td>aAN</td><td>AGU</td><td>All</td><td>Phe Thr</td><td>own</td><td>lys</td><td>Gln Cheese</td><td>lle</td><td>Pro Thir Lys</td><td></td>

<td></td><td></td><td></td><td> 525</td><td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td> 535</td><td></td>

<td>CCT</td><td>CTT</td><td>AAG</td><td>GAG</td><td>CCC</td><td>AAA</td><td>CAA ACA</td><td>AAA</td><td colspan="4">yAGTCAATGG GACyyGAyCA</td><td> 1741</td>

<td>Pro</td><td>Leu</td><td>lys</td><td>Glu</td><td>Pro</td><td>lys</td><td>Gln Thr</td><td>lys</td><td></td><td></td><td></td><td></td><td></td>

540 555

TCGACTGAAG TGCAGCACTT GTCGGATACG GCGTGATGGT TGCTTTTTAT AAACTTGGTA 1801

187 218

In the amino acid sequence shown above, in Table 3.2., HOX-1 to HOX-8 peptides are shown in bold or underlined. The bold font indicates the amino acid residues confirmed by the amino acid sequencing of the peptides. Underlining indicates amino acid residues that were obtained from the nucleotide sequence but were not confirmed by sequencing of the corresponding HOX peptides.

HOX-1 is amino acid residues 461-468, HOX-2 residues 92-114, HOX-3 residues 219-234, HOX-4 residues 189-202, HOX-5 residues 215-218, HOX-6 residues 8-22, HOX-7 residues 434-444 and HOX-8 residues 452-460.

Example 4

Production of recombinant hexose oxidase in Pichia pastoris.

4.1. Construction of the vector for the expression of recombinant hexose oxidase in Pichia pastoris.

The open reading frame encoding Chondrus crispus hexose oxidase was introduced into the expression vector Pichia pastoris, pPIC3 (Research Corporation Technologies Inc., Tucson, Arizona). The plasmid contained the alcohol dehydrogenase promoter (oax promoter 1) and the Pichia pastoris translation interrupt signal (in Figure 9 aoxp and aoxt, respectively). The his4 + gene in the vector allows selection of recombinant His + Pichia pastoris cells. When this expression cassette is transformed into Pichia pastoris, it is integrated into the chromosomal DNA. Pichia pastoris cells carrying the expression cassette with the Chondrus crispus hexose oxidase gene introduced downstream of the aoxl promoter can be induced to produce hexose oxidase by adding the aox1 promoter inductor, methanol. The mutant Pichia pastoris, KM71, which has a defect in the major alcohol oxidase gene, aox1, can be used as the recipient of the hexose oxidase gene (Cregg and Madden, 1987; Tschopp et al., 1987). However, Pichia pastoris contains one more alcohol oxidase gene, aox2, which can also be induced with methanol. Thus, recombinant Pichia pastoris transformed with a hexose oxidase expression cassette should produce two oxidases, hexose oxidase and alcohol oxidase, after adding methanol.

Prior to the introduction of the hexose oxidase gene into the pPIC3 expression vector, the 5 'and 3' sequences of the open reading frame were modified. The first strand cDNA was used as template for PCR. Synthetic oligonucleotides specific for the 5 'end of the open reading frame, Hox5-1 (Table 3.1) as a PCR primer were used together with the primer (Hox3'-1) specific for the 3' end of the Chondrus crispus oxidase coding sequence. The Hox3'-1 primer had a sequence

5'-ACCAAGTTTATAAAAAGCAACCATCAC-3 '(SEQ ID No. 32). PCR amplification was performed using the GeneAmp®PCR Reagent Kit with AmpliTaq® DNA polymerase (Perkin-Elmer Cetus. The PCR program comprised 30 cycles of 30 seconds at 94 ° C, 30 seconds at 55 ° C and 2 minutes at 72 ° C. Electrophoresis on the reaction gel showed a band of approximately 1.7 kb. This 1.7 kb fragment was introduced into the pT7 Blue vector (Novagen) (plasmid pUPO150) and sequenced.

The fragment encoding Chondrus crispus oxidase oxidase was further cloned into the expression vector Pichia pastoris pPIC3 (Clare et al., 1991) as shown in Fig. 9. Plasmid pT7 carrying the hexose oxidase gene was digested with NdeI restriction endonuclease and the ends were cleaved with Klen DNA polymerase in Sambrook et al., 1989. After reactivation of the polymerase, the DNA was digested with EcoRI restriction and the DNA fragment containing the hexose oxidase gene on an agarose gel was purified as a blunt EcoRI DNA fragment (QUIEX ™, QUIAGEN).

The expression vector Pichia pastoris pPIC3 was restriction digested with SnaBI and EcoRI enzymes and purified on an agarose gel. The purified vector and the hexose oxidase coding fragment were ligated and transformed with E. coli DH5a ligation mixture (Life Technologies) essentially as described in Sambrook et al., 1989. The resulting expression vector containing the hexose oxidase gene from Chondrus crispus, pUPO153, was subjected to DNA sequencing to ensure that no mutations occurred in the hexose oxidase gene during the cloning procedure.

Plasmid pUPO153 was purified from E. coli DH5a and introduced into Pichia pastoris using electroporation (The Pichia Yeast Expression System, Phillips Petroleum Company) or

187 218 using The Pichia Spheroplast Module (Invitrogen, San Diego, USA). Mutant Pichia pastoris with a damaged methanol utilization system, KM71 (genotype his4, aox1 :: ARG4) (Cregg and Madden 1987; Tschopp et al., 1987). Used as recipient. Recombinant Pichia pastoris colonies selected on agar plates without histidine were screened by PCR for the presence of the hexose oxidase gene.

In addition to the hexose oxidase specific primers (Table 3.1), primers specific for the alcohol oxidase promoter and translation interrupt signal Pichia pastoris (Invitrogen) were used.

A sample of Pichia pastoris KM71 containing pUPO 153 was deposited at Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124, Braunschweig, Germany, May 23, 1996 under DSM 10693.

4.2. Expression of recombinant hexose oxidase in Pichia pastoris

The KM71 Pichia pastoris strain containing the expression cassette with the hexose oxidase gene inserted between the aoxl promoter and the translation interrupt signal was grown in shaken bottles in MD (1.34 g yeast nitrogen base per liter (Difco), 0.4 mg / l biotin, 0.1 % arginine and 20 g / l glucose). 1-liter bottles containing 150 ml of culture were incubated on a rotary shaker at 30 ° C, 300 rpm. When the cells reached OD600 = 15-20, they were harvested by centrifugation at 6000 xg for 10 minutes and suspended in a similar volume (150 ml) of MM induction medium (1.34 g yeast nitrogen base per liter (Difco), 0.4 mg / l biotin, 0.1% arginine, 20 g / l glucose and 1% methanol). After culturing for two days, additional methanol (0.5%) was added to compensate for used and evaporated methanol.

Three to four days after induction, cells were harvested by centrifugation (6000 xg, 10 minutes and resuspended in approximately 1/5 volume of 50 mM Tris-Cl, pH 7.5. Suspended cells were kept cool until broken in the FRENCH® press (SLM Instrument ?, Inc., Rochester, NY) Cells were destroyed in 20K FRENCH® Pressure Cell at 20,000 psi internal pressure. The cell extract was clarified by centrifugation at 10,000 xg for 10 minutes at 5 ° C. The supernatant containing hexose oxidase was carefully removed and subjected to purification as described below.

