Biological-based catalyst to delay plant development processes

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DESCRIPTION

Bio-based catalyst to retard plant development processes

Field of the invention

The present invention relates to methods for retarding plant development which comprises exposing a plant or part of a plant to one or more enzymes. Apparatus for retarding a plant growth process are further provided.

Background of the invention

Ethylene production in plants and plant parts is induced by various external factors and stressors, including making incisions, application of hormones (e.g. auxin), anaerobic conditions, cooling, heat, drought, and infection by pathogens. . Increased ethylene production is also observed during various plant development processes, including fruit or vegetable ripening, seed germination, leaf abscission, and floral senescence.

Ethylene biosynthesis in plants is commonly represented as an enzymatic scheme involving three enzymes, traditionally called the "Yang Cycle", in which S-adenosyl-L-methionine (SAM) -syntase catalyzes the conversion of methionine to S -adenosyl-L-methionine (AdoMet); 1-aminocyclopropane-1-carboxylic acid (ACC) -syntase catalyzes the conversion of AdoMet to ACC; and ACC-oxidase catalyzes the conversion of ACC to ethylene and the by-products carbon dioxide and hydrogen cyanide, see, for example, Srivastava (2001) Plant Growth and Development: Hormones and Environment (Academic Press, New York) for a description. Overview of ethylene biosynthesis in plants and ethylene-regulated plant development processes.

Previous research has established that ripening is triggered in climacteric fruits, at least in part, by a sudden and significant increase in ethylene biosynthesis. Although a sudden increase in ethylene production is involved in the fruit ripening process, in the case of climacteric fruits, the exact mechanism, particularly in non-climacteric fruits, is not fully understood. Although there is no sudden increase in ethylene production in a non-climacteric fruit, the non-climacteric fruit will respond to ethylene. Furthermore, fruits, vegetables and other plant products vary in the amount of ethylene synthesized and also in the sensitivity of the particular product to ethylene. For example, apples show a high level of ethylene production and sensitivity to ethylene, while artichokes show a low level of ethylene biosynthesis and sensitivity to ethylene. See, for example, Cantwell (2001) “Properties and Recommended Conditions for Storage of Fresh Fruits and Vegetables” at postharvest.ucdavis.edu/Produce/Storage/index.shtml (last visited March 6, 2007). Ripening of fruits usually results in a change in color, softening of the pericarp, and changes in the sugar content and flavor of the fruit. Although ripening initially makes the fruit more edible and attractive to eat, the process ultimately leads to degradation and deterioration in the quality of the fruit, rendering it unacceptable for consumption, leading to significant commercial monetary losses. Control of the ripening process is desirable to improve the shelf life and extend the time available for transportation, storage and sale of fruit and other agricultural products undergoing ripening.

In addition to a sudden increase in ethylene biosynthesis in climacteric fruits, ripening-related changes are also associated with an increase in respiration rate. Heat is produced as a result of respiration in fruits, vegetables, and other plant products and, as a consequence, affects the shelf life and required storage conditions (eg refrigeration) for these products. Vegetable products with higher respiration rates (for example, artichokes, cut flowers, asparagus, broccoli, spinach, etc.) show shorter storage times than those with lower respiration rates (for example, walnuts, dates, apples, citrus fruits, grapes, etc.). Respiration is affected by a number of environmental factors including temperature, atmospheric composition, physical stress, light, chemical stress, radiation, water stress, growth regulators, and pathogen attack. In particular, temperature plays a significant role in the rate of respiration. For an overview of respiratory metabolism and recommended controlled atmospheric conditions for fruits, vegetables and other plant products, see, for example, Kader (2001) Postharvest Horticulture Series No. 22A: 29-70 (University of California - Davis); Saltveit (University of California - Davis) “Respiratory Metabolism at usna.usda.gov/hb66/019respiration.pdf (last viewed March 6, 2007); and Cantwell (2001) “Properties and Recommended Conditions for Storage of Fresh Fruits and Vegetables” at postharvest.ucdavis.edu/Produce/ Storage / index.shtml (last viewed March 6, 2007).

Methods and compositions for retarding the ripening process of fruits include, for example, the application of silver salts (eg, silver thiosulfate), 2,5-norbornadiene, potassium permanganate, 1-methylcyclopropene (1-MCP), cyclopropene (CP) and its derivatives. These compounds have significant disadvantages, such as the presence of heavy metals, bad odors, and explosive properties when compressed, which make them unacceptable or of limited applicability for use in the food industry. Transgenic strategies are also being investigated to control ethylene production to retard plant development processes (e.g.

ES 2 525 176 T3 example, fruit ripening) by introducing nucleic acid sequences that limit the production of ethylene, particularly by reducing the expression of the ACC-synthase or ACC-oxidase enzymes. The public response to genetically modified agricultural products, however, has not been entirely favorable.

Accordingly, there remains a significant need in the art for safe methods and apparatus for retarding plant growth processes. Such methods and apparatus could provide better control of fruit ripening, vegetable ripening, flower senescence, leaf abscission and seed germination and extend the shelf life of various agricultural products (e.g. fruits, vegetables and cut flowers), thus allowing the transport to a greater distance of these products without the need for refrigeration, increasing the attractiveness of the product to consumers and reducing the monetary costs associated with loss of products due to untimely maturation and senescence.

Brief summary of the invention

Methods are provided for retarding a plant development process including, but not limited to, fruit ripening, vegetable ripening, flower senescence, and leaf abscission. The methods of the present invention generally comprise exposing a plant or part of a plant to one or more isolated enzymes in an amount sufficient to retard the growth process of plants of interest. In some aspects of the present invention, the bacteria that provide the isolated enzymes are selected from the group comprising Rhodococcus spp., Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and mixtures thereof. The bacteria used in the practice of the present methods can be further treated with an inducing agent that includes, for example, asparagine, glutamine, cobalt, urea and their mixtures, to induce the ability of the bacteria to retard a process. development of plants of interest.

Any apparatus that allows exposure of a plant or part of a plant to the catalyst and retards the development process of a plant of interest is encompassed by the present invention. Illustrative apparatus include those in which the catalyst is immobilized in a matrix and is placed in, on, or otherwise attached to any physical structure. Various configurations of the disclosed apparatus are considered and described in more detail hereinafter. The methods and apparatus of the present invention for retarding a plant development process are particularly useful for increasing the shelf life and facilitating longer distance transportation of plant products, such as fruits, vegetables and flowers, improving consumer satisfaction with the product and reducing product losses that result from untimely ripening or senescence.

Brief description of the drawings

Having thus described the present invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and in which:

Figure 1 shows a non-limiting representation of a three-layer apparatus for retarding fruit ripening. The outer layers (designated A and B) provide structural integrity to the appliance. The catalyst layer, as defined herein below, comprises one or more of the enzymes of the invention and is located between the outer layers.

