Biological-based catalyst to delay plant development processes

(54) Title: CATALYST OF BIOLOGICAL BASIS (57) Abstract: TO SLOW DOWN PLANT DEVELOPMENT PROCESSES (30) Unionist Priority: 02/04/2007 us 11 / 695,377 (73) Holder (s): Georgia State University Research Foundation,

INC.

(72) Inventor (s): Gene K. Drago, George E. Pierce, Sangeeta

Ganguly (74) Attorney (s): Dannemann, Siemsen, Bigler &

Ipanema Moreira (86) International Order: pct us2008058286 de

26/03/2008 (87) International Publication: wo 2008 / i24307de

16/10/2008

Descriptive Report of the Patent of Invention for CATALYST OF BIOLOGICAL BASIS TO DELAY PROCESSES OF DEVELOPMENT OF PLANTS.

FIELD OF THE INVENTION

The present invention relates to methods for delaying the development of plants which comprise the exposure of a plant or part of it to one or more bacteria or enzymes. Devices for delaying the process of developing a plant are also provided. BACKGROUND OF THE INVENTION

The production of ethylene in plants and parts of plants is induced by several external stress factors and factors, including injury, the application of hormones (for example, the hormone auxin), anaerobic conditions, cooling, heat, dryness and infection by pathogen. The increase in ethylene production is also observed during the various plant development processes, including fruit and vegetable ripening, seed germination, leaf loss and flower senescence.

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

Previous research has established that in climacteric fruits, ripening is caused, at least in part, by a sudden and significant increase in ethylene biosynthesis. Although a sudden increase in ethylene production supposes the process of fruit ripening in climacteric fruits, the exact mechanism, particularly in non-climacteric fruits, is not fully understood. While there is no sudden increase in ethylene production in non-climacteric fruits, non-climatic fruits will respond to ethylene. In addition, fruits, vegetables and other vegetable products vary in the amount of ethylene synthesized and also in the sensitivity of the given product to ethylene. For example, apples exhibit a high level of production and sensitivity to ethylene, while artichokes exhibit 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 in postharvest. ucdavis.edu/Produce/Storaqe/index.shtml (last accessed March 6, 2007), which is included here as a reference in its entirety. The ripening of the fruit usually results in a change in color, softening of the pericarp and changes in the sugar content and flavor of the fruit. While ripening initially makes the fruit more edible and attractive for consumption, it leads to the degradation and deterioration of the quality of the fruit at the end of the process, rendering it unfit for consumption, generating significant commercial monetary losses. Controlling the ripening process is recommended to increase the shelf life and extend the time available for the transportation, storage and sale of fruits and other agricultural products subject to ripening.

In addition to a sudden increase in ethylene biosynthesis in climacteric fruits, changes related to ripening are also associated with an increase in respiration rate. Heat is produced as a consequence of breathing in fruits, vegetables and other vegetable products and, consequently, affects the shelf life and necessary storage conditions (for example, refrigeration) for these goods. Vegetable products with higher respiration rates (for example, artichokes, cut flowers, asparagus, broccoli, spinach, etc.) have shorter shelf lives than those with lower respiration rates (for example, nuts, dates , apples, citrus fruits, grapes, etc.). Breathing is affected by numerous environmental factors such as temperature, atmospheric composition, physical stress, light, chemical stress, radiation, water stress, growth regulators and pathogen attack. Especially, temperature plays a significant role in the rate of respiration. For a general description of respiratory metabolism and recommended controlled atmospheric conditions for fruits, vegetables and other plant products, see, for example, 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 accessed 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 accessed March 6, 2007), all of which are incorporated herein by reference in their entirety.

Methods and compositions for slowing down the fruit ripening process include, for example, the application of silver salts (for example, silver thiosulfate), 2,5-norbornadiene, potassium permanganate, 1-methylcyclopropene (1-MCP), cyclopropene (CP) and derivatives thereof. These compounds have significant disadvantages, such as the presence of heavy metals, unpleasant odor and explosive properties when compressed, which make them unacceptable or of limited applicability for use in the food industry. Transgenic approaches to control ethylene production to slow plant development processes (for example, fruit ripening) by introducing nucleic acid sequences that limit ethylene production, in particular by reducing the expression of the enzymes ACC synthase or ACC oxidase, are also under investigation. The public's response to genetically modified agricultural products, however, has not been entirely favorable.

Consequently, there remains a significant need in the art for safe methods and devices to slow plant development processes. Such methods and devices could provide better control of the ripening of fruits, the ripening of vegetables, senescence of flowers, loss of leaves and germination of seeds and increase the shelf life of various agricultural products (for example, fruits, vegetables and cut flowers), thus allowing the transport of these products over longer distances, without the need for refrigeration, increasing the suitability of the product for consumers and reducing the financial costs associated with product loss due to premature ripening and senescence.

BRIEF SUMMARY OF THE INVENTION

Methods are provided to slow the process of plant development, including, without limitation, fruit ripening, vegetable ripening, flower senescence and leaf loss. The methods of the present invention generally comprise exposing a plant or part of it to one or more bacteria in an amount sufficient to delay said plant development process. In certain aspects of the invention, the bacteria are selected from the group consisting of 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, including, for example, asparagine, glutamine, cobalt, urea and mixtures thereof, to induce the ability of the bacteria to slow down said plant development process .

The present invention, furthermore, provides apparatus for delaying the process of plant development, comprising a catalyst consisting of one or more bacteria, particularly Rhodococcus spp., Pseudomonas chloroaphis, Brevibacterium ketoglutamicum or a mixture of these. Any device that allows the exposure of a plant or part of it to the catalyst and delays said plant development process is contemplated by the present invention. Exemplary devices include those in which the catalyst is immobilized in a matrix and placed inside it, on top of it or otherwise attached to any physical structure. Various configurations of the disclosed devices are displayed and described in more detail below. The methods and apparatus of the invention for delaying the process of developing a plant are particularly useful in increasing the shelf life and facilitating the transport over longer distances of plant products such as fruits, vegetables and flowers, increasing consumer satisfaction of the product and reducing the product loss resulting from premature ripening or senescence.

BRIEF DESCRIPTION OF THE DRAWINGS

After describing the invention in general terms, reference is made to the accompanying drawings, which are not necessarily drawn according to the scale, and in which:

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

Figure 2A-C provides non-limiting descriptions of various devices for delaying fruit ripening. These devices comprise a catalyst layer, one or more layers designed to provide structural integrity and one or more layers designed to be removed before using the device. The removal of one or more of these layers may, for example, show an adhesive for fixing the device to the other physical structure.

Figures 3A-3B show a non-limiting description of an apparatus for delaying fruit ripening. The apparatus comprises a catalyst immobilized on a layer of film and affixed to a physical structure (for example, a box suitable for storing / transporting fruit).