4.3. Purification of recombinant hexose oxidase from Pichia pastoris

4.3.1. First step, anion exchange chromatography

The clarified homogenate from the FRENCH press (100-150 ml) was subjected to anion exchange chromatography in an FPLC system equipped with two 5 ml HiTrap-Q columns filled with Q-Sepharose High Performance (Pharmacia). The columns were connected in series and chromatography was carried out at room temperature. The columns were equilibrated with buffer A: 20 mM Tris-Cl, pH 7.5. The flow rate was 1.25 ml during sample introduction and 2.5 ml during washing and elution. After introducing the samples, the column was washed with 30 ml buffer A. The adsorbed proteins were eluted with 200 ml gradient buffer A to buffer B: 20 ​​mM Tris-Cl, 750 mM NaCl, pH 7.5. 2 ml fractions were collected during washing and gradient elution. Fractions were tested for hexose oxidase activity as described above in Example 1.3 (10 µL, 15 incubations). Fractions were also tested for alcohol oxidase (AOX) activity in a test that was identical to that of hexose oxidase, but 0.5% methanol instead of 0.05M glucose was used as the substrate. As can be seen in Figure 10, the activity profiles show that AOX and HOX eluted in parallel at a salt concentration of 400 mM NaCl. Fractions containing hexose oxidase were inoculated and stored at 4 ° C.

4.3.2. Stage two, gel filtration.

The pool from the first purification step (20-30 ml) was concentrated to about 3.5 ml by ultracentrifugation at 4 ° C in a Centriprep apparatus (Amicon, USA, nominal molecular weight cut-off = 30,000). The concentrated hexose oxidase preparation was clarified by centrifugation, and the supernatant was mixed with glycerin to a final concentration of 5%. The sample was loaded onto the column using the SA-5 applicator (Pharmacia) connected to the column inlet. Gel filtration was carried out at 4 ° C on a XK 26/70 column (2.6 x 66 cm, Pharmacia) with a bed volume of 350 ml. The column was filled with Sephacryl S-200 HR (Pharmacia) according to the instructions

187 218 manufacturer. The buffer was 20 mM Tris-Cl, 500 mM NaCl, pH 7.5, and the P1 peristaltic pump (Pharmacia) was set to 0.5 ml / minute. Uv absorbance at 280 nm was recorded. 2.5 ml fractions were collected and tested for hexose oxidase and alcohol oxidase activity as described above (10 pl sample, 15 minutes incubation). Activity profiles showed that AOX and HOX activities are separated, see Fig. 11. This result was expected because the alcohol oxidase from methylotropic yeast such as Pichia pastoris has a native molecular weight of approximately 600,000 (Sahm and Wagner, 1973), while HOX has a native molecular weight of approximately 11 ^^ (^^^ 130,000, like this described in section 1.8 The elution volume of recombinant HOX was identical to the elution volume previously observed on the same column for native HOX from Chondrus crispus (chapter 1.7 and

1.8.). Thus, recombinant HOX in fact had the same molecular weight as native HOX isolated from Chondrus crispus. Fractions containing hexose oxidase were pooled and stored at 4 ° C.

4.4.3. Step three, anion exchange chromatography on a Mono Q column

The pool from the above second step was further purified by anion exchange chromatography on an FPLC system equipped with a Mono Q HR 5/5 column (bed volume 1 ml). The column was equilibrated in buffer A: 20 mM Tris-Cl, pH 7.5. The flow rate was 1 ml / min. The pools from the second stage were desalted by gel filtration in buffer A on Sephadex G-25 columns (PD-10 Pharmacia). After introducing the sample, the column was washed with 30 ml buffer A. The adsorbed proteins were eluted with a 20 ml gradient from 0 to 100% buffer B: 20 ​​mM Tris-Cl, 500 mM NaCl, pH 7.5. 0.5 ml fractions were collected and tested for hexose oxidase activity as described above (10 µl sample, incubation 15 minutes). Fractions containing hexose oxidase were pooled and stored at 4 ° C.

4.3.4. Fourth stage, chromatographic focusing

The pool from the third step above was purified by chromatographic focusing on a Mono P HR 5/5 column, as described above in Example 1.13, except that the phenyl-Sepharose adsorption step was omitted. Comparing native and recombinant hexose oxidase - both forms obtained in final purification by chromatographic focusing - it was found that the specific activity of recombinant hexose oxidase from Pichia pastoris was similar to the native form isolated from Chondrus crispus. The purified recombinant hexose oxidase preparation consisted of two bands migrating as 29,000 and 40,000.

In summary, recombinant hexose oxidase can be isolated and purified from the host organism Pichia pastoris. In SDS-PAGE, the recombinant purified enzyme showed the same 40,000 and 29,000 bands as the corresponding native enzyme from Chondrus crispus.

4.4. Properties of mixed hexose oxidase from Pichia pastoris.

Preparation and analysis of the amino acid sequence of recombinant hexose oxidase (rHOX) peptide fragments.

Purified rHOX was used in preparative SDS-PAGE and electrical transfer to a PVDF membrane as described in Example 2.4. The resulting 29,000 and 40,000 bands were subjected to enzymatic digestion of PVDF-related hexose oxidase polypeptides, as described in Example 2.5. Peptide fragments were separated by reverse phase liquid phase chromatography as described in Example 2.7. Well separated and rare bands were selected for amino acid sequence analysis by automatic 10-step Edman degradation as described above in Example 2.3. The resulting amino acid sequence is shown in Table 4.1.

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Table 4.1. Peptide sequences obtained by analyzing the peptide sequences obtained by the action of Lys-C kndoprotokinase on 40,000 and 29,000 recombinant hexose oxidase expressed in Pichia pastoris.

<td>Sequence of sequential peptide</td><td>sekwewen</td><td></td><td></td>

<td></td><td>Stage No.</td><td> 1</td><td> 2</td>

<td> 40000</td><td>HOX-9 peptide</td><td>D</td><td>P</td>

<td> 29000</td><td>HOX-10 peptide</td><td>L</td><td>Q</td>

sskwek ^ I amino acid II YL

<td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>G</td><td>Y</td><td>AND</td><td>V</td><td>AND</td><td>D</td><td>V</td><td>N</td>

<td>Y</td><td>Q</td><td>T</td><td>Y</td><td>IN</td><td>Q</td><td>E</td><td>E</td>

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

<td>T</td><td>E</td><td>V</td><td>P</td><td>D</td><td>G</td><td>L</td><td>T</td>

The HOX-9 peptide sequence from the recombinant fragment 40,000 shows an identical sequence to Aspxs to Asn 7 in the amino acid sequence of hexose oxidase from Chondrus crispus as shown in Table 3.2. (SEQ ID No. 30). Sequence analysis of a peptide sample obtained from recombinant 29000 polypeptide showed two residues at each step. The amino acid sequence identifications show that the two peptides present in the sample correspond to Leu434 to Glu443 and Tyr388 to Thr397, in the Chondrus crispus hexose amino acid oxidase sequence, see Table 3.2. (SEQ ID No. 30).

It can therefore be concluded that the peptide sequences obtained from recombinant hexose oxidase were identical to the corresponding amino acid sequences of native hexose oxidase from Chondrus crispus.

In addition, it can be concluded that Pichia pastoris transformed with the hexose oxidase gene from Chondrus crispus was able to produce recombinant hexose oxidase.

4.4.1. Substrate specificity

The substrate specificity of recombinant hexose oxidase from Pichia pastoris and native hexose oxidase from Chondrus crispus was compared using multiple sugars at a final concentration of 0.1 M in the test described above. Relative quantities are shown in Table 4.2.

Table 4.2. Substrate specificity of recombinant hexose oxidase expressed in Pichia pastoris and native hexose oxidase from Chondrus crispus

<td></td><td></td><td>Relative size</td><td></td>

<td>substratum</td><td>enzyme recombinant</td><td>native enzyme this work</td><td>native enzyme Sullivan and ikawa, 1973</td>

<td>D-glucose</td><td> 100</td><td> 100</td><td> 100</td>

<td>D-galactose</td><td> 15</td><td> 15</td><td> 82</td>

<td>maltose</td><td> 51</td><td> 31</td><td> 40</td>

<td>cellobiose</td><td>5i</td><td> 33</td><td> 32</td>

<td>lactose</td><td> 38</td><td> 25</td><td> 22</td>

As shown in Table 4.2, the substrate specificity of the combination hexose oxidase oxidase was almost identical to that of the native enzyme. However, although the relative amounts of disaccharides decreased for both maltose, cellobiose and lactose enzyme forms, the recombined enzyme appeared to be less selective in the oxidation of these disaccharides. The results for the native enzyme were almost idiotical as in Sullivan et al., 1973.