Figures 2A-C provide non-limiting representations of various apparatus for retarding fruit ripening. These devices comprise a catalyst layer, one or more layers to provide structural integrity, and one or more layers that will be removed prior to use of the device. Removal of one or more of these layers may, for example, expose an adhesive for attachment of the apparatus to another physical structure.

Figures 3A-3B show a non-limiting representation of an apparatus for retarding fruit ripening. The apparatus comprises a catalyst immobilized on a layer of film and attached to a physical structure (eg a suitable box for storage / transport of fruits).

Figure 4 provides a non-limiting representation of an apparatus for retarding fruit ripening. The apparatus comprises a slotted chamber structure that allows the insertion and replacement of one or more modular catalyst elements, as defined below. The outer layers of the physical structure can be composed of a material that allows air to flow into the catalyst.

Detailed description of the invention

The present invention will now be more fully described herein with reference to specific embodiments of the invention and, particularly, to the various provided drawings. In fact, the present invention can be carried out in many different ways and should not be considered limited to the described embodiments; instead, these embodiments are provided so that this description meets applicable legal requirements. As used in the specification and the appended claims, the singular forms "a", "an", "the" include plural references, unless the context clearly indicates otherwise.

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Throughout the specification, the expression "comprising", or its grammatical variations, will be understood to imply the inclusion of an element, integer or stage, or group of elements, integers or stages indicated, but not the exclusion of any another element, integer or stage, or group of elements, integers or stages.

The present invention provides methods for retarding a growth process of a plant of interest which comprises exposing a plant or part of a plant to one or more bacteria. In particular embodiments, the methods are designed to retard a plant development process which comprises exposing a plant or part of a plant to one or more bacteria selected from the group comprising Rhodococcus spp., Pseudomonas chloroaphis, Brevibacterium ketoglutamicum and their mixtures, wherein the one or more bacteria are exposed to the plant or part of the plant in an amount sufficient to retard the development process of the plant. Also provided are apparatus for retarding a development process of a plant of interest and for practicing the methods described herein. The methods and apparatus of the present invention can be used, for example, to delay fruit / vegetable ripening or flower senescence and to increase the shelf life of fruits, vegetables or flowers, thereby facilitating transportation, distribution and the marketing of such plant products.

As used herein, "plant" or "part of a plant" is broadly defined to include intact plants and any part of a plant including, but not limited to, fruits, vegetables, flowers, seeds, leaves, nuts, embryos, pollen, ovules, branches, grains, ears, ears, shells, stems, roots, root tips, anthers and the like. In particular embodiments, the part of the plant is a fruit, vegetable or flower. In some aspects of the present invention, the plant part is a fruit, more particularly a climacteric fruit, as described in more detail below.

The methods and apparatus of the present invention are intended to retard a plant development process, such as a plant development process generally associated with increased ethylene biosynthesis. "Process of development of a plant" is intended to mean any process of growth or development of a plant or part of a plant including, but not limited to, fruit ripening, vegetable ripening, flower senescence, leaf abscission, seed germination, and the like. . In particular embodiments, the development process of a plant of interest is fruit or vegetable ripening, flower senescence or leaf abscission, more particularly fruit or vegetable ripening. As defined herein, "retarding a plant development process" and its grammatical variants, refers to any slowing, interruption, suppression or inhibition of the development process of a plant of interest or phenotypic or genotypic changes. of the plant or part of the plant usually associated with the specific development process of a plant. For example, when the development process of a plant of interest is fruit ripening, a delay in fruit ripening may include inhibition of changes generally associated with the ripening process (e.g., color change, softening of the pericarp ( i.e., ovary wall), increases in sugar content, flavor changes, general degradation / deterioration of the fruit, and eventual decreases in the attractiveness of the fruit to consumers, as described above). One skilled in the art will appreciate that the amount of time required for fruit to ripen will vary depending, for example, on the type of fruit and the specific storage conditions used (eg, temperature, humidity, air flow, etc. .). Accordingly, "delaying fruit ripening" may constitute a delay of 1 to 90 days, particularly 1 to 30 days, more particularly 5 to 30 days. Methods for evaluating a delay in a plant development process, such as fruit ripening, vegetable ripening, floral senescence, and leaf abscission, are well within the routine capabilities of those skilled in the art and can be based, for For example, in comparison with growth processes in untreated plants or plant parts. In some aspects of the present invention, delays in a plant development process resulting from the practice of the present methods can be determined with respect to untreated plants or plant parts or to plants or plant parts that have been treated with one or more agents known to retard the development process of a plant of interest. For example, a delay in fruit ripening resulting from performing a method of the present invention can be compared to the fruit ripening times of an untreated fruit or a fruit that has been treated with an anti-ripening agent, such as those described above.

In some aspects of the invention, the one or more bacteria are "induced" to display a desired characteristic (eg, the ability to retard a plant development process, such as fruit ripening) by exposure to an inducing agent. appropriate or treatment with it. Inducing agents include, but are not limited to, asparagine, glutamine, cobalt, urea, or any of their mixtures. In particular embodiments, the bacteria are exposed to or treated with the inducing agent asparagine, more particularly a mixture of the inducing agents comprising asparagine, cobalt and urea. The inducing agent can be added at any time during the cultivation of the desired cells. For example, with respect to bacteria, the culture medium can be supplemented with an inducing agent before starting the culture of the bacteria. Alternatively, the bacteria could be grown in a medium for a predetermined amount of time to grow the bacteria and the inducing agent could be added at one or more predetermined times to induce the desired enzymatic activity in the bacteria. In addition, the inducing agent could be added to the culture medium (or to a different mixture that includes the previously cultured bacteria) to induce the desired activity in the bacteria after the culture of the bacteria has been completed.

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Although not intended to be limited to a particular mechanism, "inducing" the bacteria of the present invention can result in the production (or increased production) of one or more enzymes, such as a nitrilohydratase, amidase, and / or asparaginase, and the Induction of one or more of these enzymes may play a role in retarding a developmental process of a plant of interest. The "nitrile-hydratases", "amidases" and "asparaginases" comprise families of enzymes present in cells of various organisms, including but not limited to bacteria, fungi, plants and animals. Such enzymes are well known to those of skill in the art, and each class of enzyme possesses recognized enzymatic activities. "Enzyme activity", as used herein, refers generally to the ability of an enzyme to act as a catalyst in a process, such as the conversion of one compound to another compound. In particular, nitrile hydratase catalyzes the hydrolysis of nitrile (or cyanohydrin) to the corresponding amide (or hydroxy acid). Amidase catalyzes the hydrolysis of an amide to the corresponding acid or hydroxy acid. Similarly, an asparaginase enzyme, such as asparaginase I, catalyzes the hydrolysis of asparagine to aspartic acid.

