Preventing or delaying chill injury response in plants.

(54) Title: PREVENTION OR DELAY OF RESPONSE TO DAMAGE FROM COOLING IN PLANTS.

(54) Title: PREVENTING OR DELAYING CHILL INJURY RESPONSE IN PLANTS.

(57) Summary

The present invention relates to methods and compositions for preventing or retarding a response to cooling damage or a plant or part of a plant that exhibits a response to cooling damage. The methods comprise exposing the plant or part of the plant to one or more bacteria, one or more enzymes, and / or an isolated enzyme extract from one or more bacteria. The one or more bacteria, one or more enzymes, and / or the isolated enzyme extract from one or more bacteria are exposed to the plant or part of the plant in an amount sufficient to prevent or retard the response to cooling damage to the plant or part of the plant.

(57) Abstract

Provided are methods and compositions for preventing or delaying a chill injury response of a plant or plant part that exhibits a chill injury response. The methods comprise exposing the plant or plant part to one or more bacteria, one or more enzymes, and / or an enzymatic extract isolated from one or more bacteria. The one or more bacteria, one or more enzymes, and / or the enzymatic extract isolated from one or more bacteria are exposed to the plant or plant part in a quantity sufficient to prevent or delay the chill injury response of the plant or plant part.

PATENT TITLE No. 360847

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Inventor (s)

IMPI 'MU

GEORGIA STATE UNIVERSITY RESEARCH FOUNDATION, INC.

Courtland Street, Suite 326, Atlanta, Georgia, 30303, USA

PREVENTION OR DELAY OF RESPONSE TO COOLING DAMAGE TO PLANTS.

AO1N63 / Q2rC ^ N | / (^; ^ 1 | N ^ / 07; Q12NC '

Α01Ν63 / 02> € 1> Νθ / 00 '' I /.

GEORGE E. PIERCE ·· - «.

CIP:

CPC:

42N5 / 10; C12N9 / 00

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,047 ¿5/10/19 ,01/06/201

Number:

MX / a / 2015/012453

Validity:

tional:

Country:

US

Date of V

Date

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ion:

cofffundarn * it # in * 3 *&gt; arti is awarded

In accordance with * 4ΜΙ ^ 23 of the L¿y4é ¿PropiedeJWkisjpeC from the date of prsi ^ tac ^ i of the application Aa ^^ 4a will be if fltMl ^ ymflace coirtoda ^ jnto ^ ¿or díspuieR ^ KrÉs a leeggAjqLtlF.) 27 / 06/49 ^ 1. 'Sefc ^ mq ^ a.

The patent of refere

Who subscribes to the present (Fedi Official Gazette 25/01/2006, 06/06/2009, 06/01/2019 ¾ 1W and 12th sections I and III of Regulation 07/28/2004 and 09/07/2007), Industrial Property articles (DOF 27/12 / 158½ powers in the General Directors Adjun Departmental Coordinators and others s 07/29/2004, 08/04/2004 and 09/13/2007).

1st Propte &lt;i¿ilflkistrial.

sintaA / íais JmSirorrogables, counted to ntesp0 ^ &lt;| Wos.

s 2φ lart ^ y of Industrial Property 05/05 ^ 0999, 01/26/2004, 06/16/2005,, yfcutoi'r ·, 3 'section V subsection a), 4th rowed on 07/01/2002, 07/15/2004, Mgáplco of the Instituto Mexicano de la «Finciso a) of the Agreement delegated by the regional governments. Divisional Deputy Directors, al. Kjp.F. 12/15/1999, amended on 02/04/2000,

This document is signed with advanced electronic signature (FIEL), coi ^ 0Snd0? Q || r®4g (, the aTf ^ 3os7 BIS 2 of the Industrial Property Law; 3rd of its Regulations, and 1 section III, 2 fraction V, 26 BIS and 26 TER of the Agreement that establishes the guidelines for the use of the Electronic Payment and Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated.