Figure 4 provides a non-limiting description of an apparatus for delaying fruit ripening. The apparatus comprises a slotted chamber structure that allows the insertion and replacement of one or more elements of the catalyst module, 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 described more fully with reference to the specific embodiments of the invention and, in particular, to the various drawings provided herein. In fact, the invention can be incorporated in many different forms and should not be interpreted as limited to the modalities presented here; preferably, these modalities are provided so that this disclosure meets the applicable legal requirements. As used in the specification and the appended claims, the singular forms one, one, o, a, include references in the plural, unless the context clearly dictates otherwise.

Throughout the specification, the term comprising, or its grammatical variations, will be understood as assuming the inclusion of an element, an integer or step, or a group of elements, integers or declared steps, but not the exclusion of any other element, whole number or step or group of elements, whole numbers or steps.

The present invention provides methods for delaying said plant development process comprising exposing a plant or part of the plant to one or more bacteria. In certain embodiments, the methods are designed to slow the development process of a plant, comprising exposing a plant or part of the plant to one or more bacteria selected from the group consisting of Rhodococcus spp., Pseudomonas chloroaphis, Brevibacterium ketoglutamicum and mixtures of the same, in which one or more bacteria are exposed to the plant or part of it in an amount sufficient to delay the plant's development process. Apparatus for delaying said plant development process and for practicing the methods described here are also provided. The creative methods and apparatus of the invention can be used, for example, to delay the ripening of fruits / vegetables or the senescence of flowers and to increase the shelf life of fruits, vegetables or flowers, thereby facilitating the transport, distribution and distribution. marketing of such plant products.

As used here, the terms plant and plant part are broadly defined to include intact plants or any part of a plant, including, without limitation, fruits, vegetables, flowers, seeds, leaves, nuts, embryos, pollen, ova, branches, grains, ears, ears of corn, husks, stems, roots, splinters of roots, anthers and the like. In certain modalities, the part of the plant is a fruit, vegetable or flower. In certain aspects of the invention, the plant part is a fruit, more particularly a climacteric fruit, as described in more detail below.

The methods and apparatus of the invention are aimed at slowing down the process of developing a plant, as well as the process of developing a plant generally associated with increased ethylene biosynthesis. The process of developing a plant should mean any process of growth or development of a plant or part of the plant, including, without limitation, the ripening of the fruit, the ripening of the vegetables, the senescence of the flowers, the falling of the leaves, the germination of seeds and the like. In certain modalities, the referred process of plant development is the ripening of fruits or vegetables, the senescence of flowers or the fall of leaves, more particularly, the ripening of fruits or vegetables. As defined here, the expression slowing down the process of developing a plant, and its grammatical variants, refer to any slowdown, interruption, suppression or inhibition of the referred process of plant development or to phenotypic or genotypic changes in the plant or part of the plant normally associated with the specific plant development process. For example, when the aforementioned plant development process is fruit ripening, a delay in fruit ripening may include inhibiting changes generally associated with the ripening process (for example, change in color, softening of the pericarp (that is, of the ovarian wall), the increase in sugar content, changes in flavor, general degradation / deterioration of the fruit and possible reductions in the suitability of the fruit for consumers, as described above). Someone skilled in the art will observe that the time required for fruit to ripen will vary depending on, for example, the type of fruit and the specific storage conditions used (for example, temperature, humidity, air flow, etc.). Consequently, delaying the ripening of the fruit may constitute a delay from 1 to 90 days, in particular from 1 to 30 days, more particularly, from 5 to 30 days. The methods for assessing a delay in the process of developing a plant such as the ripening of the fruit, the ripening of the greenery, the senescence of the flowers and the falling of the leaves are well within the routine capabilities of those skilled in the art and can be based, for example, in comparison with untreated or partly plant development processes. In certain aspects of the invention, the delays in a plant development process that results from the practice of the present methods can be evaluated with respect to untreated plants or parts of plants or to plants or parts of plants that have been treated with one or more more agents known to retard the said plant development process. For example, a delay in fruit ripening resulting from the performance of a method of the invention can be compared with the ripening times of untreated fruits or fruits that have been treated with an anti-ripening agent, such as those described above.

The methods of the invention for delaying a plant development process typically comprise exposing a plant or part of a plant to one or more of the following bacteria: Rhodococcus spp., Pseudomonas chloroaphis, Brevibacteríum ketoglutamicum or a mixture containing any combination of these bacteria . In certain embodiments, one or more bacteria include Rhodococcus spp., More particularly the Rhodococcus rhodochrous strain DAP 96253, the Rhodococcus sp. Strain DAP 96622, Rhodococcus erythropolis or mixtures thereof. As used here, exposure of a plant part or part to one or more of the above bacteria includes, for example, exposure to intact bacterial cells, bacterial cell lysates and bacterial extracts that have enzymatic activity (i.e., enzymatic extracts). Methods for preparing cell lysates and enzymatic extracts, including bacterial cells, are routine in the art. One or more bacteria used in the methods and apparatus of the invention may eventually be more generally mentioned here as the catalyst.

According to the methods of the invention, one or more bacteria are exposed to the plant or part of the plant in an amount sufficient to slow the process of plant development. Exposing a plant or plant part to one or more bacteria of the invention includes any method of presenting a bacterium to the plant or plant part. Indirect methods of exposure include, for example, placing the bacteria or mixture of bacteria in the general proximity of the plant or part of the plant (i.e., indirect exposure). In other modalities, bacteria can be exposed to the plant or part of the plant via closer or direct contact. In addition, as defined herein, a sufficient amount of one or more bacteria of the invention will depend on several factors, including, without limitation, the bacteria in particular used in the method, in which form the bacteria are exposed to the plant or part of the plant ( for example, as intact bacterial cells, cell lysates or enzyme extracts, as described above), the means by which bacteria are exposed to the plant or part of the plant, and the duration of exposure. It would be a matter of routine experimentation for the skilled person to determine the sufficient amount of one or more bacteria necessary to slow down the aforementioned plant development process.

Although in certain embodiments of the invention, one or more bacteria are selected from the group composed of Rhodococcus spp., Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, any bacteria that slows down a plant development process when exposed to a plant or part of the plant can be used in present methods and apparatus. For example, bacteria belonging to the genus Nocardia [see Japanese Patent Application No. 54-129190], Rhodococcus [Japanese Patent Application No. 2-470], Rhizobium [Japanese Patent Application No. 5236977], Klebsiella [Japanese Patent Application No. 5-30982], Aeromonas [Japanese Patent Application No. 5-30983], Agrobacterium [Patent Application

Japanese No. 8-154691], Bacilo [Japanese Patent Application No. 8-187092], Pseudonocardia [Japanese Patent Application No. 8-56684], Pseudomonas and Mycobacteríum are non-limiting examples of microorganisms that can be used according to the invention. Not all species within a given genus can exhibit the same properties. Thus, it is possible to have a genus generally known to include strains capable of exhibiting a desired activity (for example, the ability to slow down a given plant development process such as, for example, fruit ripening), but to have one or more species that generally do not exhibit the desired activity. In the light of the disclosure provided here and the general knowledge of the technique, however, it would be a matter of routine experimentation for the professional versed in the technique to perform an experiment to determine whether a particular species has one or more desired activities.