In some aspects of the invention, enzyme activity can be referred to in terms of "units" per mass of enzyme or cells (typically based on dry weight of cells, eg, units / mg psc). A "unit" generally refers to the ability to convert a specific amount of a compound to a different compound under a defined series of conditions as a function of time. In specific embodiments, a "unit" of nitrile hydratase activity can refer to the ability to convert one pmol of acrylonitrile to its corresponding amide per minute, per milligram of cells (dry weight) at pH 7.0 and at a temperature of 30 ° C. Similarly, a unit of amidase activity can refer to the ability to convert one pmol of acrylamide to its corresponding acid per minute, per milligram of cells (dry weight) at a pH of 7.0 and a temperature of 30 ° C. Furthermore, one unit of asparaginase activity can refer to the ability to convert one pmol of asparagine to its corresponding acid per minute, per milligram of cells (dry weight) at a pH of 7.0 and a temperature of 30 ° C. Assays to measure nitrile hydratase activity, amidase activity or asparaginase activity are known in the art and include, for example, the detection of free ammonia. See Fawcett and Scott (1960) J. Clin. Pathol. 13: 156-159.

Also included in the present invention are methods of retarding a plant development process that comprise exposing a plant or part of a plant to one or more enzymes selected from the group comprising nitrile-hydratase, amidase, asparaginase or one of its mixtures, in which the one or more enzymes are exposed to the plant or part of the plant in an amount or level of enzymatic activity sufficient to retard the growth process of a plant.

Nitrile-hydratase, amidase and / or asparaginase are isolated, purified or semi-purified from any of the above cells and exposed to the plant or part of the plant in a more isolated form. See, for example, Goda et al., (2001) J. Biol. Chem. 276: 23480-23485; Nagasawa et al., (2000) Eur. J. Biochem. 267: 138-144; Soong et al., (2000) Appl. Environ. Microbiol. 66: 1947-1952; Kato et al., (1999) Eur. J. Biochem. 263: 662-670. One skilled in the art will further appreciate that a single cell type may be capable of producing (or being induced or genetically modified to produce) more than one of the enzymes of the invention. Said cells are suitable for use in the methods and apparatus described.

The nucleotide and amino acid sequences for various nitrile hydratases, amidases, and asparaginases from various organisms are described in publicly available sequence databases. A non-limiting list of representative nitrile hydratases and aliphatic amidases known in the art is set forth in Tables 1 and 2 and in the sequence listing. The "protein value" mentioned in Tables 1 and 2 provides an overview of the percentage confidence intervals (% confidence interval) of the identification of isolated proteins based on mass spectroscopy data.

Table 1: Amino Acid Sequence Information for Representative Nitrile Hydratases

<td>Source organism</td><td>Access number</td><td>Sequence identifier</td><td>Protein value (% confidence interval)</td>

<td>Rhodococcus sp.</td><td> 806580</td><td>SEQ ID NO: 1</td><td> 100%</td>

<td>Nocardia sp.</td><td> 27261874</td><td>SEQ ID NO: 2</td><td> 100%</td>

<td>Rhodococcus rhodochrous</td><td> 49058</td><td>SEQ ID NO: 3</td><td> 100%</td>

<td>Uncultured bacteria (BD2); nitrile hydratase beta subunit</td><td> 27657379</td><td>SEQ ID NO: 4</td><td> 100%</td>

<td>Rhodococcus sp.</td><td> 806581</td><td>SEQ ID NO: 5</td><td> 100%</td>

<td>Rhodococcus rhodochrous</td><td> 581528</td><td>SEQ ID NO: 6</td><td> 100%</td>

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<td>Source organism</td><td>Access number</td><td>Sequence identifier</td><td>Protein value (% confidence interval)</td>

<td>Bacterium not cultured (SP1); nitrile hydratase alpha subunit</td><td> 7657369</td><td>SEQ ID NO: 7</td><td> 100%</td>

Table 2: Amino Acid Sequence Information for Representative Aliphatic Amidases

<td>Source organism</td><td>Access number</td><td>Sequence identifier</td><td>Protein value (% confidence interval)</td>

<td>Rhodococcus rhodochrous</td><td> 62461692</td><td>SEQ ID NO: 8</td><td> 100%</td>

<td>Nocardia farcinica IFM 10152</td><td> 54022723</td><td>SEQ ID NO: 9</td><td> 100%</td>

<td>Pseudomonas aeruginosa PAO1</td><td> 15598562</td><td>SEQ ID NO: 10</td><td> 98,3%</td>

<td>Helicobacter pylori J99</td><td> 15611349</td><td>SEQ ID NO: 11</td><td> 99,6%</td>

<td>Helicobacter pylori 26695</td><td> 2313392</td><td>SEQ ID NO: 12</td><td> 97,7%</td>

<td>Pseudomonas aeruginosa</td><td> 150980</td><td>SEQ ID NO: 13</td><td> 94%</td>

Generally, any bacterial, fungal, plant or animal cell capable of producing or being induced to produce nitrile hydratase, amidase, asparaginase, or any of their combinations can be used in the practice of the invention. A nitrile-hydratase, amidase, and / or asparaginase can be constitutively produced in a cell of a particular organism (e.g., a bacterium, a fungus, a plant cell, or an animal cell) or, alternatively, a cell can produce the enzyme or desired enzymes, only after "induction" with a suitable inducing agent. "Constitutively" is intended to mean that at least one enzyme of the invention is continuously produced or expressed in a particular cell type. Other types of cells, however, may need to be "induced", as described above, to express nitrile-hydratase, amidase and / or asparaginase in an amount or level of enzymatic activity sufficient to retard a process of development of a plant of interest. That is, an enzyme of the invention can only be produced (or produced at sufficient levels) after exposure to a suitable inducing agent or treatment with it. Such inducing agents are known in the art and have been outlined above. For example, in some aspects of the invention, the one or more bacteria are treated with an inducing agent, such as asparagine, glutamine, cobalt, urea, or any of their mixtures, more particularly a mixture of asparagine, cobalt, and urea. In addition, as described in pending US application No. 11 / 669,011, entitled "Induction and Stabilization of Enzymatic Activity in Microorganisms, filed January 30, 2007, asparaginase I activity can be induced in Rhodococcus rhodochrous DAP 96622 (Gram positive) or Rhodococcus sp. DAP 96253 (Gram positive), in medium supplemented with amino acids containing amide, or its derivatives. Other Rhodococcus strains can also preferably be similarly induced to show asparaginase I enzymatic activity using amide-containing amino acids, or derivatives thereof.

In other aspects of the invention, P. chloroaphis (ATCC Deposit No. 43051), which produces asparaginase I activity in the presence of asparagine, and B. kletoglutamicum (ATCC Deposit No. 21533), a gram positive bacterium that has also been shown to produce asparaginase activity, they are used in the methods described. Fungal cells, such as those of the genus Fusarium, plant cells and animal cells, which express a nitrilohydratase, amidase and / or asparaginase, can also be used in the methods and apparatus described herein, as whole cells or as a source to from which to isolate one or more of the above enzymes.