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4.4.2. Inhibition of sodium diethyldithiocarbamate

Sullivan and Ikawa (1973) report that hexose oxidase from Chondrus crispus is strongly inhibited by sodium diethyldithiocarbamate. Recombinant hexose oxidase from Pichia pastoris and the native enzyme from Chondrus crispus were compared for inhibition by this copper binding compound. The inhibitor was included in the enzyme assay at two concentrations, 0.1 mM and 0.01 mM, as described in Sullivan and Ikawa (1973). The results are shown in Table 4.3.

Table 4.3. Comparison of the inhibitory effect of sodium diethyldithiocarbamate on the enzymatic activity of recombinant hexose oxidase from Pichia pastoris and native hexose oxidase from Chondrus crispus

Inhibition (%)

Inhibitor concentration. Recombinant native enzyme enzyme

0.1 mM 9 (5 95

0.01 mM 39 41

Table 4.3 shows that recombinant and native hexose oxidase was equally sensitive to sodium diethyldithiocarbamate inhibition. In addition, the results were similar to native hexose oxidase data reported by Sullivan and Ikawa (1973).

Example 5

Production of recombinant hexose oxidase in Escherichia coli

5.1. Construction of a vector for the expression of recombinant hexose oxidase in E. coli.

Open reading frame coding for Chondrus crispus hexose oxidase shown in Table 3.2. (SEQ ID NO: 30) was introduced into the E. coli expression vector, pET17b (Novagen). The plasmid contained the strong, inducible T7 bacteriophage promoter and the T7 translation interrupting signal. Genes inserted between these control elements can be expressed by the addition of isopropyl PD-thiogalactopranoside (1PTG) if the plasmid is propagated in a special E. coli host, e.g. strain BL21 (DE3) (Novagen).

The hexose oxidase gene has been modified at the 5 'and 3' ends to introduce the gene into the expression vector -pET17b. The hexose oxidase gene was isolated by PCR using primers specific for the 5 'and 3' ends of the hexose oxidase gene. The 5 'primer (Hox5'-2) had the 5'-ATGAATI'CGTGGGTCGAAGAGCCC-3' sequence (SEQ ID No. 33), and the 3 'end specific primer was Hox3'-1. The first strand cDNA from Chondrus crispus was used as the template. PCR amplification was performed using AmpliTaq® DNA polymerase (Perkin-Elmer Cetus) as described in Example 4.1. Agarose gel electrophoresis of the reaction mixture showed a band about 1.7 kb in size. This 1.7 kb fragment was introduced into the pT7 Blue vector (Novagen) giving rise to the plasmid pUP0161.

Modification of the 5 'end of the hexose oxidase gene and cloning of the gene into the E. coli expression vector is shown in Figure 12. The 5' end was modified by PCR to introduce the Ndel site at the origin of ATG translation of the Hox5'-4 oligonucleotide, sequence 5'-CAGGAATTCATATGGCTACTCTTCCCCAGAAAG -3 '(SEQ ID No. 34) was used together with the Hox13 oligonucleotide (SEQ ID No. 28) (Table 3.1.) PCR amplification was described in Example 4.1. The reaction mixture was fractionated on a 2% agarose gel, and the 180 bp hexose oxidase specific fragment was purified according to Example 3.4. The 180 bp fragment was restriction digested with ClaI and EcoRI endonuclease, and then ligated with pUP0161 digested with the same enzymes, giving rise to plasmid pUP0167.

The hexose oxidase gene in plasmid pUPO167 was further cloned to construct the E. coli hexose oxidase expression vector. Plasmid pUPO167 was restriction digested with Ndel and BamHI enzymes, and with BamHI and SalI enzymes. First reaction

187 218 gave the 1.6 kb fragment encoding the 5 'fragment and the middle portion of the hexose oxidase gene, while the reaction with the BamHI and Sal enzymes gave a 200 bp fragment encoding the 3' end of the hexose oxidase gene. Two fragments specific for the hexose oxidase gene were purified on an agarose gel as described in Example 3.4. and ligated to plasmid pET17b digested with Ndel and Xhol endonucleases. Plasmid pET17b carrying the hexose oxidase gene was designated pUPO181. DNA sequencing showed that no mutation was introduced in the hexose oxidase gene during the isolation and cloning process.

5.2. Expression of recombinant hexose oxidase in Escherichia coli ..

Plasmid pUPO181 was introduced into E. coli strain BL21 (DE3) (Novagen) by standard transformation procedure (Sambrook et al., 1989). Cells were grown in shake bottles in LB medium (Sambrook et al., Supra). At OD600 = 0.5, the cells were induced to express recombinant hexose oxidase by adding 10 '³ M IPTG One hour after IPTG addition, cells were harvested by centrifugation and resuspended in sample buffer and subjected to SDS-PAGE as described above in Example 1.10.

The result of electrophoresis is shown in Figure 13. Surow181 showed a clear protein band of 62000 size. This band of 62000 had the same molecular weight as the translation product predicted from the open reading frame. Non-transformed E. coli cells did not show 62000 protein.

An E. coli BL21 (DE3) sample containing UPO181 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124, Braunschweig, Germany, May 23, 1996 under DSM 10692.

Example 6

Production of recombinant hexose oxidase in Saccharomyces cerevisiae

6.1. Construction of a vector for the expression of recombinant hexose oxidase in Saccharomyces cerevisiae

The open reading frame coding for Chondrus crispus hexose oxidase, shown in Table 3.2 (SEQ ID NO: 30), was introduced into the yeast expression vector, pYES2 (Invitrogen). Plasmid pYES2 is a high-copy episomal vector for inducible expression of recombinant proteins in Saccharomyces cerevisiae. The vector contains activating and promoter sequences from the Gall S. cerevisiae gene for strong, tightly regulated transcription. The transcription termination signal is from the CYC1 gene.

The hexose oxidase gene from Chondrus crispus was modified at the 5 'and 3' ends to introduce the gene into the pYES2 expression vector. The hexose oxidase gene was isolated from the pUPO150 plasmid as described in Example 4.1. (Figure 9). The hexose oxidase gene was isolated as a DNA blunt fragment with EcoRI and introduced into the plasmid pYES2 digested with the restriction PvuII and EcoRI enzymes (Figure 14). The resulting plasmid, pUPO155 was subjected to DNA sequencing to ensure that no mutations occurred during cloning.

Plasmid pUPO155 was purified from E. coli DH5a and transformed into S. corovisiao by electroporation (Gray and Brendel, 1992). Strain PAP1500 (genotype at, ura3-52, trp1 :: GAL10-GAL4, lys2-801, leu2A1, his3A200, pep4 :: HIS3, prblA1.6R, can1, GAL) was used as the recipient (Pedersen et al., 1996).

6.2. Expression of recombinant hexose oxidase in Saccharomyces cerevisiae

The S. corevisiao 1500 strain containing plasmid pUP0155 was cultured and induced with 2% galactose as described in Pedersen et al., 1996. Three days after induction, the cells were harvested by centrifugation and lysed as described in Example 4.2 above. The crude extract was tested for hexose oxidase activity using the o-dianisidine test described in Example 1.3 above. Table 6.1 shows that S. cerevisiao cells carrying the hexose oxidase gene are able to express active hexose oxidase.