In addition, specific examples of bacteria useful according to the invention include, without limitation, Nocardia sp., Rhodococcus sp., Rhodococcus rhodochrous, Klebsiella sp., Aeromonas sp., Citrobacter freundii, Agrobacteríum rhizogenes, Agrobacteríum tumefaciens, Xanthobacter fiavas, Erwinia nigrífens, Erwinia nigrífens Enterobacter sp., Streptomyces sp., Rhizobium sp., Rhizobium loti, Rhizobium legminosarum, Rhizobium merioti, Candida guilliermondii, Pantoea agglomerans, Klebsiella pneumoniae subsp. pneumoniae, Agrobacteríum radiobacter, Bacilo smithii, Pseudonocardia thermophila, Pseudomonas chloroaphis, Pseudomonas erythropolis, Brevibacterium ketoglutamicum, Rhodococcus erythropolis, Nocardia farcinica, Pseudomonas aeruginosa and Heliobacter pylori. In certain embodiments, bacteria of the genus Rhodococcus, more specifically of the strain DAP 96253 of Rhodococcus rhodochrous (ATCC Deposit No. 55899; deposited with the ATCC on December 11, 1996), strain DAP 96622 of Rhodococcus sp. (ATCC Deposit No. 55898; filed with the ATCC on December 11, 1996), Rhodococcus erythropolis or mixtures thereof, are used in the methods and apparatus of the invention.

In certain aspects of the invention, one or more bacteria are induced to exhibit a desired characteristic (for example, the ability to delay a plant development process, such as fruit ripening) by exposure to or treatment with a suitable inducing agent. Inducing agents include, without limitation, asparagine, glutamine, cobalt, urea or any mixture thereof. In certain 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 the bacteria start to grow. Alternatively, the bacteria can be grown in a medium for a predetermined period of time for the growth of the bacteria and the inducing agent can be added on one or more specific occasions to induce the desired enzymatic activity in the bacteria. In addition, the inducing agent can be added to the growth medium (or to a separate mixture including previously grown bacteria) to induce the desired activity in the bacteria after the growth of the bacteria has stopped.

While there is no intention to limit itself to a certain mechanism, the induction of the bacteria of the invention can result in the production (or increased production) of one or more enzymes, such as nitrile hydratase, amidase and / or asparaginase and the induction of a or more of these enzymes may play a role in delaying the said plant development process. Nitrile hydratases, amidases and asparaginases comprise families of enzymes present in the cells of various organisms, including, without limitation, bacteria, fungi, plants and animals. Such enzymes are well known to those skilled in the art and each class of enzyme has recognized enzyme activities . The term enzyme activity, as used here, generally refers to an enzyme's ability to act as a catalyst in a process, such as the conversion of a compound into another compound. In particular, the enzyme nitrile hydratase catalyzes the hydrolysis of nitrile (or cyanohydrin) to the corresponding amide (or hydroxy acid). The 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 certain aspects of the invention, enzymatic activity can be referred to in terms of units per mass of enzyme or cells (typically based on the dry weight of the cells, for example, units / mg cdw). A unit generally refers to the ability to convert a specific amount of a compound to a different compound under a defined set of conditions as a function of time. In specific modalities, a unit of nitrile hydratase activity can relate to the ability to convert one pmol of acrylonitrile to its corresponding amide per minute, per milligram of cells (dry weight) at a pH of 7.0 and a temperature of 30 ° C. Similarly, a unit of the activity of the enzyme amidase can relate to the ability to convert one pmol of acrylamide into its corresponding acid per minute, per milligram of cells (dry weight) at a pH of 7.0 and at a temperature of 30 ° C . In addition, a unit of asparaginase activity can be related 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 at a temperature of 30 ° Ç. Assays for measuring nitrile hydratase, 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, which is incorporated as a reference in its entirety to the present.

Methods for delaying a plant development process comprising exposing a plant or part of the plant to one or more enzymes selected from the group consisting of nitrile hydratase, amidase, asparaginase or a mixture of these, in which one or more enzymes are exposed to the plant or part of the plant in an amount or level of enzyme activity sufficient to slow the process of plant development are further contemplated by the present invention. For example, whole cells that produce, are induced to produce or are genetically modified to produce one or more of the above enzymes (i.e., nitrile hydratase, amidase and / or asparaginase) can be used in methods to slow a process of developing a plant. Alternatively, the enzymes nitrile hydratase, amidase and / or asparaginase can be isolated, purified or semipurified from any of the cells above 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: 2348023485; 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, all of which are incorporated herein by reference in their entirety. One skilled in the art will still note that a single cell type may be able to produce (or be induced to or genetically modified to produce) more than one of the enzymes of the invention. Such cells are suitable for use in the disclosed methods and apparatus.

The nucleotide and amino acid sequences for the various enzymes nitrile hydratases, amidases and asparaginases of various organisms are disclosed in publicly available sequence databases. A non-limiting list of nitrile hydrazides and aliphatic amidases known in the art is presented in Tables 1 and 2 and in the sequence listing. The protein score referred to in Tables 1 and 2 provides an overview of the percentage confidence intervals (% of the Confidence Interval) of the identification of the isolated proteins based on mass spectroscopy data.

Table 1: Information on amino acid sequences for representative nitrile hydratases

<td>Source organism i- · ·, * · '&gt;; ~ í '„.</td><td>AccessN</td><td>'String' identifier *</td><td>Escg</td><td></td>

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

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

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

<td>Bacteria without culture (BD2); beta-subunit of nitrile hydratase</td><td> 27657379</td><td>ID. SEQ No.: 4</td><td></td><td> 100%</td>

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

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

<td>Bacteria without culture (SP1); alpha subunit of nitrile hydratase</td><td> 7657369</td><td>ID. SEQ No.: 7</td><td></td><td> 100%</td>

Table 2: Information on the amino acid sequence of representative aliphatic amidases

<td colspan="2">Ί 'S⁵’</td><td colspan="2">SeequehW3- «mJ« SOlntet ^ HQiOe con - ty</td>

<td>Rhodococcus rtiodochrous</td><td> 62461692</td><td>ID. SEQ No.: 8</td><td> 100%</td>

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

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

<td>Heiicobacter pylori J99</td><td> 15611349</td><td>ID. SEQ No.: 11</td><td> 99,6%</td>

<td>Heiicobacter pylori 26695</td><td> 2313392</td><td>ID. SEQ No.: 12</td><td> 97,7%</td>

<td>Pseudomonas aeruginosa</td><td> 150980</td><td>ID. SEQ 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 combination thereof can be used in the practice of the invention. An enzyme nitrile hydratase, amidase and / or asparaginase can be produced constitutively in a cell of a certain organism (for example, from a bacterium, fungus, plant cell or animal cell) or, alternatively, a cell can produce the enzyme or desired enzymes only after induction with a suitable inducing agent. The term constitutively means 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 the enzyme nitrile hydratase, amidase and / or asparaginase in an amount sufficient or the level of enzymatic activity to slow the said process of plant development. That is, an enzyme of the invention can only be produced (or produced at sufficient levels) after exposure to or treatment with a suitable inducing agent. Such inducing agents are known in the art and detailed above. For example, in certain aspects of the invention, one or more bacteria are treated with an inducing agent, such as asparagine, glutamine, cobalt, urea or any mixture thereof, more particularly a mixture of asparagine, cobalt and urea. In addition, as disclosed in pending North American Order No. 11 / 669.011, entitled Induction and Stabilization of Enzyme Activity in Microorganisms, deposited on January 30, 2007, asparaginase I activity can be induced in