In additional embodiments, host cells that have been genetically engineered to express a nitrile-hydratase, amidase, and / or asparaginase can be used exposed to a plant or part of a plant in accordance with the present methods and apparatus to retard a process of growth of a plant. Specifically, a polynucleotide encoding a nitrile-hydratase, amidase, or asparaginase (or multiple polynucleotides each encoding a nitrile-hydratase, amidase, or asparaginase) can be introduced by standard molecular biology techniques into a host cell to produce a cell. transgenic that expresses one or more of the enzymes of the invention. The use of the terms "polynucleotide", "polynucleotide construct", "nucleotide" or "nucleotide construct" is not intended to limit the present invention to DNA comprising polynucleotides or nucleotides. Those skilled in the art will recognize that polynucleotides and nucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides.

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Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of the invention also encompass all sequence forms including, but not limited to, single-stranded forms, double-stranded forms, and the like.

Polynucleotide variants and fragments that encode polypeptides that retain the desired enzymatic activity (ie, nitrile-hydratase, amidase, or asparaginase activity) can also be used in the practice of the invention. By "fragment" is meant a part of the polynucleotide and therefore also encodes a part of the corresponding protein. Polynucleotides that are fragments of a nucleotide sequence of an enzyme generally comprise at least 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 , 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 contiguous nucleotides, or up to the number of nucleotides present in the polynucleotide sequence of a full-length enzyme. A polynucleotide fragment will encode a polypeptide with a desired enzymatic activity and will generally encode at least 15, 25, 30, 50, 100, 150, 200, or 250 contiguous amino acids or up to the total number of amino acids present in the amino acid sequence of an enzyme. full length of the invention. "Variant" is intended to mean substantially similar sequences. Generally, variants of a sequence of a particular enzyme of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference enzyme sequence, as determined by standard alignment programs of sequences. Variant polynucleotides encompassed by the invention will encode polypeptides with the desired enzymatic activity.

As used in the context of the production of transgenic cells, the term "introduce" is intended to mean presenting to a host cell, particularly a microorganism such as Escherichia coli, a polynucleotide that encodes a nitrile-hydratase, amidase and / or asparaginase. In some embodiments, the polynucleotide will be presented such that the sequence can access the interior of a host cell, including its potential insertion into the genome of the host cell. The methods of the invention do not depend on a particular method of introducing a sequence into a host cell, only on the polynucleotide accessing the interior of at least one host cell. Methods for introducing polynucleotides into host cells are well known in the art including, but not limited to, stable transfection methods, transient transfection methods, and virus-mediated methods. "Stable transfection" is intended to mean that the polynucleotide construct introduced into a host cell integrates into the host's genome and is capable of being inherited by its progeny. "Transient transfection" or "transient expression" is intended to mean that a polynucleotide is introduced into the host cell but does not integrate into the host genome.

Furthermore, the nucleotide sequence of nitrile-hydratase, amidase or asparaginase can be contained in, for example, a plasmid for introduction into the host cell. Typical plasmids of interest include vectors that have defined cloning sites, origins of replication, and selectable markers. The plasmid may further include transcriptional and translation initiation sequences and transcriptional and translational terminators. Plasmids can also include gene expression cassettes containing at least one independent terminator sequence, sequences that allow replication of the cassette in eukaryotes or prokaryotes or both, (eg, shuttle vectors), and selection markers for both prokaryotic and prokaryotic systems. eukaryotes. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or optimally both. For general descriptions of cloning, packaging and expression systems and methods, see Giliman and Smith (1979) Gene 8: 81-97; Roberts et al., (1987) Nature 328: 731-734; Berger and Kimmel (1989) Guide to Molecular Cloning Techniques, Methods in Enzymology Vol. 152 (Academic Press, Inc., San Diego, California); Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual Vols. 1-3 (2d ed; Cold Spring Harbor Laboratory Press, Plainview, New York); and Ausubel et al., eds. (1994) Current Protocols in Molecular Biology, Current Protocols (Greene Publishing Associates, Inc., and John Wiley &amp; Sons, Inc., New York; 1994 Supplement). Transgenic host cells expressing one or more of the enzymes of the present invention can be used in the methods and apparatus described as whole cells or as a biological source from which one or more enzymes of the invention can be isolated.

In addition, apparatus are provided for retarding a plant growth process and for carrying out the methods of the invention. The catalyst comprises one or more enzymes (ie nitrile hydratase, amidase and / or asparaginase) in an amount or at a level of enzymatic activity sufficient to retard a plant growth process. Sources of the desired enzymes for use as a catalyst in the apparatus of the invention have also been described in detail above. The catalyst comprises the enzyme (s) itself in an isolated, purified or semi-purified form.

Apparatus for retarding a plant growth process according to the present invention may be supplied in various suitable formats and may be suitable for single use or multiple uses (eg "rechargeable"). Furthermore, the apparatus of the invention will be useful in both domestic and commercial settings. For example, said appliances can be integrated into domestic or commercial refrigerators, including in trains, trucks, etc., for long-distance transport of fruits, vegetables or flowers, or used as autonomous cabins for the storage or transport of said plant products. Illustrative non-limiting apparatus of the invention are described hereinafter and depicted in Figures 1-4.

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In particular embodiments, the catalyst is supplied in an immobilized format. Any process or matrix can be used to immobilize the catalyst, as long as the ability of the one or more enzymes to retard a plant development process is preserved. For example, the catalyst can be immobilized in a matrix comprising alginate (eg, calcium alginate), carrageenans, DEAE-cellulose, or polyacrylamide. Other such matrices are well known in the art and may further be cross-linked with any suitable cross-linking agent including, but not limited to, glutaraldehyde or polyethyleneimine, to increase the strength of the catalyst matrix. In one aspect of the invention, the catalyst is immobilized on a DEAE-cellulose matrix crosslinked with glutaraldehyde. The catalyst, particularly the catalyst in immobilized form, can further be presented as a "modular catalyst element. A modular catalyst element comprises a catalyst, such as an immobilized catalyst, within an additional structure that, for example, reduces potential contact with the catalyst, facilitates replacement of the catalyst, or allows air to flow through the catalyst.