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Table 6.1 Preparation of recombinant hexose oxidase in Saccharomycos corovisiae Saccharomyces cerevisiao

<td>substratum</td><td>+ hexose oxidase gene non-recombinant control</td>

<td>D-glucose D-galactose no substrate</td><td> + + 0 + 0 0 0</td>

= no detectable activity

A sample of Saccharomyces cerevisiae strain 1500 containing pUP0155 was deposited at Deutsche Sammlung von Mikroorganismen und Zollkulturen GmbH (DSM), Mascherodor Weg 1b, D-38124, Braunschwoig, Germany, May 23, 1996 under DSM 10694.

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Literature:

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2. Bean, RC and WZ Hassid 1956. J. Biol. Chem. 218: 425-436.

3. Clare, J J., FB Rayment, SP Ballantine, K. Sreekrishna and MA Romanos 1991. High-leyel expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Bio / Technology 9: 455-460.

4. Cregg, JM and KN Madden 1987. Development of transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption. In: Biological Research on Industrial Yeast, Vol III. Stewart, GG et al. (Eds.). pp 1-18. CRC Press, Boca Raton, FL.

5. Fernandez, J. et al. 1992. Internal Protein Sequence Analysis: Enzymatic Digestion for Less Than 10 pg of Protein Bound to Polyvinylidene Difluoride or Nitrocellulose Membranes. Analytical Biochemistry, 201: 255-264.

6. Fernandez, J. et al. 1994. An Improved Procedure for Enzymatic Digestion of Polyvinylidene Difluoride-Bound Proteteins for Internal Sequence Analysis. Analytical Biochemistry, 218: 112-117.

7. Groppe, JC and DE Morse 1993. Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans, Anal. Biochem., 210: 337-343.

8. Kerschensteiner, DA and GL. Klippenstein 1978. Purification, Mechanism, and State of Copper in Hexose Oxidase. Federation Proceedings 37: 1816 abstract.

9. Laemmli, UK 1970. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature (London) 227: 680-685.

10. Pedersen PA, JH Rasmussen, and PL. Jorgensen. 1996. Expression in high yield of pig α1β1 Na, K-ATPase and inactive mutants D369N and D807N in Sacctiaromyces cerevisiae, J. Biol. Chem. 271: 2514-2522.

11. Rand, AG 1972. Direct enzymatic conversion of lactose to acid: glucose oxidase and hexose oxidase. Journal of Food Science 37: 698-701.

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13. Sambrook, J., EF Fritsch and T. Maniatis 1989. Molecular Cloning, A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

14. Schagger, H. and G von Jagow 1987. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Rank from 1 to 100 kDa. Analytical Biochemistry 166: 368-379.

15. Sock, J. and R. Rohringer 1988. Activity Staining of Blotted Enzymes by Reaction Coupling with Transfer Membrane- Immobilized Auxiliary Enzymes. Analytical Biochemistry 171: 310-319.

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18. Yeh, KW, RH Juang and JC. Su. A rapid and efficient method for RNA isolation from plants with high carbohydrate content. Focus 13: 102-103

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TCGCTTGCAC ACTGAACTTC ACG ATG GCT ACT CTT CCT CAG AAA GAC CCC 110

Met Ala Thr Leu Pro Gln Lys Asp Pro

5

<td rowspan="2">GGT Gly 10</td><td rowspan="2">TAT Tyr</td><td rowspan="2">ATT How much</td><td rowspan="2">GTA val</td><td rowspan="2">ATT How much</td><td rowspan="2">GAT Asp 15</td><td rowspan="2">GTC val</td><td rowspan="2">AAC own</td><td rowspan="2">GCG ala</td><td colspan="2">GGC ACC</td><td rowspan="2">GCG ala</td><td rowspan="2">GAC Asp</td><td rowspan="2">AAG lys</td><td rowspan="2">CCG Pro</td><td rowspan="2">GGC. N s&gt; 25</td><td rowspan="2"> 150</td>

<td>Gly</td><td>Thr twenty</td>

<td>CCA</td><td>CGT</td><td>CTC</td><td>CCC</td><td>TCC</td><td>ATG</td><td>AAG</td><td>CCAS</td><td>GGC</td><td>TTC</td><td>GA ^ C</td><td>CGC</td><td>CGC</td><td>TGG</td><td>ATT</td><td>GGA</td><td> 200</td>

<td>Pro</td><td>Arg</td><td>Leu</td><td>Pro</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Gln</td><td>Gly</td><td>phe</td><td>own</td><td>Arg</td><td>AOG</td><td>Trp</td><td>How much</td><td>Bullfinch</td><td></td>

<td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td>

<td>ACT</td><td>AAT</td><td>ATT</td><td>GAT</td><td>TTC</td><td>GTT</td><td>TAT</td><td>GTC</td><td>GTG</td><td>TAC</td><td>ACT</td><td>CCT</td><td>CAA</td><td>(GGT</td><td>GCT</td><td>TTG</td><td> 254</td>

<td>Thr</td><td>own</td><td>How much</td><td>Asp</td><td>phe</td><td>val</td><td>Tyr</td><td>val</td><td>val</td><td>Tyr</td><td>Thr</td><td>Pro</td><td>Gln</td><td>Gly</td><td>ala</td><td>CCs</td><td></td>

<td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 5 !5</td><td></td><td></td><td></td>

<td>ACT</td><td>GCA</td><td>CTT</td><td>GAC</td><td>CGT</td><td>GCT</td><td>ATG</td><td>GAA</td><td>(AAC</td><td>TGT</td><td>TCT</td><td>CCC</td><td>GGT</td><td>ACA</td><td>GTC</td><td>AGG</td><td> 302</td>

<td>Thr</td><td>ala</td><td>Leu</td><td>Asp</td><td>Arg</td><td>ala</td><td>Underworld</td><td>Glu</td><td>lys</td><td>Cys</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Thr</td><td>val</td><td>Oh</td><td></td>

<td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td>

<td>ATC</td><td>GTC</td><td>TCT</td><td>GGC</td><td>G (^ &lt;C</td><td>CAT</td><td>TGC</td><td>TAC</td><td>GAG</td><td>GAC</td><td>TTC</td><td>GTA</td><td>TTT</td><td>CAC</td><td>GAA</td><td>TGG</td><td> 350</td>

<td>How much</td><td>val</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>His</td><td>Cys</td><td>Tyr</td><td>GGN</td><td>Asp</td><td>phe</td><td>val</td><td>phe</td><td>Asp</td><td>Glu</td><td>CCs</td><td></td>

<td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td></td>

<td>GTC</td><td>AAG</td><td>GCC</td><td>atc</td><td>ATC</td><td>AAC</td><td>GTC</td><td>ACT</td><td>GGT</td><td>CTC</td><td>GTT</td><td>GAG</td><td>AGI</td><td>GGT</td><td>TAT</td><td>GAG</td><td> 390</td>

<td>val</td><td>lys</td><td>AAa</td><td>How much</td><td>How much</td><td>own</td><td>val</td><td>Thr</td><td>Gly</td><td>Leu</td><td>val</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Tyr</td><td>acs&gt;</td><td></td>

<td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 105</td><td></td>

<td>GAC</td><td>GAT</td><td>AGG</td><td>GGT</td><td>TAC</td><td>TTC</td><td>GTC</td><td>AGC</td><td>AGT</td><td>(GGA</td><td>GAT</td><td>ACA</td><td>AM</td><td>TGG</td><td>GGC</td><td>TCC</td><td> 440</td>

<td>Asp</td><td>Asp</td><td>AXG</td><td>Gly</td><td>Tyr</td><td>phe</td><td>val</td><td>Cheese</td><td>See</td><td>dy</td><td>Asp</td><td>Thr</td><td>own</td><td>Trp</td><td>Gly</td><td>Cheese</td><td></td>

<td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 111</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>TTC</td><td>AAG</td><td>ACC</td><td>TTG</td><td>TTC</td><td>AGA</td><td>GAC</td><td>CAC</td><td>GGA</td><td>AGA</td><td>GTT</td><td>CTT</td><td>CCC</td><td>GGG</td><td>GGT</td><td>TTC</td><td> 494</td>