THE DIVISIONAL DIRECTOR OF PATENTS

NAHANNY CANAL REYES

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NAHANNY MARISOL CANAL REYES | 00001000000403252793 | Administration Service

Tax | 1695 || MX / 2019/3227 | MX / a / 2015/012453 | PCT patent title | 1223 | GAGV | Page (s) | xQyPkrOyyC1k7DTJyo8ujyCxRJw =

Digital stamp;

cJCZQjC5q¡6sEWPg3RfXDDdbvPWb06DlkX3z / A + GEVF / 9Qht5B0xai / 2Abjt5 + T66mjyHBAbF91TggLuXJyK6hqPHs ibsAzCM4CL8woTfsfY0zxQaDP2lgwA3zvd4O IWArgFFUZCED5vwNmxs6K + / + 2bMTAna R9yHGee2fjnYzKtAQkatZjf UYtRPxEegKOB76t81gf4DYD60wpW3VJZC5zFAs2D / eNSje / OQR / 0kKnlz3V52AiuXP9b9Zb0t3Uo3F4qZlhjsOWwtg 5HzZgh + 3IYsNiyhLllfr4Mml3Vey4y1SzQQOWpQIMYycTY + klSr37yXU4AK3q1hM / == Eu6jV6g

Arenal No 550, Pso 1, Pueblo Sania María Tepepan, Xochintilco, 15020, Mexico City.

(55) 53340700 www gob.mx/impi

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<img file="MX360847B_D0005.tif" />

PREVENTION OR DELAY OF RESPONSE TO DAMAGE BY

<img file="MX360847B_D0006.tif" />

BACKGROUND OF THE INVENTION

Cooling damage in a plant causes a molecular response, which results in the production of signaling compounds in the plant (ethylene, hydrogen cyanide (HCN), and carbon dioxide), which serve as part of a system of cascade into the plant to cause the plant to exhibit a response to exposure to colder temperatures. Examples of plants that exhibit a response to cooling damage include fruits, vegetables, and flowers.

The response to cooling damage can be negative in plants. In fruits and vegetables, the response to cooling damage can result in irreparable damage to the fruit or vegetable. The response to cooling damage on fruits and vegetables can produce undesirable results such as fermented taste, fermented odor, discoloration, water-soaked appearance, wilting, mark formation, browning, softening, roughness (russeting) and decomposition of the fruit or vegetable. The response to cooling damage in a flower can result in a darkening and appearance of having been soaked in water; discoloration of the stem, sepals and petals; or wilting of the flower. Interference with plant signaling systems that cause the response to damage by cooling can allow increased and prolonged exposure to cooler temperatures, which is critical for the transport of fruits, vegetables and flowers, as these are usually refrigerated. during transportation.

SUMMARY OF THE INVENTION

Methods are provided herein to prevent or retard a response to cooling damage from a plant or part of a plant that exhibits a response to cooling damage. The methods comprise exposing the plant or part of the plant to one or more bacteria, one or more enzymes, an isolated enzyme extract from one or more bacteria, or a combination thereof, in an amount sufficient to prevent or delay the response. to damage

<img file="MX360847B_D0007.tif" />

IMPI

... . ,,. . . . .. MEXICAN INSTITUTE for cooling the plant or part of the plant. of the property^rr INDUSTRIAL

The details of one or more aspects are explained later in the accompanying drawings and subsequent description. Other features, objects and advantages will be apparent from the description and drawings and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an image of the control peaches stored at 4 ° C for 3 weeks.

Figure 2 shows an image of the peaches stored at 4-7 ° C for 3 weeks, and subsequently exposed to the catalyst for 7 days, where the catalyst cells were grown in medium with cobalt, urea, and asparagines.

Figure 3 shows an image of the peaches stored at 4-7 ° C for 3 weeks, and subsequently exposed to the catalyst for 7 days, where the catalyst cells were grown in medium with cobalt and urea.