DAP 96622 Rhodococcus rhodochrous (Gram-positive) or in DAP 96253 Rhodococcus sp. (Gram-positive), in a medium supplemented with amide containing amino acids or derivatives thereof. Other strains of Rhodococcus may also preferably be similarly induced to exhibit enzymatic activity of asparaginase I 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, are used in the disclosed methods. Fungal cells, such as those of the Fusarium genus, plant cells and animal cells, which express a nitrile hydratase, amidase and / or an asparaginase, can also be used in the methods and apparatus disclosed here, either as whole cells or as its source for one or more of the above isolated enzymes.

In additional embodiments, host cells that have been genetically produced to express a nitrile hydratase, amidase and / or asparaginase can be used exposed to a plant or part of the plant in accordance with the present methods and apparatus to delay a plant development process. Specifically, a polynucleotide that encodes a nitrile hydratase, amidase or asparaginase enzyme (or multiple polynucleotides each that encodes a nitrile hydratase, amidase or asparaginase enzyme) can be introduced by standard molecular biology techniques into a host cell to produce a transgenic cell that expresses a transgenic cell one or more 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 polynucleotides or nucleotides comprising DNA. Those skilled in the art will recognize that polynucleotides and nucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include molecules16

Naturally occurring and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences, including, without limitation, thermal tape forms, double tape forms and the like.

Variants and fragments of polynucleotides encoding polypeptides that retain the desired enzyme activity (i.e., the activity of the enzymes nitrile hydratase, amidase or asparaginase) can also be used in the practice of the invention. A fragment is defined as a portion of polynucleotide and therefore also encodes a portion of the corresponding protein. Polynucleotides that are fragments of a nucleotide sequence of enzymes 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 even the number of nucleotides present in a full-length enzyme polynucleotide sequence. A polynucleotide fragment will encode a polypeptide with a desired enzyme activity and will generally encode at least 15, 25, 30, 50, 100, 150, 200 or 250 contiguous amino acids, or even the total number of amino acids present in an enzyme amino acid sequence full length of the invention. Substantially similar sequences are defined by variants. Generally, variants of a given enzyme sequence of the invention will have at least approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the reference enzyme sequence, as determined by standard sequence alignment programs. The variant polynucleotides contemplated by the invention will encode the polypeptides with the desired enzymatic activity.

As used in the context of transgenic cell production, the term introduction means presenting a host cell with a particular microorganism, such as Escherichia coli, with a polynucleotide encoding a nitrile hydratase, amidase and / or asparaginase enzyme. In some embodiments, the polynucleotide will be presented in such a way that the sequence gains access to the interior of a host cell, including its potential insertion into the host cell's genome. The methods of the invention do not depend on a particular method for introducing a sequence into a host cell, only that the polynucleotide gains access to the interior of at least one host cell. Methods for introducing polynucleotides into host cells are well known in the art including, without limitation, stable transfection methods, transient transfection methods and virus-mediated methods. By stable transfection it is defined that the polynucleotide construct introduced in a host cell integrates with the host's genome and is capable of being inherited by its offspring. Transient transfection or transient expression means that a polynucleotide is introduced into the host cell, but does not integrate into the host's genome.

In addition, 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 referred to include vectors containing defined cloning sites, origins of the replica and selectable markers. The plasmid can further include transcription and translation initiation sequences and transcription and translation terminators. Plasmids can also include generic expression cassettes containing at least one independent terminator sequence, sequences that allow replication of the cassette in eukaryotes or prokaryotes, or both (for example, propagating vectors) and selection markers for both prokaryotic and eukaryotic systems . The vectors are suitable for replication and integration in prokaryotes, eukaryotes or ideally 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 (2nd ed; Cold Spring Harbor Laboratory Press, Plainview, 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 that express one or more enzymes of the invention can be used in the methods and apparatus disclosed as whole cells or as a biological source from which one or more enzymes of the invention can be isolated.

Apparatus for delaying a plant development process and for carrying out the methods of the invention are also provided. In certain embodiments, an apparatus for delaying a plant development process, in particular the ripening of the fruit, comprising a catalyst comprising one or more bacteria selected from the group composed of Rhodococcus spp., Pseudomonas chloroaphis, Brevibacterium ketoglutamicum and mixtures of these is contemplated by the present invention. The DAP strain 96253 of Rhodococcus rhodochrous, the DAP strain of 96622 Rhodococcus sp., Rhodococcus erythropolis or mixtures thereof can be used in certain aspects of the invention. One or more bacteria from an apparatus of the invention are provided in an amount sufficient to delay said plant development process, as defined above. In other aspects of the invention, the catalyst comprises one or more enzymes (i.e., nitrile hydratase, amidase and / or asparaginase) in an amount or level of enzyme activity sufficient to delay a plant development process. The sources of the desired enzymes for use as a catalyst in the apparatus of the invention are also described in detail above. For example, the catalyst can be used in the form of whole cells that produce (or are induced to or genetically modified to produce) one or more of the enzymes of the invention or can comprise the enzyme (s) itself in form isolated, purified or semi-purified.

Apparatus for delaying a plant development process contemplated by the present invention may be provided in a number of suitable formats and may be suitable for single use or multiple uses (for example, refillable). In addition, the devices of the invention find use in both residential and commercial environments. For example, such devices can be integrated into residential or commercial refrigerators, including trains, trucks, etc. for the long-distance transport of fruits, vegetables or flowers or used in independent compartments for the storage or transport of such plant products. Exemplary, non-limiting devices of the invention are described here below and illustrated in figures 1-4.

In certain embodiments, the catalyst is provided in an immobilized format. Any process or matrix for immobilizing the catalyst can be used as long as the ability of one or more bacteria (or enzymes) to slow down a plant development process is conserved. For example, the catalyst can be immobilized in a matrix that comprises alginate (for example, calcium alginate), carrageenan, DEAE-cellulose or polyacrylamide. Other such matrices are well known in the art and may further crosslink with any appropriate crosslinking agent, including, without limitation, glutaraldehyde or polyethyleneimine, to increase the mechanical strength of the catalyst matrix. In one aspect of the invention, the catalyst is immobilized in a DEAE-cellulose matrix cross-linked by glutaraldehyde. The catalyst, in particular, the catalyst in immobilized form, can further be presented as a catalyst module element. A catalyst module element comprises a catalyst, such as an immobilized catalyst, within an additional structure which, for example, reduces potential contact with the catalyst, facilitates the replacement of the catalyst or allows air to flow through the catalyst.