<td>phe</td><td>lys</td><td>Thr</td><td>Leu</td><td>phe</td><td>Arg</td><td>Asp</td><td>His</td><td>Gly</td><td>.Arg</td><td>l</td><td>Leu</td><td>Pro</td><td>Gly</td><td>Gly</td><td>See</td><td></td>

<td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 133</td><td></td><td></td><td></td><td></td><td>H5</td><td></td><td></td><td></td>

<td>TGC</td><td>TAC</td><td>TCC</td><td>GTC</td><td>GGC</td><td>CTC</td><td>(GGT</td><td>GGC</td><td>CAC</td><td>ATT</td><td>GTC</td><td>GGC</td><td>(GGA</td><td>GGT</td><td>GAC</td><td>GH</td><td> 542</td>

<td>Cys</td><td>Tyr</td><td>Cheese</td><td>val</td><td>Gly</td><td>Leu</td><td>Gly</td><td>Gly</td><td>His</td><td>Ilee</td><td>l</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Asp</td><td>GGU</td><td></td>

<td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td> 114</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td>

<td>ATT</td><td>TTG</td><td>GCC</td><td>CGC</td><td>TTG</td><td>CAT</td><td>GGC</td><td>CTC</td><td>CCC</td><td>GTC</td><td>GG ^ T</td><td>T (GG</td><td>CCC</td><td>AGC</td><td>(HC</td><td>GGG</td><td> 590</td>

<td>How much</td><td>Leu</td><td>ala</td><td>Arg</td><td>Leu</td><td>His</td><td>Gly</td><td>Leu</td><td>iDP</td><td>val</td><td>aop</td><td>Trp</td><td>llu</td><td>Cheese</td><td>Gly</td><td>wi</td><td></td>

<td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 116</td><td></td><td></td><td></td><td></td><td></td>

<td>GAG</td><td>GTC</td><td>GTC</td><td>GTT</td><td>AAG</td><td>CCA</td><td>GTC</td><td>CCC</td><td>AtZC</td><td>GGAr</td><td>d ^ C</td><td>TCG</td><td>GTA</td><td>CCC</td><td>(ΓΗ</td><td>TTA</td><td> 638</td>

<td>Glu</td><td>val</td><td>val</td><td>val</td><td>lys</td><td>iDP</td><td>l</td><td>Leu</td><td>tlir</td><td>CG.u</td><td>aop</td><td>(er</td><td>l</td><td>Leu</td><td>lys</td><td>tts</td><td></td>

<td> 170</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 185</td><td></td>

<td>GTG</td><td>CAC</td><td>AAA</td><td>GAT</td><td>TCC</td><td>GAA</td><td>GGC</td><td>AAC</td><td>GAC</td><td>GGG</td><td>GAG</td><td>CTC</td><td>CCC</td><td>TGG</td><td>GCA</td><td>CAC</td><td> 60 6</td>

187 218

<td>val</td><td>His</td><td>lys</td><td>Asp</td><td>Cheese 190</td><td>Alu</td><td>nly</td><td>own</td><td>Asp</td><td>nly 195</td><td>llu</td><td>Leu</td><td>phu</td><td>Trp</td><td>ala 200</td><td colspan="2">His</td>

<td>ACA</td><td>GGT</td><td>GGC</td><td>GGT</td><td>AAG</td><td>GGA</td><td>AAC</td><td>TTT</td><td>GGA</td><td>ATCC</td><td>ATC</td><td>AGC</td><td>CCA</td><td>TAC</td><td>TAC</td><td>TTC</td><td> 304</td>

<td>Thr</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>own</td><td>phe</td><td>Gly</td><td>How much</td><td>How much</td><td>Thr</td><td>lys</td><td>Tyr</td><td>Tyr</td><td>phe</td><td></td>

<td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td>

<td>AAG</td><td>GAT</td><td>TTG</td><td>CCC</td><td>ATG</td><td>TCT</td><td>CCCC</td><td>CGG</td><td>GGC</td><td>GTC</td><td>ATC</td><td>AGA</td><td>TCA</td><td>AAT</td><td>TTA</td><td>CAC</td><td> 822</td>

<td>lys</td><td>Asp</td><td>Leu</td><td>Pro</td><td>Underworld</td><td>Cheese</td><td>Pro</td><td>Arg</td><td>Gly</td><td>val</td><td>How much</td><td>ala</td><td>Cheese</td><td>own</td><td>Leu</td><td>His</td><td></td>

<td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td>

<td>TTC</td><td>AGC</td><td>TGG</td><td>GAC</td><td>GAT</td><td>TTC</td><td>ACG</td><td>AGA</td><td>GAT</td><td>GCC</td><td>TTG</td><td>CAG</td><td>(ACT</td><td>Tr ^ G</td><td>TTG</td><td>ACA</td><td> 830</td>

<td>phe</td><td>Cheese</td><td>Trp</td><td>Asp</td><td>Gly</td><td>phe</td><td>Thr</td><td>Arg</td><td>Asp</td><td>ala</td><td>Leu</td><td>Gln</td><td>Asp</td><td>Leu</td><td>Leu</td><td>Thr</td><td></td>

<td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td><td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td></td>

<td>AAG</td><td>TAC</td><td>TTC</td><td>CCAC</td><td>CTT</td><td>GCC</td><td>AGA</td><td>TGT</td><td>GAT</td><td>TGG</td><td>AAG</td><td>TCA?</td><td>ACG</td><td>GTT</td><td>GGC</td><td>TCCG</td><td> 878</td>

<td>lys</td><td>Tyr</td><td>phe</td><td>lys</td><td>Leu</td><td>ala</td><td>AOG</td><td>Cys</td><td>Asp</td><td>Trp</td><td>lys</td><td>own</td><td>Thr</td><td>val</td><td>Gly</td><td>lys</td><td></td>

<td> 250</td><td></td><td></td><td></td><td></td><td> 25 5</td><td></td><td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td>

<td>TTT</td><td>CAA</td><td>ATC</td><td>TTC</td><td>CAT</td><td>CAG</td><td>GCA</td><td>GCG</td><td>G (CC</td><td>GAG</td><td>TTT</td><td>GTC</td><td>ATG</td><td>TAC</td><td>TTG</td><td>TAT</td><td>2T 6</td>

<td>phe</td><td>Gln</td><td>How much</td><td>phe</td><td>His</td><td>Gln</td><td>ala</td><td>ala</td><td>Glu</td><td>Glu</td><td>phe</td><td>val</td><td>Underworld</td><td>Tyr</td><td>Leu</td><td>Tyr</td><td></td>

<td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 225,</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td>

<td>ACA</td><td>TCC</td><td>TAC</td><td>TCG</td><td>AAC</td><td>CGAC</td><td>GCC</td><td>GAG</td><td>CGC</td><td>GTCC</td><td>GTT</td><td>GCC</td><td>CCCC</td><td>GAC</td><td>CGT</td><td>SCAC</td><td>9T 4</td>

<td>Thr</td><td>Cheese</td><td>Tyr</td><td>Cheese</td><td>own</td><td>Asp</td><td>ala</td><td>Glu</td><td>Arg</td><td>Glu</td><td>val</td><td>ala</td><td>Gln</td><td>Asp</td><td>TCRG</td><td>His</td><td></td>

<td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 29 5</td><td></td><td></td><td></td>

<td>TAT</td><td>CAT</td><td>TTG</td><td>GAG</td><td>GCT</td><td>GAC</td><td>ATA</td><td>GGCC</td><td>CAG</td><td>ATC</td><td>TAC</td><td>TCCC</td><td>ACA</td><td>TGC</td><td>GAG</td><td>CCC</td><td> 1022</td>

<td>Tyr</td><td>His</td><td>Leu</td><td>Glu</td><td>ala</td><td>Asp</td><td>How much</td><td>Glu</td><td>Gln</td><td>How much</td><td>Tyr</td><td>lys</td><td>Thr</td><td>Cys</td><td>Glu</td><td>Pro</td><td></td>