Figure 4A shows an image of the control peaches stored at 4-7 ° C for 3 weeks.

Figure 4B shows an image of the peaches stored at 4-7 ° C for 3 weeks and subsequently exposed to the catalyst, where the catalyst cells were induced with cobalt and urea.

Figure 4C shows an image of the peaches stored at 4-7 ° C for 3 weeks and subsequently exposed to the catalyst, where the catalyst cells were induced with cobalt, urea and asparagines.

Figure 5 shows a non-limiting representation of a three-layer apparatus for preventing or delaying cooling damage. The outer layers provide structural integrity to the apparatus. The catalyst layer, as defined hereinafter, comprises one or more of the disclosed enzymes and is located between the outer layers.

Figures 6A-6C provide non-limiting representations of various apparatuses to prevent or delay cooling damage. These devices comprise a catalyst layer,

IMPI

<img file="MX360847B_D0008.tif" />

MEXICAN PROPERTY INSTITUTE \ one or more layers intended to provide structural integrity, and a WWaBMfiapi to be removed prior to use of the device. The romnHAn ..no_nmThese layers can, for example, expose an adhesive to couple the device to another physical structure.

Figures 7A and 7B show a non-limiting representation of an apparatus for preventing or delaying cooling damage. The apparatus comprises a catalyst immobilized on a film layer and coupled to a physical structure (for example, a box suitable for storing / transporting the fruits).

Figure 8 provides a non-limiting representation of an apparatus for preventing or retarding damage from cooling. 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 made of a material that allows air to flow into the catalyst.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms "a", "one" and "the" include references to the plural unless the context clearly indicates otherwise.

Throughout the description, the term "comprises" and variations thereof are non-limiting, open terms, and are understood to imply the inclusion of an element, integer or declared stage, or group of elements, integers or stages, but not the exclusion of any other element, integer or stage, or group of elements, integers or stages. The term "includes" and variations thereof as used herein means "comprises" and variations thereof.

Methods and compositions are provided herein to prevent or retard a response to cooling damage from a plant or part of a plant that exhibits a response to cooling damage. The methods comprise exposing the plant or part of the plant to one or more bacteria, where the one or more bacteria are exposed to the plant or part of the plant in an amount sufficient to prevent or delay the response to damage by cooling of the plant or part of the plant. In some embodiments, the methods include exposing the plant or part ^ 5

IMPI

<img file="MX360847B_D0009.tif" />

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY from the plant to an enzyme extract isolated from one or more bacteria, where the enzyme extract is exposed to the plant or part of the plant in an amount that will provide or retard the response to damage by cooling of the plant or part of the plant. In some embodiments, the methods comprise exposing the plant or part of the plant to one or more enzymes as described herein, wherein the one or more enzymes are exposed to the plant or part of the plant in an amount sufficient to prevent or delay the response to damage by cooling the plant or part of the plant. Optionally, the methods are carried out in a refrigerated device.

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 fruit, vegetables, flowers, seeds, leaves, nuts, germ , pollen, ovules, branches, grains, ears, ears, husks, stems, roots, root tips, anthers, and the like. In particular modalities, the part of the plant is a fruit, vegetable or flower (including cut flowers). In certain respects, the part of the plant is a fruit, vegetable, or flower.

The methods and compositions are directed at preventing or retarding a response to cooling damage from a plant or part of a plant that exhibits a response to cooling damage. The response to cooling damage is generally associated with the production of signaling compounds in the plant such as ethylene, HCN, and carbon dioxide in the plant and is triggered by exposing a plant to a temperature that is below normal climatic temperature. in which the plant grows, but not so low as to cause the cells of the plant or part of the plant to freeze. In some modalities, the response to cooling damage is generally associated with increased ethylene biosynthesis. As defined herein, “preventing or retarding a response to cooling damage,” and grammatical variants thereof, refers to any deceleration, interruption, suppression, or inhibition of the response to cooling damage of a plant or part of a plant that exhibits the response to cooling damage. For example, preventing or delaying the response to cooling damage in a fruit or vegetable may include preventing or delaying a fermented taste, a fermented odor, a discoloration, an appearance of having been soaked,