In one embodiment, the matrix comprises alginate or salts thereof. Alginate is a linear copolymer with homopolymer blocks of Dmanuronate (M) (1-4)-linked β and its residues of C-5 epimer a-Lguluronate (G), respectively, covalently linked together in different sequences or blocks . Monomers can appear in homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M-blocks), alternating M and G residues (MG-blocks) or randomly arranged blocks. In one embodiment, calcium alginate is used as the substrate, more particularly the calcium alginate which has been crosslinked, as with polyethyleneimine, to form a hardened calcium alginate substrate. A more detailed 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.) , which is incorporated here as a reference. An exemplary method of immobilization using polyethyleneimine crosslinked calcium alginate is also described in Example 5. In another embodiment, the matrix comprises an amide-containing polymer. Any polymer comprising one or more amide groups can be used according to the invention. In one embodiment, the substrate comprises a polyacrylamide polymer.

The increase in mechanical strength of an immobilized catalyst matrix can be achieved through crosslinking. For example, cells can be chemically cross-linked 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 the addition of polyethyleneimine until maximum flocculation is achieved. The cross-linked cells (typically in the form of particles formed by countless cells) can be harvested by simple filtration. Another description of such techniques is provided in LopezGallego et al. (2005) J. Biotechnol. 119: 70-75, which is hereby incorporated by reference in its entirety. A general protocol for immobilizing cells, in particular Rhodococcus spp. Cells, in DEAE-cellulose cross-linked with glutaraldehyde is also outlined below in Example 4.

In certain aspects of the invention, the immobilized catalyst or one or more modular elements of the catalyst are placed inside the, placed on, or attached to a physical structure. The physical structure includes, without limitation, a film, blade, coating layer, box, pouch, bag, or slotted chamber capable of retaining one or more modular elements of the catalyst. In certain embodiments, the physical structure comprises a container suitable for transporting or storing fruits, vegetables or flowers. The physical structure can further comprise more than one individual structure, whereby all individual structures are joined to a central catalyst or to a modular element of the catalyst. A physical structure described here above may optionally be cooled by external means or comprise a cooling unit within the physical structure itself.

The elements for monitoring the effectiveness of the catalyst to slow down said plant development process (for example, to assess when the catalyst or catalyst module should be replaced) or to measure or control air flow, moisture / moisture content and carbon dioxide levels can optionally be included in an apparatus of the invention. Any apparatus for delaying a plant development process may further comprise one or more elements to allow air to flow through or through the catalyst or the modular catalyst element. The skilled person would readily view other possible modifications to the devices described here to monitor and control the atmospheric conditions (for example, airflow, humidity and carbon dioxide levels) of the catalyst, the modular element of the catalyst or the physical structure. Conditions, such as temperature, atmospheric composition (for example, relative humidity, O levels₂ and CO2, 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 or other related plant products . Although the temperature and atmospheric conditions of storage vary depending on the fruit, vegetable or other related plant product, the recommended storage temperatures typically reside in the range of approximately 0 ° to approximately 20 ° C with O levels₂ and CO₂ in the approximate ranges of 1-10% and 0-20%, respectively. A relative humidity of approximately 50% to approximately 100%, in particular 85% to approximately 95%, more particularly from approximately 90% to approximately 95% is generally recommended for the storage of fruits, vegetables and related vegetable products. Considering the significant correlation between the respiration rate and the shelf life of plant products, the control of the factors mentioned above is important to delay the deterioration of such products. Consequently, a carbon dioxide sequestrant can be provided in the apparatus to reduce the carbon dioxide content.

In certain embodiments of the invention, air permeable catalyst apparatus for delaying a plant development process comprising multiple layers is provided. For example, as shown in figure 1, a catalyst apparatus 10 can include outer layers 12 and 14 and an intermediate catalyst layer 16 located between outer layers 12 and 14. The catalyst layer 16 comprises one or more bacteria (for example, Rhodococcus spp., Pseudomonas chloroaphis, Brevibacterium ketoglutamicum and mixtures thereof) or enzymes (nitrile hydratase, amidase, asparaginase and mixtures thereof), wherein one or more bacteria or Enzymes are provided in sufficient quantity to slow the related plant development process 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 to flow into 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 device forms the side of the box and there is a recommendation not to allow the outer layer of the box to expose the catalyst layer to the environment. The catalyst apparatus 10 can be provided in reusable or non-reusable bags or bags according to the invention. In one embodiment, the catalyst layer 16 comprises Rhodococcus spp. Cells, in particular the Rhodococcus rhodochrous strain DAP 96253, the Rhodococcus sp. Strain DAP 96622, Rhodococcus erythropolis or mixtures thereof. The bacterial cells used as a catalyst in an apparatus of the invention can be induced with one or more inducing agents (for example, asparagine, glutamine, cobalt, urea or a mixture thereof), as described in detail23 above.

Figures 2A-2C illustrate alternative apparatus in accordance with the invention to delay a plant development process. These devices comprise multiple layers, where one or more of the layers are removable. As shown in figure 2A, the apparatus can include an air-permeable structural layer 22 and a catalyst layer 24. Removable layers 26 and / or 28 can be provided along structural layer 22 and / or the catalyst layer 24 and are typically intended to be removed prior to use or activation of the catalyst. In certain aspects of the invention, removing the removable layers 26 and 28 exposes an adhesive that facilitates the placement or affixing of the catalyst structure to a separate physical structure. Figure 2B illustrates an alternative embodiment in which the 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 the apparatus 40 includes two layers air-permeable structural elements 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 affixed to the inside of a container, such as a cardboard box. As shown in figure 3A, one side 62 of the container includes a layer of catalyst 64 affixed to it using an adhesive layer 66. A peelable film 68 can be provided adjacent to the catalyst layer 64 to protect the catalyst layer from exposure to environment. The peelable film 68 can be removed to activate the catalyst in the catalyst layer 64 by exposing the catalyst to a plant part supplied in the container to hereby delay an unwanted plant development process.

Figure 3B shows a catalyst structure 70 before affixing the catalyst structure to the inside of a container in the manner illustrated in figure 3A. In addition to the catalyst layer 64, the adhesive layer 66 and the peelable film 68, the catalyst structure 70 includes an additional peelable film 24. The peelable film 72, like the peelable film 68, protects the catalyst structure 70 when it is packed, shipped or stored. The peelable film 72 can be removed to expose the adhesive layer 66 to allow the catalyst structure 70 to be affixed 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 (e.g., cassette 86). A catalyst cassette 86 is air permeable and can be easily inserted into or removed from slot 84. Thus, catalyst cassette 86 can be readily replaced if a new catalyst cassette is recommended for use in catalyst structure 80. Catalyst cassette 86 includes a catalyst as described herein and this is preferably immobilized in a matrix. The catalyst structure 80 can include opposing air-permeable surfaces 88 and 90, such as mesh screens to allow air to flow through catalyst cassette 86. The catalyst structure 80, in alternative embodiments, may include only one air-permeable surface, two non-opposing air-permeable surfaces or more than two air-permeable surfaces as would be understood by one skilled in the art. Although figure 4 includes two slots 82 and 84 for receiving a catalyst cassette (for example, cassette 86), it would be understood by one skilled in the art that the catalyst structure 80 can include one or more slots for receiving a cassette. The catalyst structure 80 can be provided within a container used to transport a part of the plant, such as fruit or flowers, or can be affixed to a container, for example, by using an adhesive layer as discussed here.