In one embodiment, the matrix comprises alginate, or its salts. Alginate is a linear copolymer with homopolymeric blocks of β-D-mannuronate (M) linked at (1-4) and its epimeric residues of C-5 α-L-guluronate (G), respectively, covalently linked in different sequences or blocks. Monomers can appear in homopolymeric blocks of consecutive G residues (G blocks), consecutive M residues (M blocks), alternate M and G residues (MG blocks), or randomly arranged blocks. In one embodiment, calcium alginate is used as the substrate, more particularly calcium alginate that has been cross-linked, such as with polyethyleneimine, to form a hardened calcium alginate substrate. A further description of such immobilization techniques can be found in Bucke (1987) "Cell Immobilization in Calcium Alginate" in Methods in Enzymology, Vol. 135 (B) (Academic Press, Inc., San Diego, California; Mosbach, ed. ). An illustrative immobilization method using calcium alginate crosslinked with polyethyleneimine is also described below in Example 5. In another embodiment, the matrix comprises an amide-containing polymer. In accordance with the invention any polymer comprising one or more amide groups could be used. In one embodiment, the substrate comprises a polyacrylamide polymer.

Higher strength can be achieved from an immobilized catalyst matrix by crosslinking. For example, cells can chemically crosslink to form cell clumps. In one embodiment, the harvested cells are cross-linked using glutaraldehyde. For example, cells can be suspended in a mixture of deionized water and glutaraldehyde followed by addition of polyethyleneimine until maximum flocculation is reached. Cross-linked cells (typically in the form of multi-cell particles) can be collected by simple filtration. A further description of these techniques is provided in López-Gallego et al., (2005) J. Biotechnol. 119: 70-75. A general protocol for the immobilization of cells, particularly Rhodococcus spp. Cells, in DEAE-cellulose cross-linked with glutaraldehyde is also summarized below in Example 4.

In some aspects of the invention, the immobilized catalyst or one or more modular catalyst elements are placed in, placed on, or attached to a "physical framework." The physical structure includes, but is not limited to, a film, sheet, cover layer, box, sachet, bag or slotted chamber capable of housing one or more modular catalyst elements. In some embodiments, the physical structure comprises a container suitable for transporting or storing fruits, vegetables, or flowers. The physical structure may further comprise more than one individual structure, whereby all individual structures are connected to a catalyst or core catalyst modular element. A physical structure described hereinabove may optionally be cooled by external means or comprise a cooling unit within the physical structure itself.

In an apparatus of the invention, elements may be included to monitor the efficiency of the catalyst to retard a development process of a plant of interest (for example, to determine when the catalyst or catalyst module should be replaced) or to measure or control the air flow, moisture content / humidity and carbon dioxide levels. Any apparatus for retarding a plant growth process may further comprise one or more elements to allow air to flow to or through the catalyst or the modular catalyst element. One skilled in the art will readily envision other possible modifications of the apparatus described herein to monitor and control atmospheric conditions (eg, air flow, humidity, and carbon dioxide levels) of the catalyst, the modular element of the catalyst, or the physical structure. Conditions such as temperature, atmospheric composition (e.g. relative humidity, O2 and CO2 levels), physical stress, light, chemical stress, radiation, water stress, growth regulators, and pathogen attack play an important role in respiration rates. and significantly affect the shelf life of fruits, vegetables, flowers and other plant-related products. Although the temperature and atmospheric conditions for storage vary depending on the fruit, vegetable or other plant product of interest, the recommended storage temperatures are usually in the range of about 0 ° to about 20 ° C with the O2 and CO2 levels at the approximate ranges of 1-10% and 0-20%, respectively. For the storage of fruits, vegetables and related plant products, a relative humidity of from about 50% to about 100%, particularly from 85% to about 95%, more particularly from about 90% to about 95%, is generally recommended. Given the significant correlation between respiration rate and

ES 2 525 176 T3 the preservation period of plant products, the control of the above factors is important to delay the deterioration of said products. Accordingly, a carbon dioxide scavenger can be arranged in the apparatus to reduce the carbon dioxide content.

In particular embodiments of the invention, air-permeable catalyst apparatus are provided to retard a plant growth process comprising multiple layers. For example, as shown in Figure 1, a catalyst apparatus 10 may include outer layers 12 and 14 and an intermediate catalyst layer 16 positioned between outer layers 12 and 14. The catalyst layer 16 comprises one or more bacteria (eg, Rhodococcus spp., Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and their mixtures) or enzymes (a nitrile-hydratase, amidase, asparaginase, and mixtures thereof), wherein the one or more bacteria or enzymes are provided in an amount sufficient to retard the development process of a plant of interest, and a third layer. In this embodiment, one or more of the outer layers 12 and 14 provide structural integrity to the catalyst apparatus 10. The outer layers 12 and 14 typically allow air flow to the catalyst layer 16 although, in some embodiments, it may be advantageous to have an outer layer that is not permeable to air, for example if the apparatus forms the side of the box and it is not desired to allow the outermost layer of the box to expose the catalyst layer to the environment. The catalyst apparatus 10 may be provided in reusable or non-reusable bags or pouches in accordance with the invention. In one embodiment, the catalyst layer 16 comprises cells of Rhodococcus spp., Particularly the Rhodococcus rhodochrous strain DAP 96253, the Rhodococcus sp. Strain. DAP 96622, Rhodococcus erythropolis or their mixtures. Bacterial cells used as a catalyst in an apparatus of the present invention can be induced with one or more inducing agents (eg, asparagine, glutamine, cobalt, urea, or a mixture thereof), as described in detail above.

Figures 2A-2C illustrate alternative apparatus in accordance with the invention for retarding a plant growth process. These appliances comprise multiple layers, in which one or more of the layers are removable. As shown in Figure 2A, the apparatus may include an air-permeable structural layer 22 and a catalyst layer 24. Removable layers 26 and / or 28 may be disposed along structural layer 22 and / or catalyst layer 24 and are typically intended to be removed prior to using or activating the catalyst. In some aspects of the invention, removal of removable layers 26 and 28 exposes an adhesive that facilitates placement or attachment of the catalyst structure to a separate physical structure. Figure 2B illustrates an alternative embodiment in which apparatus 30 includes two air-permeable structural layers 32 and 34, an intermediate catalyst layer 36, and a removable layer 38. Figure 2C illustrates yet another embodiment in which apparatus 40 includes two air-permeable structural layers 42 and 44, an intermediate catalyst layer 46, and two removable layers 48 and 50.

Figures 3A-3B illustrate an alternative embodiment 60 in which the catalyst is attached to the interior of a container, such as a cardboard box. As shown in Figure 3A, one side 62 of the container includes a catalyst layer 64 attached thereto by use of an adhesive layer 66. A release film 68 may be disposed adjacent the catalyst layer 64 to protect the layer. catalyst from exposure to the environment. The release film 68 can be removed to activate the catalyst in the catalyst layer 64 by exposing the catalyst to the part of a plant provided in the container to thereby retard an unwanted plant growth process.