ΙΜΡΙ «?&gt; Ζ

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL ^ uírL-S * '' wilt, mark formation, browning, softening, roughness (russeting), and / or zr.n.UÍ. decomposition of the fruit or vegetable. By way of another example, the prevention or delay of a response to cooling damage in a flower may comprise the prevention or delay of darkening and appearance of soaking; discoloration of the stem, sepals and petals; or wilt.

In certain embodiments, methods and compositions are provided to retard a response to cooling damage in a fruit and / or vegetable. A “fruit” or “vegetable” that exhibits a response to cooling damage may include, but is not limited to, apples, apricots, asparagus, avocados, bananas, beans, melon, cucumbers, eggplant, grapefruit, Chinese melon, lemons, lima beans, limes, mangoes, nectarines, okra, oranges, papayas, peaches, peppers, pineapples, potatoes, pumpkins, soybeans, spinach, summer squash, sweet potatoes, tomatoes, watermelons, winter squash, and zucchini. In some modalities, the fruit is a climacteric fruit. In some modalities, the fruit is a non-climacteric fruit. Without intending to be bound by theory, while non-climacteric plants do not produce ethylene, non-climacteric plants do respond to ethylene. Therefore, when non-climacteric plants are exposed to cold, they can exhibit cooling damage. Non-climacteric plants can be exposed to the one or more enzymes, enzyme extract, or one or more bacteria to slow the response to cooling damage.

In certain embodiments, methods and compositions are provided to retard a response to cooling damage in a flower. A "flower" exhibiting a response to cooling damage may include, but is not limited to, anthurium, basil, worts, orchids, and poinsettia flowers. In some embodiments, the methods and compositions are used to retard a response to cooling damage in an ornamental plant. Examples of ornamental plants include, but are not limited to, Acacia, Achillea, African Boxwood, African Lily, African Violet, Agapanthus, Ageratum, Ageratum houstonim, Allium, Alpina, Alstroemeria, Amaranthus hypochondriacus, Amaryllis, Ammi majus, Anconitum, Anemone , Anigozanthus, Annual delphinium, Anthuríum, Antirrhinum majus, Asparagus, Aster spp., Astilbe, Azalea, Baby's breath, Tile, Banksia, Begonia, Tinker Bell, Bells of Ireland, Large Linen, Craspedias, Burning Star, impif '

<img file="MX360847B_D0010.tif" />

INSTITUTO MEXICANO 'faú Dt LA PROPIEDAD Λ Bleeding Heart, Boronia, Bouvardia, Escoba, Buddleia, Bupleururrt * fífá9l &amp; to dw

Butterfly Orchid, California Pepper, Alcatraz, fl »πιρόη ·· ι» Penanhn de Qflpynein

Bluebells of Canterbury, Carnation, Carthamus, Caspia, Cattleya, Lattice, Argent Lattice,

Centaurea cyanus, Chamelaucium, Chimney Chimes, Chrysanthemum, Chrysanthemum x morífolium, Clarkia, Consolida ambigua, Convallaria, Coral Bells, Cordilina, Coreopsis,

Cornflower, Craspedia, Willow Crespo, Cyclamen, Cymbidíum, Cymbidium Orchid, Narcissus, Daisy, Chrysanthemum Daisies, Day Lily, Delphinium, Dendrobium, Dendrobium Orchid, Dianthus barbatus, Díanthus caryophyllus, Dianthus caryophyllus nana, Baboon Dragons Erica spp, Eustoma grandiflorum, False Bird of Paradise, False Spirea, 10 Godetia, Anthurium, Agerate, Freesia, Freesia x hybrida, Fuji or Spider Chrysanthemums, Cachaña Flower,