The present methods and devices can be used to slow down a development process for any plant or part of the related plant. In certain embodiments, the methods and apparatus of the invention are aimed at delaying ripening and the plant part is a fruit (climacteric or non-climacteric), greenery or another part of the plant subject to ripening. Someone skilled in the art will recognize that climacteric fruits exhibit a sudden increase in ethylene production during fruit ripening, whereas non-climacteric fruits are generally not believed to experience a significant increase in ethylene biosynthesis during the ripening process. Fruits, vegetables and other related vegetable products include, but are not limited to: apple, apricot, biriba, breadfruit, cherimoya, mountain guava, fig, guava, jackfruit, kiwi, banana, peach, avocado, apple, cantaloupe melon, mango, melon, nectarine, persimmon, sapodilla, earl fruit, olive, papaya, passion fruit, pear, plum, tomato, pepper, blackberry, cocoa, cashew, cucumber, grapefruit, lemon, lemon, pepper, cherry, orange, grape, pineapple, strawberry, watermelon, tamarillo and nuts.

In other aspects of the invention, methods and apparatus are designed to delay the senescence of the flowers, the stolification, the fall of leaves or the closing of the petal. Any flower can be used in the practice of the invention. Related flowers include, but are not limited to: rose, carnation, orchid, eleven o'clock, mallow and begonia. Cut flowers, more particularly, commercially important cut flowers, such as roses and carnations, are particularly related. In certain embodiments, flowers that are sensitive to ethylene are used in the practice of the invention. Ethylene-sensitive flowers include, without limitation, flowers of the genera Alstroemeria, Aneomone, Anthurium, Antirrhinum, Aster, Astilbe, Cattleya, Cymbidium, Dahlia, Dendrobium, Dianthus, Eustoma, Freesia, Gerbera, Gypsophila, Iris, Lathyrus, Lilium, Limium , Nerine, Rosa, Syringa, Tulipa and Zinia. Representative ethylene-sensitive flowers also include those from the Amarylidaceae, Alliaceae, Convallariaceae, Hemerocallidaceae, Hyacinthaceae, Liliaceae, Orchidaceae, Aizoaceae, Cactaceae, Campanulaceae, Caryophyllaceae, Crassulaceae, Gentianaceae, Solaceae, Malaceae, Malaceae, Malaceae, Begoniaceae, Caprifoliaceae, Dipsacaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Myrtaceae, Onagraceae, Saxifragaceae and Verbenaceae. See, for example, Van Doorn (2002) Annals of Botany 89: 689-693; and Elgar (1998) Cut Flowers and Foliage - Cooling Requirements and Temperature Mana26 gement at hortnet.co.nz/publications/hortfacts/hf305004.htm (last accessed 20 March 2007), all of which are incorporated herein by reference in its entirety. Methods and apparatus for delaying leaf fall are also contemplated by the present invention. There is great commercial interest in the sector of plants, fruits, vegetables and flowers in methods and devices that regulate the development processes of plants such as ripening, senescence and leaf fall.

The skilled practitioner will also recognize that any of the methods or apparatus disclosed herein can be combined with other known methods and apparatus for slowing down a plant development process, particularly those processes generally associated with increased ethylene biosynthesis (for example, the ripening of fruits / vegetables, senescence of flowers and fall of leaves). In addition, as described above, the increase in ethylene production was also observed during the attack of plants or parts of plants by pathogenic organisms. Consequently, the methods and apparatus of the invention may find another use in improving the response of plants to pathogens.

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

EXPERIMENTS

The present invention will now be described with specific reference to several examples. The following examples are not limiting the invention and are, in fact, provided as exemplary embodiments.

Example 1: Delayed fruit ripening after exposure to Rhodococcus spp. induced

Rhodococcus spp. induced with asparagine, acrylonitrile or acetonitrile were immobilized in a cross-linked glutaraldehyde matrix from DEAE-cellulose. The methods of inducing cells and preparing the matrix above are described here in more detail below.

The cross-linked DEAE-cellulose catalyst matrix was placed 27 each in three separate paper bags (approximately 1-2 grams of wet cell pack weight per bag), with each bag containing bananas, peaches or green avocados. As negative controls, the same fruits were placed in separate paper bags without the presence of the catalyst matrix. The paper bags were kept at room temperature and the product was observed daily for signs of ripening and degradation of the fruits.

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

Example 2: General fermentation and induction protocols

Fermentation process

The following general protocols and culture media were used for the fermentation of Rhodococcus spp. and the DAP 96622 strains of Rhodococcus sp. and Rhodococcus rhodochrous DAP 96523 for use in other experiments:

The fermentation vessels were configured with probes to measure dissolved oxygen (DO) and pH, as well as sampling devices to measure glucose concentration (inactive). Additional openings were used to add concealers (for example, acid, base, or defoamer), inducers, nutrients and supplements. Pre-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 shown below. Certain substitutions of media content have been made in certain experiments. For example, Proflo® (Trader's Protein, Memphis, TN) was eventually used in place of proteose peptone acids and / or casamino acid. In addition, in certain experiments, Hy-Cotton 7803® (Quest International, Hoffman Estates, IL), ultrafiltered cottonseed hydrolyzate (Marcor Devolpment Corp., Carlstadt, NJ) was used instead of Proflo® (Trader's Protein, Memphis, TN).

A feed profile for nutritional supplements has been established to gradually replace the R2A or R3A base medium with a richer medium, namely 2X YEMEA, whose components are also described in more detail below. Other optional nutritional supplements included 50% maltose (w / v) and 50% dextrose (w / v). Commercial products containing dextrose equivalents (glucose, maltose and superior polysaccharides) have occasionally been used in place of maltose and dextrose.

Inocula were prepared from cultures of the DAP 9662 strains of Rhodococcus sp. and Rhodococcus rhodochrous DAP 96523 in an appropriate solid medium and incubated at its appropriate temperature (for example, 30 ° C). In certain embodiments, cells were cultured on YEMEA agar plates for 4-14 days, preferably 07 days. Alternatively, the inoculants were prepared from concentrates of frozen cells from previous fermentation cycles. Cell concentrates were typically prepared at a concentration of 20X above that present in the fermenter. In addition, the inoculum was eventually prepared from a suitable two-phase medium (i.e., a combination of the liquid medium that covers a solid medium of the same or different composition). When a biphasic medium was used, the medium generally contained YEMEA in both liquid and solid layers.