Figure 3B illustrates a catalyst structure 70 prior to attaching the catalyst structure to the interior of a container in the manner shown in Figure 3A. In addition to catalyst layer 64, adhesive layer 66, and release film 68, catalyst structure 70 includes an additional release film 72. Release film 72, like release film 68, protects catalyst structure 70 when it is packaged, shipped or stored. Release film 72 can be removed to expose adhesive layer 66 to allow catalyst structure 70 to attach to the interior of the container in the manner illustrated in Figure 3A.

Figure 4 illustrates a catalyst structure 80 that includes two slots 82 and 84 for receiving a catalyst cassette (eg, cassette 86). The catalyst cassette 86 is permeable to air and can be easily inserted into or removed from slot 84. Therefore, the catalyst cassette 86 can be easily replaced if a new catalyst cassette is desired for use in the catalyst structure 80. The catalyst cassette 86 includes a catalyst as described herein and which is preferably immobilized in a matrix. The catalyst structure 80 may include opposing air-permeable surfaces 88 and 90, such as mesh screens, to allow air flow through the catalyst cassette 86. The catalyst structure 80 may include, in alternative embodiments, only one air-permeable surface, two non-opposing air-permeable surfaces, or more than two air-permeable surfaces as would be understood by one of ordinary skill in the art. Although Figure 4 includes two slots 82 and 84 for receiving a catalyst cassette (eg, cassette 86), one skilled in the art would understand that catalyst frame 80 could include one or more slots for receiving a cassette. The catalyst structure 80 may be disposed within a container used to transport the part of a plant, such as fruit or flowers, or it may be attached to a container, for example, by the use of an adhesive layer, as described. In the present memory.

ES 2 525 176 T3

The present methods and apparatus can be used to retard a plant development process of any plant or part of a plant of interest. In particular embodiments, the methods and apparatus of the present invention are intended to delay ripening and the part of the plant is a fruit (climacteric or non-climacteric), vegetable or other part of the plant subjected to ripening. One skilled in the art will recognize that "climacteric fruits" show a sudden increase in ethylene production during fruit ripening, whereas "non-climacteric fruits" are not generally believed to experience a significant increase in ethylene biosynthesis. during the ripening process. Fruits, vegetables, and other plant products of illustrative interest include, but are not limited to: apples, apricots, biriba, breadfruit, custard apple, feijoa, fig, guava, jackfruit, kiwi, bananas, peaches, avocados, apples, cantaloupes, mangoes, musk melons, nectarines, persimmon, sapote, soursop, olives, papaya, passion fruit, pears, plums, tomatoes, paprika, blueberries, cocoa, cashew, cucumbers, grapefruit, lemons, limes, peppers, cherries, oranges, grapes, pineapples, strawberries, watermelons, tamarillos and walnuts.

In other aspects of the invention, methods and apparatus are provided for retarding flower senescence, wilting, abscission or closure of petals. Any flower can be used in the practice of the invention. Illustrative flowers of interest include, but are not limited to, roses, carnations, orchids, purslane, mallow, and begonias. Cut flowers, more particularly commercially important cut flowers, such as roses and carnations, are of particular interest. In some embodiments, flowers that are sensitive to ethylene are used in the practice of the present invention. Ethylene sensitive flowers include, but are not limited to, flowers of the genera Alstroemeria, Aneomone, Anthurium, Antirrhinum, Aster, Astilbe, Cattleya. Cymbidium, Dahlia, Dendrobium, Dianthus, Eustoma, Freesia, Gerbera, Gypsophila, Iris, Lathyrus, Lilium, Limonium, Nerine, Rosa, Syringa, Tulipa and Zinnia. Representative ethylene-sensitive flowers also include those of the families Amarylidaceae, Alliaceae, Convallariaceae, Hemerocallidaceae, Hyacinthaceae, Liliaceae, Orchidaceae, Aizoaceae, Cactaceae, Campanulaceae, Caryophyllaceae, Crassulaceae, Gentianaceae, Malvaceae, Plumbaginaceae, Aspalaceaceae, Portulacalaceeaceae, Portulacaceae, Soalaceeaceae Asparagaceae, Begoniaceae, Caprifoliaceae, Dipsacaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Myrtaceae, Onagraceae, Saxifragaceae and Verbenaceae. See, for example, Van Doorn (2002) Annals of Botany 89: 375-383; Van Doorn (2002) Annals of Botany 89: 689-693; and Elgar (1998) "Cut Flowers and Foliage - Cooling Requirements and Temperature Management" at hortnet.co.nz/publications/hortfacts/hf305004.htm (last visited March 20, 2007). Methods and apparatus for retarding sheet abscission are also encompassed by the present invention. There is significant commercial interest in the plant, fruit, vegetable, and flower industries in methods and apparatus for regulating plant development processes, such as ripening, senescence, and abscission.

The person skilled in the art will further recognize that any of the methods or apparatus described herein can be combined with other known methods and apparatus to retard a plant growth process, particularly processes generally associated with increased ethylene biosynthesis (e.g. example, fruit / vegetable ripening, flower senescence and abscission of leaves). Furthermore, as described above, increased ethylene production has also been observed during attack on plants or parts of a plant by pathogenic organisms. Accordingly, the methods and apparatus of the invention may further be useful in enhancing the response of the plant to pathogens.

The following examples are offered by way of illustration and not by limitation:

Experimental part

The present invention will now be described with specific reference to various examples. The following examples are not intended to be limiting of the invention and are instead provided as illustrative embodiments.

Example 1: Delayed fruit ripening after exposure to Rhodococcus spp. Induced (comparative)

Cells of Rhodococcus spp. induced with asparagine, acrylonitrile or acetonitrile were immobilized on a DEAE-cellulose matrix crosslinked with glutaraldehyde. The methods of cell induction and matrix preparation above are described in greater detail hereinafter.

The crosslinked DEAE-cellulose catalyst matrix was placed in three separate paper bags (cell pack wet weight about 1-2 grams per bag), each bag containing unripe bananas, peaches, or avocados. As negative controls, the same fruits were placed in separate paper bags in the absence of the catalyst matrix. The paper bags were stored at room temperature and the product was observed daily for signs of ripening and degradation of the fruit.

All products exposed to the catalyst matrix showed significant delays in fruit ripening. In particular, the firmness and integrity of the peach skin were preserved for a longer time in the presence of the catalyst matrix. Similarly, with bananas, the appearance of brown spots was delayed and firmness was preserved for a longer time with respect to negative controls.