Genista spp., Geranium, Gerbera, Gerbera spp., Ginger, Gladiolus, Gladíolus hybrid nanus, Goat's Beard, Godetia, Solidago, Guersney's Lily, Gypsylata, Gypsophila paniculata, Heliconia Hanging, Heather, Heather, Helianthus annuus, Hel , Hippeastrum, Hosta, Hydrangea, Iberis amara, Impatiens, Lily of the Incas, Iris, Iris spp., White Lily, 15 Jade Plant, Jaffet Orchid, Narcissus, Calanchoe, Kangaroo Paw, Centaurea, Larkspur, Lathyrus odoratus, Lavandula , Lavender, Liatrís, Lila, Lilium spp., Lily of the Valley, Lily, Lily of the Field, Lily of the Nile, Limonium, Limonium spp., Lisianthus, Lobster Pincer, Love in the Mist, Mattholia incana, Mimosa, Miniature Carnation , Miniclavel, Miniature Gladiola, Moluccella laevis, Aconite, Mother-in-law's Tongue, Musa, Mirsina, Myrtle, Myrtus, Narcissus, 20 Nephrolepis, Nerina, Azucena Nerina, Nigella, Orchid, Ornamental Onion, Ornithogalum,

Paeonia, Painted Tongue, Peony, Peruvian Lily, Petunia, Phalaenopsis, Philodendron, Phlox, Pincushion Flower, Pitosporo, Pittosporum, Dwarf Carnation, Christmas Eve, Polianthes tuberosa, Poppy Anemone, Poríum, Protea spp., Echinacea , Queen Anne's Lace, Ranunculus, Rattlesnake, Red Ribbon Rose, Rosa spp., Rosa, 25 Rudbequia, Safflower, Salix, Salvia, Sansevieria, Huilmos, Scabiosa, Schínus, Siempreviva, Sedum, i

Shell Flowers, Snake Plant, Dragon's Mouth, Solidago, Solidaster spp., Veronica, Lily

Spider, Spider Chrysanthemums, Multiflora Carnation, Star of Bethlehem, Static, Stenamezón, Wallflower, Summer Azalea, Sunflower, Sweet Pea, Carnation of the Poet, Helécho Espada, Syringa vulgaris,

Anthocercis, Amaranth, Bupleuro, Widow's Flower, Trachelium,

IMPI iiNSTUUIo MaucANci industrial

<img file="MX360847B_D0011.tif" />

Tuberose, Tulip, Tulipa, Veronica, Wattle, Wax Flower, Wild Plantain, Wind Flower, Aconite, Paper Flower, Zantedeschia, Zinna, Zínnia elegans, and Zygocactus.

In certain embodiments, methods and compositions to prevent or retard a response to cooling damage in a plant include exposing the plant or part of the plant to one or more bacteria selected from the group consisting of Rhodococcus spp., Brevibacterium ketoglutamicum, Pseudomonas chloroaphis, Nocardia, Pseudonocardia and combinations thereof. The one or more bacteria can, for example, include Rhodococcus spp. Rhodococcus spp can, for example, include the Rhodococcus rhodochrous DAP strain 96253, the Rhodococcus rhodochrous DAP strain 96622, Rhodococcus erythropolis, or combinations thereof. Exemplary organisms include, but are not limited to, Pseudomonas chloroaphis (ATCC 43051) (Gram negative), Pseudomonas chloroaphis (ATCC 13985) (Gram negative), Rhodococcus erythropolis (ATCC 47072) (Gram positive), and Brevibacterium ketoglutamicum (ATCC 21533 ) (Gram positive). Examples of the Nocardia and Pseudonocardia species have been described in European Patent No. 0790310; Collins and Knowles J. Gen. Microbiol. 129: 711-718 (1983); Harper Blochem. J. 165: 309-319 (1977); Harper Int. J. Biochem. 17: 677-683 (1985); Linton and Knowles J Gen. Microbiol. 132: 1493-1501 (1986); and Yamaki et al., J. Ferm. Bioeng. 83: 474-477 (1997).