<td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td><td> 3 30</td><td></td><td></td><td></td><td></td>

<td>ACC</td><td>AAA</td><td>GCG</td><td>CTT</td><td>GAG</td><td>GGG</td><td>CAT</td><td>GCT</td><td>GGG</td><td>TGG</td><td>GCG</td><td>CCG</td><td>TTC</td><td>CCC</td><td>GTG</td><td>CGC</td><td> 10 20</td>

<td>Thr</td><td>lys</td><td>ala</td><td>Leu</td><td>Gly</td><td>gly</td><td>hii</td><td>ala</td><td>Gly</td><td>Trp</td><td>Tcl</td><td>Pro</td><td>phe</td><td>Pro</td><td>val</td><td>Arg</td><td></td>

<td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 332</td><td></td><td></td><td></td><td></td><td></td>

<td>CCG</td><td>CGC</td><td>aac;</td><td>TkGG</td><td>CAC</td><td>ACA</td><td>TCC</td><td>TCCG</td><td>ACG</td><td>TCG</td><td>TAT</td><td>ATG</td><td>CAC</td><td>GAC</td><td>GAG</td><td>ACG</td><td> 1188</td>

<td>Pro</td><td>Arg</td><td>lys</td><td>Arg</td><td>His</td><td>Thr</td><td>Cheese</td><td>LLT</td><td>TTR</td><td>Seu</td><td>Tyr</td><td>Underworld</td><td>His</td><td>Asp</td><td>Glu</td><td>TTR</td><td></td>

<td> 330</td><td></td><td></td><td></td><td></td><td> 333</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 330</td><td></td>

<td>ATG</td><td>GAC</td><td>TAC</td><td>CCC</td><td>TTC</td><td>TAC</td><td>GCG</td><td>CCT</td><td>ACC</td><td>TGG</td><td>ACG</td><td>ATC</td><td>TCCC</td><td>GGC</td><td>TCC</td><td>GGG</td><td>ZL16 6</td>

<td>Underworld</td><td>Asp</td><td>Tyr:</td><td>iDP</td><td>phe</td><td>TTR</td><td>ala</td><td>llu</td><td>TTH</td><td>Glu</td><td>Thr</td><td>tIL;</td><td>own</td><td>Gly</td><td>Cheese</td><td>Gly</td><td></td>

<td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td>

<td>CCG</td><td>AAT</td><td>cac;</td><td>AGL</td><td>AGC</td><td>TCCG</td><td>TAC</td><td>TCCG</td><td>TCT</td><td>TLG</td><td>TAC</td><td>ATG</td><td>ATC</td><td>TCCG</td><td>GAT</td><td>TTT</td><td> 1214</td>

<td>Pro</td><td>own</td><td>Gln</td><td>tag</td><td>Gly</td><td>LLS</td><td>TTR</td><td>TLY</td><td>See</td><td>ala</td><td>tyy</td><td>Underworld</td><td>Tla</td><td>LLT</td><td>Asp</td><td>phr</td><td></td>

365 370 375

<td>CCl</td><td>GAT</td><td>TTC</td><td>CCC</td><td>TTG</td><td>GAC</td><td>TAT</td><td>GCC</td><td>TTA</td><td>TAC.</td><td>TAC</td><td>TGT</td><td>ACG</td><td>GAG</td><td>GLT</td><td>CCG</td>

<td>HRO</td><td>Asp</td><td>PHH</td><td>Uln</td><td>lle</td><td>alp</td><td>val</td><td>Tla</td><td>TTP</td><td>tsy</td><td>Ti ^ r</td><td>tlu</td><td>TTR</td><td>TAN</td><td>val</td><td>iDP</td>

<td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td><td> 338</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td>

187 218

<td>GAC Asp</td><td colspan="2">GGC TTG Gly Leu 395</td><td>ACT Thr</td><td>AGT Cheese</td><td>GCC ala</td><td colspan="2">GAA ATG Glu Met 400</td><td>AAG lys</td><td>GAT Asp</td><td>GCC ala</td><td>TTA Leu 40 5</td><td>CTC llu</td><td>CAG Gln</td><td>GTG val</td><td>GTAC Asp</td><td> 1310</td>

<td>ATG</td><td>TTT</td><td>GGT</td><td>GGT</td><td>GAG</td><td>ATT</td><td>CAC</td><td>AAG</td><td>GTG</td><td>GTC</td><td>TGG</td><td>GAT</td><td>GCG</td><td>ACG</td><td>GCA</td><td>CCC</td><td> 1358</td>

<td>Underworld</td><td>phe</td><td>Gly</td><td>Gly</td><td>Glu</td><td>How much</td><td>His</td><td>lys</td><td>val</td><td>val</td><td>Trp</td><td>Asp</td><td>ala</td><td>Thr</td><td>ala</td><td>val</td><td></td>

<td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 425</td><td></td>

<td>GCG</td><td>CAG</td><td>CGC</td><td>GAG</td><td>TAC</td><td>ATC</td><td>ATC</td><td>AAA</td><td>CTG</td><td>CAG</td><td>TAC</td><td>CAG</td><td>ACA</td><td>TAC</td><td>TGG</td><td>CAG</td><td> 1406</td>

<td>ala</td><td>Gln</td><td>Arg</td><td>Glu</td><td>Tyr</td><td>How much</td><td>How much</td><td>lys</td><td>Leu</td><td>Gln</td><td>Tyr</td><td>Gln</td><td>Thr</td><td>Tyr</td><td>Trp</td><td>Gln</td><td></td>

<td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td><td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 400</td><td></td><td></td>

<td>GAA</td><td>GAA</td><td>GAC</td><td>AAG</td><td>GAT</td><td>GCA</td><td>GTG</td><td>AAC</td><td>CTC</td><td>AAG</td><td>TGG</td><td>ATT</td><td>AGA</td><td>GAC</td><td>ttt</td><td>TAC</td><td> 1454</td>

<td>Glu</td><td>Glu</td><td>Asp</td><td>lys</td><td>Asp</td><td>ala</td><td>val</td><td>own</td><td>Leu</td><td>lys</td><td>Τη?</td><td>How much</td><td>Arg</td><td>Asp</td><td>phe</td><td>Tyr</td><td></td>

<td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td><td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 4!55</td><td></td><td></td><td></td>

<td>GAG</td><td>GAG</td><td>ATG</td><td>TAT</td><td>GAG</td><td>CCG</td><td>TAT</td><td>GGC</td><td>GGG</td><td>GTT</td><td>CCA</td><td>CGCC '</td><td>CCC</td><td>TCCC</td><td>ACG</td><td>(.CAG</td><td> 150 2</td>

<td>Glu</td><td>Glu</td><td>Underworld</td><td>Tyr</td><td>Glu</td><td>Pro</td><td>Tyr</td><td>Gly</td><td>Gly</td><td>val</td><td>Pro</td><td>Asp</td><td>Pro</td><td>own</td><td>Tho</td><td>Gln</td><td></td>

<td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td><td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td>

<td>GTG</td><td>GAG</td><td>AGT</td><td>GGT</td><td>AAA</td><td>GGT</td><td>GTG</td><td>TTT</td><td>GAG</td><td>GGA</td><td>TGC</td><td>TTCC</td><td>TTC</td><td>TACC</td><td>TAC</td><td>CCG</td><td> 1150</td>

<td>val</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>lys</td><td>Gly</td><td>val</td><td>phe</td><td>Glu</td><td>Gly</td><td>Cys</td><td>Tyr</td><td>phe</td><td>own</td><td>tyo</td><td>Pro</td><td></td>

<td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td><td></td><td></td><td></td><td></td><td> 448</td><td></td><td></td><td></td><td></td><td></td>