For the induction of nitrile hydratase, at hour t = 0, sterile CoCh efUO and urea were added to achieve concentrations of 5-200 ppm CoCI₂ and 750 mg / l - 10 g / l urea, with 10-50 ppm CoCl₂ and 7500 mg / l 7.5 g / l of urea generally preferred. In a given modality, urea and / or cobalt were added again during fermentation. For example, an equivalent volume of urea and 150 ppm C0Cl2 was added in 4 - 6 hours or in 24 - 30 hours. In addition to urea, a final concentration of 300 - 500 ppm acrylonitrile / acetonitrile or 0.1 M - 0.2 M asparagine was added gradually or at a constant speed, starting at various times. Fermentation cycles were completed when cell mass and enzyme concentrations were acceptable, typically within 24-96 hours.

The cells were then harvested by any acceptable method, including, without limitation, batch or continuous centrifugation, decantation or filtration. The harvested cells were resuspended in a concentrated 20X volume in a suitable buffer, such as 50 mM phosphate buffered saline (PBS) supplemented with the inducer during the fermentation process. The cell concentrates were then frozen, in particular by rapid freezing. The frozen cells were stored at -20 ° C-80 ° C or under liquid nitrogen for later use.

Description of culture media

R2A medium (See Reasoner and Geldreich (1985) Appl. Environ. Microbiol. 49: 17-)

Yeast extract Proteose peptone No. 3 Casamino Glucose acids

Soluble starch K₂HPO₄MgSO₄7H₂Sodium pyruvate Dl or H₂The dest.

0.5 g 0.5 g 0.5 g 0.5 g 0.5 g 0.3 g 0.05 g 0.3 g 1.0 liter

R3A medium (See Reasoner and Geldreich, supra.)

Yeast extract Proteose peptone No. 3 Casamino Glucose acids

Soluble starch K₂HPO₄MgSO₄7H₂Sodium pyruvate Dl or H₂The dest.

1.0 g 1.0 g 1.0 g 1.0 g 1.0 g 0.6 g 0.1 g 0.5 g 1.0 liter

YEMEA medium

<td></td><td>IX</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>Dl or H₂The dest.</td><td>1.0 liter</td><td>1.0 liter</td>

Induction

The following general protocol was used to induce strains of Rhodococcus spp., DAP 96622 of Rhodococcus sp. and Rhodococcus rhodochrous · DAP 96523.

Volatile inducing liquids (eg acrylonitrile / acetonitrile) were added volumetrically as sterile liquid inductors in a filter based on the density of the given liquid inductor. In the case of solid inducers (for example, asparagine / glutamine), the solids were weighed and added directly to the culture medium. The resulting media was autoclaved. When sterilized liquid filter inductors were used, the separate culture medium was autoclaved and cooled to 40 ° C before the liquid inductor was added. Typical concentrations for related inducers were: 500 ppm acrylonitrile / acetonitrile; 500 ppm asparagine / glutamine; and 50 ppm succinonitrile. The cells were then grown in specified media and, furthermore, analyzed for certain enzymatic activities and biomass. Example 3; Analysis of the activity of nitrile hydratase, amidase and asparaqinase and biomass in cells of Rhodococcus spp. induced by asparagine

The activity of nitrile hydratase, amidase and asparaginase and biomass were evaluated in asparagine-induced cells of the strains of Rhodococcus spp., DAP 96622 of Rhodococcus sp. and Rhodococcus rhodochrous DAP 96523. Various changes in components of culture media, methods of administration, proportions and concentrations of asparagine supplied to the cells and the source of the cells were analyzed with respect to their effects on the activities of the enzymes mentioned above and on the biomass. Sections A to G of this example describe the specifications for each set of test conditions and provide a summary of the enzyme and biomass activities obtained under each of the specified conditions.

A. Essentially as described above in Example 2, a 20 liter fermenter inoculated using Rhodococcus rhodochrous strain DAP 96253 cells harvested from the solid medium was continuously supplemented with inducing asparagine (120 μΙ / minute of a 0.2 Μ solution). Hy-Cotton 7803® was used in place of proteose peptone No. 3 in the R3A medium described above. At the end of the fermentation cycle, the activity of nitrile hydratase specific to acrylonitrile, amidase activity and biomass were measured according to standard techniques known in the art.

The results for nitrile hydratase activity, amidase activity and biomass are shown below in Table 3, with activities provided in units / mg cps (dry weight cell). A unit of nitrile hydratase activity refers to the ability to convert 1 pmol of acrylonitrile into its corresponding amide per minute, per milligram of cells (dry weight) at a pH of 7.0 and a temperature of 30 ° C. One unit of amidase activity refers to the ability to convert 1 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. Biomass is reported as cells packed in g / l cpu (wet weight cell).

Table 3: Enzymatic activities and cell biomass of the strain

Rhodococcus rhodochrous DAP 96523 after induction with asparagine

Nitrile hydratase activity Amidase activity Biomass _ (units / mg cps) (units / mg cps) &lt;(g / l cpu)

168 2 36

B. Essentially as described above in Example 3A, with modifications to the medium as noted below, enzyme activities and biomass were evaluated with cells from the DAP 96523 strain of Rhodococcus rhodochrous. In particular, YEMEA, dextrose or maltose were added to a modified R3A medium, still containing Hy-Cotton 7803® used in place of proteose peptone No. 3. A 0.2 M solution of aspa32 ragin was added at a continuous rate of 120 pl // minute starting at t = 8 hours. At the end of the fermentation cycle, specific acrylonitrile nitrile hydratase activity, amidase activity and biomass were measured. The results are summarized in Table 4. The increase in biomass yield was observed with the addition of YEMEA, dextrose or maltose to the medium.

Table 4: Enzymatic activities and cell biomass of the DAP96523 strain of Rhodococcus rhodochrous after continuous induction with asparagine

Nitrile hydratase activity Amidase activity Biomass _ (units / mg cps) _ (units / mg cps). (g / l cpu)

155 6 52

C. The cells of the DAP 96622 strain of Rhodococcus sp. of the solid medium were used as the source of the inoculum of a 20 liter fermentation cycle (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, for 50-70 minutes at a rate of 2 ml / minute. Hy-Cotton 7803® was used in place of proteose peptone No. 3 in a modified R3A medium. At the end of the fermentation cycle, specific acrylonitrile nitrile hydratase activity, amidase activity and biomass were measured. The results are summarized in Table 5.

Table 5: Enzymatic activities and cell biomass of the DAP 96622 strain of Rhodococcus sp. after semicontinuous induction with asparagine

Nitrile hydratase activity Amidase activity Biomass __ (units / mg cps) _ (units / mg cps) _ (g / l cpu)

172 2 44

D. Cells of the DAP 96622 strain of Rhodococcus sp. of the solid medium were used as the source of the inoculum of the 20 liter fermentor cycle. A 0.2 M solution of asparagine was added semi-continuously every 6 hours, starting at t = 12 hours, for 12-85 minutes at a speed of 2.5 ml / minute. Cottonseed Hydrolyzate was used instead of proteose peptone No. 3 in a modified R3A medium. At the end of the fermentation cycle, specific acrylonitrile nitrile hydratase activity, amidase activity and biomass were measured, and the results are summarized in Table 6.