Although in some embodiments the one or more bacteria are selected from the group consisting of Rhodococcus spp., Brevibacterium ketoglutamicum, and Pseudomonas chloroaphis, any bacteria that prevents or retards a response to cooling damage when exposed to a plant or part of a plant can be used in the present methods. For example, bacteria belonging to the genus Nocardia [see Japanese Patent Application No. 54-129190], Rhodococcus [see Japanese Patent Application No. 2-470], Rhizobium [see Japanese Patent Application No. 5-236977], Klebsiella [Japanese Patent Application No. 5-30982], Aeromonas [Japanese Patent Application No. 5-30983], Agrobacterium [Japanese Patent Application No. 8-154691], Bacillus [Japanese Patent Application No. 8-187092],

Pseudonocardia [Japanese Patent Application No. 8-56684], Burkholderia, Corynebacterium, and

Τ Μ ΡΙ

Pseudomonas are non-limiting examples of bacteria that piS ^ 3l§bm® ^^ acl ^^^ roas species within a given genus exhibit the same type of enzyme activity v / o production. Therefore, it is possible to have a genus generally known to include strains capable of exhibiting a desired activity but to have one or more strains that do not naturally exhibit the desired activity or one or more strains that do not exhibit activity when grown in the same medium as the species that exhibit this activity. Thus, the host microorganisms may include strains of bacteria that are not specifically known to have the desired activity but are of a genus known to have specific strains capable of producing the desired activity. Such strains may have transferred to them one or more useful genes to elicit the desired activity. Non-limiting examples of such strains include Rhodococcus equi and Rhododoccus globerulus PWD1.

In addition, specific examples of bacteria include, but are not limited to, Nocardia sp., Rhodococcus sp., Rhodococcus rhodochrous, Klebsiella sp., Aeromonas sp., Citrobacter freundii, Agrobacterium rhizogenes, Agrobacterium tumefaciens, Xanthobacter flavas, Erwinia nigrbacter ., Streptomyces sp., Rhizobium sp., Rhizobium loti, Rhizobium legminosarum, Rhizobium merioti, Pantoea agglomerans, Klebsiella pneumoniae subsp.

pneumoniae, Agrobacterium radiobacter, Bacillus smithii, Pseudonocardia thermophila, Pseudomonas chloroaphis, Rhodococcus erythropolis, Brevibacteríum ketoglutamicum, and Pseudonocardia thermophila. Optionally, the microorganisms used may, for example, comprise Rhodococcus rhodochrous DAP 96253 and Rhodococcus rhodochrous DAP 96622, and combinations thereof.

As used herein, exposing the plant or part of the plant to one or more bacteria includes, for example, exposing it to intact bacterial cells, bacterial cell lysates, bacterial extracts that possess enzymatic activity (ie, "extracts enzymatic), or any combination thereof. Methods for preparing lysates and enzyme extracts from cells, including bacterial cells, are routine in the art. Optionally, the one or more bacteria or enzyme extracts are fixed with glutaraldehyde and are crosslinked. Optionally, the bacterium or the crosslinked extract, fixed to glutaraldehyde,

<img file="MX360847B_D0012.tif" />

It is formulated with a vehicle in an aerosol.

IMPIí

MEXICAN INSTITUTE of the ηοριεοΑΓ INDUSTRIAL

In certain embodiments, the methods and compositions to prevent or retard a response to cooling damage in a plant or in &lt;