<td>GAT</td><td>GTG</td><td>GAC</td><td>TTG</td><td>AAC</td><td>AAC</td><td>TGG</td><td>AAG</td><td>AAC</td><td>GGC</td><td>AAG</td><td>TAT</td><td>GGT</td><td>GCC</td><td>CTC</td><td>GTAC</td><td> 1198</td>

<td>Asp</td><td>val</td><td>Asp</td><td>Leu</td><td>own</td><td>own</td><td>Trp</td><td>lys</td><td>own</td><td>Gly</td><td>LyL</td><td>lyy</td><td>Gly</td><td>ala</td><td>Leu</td><td>Glu</td><td></td>

<td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td><td></td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td></td><td> 505</td><td></td>

<td>CTT</td><td>TAC</td><td>TTT</td><td>TTG</td><td>GGT</td><td>AAC</td><td>CTG</td><td>AAC</td><td>CGC</td><td>CTC</td><td>ATC</td><td>CCA</td><td>GCC</td><td>TAAC</td><td>TGG</td><td>TTG</td><td> 1646</td>

<td>Leu</td><td>Tyr</td><td>phe</td><td>Leu</td><td>Gly</td><td>own</td><td>Leu</td><td>own</td><td>Arg</td><td>Leu</td><td>How much</td><td>Lyt</td><td>ala</td><td>lys</td><td>Trp</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 510</td><td></td><td></td><td></td><td></td><td> 551</td><td></td><td></td><td></td><td></td><td> 520</td><td></td><td></td>

<td>TGG</td><td>GAT</td><td>CCC</td><td>AAC</td><td>GAG</td><td>ATC</td><td>TTC</td><td>ACA</td><td>AAC</td><td>AAA</td><td>CAC</td><td>GAC</td><td>ATC</td><td>CCT</td><td>ACT</td><td>AAA</td><td> 1694</td>

<td>Trp</td><td>Asp</td><td>Pro</td><td>own</td><td>Glu</td><td>How much</td><td>phe</td><td>Thr</td><td>own</td><td>lys</td><td>Gln</td><td>See</td><td>ILL</td><td>iDP</td><td>Thr</td><td>lys</td><td></td>

<td></td><td></td><td></td><td> 525</td><td></td><td></td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td></td><td> 553</td><td></td><td></td><td></td>

<td>CCT</td><td>CTT</td><td>AAG</td><td>GAG</td><td>CCC</td><td>AAG</td><td>CAG</td><td>ACG</td><td>AAA</td><td colspan="6">TAGTAGGTCA CAATTAGTCA</td><td></td><td> 1774</td>

<td>Pro</td><td>Leu</td><td>lys</td><td>Glu</td><td>Pro</td><td>lys</td><td>Gln</td><td>Thr</td><td>lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td><td> 555</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

TCGACTGAAG TGCAGCACTT GTCGGATACG GCGTGATGGT TGCTTTTTAT AAACTTGGTA 1801 (2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 546 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

187 218 (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

<td>Underworld 1</td><td colspan="3">Ala Thh Leu</td><td>poo 5</td><td>U1o</td><td>You about</td><td>Aso</td><td colspan="2">roo uso 10</td><td>You about</td><td colspan="2">llo vao</td><td>ie o</td><td>Aso 15</td><td>val</td>

<td>own</td><td>ala</td><td>Gly</td><td>Thr</td><td>ala</td><td>Asp</td><td>lys</td><td>Pro</td><td>Asp</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Pro</td><td>Cheese</td><td>Underworld</td><td>lys</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gln</td><td>Gly</td><td>phe</td><td>own</td><td>Arrg</td><td>Arg</td><td>Trp</td><td>How much</td><td>Gly</td><td>Thr</td><td>own</td><td>How much</td><td>Asp</td><td>phe</td><td>val</td><td>Tyr</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>val</td><td>val</td><td>Tyr</td><td>Thr</td><td>Pro</td><td>Gln</td><td>Gly</td><td>ala</td><td>Cys</td><td>Thr</td><td>ala</td><td>Leu</td><td>Asp</td><td>AOG</td><td>ala</td><td>Underworld</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Glu</td><td>lys</td><td>Cys</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Thr</td><td>val</td><td>AOrg</td><td>How much</td><td>val</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>His</td><td>Cys</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Tyr</td><td>Glu</td><td>Asp</td><td>phe</td><td>val</td><td>phe</td><td>Asp</td><td>Glu</td><td>cyo</td><td>val</td><td>lys</td><td>ala</td><td>How much</td><td>11 e</td><td>own</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>Gly</td><td>Leu</td><td>val</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Tyr</td><td>Asp</td><td>Asp</td><td>Asp</td><td>Arg</td><td>Gly</td><td>Tyr</td><td>phe</td><td>val</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Cheese</td><td>Cheese</td><td>Gly</td><td>Asp</td><td>Thr</td><td>own</td><td>Trp</td><td>Gly</td><td>Cheese</td><td>phe</td><td>lys</td><td>Thr</td><td>Leu</td><td>phe</td><td>Arg</td><td>Asp</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td>

<td>His</td><td>Gly</td><td>.Arg</td><td>val</td><td>Leu</td><td>Pro</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>val</td><td>Gly</td><td>Leu</td><td>Gly</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>His</td><td>How much</td><td>val</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Asp</td><td>Gly</td><td>How much</td><td>Leu</td><td>ala</td><td>Arg</td><td>Leu</td><td>His</td><td>Gly</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Leu</td><td>Pro</td><td>val</td><td>Asp</td><td>Trp</td><td>Leu</td><td>Cheese</td><td>Gly</td><td>a ^ «^ and</td><td>GGN</td><td>val</td><td>val</td><td>val</td><td>lys</td><td>Pro</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Leu</td><td>Thr</td><td>Glu</td><td>Asp</td><td>S er</td><td>val</td><td>Leu</td><td>Ll ^ sj</td><td>Tyr</td><td>val</td><td>His</td><td>lys</td><td>Asp</td><td>Cheese</td><td>Glu</td><td>Gly</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 118</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>own</td><td>Asp</td><td>Gly</td><td>Glu</td><td>Leu</td><td>phe</td><td>Trp</td><td>ala</td><td>His</td><td>Thr</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>own</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 2205</td><td></td><td></td><td></td>

<td>phe</td><td>Gly</td><td>11 e</td><td>How much</td><td>Thr</td><td>lys</td><td>Tyr</td><td>Tyr</td><td>phe</td><td>lys</td><td>Asp</td><td>Leu</td><td>iDP</td><td>Underworld</td><td>SSr</td><td>iDP</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 22^5</td><td></td><td></td><td></td><td></td><td> 222</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>Gly</td><td>val</td><td>How much</td><td>ala</td><td>SEZ</td><td>own</td><td>Leu</td><td>Hi.s</td><td>PP.h</td><td>SEZ</td><td>tHP</td><td>Asp</td><td>Gly</td><td>phe</td><td>Thr</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 220</td>

187 218

<td colspan="2" rowspan="2">Arg Asp</td><td rowspan="2">ala</td><td colspan="7">Leu Gln Asp Leu Leu Thr Lys</td><td rowspan="2">Tyr</td><td rowspan="2">phe</td><td rowspan="2">lys</td><td rowspan="2">Leu</td><td rowspan="2">ala 255</td><td rowspan="2">Arg</td>

<td colspan="3"> 245</td><td colspan="4"> 2250</td>

<td>Cys</td><td>Asp</td><td></td><td>lys</td><td>own</td><td>Ohr</td><td>val</td><td>Gly</td><td>lys</td><td>phe</td><td>Gln</td><td>How much</td><td>phe</td><td>His</td><td>Gln</td><td>ala</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td>

<td>ala</td><td>Glu</td><td>Glu</td><td>Ph.e</td><td>vil</td><td>Underworld</td><td>Tyr</td><td>Leu</td><td>Tyr</td><td>Thr</td><td>Cheese</td><td>Oyr</td><td>Cheese</td><td>own</td><td>Asp</td><td>ala</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 263 25</td><td></td><td></td><td></td>