Table 6: Enzymatic activities and cell biomass of the DAP 96622 strain of Rhodococcus sp. after semicontinuous induction with asparagine

Nitrile hydratase activity Amidase activity - Biomass (units / mg cps) _ (units / mg cps), (g / l cpu)

165 2 57

E. Cells from the previously frozen Rhodococcus rhodochrous strain DAP 96253 were used as the source of the inoculum of a 20 liter fermentation cycle. YEMEA, dextrose or maltose were added to a modified R3A medium that still contained Hy-Cotton 7803® as a substitute for proteose peptone No. 3. A 0.15 M asparagine solution was added at a continuous speed of 120 μΙ / minute starting at t = 8 hours. At the end of the fermentation cycle, specific acrylonitrile nitrile hydratase activity, amidase activity and biomass were measured. The results are summarized in Table 7.

Table 7: Enzymatic activities and cell biomass of the DAP 96523 strain of Rhodococcus rhodochrous after continuous induction with asparagine

Nitrile hydratase activity Amidase activity Biomass (units / mg cps) _ (units / mg cps) (g / l cpu)

171 4 74

F. The cells of the DAP 96253 strain of Rhodococcus rhodochrous grown in biphasic medium were used as the source of inoculum for a 20 liter fermentation cycle. A modified R3A medium was used, which was complemented by the addition of a carbohydrate (ie, YEMEA, dextrose or maltose), and still containing Cottonseed Hydrolyzate in place of proteose peptone No. 3. A 0.15 M asparagine solution was added at a continuous speed of 1000 μΙ / minute starting at t = 10 hours. At the end of the fermentation cycle, specific acrylonitrile nitrile hydratase activity, amidase activity, asparaginase I activity and biomass were measured. The results are summarized in Table 8.

Table 8: Enzymatic activities and cell biomass of the DAP 96523 strain of Rhodococcus rhodochrous after continuous induction with asparagine

Nitrile hydra activity- Amidase activity Asparagine activity- Biomass tase (units / mgcps) (units / mgcps) sei (units / mgcps) (g / l cpu)

159 22 16 16

G. The cells of the Rhodococcus rhodochrous strain DAP 96253 grown in the biphasic medium were used as a source of inoculum for a 20 liter fermentation cycle. A modified R3A medium was used, which contained maltose (in place of dextrose) and Hy-Cotton 7803® as a substitute for proteose peptone No. 3. A 0.15 M asparagine solution was added at a continuous speed of 476 μΙ / minute starting at t = 8 hours. At the end of the fermentation cycle, nitrile hydratase activity, amidase activity, and biomass were measured, and the results are summarized in Table 9.

Table 9: Enzymatic activities and cell biomass of the DAP 96523 strain of Rhodococcus rhodochrous after continuous induction with asparagine

Nitrile hydratase activity Amidase activity Biomass (units / mg cps) (units / mg cps) (g / l cpu)

137 6 35

Example 4: Immobilization of Rhodococcus spp cells. in DEAE-cellulose cross-linked with glutaraldehyde

A modified process derived from the methods described in U.S. Patent No. 4,229,536 and in Lopez-Gallego et al. (2005) J. Biotechnol. 119: 70-75 is used to immobilize Rhodococcus spp cells. in a matrix comprising DEAE-cellulose cross-linked by glutaraldehyde. Preparation of cells

Rhodococcus cells are grown in an appropriate culture medium (for example, YEMEA-maltose + inducers, biphasic cultures, etc.) and harvested by centrifugation at 8,000 streams per minute for 10 minutes. The resulting cell pellet is resuspended in 100 ml of the 50 mm phosphate buffer solution (pH 7.2) and centrifuged at 8,000 revolutions per minute for 10 minutes. This process of resuspending the cell sphere and centrifuging at 8,000 revolutions per minute for 10 minutes is repeated twice. The wet packaged weight (pu) of the final cell sample is observed. The activity of a small sample of cells is performed to assess the enzymatic activity of whole cells.

Cell Immobilization

An amount of DEAE-cellulose equivalent to that of Rhodococcus spp. harvested is obtained and the cells and DEAE-cellulose are resuspended in 100 ml of Η₂It is deionized. A volume of a 25% glutaraldehyde solution sufficient to obtain a final concentration of 0.5% is added with stirring to the cell / DEAE-cellulose mixture. The mixture is stirred for 1 hour, after which 400 ml of deionized H2O are added with further mixing. During stirring, 50% (solution by weight) of polyethyleneimine (PEI; MW 750,000) is added. Stirring is continued until flocculation is complete. The flocculated mixture is filtered and expelled through an appropriately sized syringe. The immobilized cells are broken into small pieces, dried overnight, and cut into granules approximately 2-3 mm before use.

Example 5: Immobilization of Rhodococcus spp. in calcium alginate and hardening of calcium alginate beads

A process adapted from the method described in Bucke (1987) Celi Immobilization in Calcium Alginate in Methods in Enzymology, Vol. 135 (B) (Academic Press, Inc., San Diego, California; Mosbach, ed.) Is used to immobilize cells Rhodococcus spp. in calcium alginate. Preparation of cells

Rhodococcus spp. are prepared as described above in Example 4.

Immobilization of g cells of a 4% solution of sodium alginate are produced by dissolving 1 g of sodium alginate in 24 ml of 50 mM Tris-HCI (pH 7.2). 25 mg of sodium metaperiodate is added to the alginate solution and stirred at 25 ° C for 1 hour or until the alginate is completely dissolved. The cells prepared as described above are resuspended to a final volume of 50 ml in 50 mM Tris-HCI (pH 7.2) and then added to the sodium alginate solution with stirring. The resulting beads are extruded through a 27 gauge needle into 500 ml of a CaCI solution₂ to 0.1 Μ. The needle is usually placed approximately 5.08 cm (two inches) above the solution to prevent air from entering the small beads and to prevent beads from sticking. The beads are cured for 1 hour in the CaCI solution₂ and the beads are then rinsed with water and stored at 4 ° C in a CaCI solution₂ to 0.1 M before use.

Hardening of calcium alginate beads comprising cells of Rhodococcus spp.

Calcium alginate beads prepared as described above can be further strengthened by crosslinking with PEI. The beads are incubated in 2 L of 0.5% PEI in a CaCI solution₂ to 0.1 M (20 g of 50% PEI in a CaCI solution₂ of 0.1 Μ). The pH of the final solution is adjusted to 7.0 with HCI or NaOH, if necessary and the beads are incubated for 24 hours. The beads are then rinsed with water and stored at 4 ° C in a CaCI solution₂ to 0.1 M before use.

Many modifications and other modalities of the inventions presented here will come to the mind of one skilled in the art to which these inventions relate, together with the benefit of the teachings presented in the preceding descriptions. Therefore, it should be understood that the inventions are not limited to the specific modalities disclosed and that modifications and other modalities should be included in the scope of the attached claims. Although specific terms are used here, they are used in a generic and descriptive sense only and not for purposes of limitation.