exposing the plant or part of the plant to an enzyme. The enzyme can be selected from the group consisting of nitrile hydratase, amidase, asparaginase, ACC (1-amynocloclopan-1-carboxylic acid) deaminase, enzyme similar to cyanoalanine synthase, alkane monooxygenase, ammonium monooxygenase, methane monooxygenase , toluene dioxygenase, cyanidase, and / or a combination thereof. The enzyme can be provided within a composition to be exposed to the plant or part of the plant. The enzyme can also be a purified enzyme or can be provided as an enzyme extract as described above. Optionally, methods of preventing or retarding a response to cooling damage in a plant or part of a plant comprise exposing the plant or part of a plant to a composition comprising an enzyme, the enzyme being selected from one or more of the nitrile hydratase, amidase, asparaginase, ACC deaminase, cyanoalanine synthase-like enzyme, alkane monooxygenase, ammonium monooxygenase, methane monooxygenase, toluene dioxygenase, and cyanidase. The one or more bacteria, enzyme extract, or enzymes used in the methods at times may be more generally referred to herein as the catalyst.

In the methods provided herein, the plant or part of the plant is exposed to one or more bacteria, one or more enzymes, enzyme extract isolated from or derived from the one or more bacteria, or any combination thereof, in a Enough to slow response to cooling damage. In some embodiments, the plant or part of the plant is exposed to one or more bacteria in combination with one or more exogenous enzymes and / or enzyme extracts. Exogenous refers to enzymes or enzyme extracts that are isolated and / or purified ex situ and is distinguished from enzymes produced by bacteria in situ. This combined exposure can take place simultaneously and / or sequentially. For example, the plant can be exposed to exogenous enzymes and / or enzyme extracts from 1 to 60 minutes, from 1 to 24 hours, or from 1 to 7 days after exposure to bacteria.

The "exposure of a plant or part of a plant to one or more bacteria, one or more

IMPIOS. ,. . . ,. , Mexican iNsirruro enzymes, and / or an enzymatic extract includes any method dg Bi [gg ^ ñtar »enzyme, and / or extract before the plant or part of the plant. Optionally, the plant or part of the plant is indirectly exposed to the one or more bacteria, one or more enzymes, and / or the enzyme extract. Indirect methods of exposure include, for example, placing the one or more bacteria, one or more enzymes, and / or enzyme extract in general proximity to the plant or part of the plant (ie, indirect exposure). Optionally, the plant or part of the plant is directly exposed to one or more bacteria, one or more enzymes, and / or the enzyme extract, whereby the one or more bacteria, one or more enzymes, and / or enzyme extract are in direct contact with the plant or part of the plant.

In certain embodiments, exposure of the bacterium, enzyme, and / or the enzyme extract isolated from the bacterium can occur, for example, by providing the bacteria, enzyme, and / or enzyme extract in liquid form and spraying it on or near the plant or part of the plant. The bacteria, enzyme, and / or enzyme extract may, for example, further comprise a liquid vehicle. Liquid carriers can be selected from the group consisting of an aromatic hydrocarbon, a substituted naphthalene, an italic acid ester, an aliphatic hydrocarbon, an alcohol, and a glycol. Optionally, the liquid vehicle may be a wax or coating of a similar type material, which could be applied to the plant as a liquid, but which would be solid at ambient or lower temperatures.

In certain embodiments, exposure of the one or more bacteria, one or more enzymes, and / or the enzyme extract isolated from the bacteria can occur, for example, by providing the bacteria, enzyme, and / enzyme extract in solid form and dusting it on or near the plant or part of the plant. The bacteria, enzyme, and / or enzyme extract may, for example, further comprise a solid vehicle. The solid vehicle can be selected from the group consisting of a powder, a wettable powder, a water dispersible granule, and mineral diluents. Optionally, the solid vehicle is a mineral diluent. Mineral diluents can, for example, be selected from the group consisting of a calcite, a silica, a talc, a kaolin, a montmorillonite, and an attapulgite. Other solid supports for use with the bacteria, enzyme, and / or enzyme extract are described herein.