Example 2: General fermentation and induction protocols

Fermentation process

ES 2 525 176 T3

The following general protocols and culture media were used for the fermentation of the Rhodococcus spp., Rhodococcus sp. DAP 96622 and Rhodococcus rhodochrous DAP 96253, for use in other experiments:

The fermentation vessels were set up with probes to measure dissolved oxygen (DO) and pH, as well as sampling devices to measure glucose concentration (off-line). Additional holes were used to add correctors (eg acid, base or defoamer), inducers, nutrients and supplements. The previously cleaned containers were sterilized on site. A suitable base medium (1 or 1.5X) R2A or R3A was used. The specific components of these culture media are described below. Some substitutions of the media contents were made in some experiments. For example, Proflo® (Trader's Protein, Memphis, TN) was sometimes used in place of proteose peptone and / or casamino acids. Additionally, in some experiments, Hy-Cotton 7803® (Quest International, Hoffman Estates, IL), cottonseed hydrolyzate, cottonseed hydrolyzate-ultrafiltrate (Marcor Devolpment Corp., Carlstadt, NJ) was used in place of Proflo ® (Trader's Protein, Memphis, TN).

A feeding profile for nutrient supplementation was established to gradually replace the R2A or R3A base medium with a richer medium, specifically 2X YEMEA, the components of which are also described in greater detail below. Other optional nutrient supplements included maltose 50% (w / v) and dextrose 50% (w / v). Commercial products containing dextrose equivalents (glucose, maltose, and higher polysaccharides) were sometimes used in place of maltose and dextrose.

Inocula were prepared from cultures of the Rhodococcus sp. DAP 96622 and Rhodococcus rhodochrous DAP 96253 on a suitable solid medium and incubated at their appropriate temperature (eg, 30 ° C). In particular embodiments, cells were grown on YEMEA agar plates for 4-14 days, preferably 7 days. Alternatively, inocula were prepared from frozen cell concentrates from previous fermentation runs. Cell concentrates were typically prepared at a concentration of 20X relative to that present in the fermenter. In addition, the inoculum was sometimes prepared from a suitable biphasic medium (ie, a combination of liquid medium coating a solid medium of the same or different composition). When a biphasic medium was used, the medium generally contained YEMEA in both the liquid and solid layers.

For the induction of nitrile-hydratase, at t = 0 hours, sterile CoCt6H2O and urea were added until reaching concentrations of 5-200 ppm of CoCl2 and 750 mg / L - 10 g / L of urea, generally preferred 10-50 ppm of CoCl2 and 7500 mg / L - 7.5 g / L of urea. In a particular embodiment, urea and / or cobalt were added again during fermentation. For example, an equivalent volume of urea and 150 ppm of CoCl2 were added at 4-6 hours or at 24-30 hours. In addition to urea, 0.1M - 0.2M acrylonitrile / acetonitrile or asparagine was added stepwise to a final concentration of 300-500 ppm or at a constant flow rate, starting at various times. Fermentation operations were terminated when cell mass and enzyme concentrations were acceptable, typically 24-96 hours.

Cells were then harvested by any acceptable method including, but not limited to, batch or continuous centrifugation, decantation, or filtration. The harvested cells were resuspended to a 20X concentrated volume in a suitable buffer, such as 50 mM phosphate buffered saline (PBS), supplemented with the inducer used during the fermentation process. The cell concentrates were then frozen, particularly by flash freezing. Frozen cells were stored at -20 ° C-80 ° C or in liquid nitrogen for later use.

Description of the culture media

R2A medium (See Reasoner and Geldreich (1985) Appl. Environ. Microbiol. 49: 1-7)

ES 2 525 176 T3

<td>Yeast extract</td><td>0.5 g</td>

<td>Proteose Peptone n ° 3</td><td>0.5 g</td>

<td>Casamino acids</td><td>0.5 g</td>

<td>Glucose</td><td>0.5 g</td>

<td>Soluble starch</td><td>0.5 g</td>

<td>K2HPO4</td><td>0.3 g</td>

<td>MgSO4-7H2O</td><td>0.05 g</td>

<td>Sodium pyruvate</td><td>0.3 g</td>

Deionized or distilled H2O 1.0 liter

R3A medium (See Reasoner and Geldreich, supra.)

<td>Yeast extract</td><td>1.0 g</td><td></td>

<td>Proteose peptone n ° 3</td><td>1.0 g</td><td></td>

<td>Casamino acids</td><td>1.0 g</td><td></td>

<td>Glucose</td><td>1.0 g</td><td></td>

<td>Soluble starch</td><td>1.0 g</td><td></td>

<td>K2HPO4</td><td>0.6 g</td><td></td>

<td>MgSO4-7H2O</td><td>0.1 g</td><td></td>

<td>Sodium pyruvate</td><td>0.5 g</td><td></td>

<td>Deionized or distilled H2O</td><td>1.0 liter</td><td></td>

<td>Half YEMEA</td><td></td><td></td>

<td></td><td>1X</td><td>2X</td>

<td>Yeast extract</td><td>4.0 g</td><td>8.0 g</td>

<td>Malt extract</td><td>10.0 g</td><td>20.0 g</td>

<td>Glucose</td><td>4.0 g</td><td>8.0 g</td>

<td>Deionized or distilled H2O</td><td>1.0 liter</td><td>1.0 liter</td>

Induction

The following general protocol was used for the induction of Rhodococcus spp., Rhodococcus sp. DAP 96622 and Rhodococcus rhodochrous DAP 96253:

Volatile inducer liquids (eg, acrylonitrile / acetonitrile) were added volumetrically as filter sterilized liquid inducers based on the density of the particular liquid inducer. In the case of solid inducers (eg, asparagine / glutamine), the solids were weighed and added directly to the culture medium.

The resulting media was sterilized in an autoclave. When filter sterilized liquid inducers were used, only the culture medium was autoclaved and cooled to 40 ° C before the liquid inducer was added. Typical concentrations for inducers of interest were: 500 ppm acrylonitrile / acetonitrile;

500 ppm asparagine / glutamine; and 50 ppm succinonitrile. The cells were then cultured in specified media and subsequently analyzed for particular biomass and enzyme activities.

Example 3: Analysis of nitrile-hydratase, amidase and asparaginase activity and biomass in cells of Rhodococcus spp. asparagine-induced

ES 2 525 176 T3

Nitrile-hydratase, amidase and asparaginase activity and biomass were evaluated in asparagine-induced cells of Rhodococcus spp., Rhodococcus sp. DAP 96622 and Rhodococcus rhodochrous DAP 96253. Various modifications in the components of the culture media, the methods, the flow rates and the asparagine administration concentrations provided to the cells, and the source of the cells were analyzed for their effects on the activities of the above enzymes and on biomass. Sections A to G of this example describe the specifics of each set of test conditions and provide a summary of the enzymatic activities and biomasses obtained under each of the specified conditions.

A. Essentially as described above in Example 2, a 20-liter fermenter inoculated using Rhodococcus rhodochrous DAP 96253 cells harvested from solid medium was continuously supplemented with the inducer asparagine (120 gL / minute of a 0.2 M solution ). Hy-Cotton 7803® was used in place of proteose peptone # 3 in the R3A medium described above. At the end of the fermentation run, acetonitrile-specific nitrile hydratase activity, amidase activity, and biomass were measured according to standard methods known in the art.