<td>Glu</td><td>Arg</td><td>Glu</td><td>val</td><td>ala</td><td>Gln</td><td>alp</td><td>ppg</td><td>His</td><td>Oyr</td><td>hSS</td><td>Leu</td><td>Glu</td><td>ALa</td><td>Asp</td><td>How much</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Glu</td><td>Gln</td><td>How much</td><td>Oyr</td><td>lys</td><td>Thr</td><td>Cys</td><td>Glu</td><td>Pro</td><td>oho</td><td>lys</td><td>ala</td><td>Leu</td><td>Gl y</td><td>Gly</td><td>His</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 330</td>

<td>ala</td><td>Gly</td><td>ORP</td><td>ala</td><td>Pro</td><td>phe</td><td>Pro</td><td>VAL</td><td>Arg</td><td>Pro</td><td>Arg</td><td>lys</td><td>Arg</td><td>His</td><td>Thr</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>lys</td><td>Thr</td><td>Cheese</td><td>You_r</td><td>Underworld</td><td>His</td><td>Asp</td><td>Glu</td><td>oho</td><td>Underworld</td><td>Asp</td><td>Tyr</td><td>iDP</td><td>phe</td><td>Oyr</td><td>ala</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td>

<td>Leu</td><td>Thr</td><td>Glu</td><td>Thr</td><td>How much</td><td>own</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Pro</td><td>own</td><td>Gln</td><td>Arg</td><td>Gly</td><td>lys</td><td>Tyr</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 3 &lt;50</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td>

<td>lys</td><td>Cheese</td><td>ala</td><td>Tyr</td><td>mee</td><td>How much</td><td>iys</td><td>.asp</td><td>plie</td><td>Pro</td><td>Asp</td><td>phe</td><td>Gln</td><td>hu</td><td>Asp</td><td>val</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td>

<td>How much</td><td>Trp</td><td>lys</td><td>Tyr</td><td>Leu</td><td>Thr</td><td>Glu</td><td>val</td><td>Pro</td><td>Asp</td><td>Gly</td><td>Leu</td><td>Thr</td><td>Cheese</td><td>ala</td><td>Glu</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 399</td><td></td><td></td><td></td><td></td><td> 440</td>

<td>Underworld</td><td>lys</td><td>Asp</td><td>ali</td><td>Leu</td><td>Leu</td><td>Gln</td><td>val</td><td>Asp</td><td>Underworld</td><td>phe</td><td>Gly</td><td>Gly</td><td>Glu</td><td>IIn;</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>lys</td><td>val</td><td>val</td><td>Trp</td><td>Asp</td><td>ala</td><td>Thr</td><td>ali</td><td>ViL</td><td>ala</td><td>Gln</td><td>Arg</td><td>Glu</td><td>Tyr</td><td>How much</td><td>He</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 442</td><td></td><td></td><td></td><td></td><td> 443</td><td></td><td></td>

<td>lys</td><td>Leu</td><td>Gln</td><td>Tyr</td><td>Gln</td><td>Thr</td><td>Tyr</td><td>Τη?</td><td>Gln</td><td>Glu</td><td>Glu</td><td>Asp</td><td>Ll.</td><td>agp</td><td>ali</td><td>val</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 444</td><td></td><td></td><td></td>

<td>own</td><td>Leu</td><td>lys</td><td>tro</td><td>And others</td><td>Arg</td><td>Asp</td><td>PPe</td><td>Tyr</td><td>Glu</td><td>Alu</td><td>M ^ t;</td><td>Ayr</td><td>Alu</td><td>Pro</td><td>Tyr</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 445)</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Gly</td><td>val</td><td>Pro</td><td>Asp</td><td>iDP</td><td>own</td><td>Thr</td><td>Gln</td><td>vil</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>lys</td><td>Gln</td><td>val</td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 440</td>

<td>phe</td><td>Glu</td><td>Gly</td><td>Cys</td><td>tyo</td><td>PPe</td><td>own</td><td>Ie.</td><td>iDP</td><td>Asp</td><td>val</td><td>Asp</td><td>Leu</td><td>own</td><td>aap</td><td>aop</td>

485 490 495

Lys Asn Gly Lys Tyr Gly Ala Leu Glu Leu Tyr Phe Leu Gly Asn Leu 500 505 510

187 218

<td colspan="3">Asn Arg Leu</td><td rowspan="2">How much</td><td colspan="2" rowspan="2">Lys Ala</td><td rowspan="2">lys</td><td rowspan="2">Trp 520</td><td rowspan="2">Leu</td><td colspan="2" rowspan="2">Trp Asp</td><td rowspan="2">Pro</td><td rowspan="2">own 525</td><td rowspan="2">Glu</td><td rowspan="2">How much</td><td rowspan="2">phe</td>

<td colspan="2"></td><td> 515</td>

<td>Thr</td><td>own</td><td>lys</td><td>Gln</td><td>Cheese</td><td>How much</td><td>Pro</td><td>Thr</td><td>lys</td><td>Pro</td><td>Leu</td><td>lys</td><td>Glu</td><td>Pro</td><td>lys</td><td>Gln</td>

<td></td><td> 530</td><td></td><td></td><td></td><td></td><td> 535</td><td></td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

545 (2) INFORMATION FOR THE SEQ ID. NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) THREADS: single (D) TOPOLOGY: linear (ii) PARTICLE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

ACCAAGTTTA TAAAAAGCAA CCATCAC 27 (2) INFORMATION FOR THE SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) THREADS: single (D) TOPOLOGY: linear (ii) PARTICLE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

ATGAATTCGT GGGTCGAAGA GCCC 24 (2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) THREADS: single

187 218 (D) TOPOLOGY: linear (ii) PARTICLE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

CAGGAATTCA TATGGCTACT CTTCCCCAGA AAG 33

187 218

3 4

<img file="PL187218B1_D0001.tif" />

Fig. 2

187 218

<img file="PL187218B1_D0002.tif" />

187 218

<img file="PL187218B1_D0003.tif" />

Fig. 5

187 218

<img file="PL187218B1_D0004.tif" />

BCoueqjosqy c

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c jo

UJ

Elution time (min.

187 218

<img file="PL187218B1_D0005.tif" />

Bpoueqjosqv φ

E c

.2 zj

UJ

Elution time (min.

187 218

2

<img file="PL187218B1_D0006.tif" />

Fig. 8

187 218

1. strand cDNA 1 strand cDNA

<img file="PL187218B1_D0007.tif" />

Fig. 9

187 218

Figure 10

<img file="PL187218B1_D0008.tif" />

glucose methanol

Oxidase activity (10 μ1 / 15 min.

Oxidase acth / ity (A₄₁₀ / 10 // I / 15min) fraction number (2.0 ml)

Number of fractions (2.0 ml)

187 218

Oxidase activity (^Α41θ / 10 μΙ / 15 min)

Oxidase activity (A_41o/ 10 μΙ / 15 min)

<img file="PL187218B1_D0009.tif" />

Number of fractions (2.5 ml)

187 218

PCR EcjRI + Clal

<img file="PL187218B1_D0010.tif" />

pUPolol .EcoRI + Clal

EcoRI Clal Clal

<img file="PL187218B1_D0011.tif" />

<img file="PL187218B1_D0012.tif" />

187 218

2

<img file="PL187218B1_D0013.tif" />

Fig. 13

187 218

<img file="PL187218B1_D0014.tif" />

Ndel + Kle no in EcoRI

Ligation

Ligation

<img file="PL187218B1_D0015.tif" />

/ © URI

<img file="PL187218B1_D0016.tif" />

pUPO155

Fig 14

187 218

Purification of hexose oxidase from Chondrus crispus followed by the production and separation of peptide fragments for amino acid sequence analysis

<img file="PL187218B1_D0017.tif" />

FIG. 1

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