The results for nitrile hydratase activity, amidase activity and biomass are provided below in Table 3, the activities being expressed in units / mg psc (dry weight of cells). A unit of nitrile-hydratase activity refers to the ability to convert 1 gmol of acrylonitrile to its corresponding amide per minute, per milligram of cells (dry weight) at pH 7.0 and at a temperature of 30 ° C. One unit of amidase activity refers to the ability to convert 1 gmol of acrylamide to its corresponding acid per minute, per milligram of cells (dry weight) at pH 7.0 and at a temperature of 30 ° C. Biomass is described as cells packed in g / L phc (wet weight of cells).

Table 3: Enzyme activities and cell biomass of Rhodococcus rhodochrous DAP 96253 after induction with asparagine

<td>Nitrile-hydratase Activity (Units / mg psc)</td><td>Amidase activity (Units / mg psc)</td><td>Biomass (g / L of phc)</td>

<td> 168</td><td> 2</td><td> 36</td>

B. Essentially as described above in Example 3A, with changes of the medium as indicated below, the enzymatic activities and biomass were determined with cells of Rhodococcus rhodochrous DAP 96253. In particular, YEMEA, dextrose or maltose to a modified R3A medium, which additionally contained Hy-Cotton 7803® as a substitute for proteose peptone no. 3. A 0.2 M solution of asparagine was added at a continuous flow rate of 120 gL / minute starting at t = 8 hours. At the end of the fermentation operation, acrylonitrile-specific nitrile-hydratase activity, amidase activity and biomass were measured. The results are summarized in Table 4. A higher biomass yield was observed with the addition of YEMEA, dextrose or maltose to the medium.

Table 4: Enzyme activities and cell biomass of Rhodococcus rhodochrous DAP 96253 after continuous induction with asparagine

<td>Nitrile-hydratase Activity (Units / mg psc)</td><td>Amidase Activity (Units / mg psc)</td><td>Biomass (g / L of phc)</td>

<td> 155</td><td> 6</td><td> 52</td>

C. Rhodococcus sp. Cells were used. Solid medium DAP 96622 as inoculum source for a 20 liter fermenter run (see Example 2 for details of the fermentation process). A 0.2 M solution of asparagine was added semi-continuously every 6 hours, starting at t = 24 hours, for 50-70 minutes at a flow rate of 2 mL / minute. Hy-Cotton 7803® was used in place of proteose peptone # 3 in a modified R3A medium. At the end of the fermentation operation, acrylonitrile-specific nitrile-hydratase activity, amidase activity and biomass were measured. The results are summarized in Table 5.

Table 5: Enzyme activities and cell biomass of Rhodococcus sp. DAP 96622 after semi-continuous induction with asparagine

ES 2 525 176 T3

<td>Nitrile-hydratase Activity (Units / mg psc)</td><td>Amidase Activity (Units / mg psc)</td><td>Biomass (g / L of phc)</td>

<td> 172</td><td> 2</td><td> 44</td>

D. Rhodococcus sp. Cells were used. DAP 96622 of solid medium as inoculum source for a 20 liter fermenter operation. A 0.2 M asparagine solution was added semi-continuously every 6 hours, starting at t = 12 hours, for 12-85 minutes at a flow rate of 2.5 mL / minute. Cottonseed hydrolyzate was used in place of proteose peptone # 3 in modified R3A medium. At the end of the fermentation operation, acrylonitrile-specific nitrile-hydratase activity, amidase activity, and biomass were measured, and the results are summarized in Table 6.

Table 6: Enzyme activities and cell biomass of Rhodococcus sp. DAP 96622 after semi-continuous induction with asparagine

<td>Nitrile-hydratase Activity (Units / mg psc)</td><td>Amidase Activity (Units / mg psc)</td><td>Biomass (g / L of phc)</td>

<td> 165</td><td> 2</td><td> 57</td>

E. Pre-frozen Rhodococcus rhodochrous DAP 96253 cells were used as the inoculum source for a 20 liter fermenter run. YEMEA, dextrose or maltose was added to a modified R3A medium that additionally contained Hy-Cotton 7803® as a substitute for proteose peptone # 3. A 0.15 M solution of asparagine was added at a continuous flow rate of 120 pL / minute started at = 8 hours. At the end of the fermentation operation, acrylonitrile-specific nitrile-hydratase activity, amidase activity and biomass were measured. The results are summarized in Table 7.

Table 7: Enzyme activities and cell biomass of Rhodococcus rhodochrous DAP 96253 after continuous induction with asparagine

<td>Nitrile-hydratase Activity (Units / mg psc)</td><td>Amidase Activity (Units / mg psc)</td><td>Biomass (g / L of phc)</td>

<td> 171</td><td> 4</td><td> 74</td>

F. Rhodococcus rhodochrous DAP 96253 cells grown in biphasic medium were used as an inoculum source for a 20 liter fermenter run. A modified R3A medium was used that was supplemented by the addition of a carbohydrate (that is, YEMEA, dextrose or maltose) and that also contained cottonseed hydrolyzate instead of the proteose peptone No. 3. A 0.15 M solution of asparagine was added at a continuous flow rate of 1000 pL / minute starting at t = 10 hours. At the end of the fermentation operation, acrylonitrile-specific nitrile-hydratase activity, amidase activity, asparaginase I activity, and biomass were measured. The results are summarized in Table 8.

Table 8: Enzyme activities and cell biomass of Rhodococcus rhodochrous DAP 96253 after continuous induction with asparagine

<td>Activity of</td><td>Activity of</td><td>Activity of</td><td>Biomass</td>

<td>nitrile-hydratase</td><td>amidase</td><td rowspan="2">Asparaginase I (Units / mg psc)</td><td>(g / L of phc)</td>

<td>(Units / mg psc)</td><td>(Units / mg psc)</td><td></td>

159 22 16 16

G. Rhodococcus rhodochrous DAP 96253 cells grown in biphasic medium were used as an inoculum source for a 20 liter fermenter run. A modified R3A medium containing maltose (instead of dextrose) and Hy-Cotton 7803® was used as a substitute for proteose peptone # 3. A 0.15 M solution of asparagine was added at a continuous flow rate of 476 pL / minute starting at t = 8 hours. At the end of the operation

ES 2 525 176 T3 fermentation, acrylonitrile specific nitrile hydratase activity, amidase activity and biomass were measured, and the results are summarized in Table 9.

Table 9: Enzyme activities and cell biomass of Rhodococcus rhodochrous DAP 96253 after continuous induction with asparagine

<td>Nitrile-hydratase Activity (Units / mg psc)</td><td>Amidase Activity (Units / mg psc)</td><td>Biomass (g / L of phc)</td>

<td> 137</td><td> 6</td><td> 35</td>