<img file="MX360847B_D0013.tif" />

IMPI '' WHTUTOMEXICano, THE PROPERTY

In certain embodiments, the one or more bacteria, an enzymatic phrasase, further comprise a coating ^doT? Lféotp ·· ^do arid ar ^ⁿ hydrophobic, where the hydrophobic fatty acid polyester coating makes the bacteria or enzyme extract water resistant. Optionally, the hydrophobic fatty acid polyester coating is a long chain fatty acid polyester derived from sucrose, sorbitol, sorbinose, glycerol, or raffinose.

Compositions for preventing or retarding a response to cooling damage from a plant or part of a plant that exhibits a response to cooling damage are also provided herein. The compositions may, for example, comprise one or more bacteria, one or more enzymes, and / or one or more enzyme extracts capable of retarding a response to cooling damage from a plant or part of a plant that exhibits a response to damage by cooling. The compositions may further comprise solid, liquid, and gelatinous vehicles, as described above, and / or means and components of means for inducing and stabilizing the one or more bacteria, one or more enzymes, and / or enzyme extracts, as described later.

The methods and compositions provided to prevent or delay a response to cooling damage in a plant or part of a plant can be combined with other known agents to delay the response to cooling damage. Thus, for example, the methods provided may further comprise exposing a plant or part of a plant to an agent that retards or prevents a response to cooling damage. Such agents include, for example, synthetic phytohormone analogs. Likewise, the provided compositions may further comprise an agent that retards or prevents a response to cooling damage, such as a synthetic analog of a phytohormone.

As defined herein, a sufficient amount or effective amount of the bacterium, enzyme, and / or enzyme extract will depend on a variety of factors, including, but not limited to, the particular bacterium, enzyme, and / or extract. particular enzyme used in the method, the way in which the bacterium is exposed to the plant or part of the plant (for example, as intact bacterial cells (live or dead), used cell phones, enzyme extracts, or enzymes

<img file="MX360847B_D0014.tif" />

tract

IMPI ^ as described above), the means by which WH ^ j ^ i ^^ NLe

INDUSTRIAL enzymatic is exposed to the plant or part of the plant, the time period of exposure, and the type and amount of plant signaling compounds that produce the response to cooling damage. Optionally, the amount of bacteria exposed to the plant or part of the plant is in the range of 1 to 250 mg dry cell weight (per pound of plant [ie fruit, etc.]) or the equivalent thereof for enzyme extracts and enzymes. For 1 mg dry cell weight, there are typically 150-300 units of nitrile hydratase, 10-25 units of amidase, 7 units of cyanidase, 7-20 units of ACC deaminase, and 7-20 units of the enzyme similar to that of cyanoalanine synthase. By way of other examples, the amount of bacteria exposed to the plant or part of the plant is in the range of 0.1 to 400 mg, 1 to 200 mg, 1 to 80 mg, or 1 to 10 mg of dry weight of cells or the equivalent of the same for enzymatic extracts and enzymes. Optionally, the amount of bacteria exposed to the plant or part of the plant is 1-3 mg dry weight of cells per kilogram of plant or part of the plant or the equivalent thereof for enzyme extracts and enzymes. By way of other examples, the amount of bacteria exposed to the plant or part of the plant is 10 pg-100 mg, 100 pg-50 mg, 100 pg-25 mg, or 1-10 mg dry cell weight per kilogram of plant or part of the plant or the equivalent thereof for enzyme extracts and enzymes. It would be a matter of routine experimentation for the person skilled in the art to determine enough "of the one or more bacteria, one or more enzymes, or enzyme extract necessary to retard a response to cooling damage in a plant or part of a plant exhibiting a response to cooling damage.

In certain embodiments, the one or more bacteria are induced to exhibit a desired characteristic (for example, the ability to retard a response to cooling damage in a plant or part of a plant that exhibits a response to cooling damage, the expression of a desired level of activity of a bacterial enzyme, and / or the ability to reduce the level of ethylene and / or hydrogen cyanide produced by the plant) by exposure or treatment with a suitable inducing agent. Inducing agents include, but are not limited to urea, methylcarbamate, cobalt, asparagine, glutamine, and combinations thereof. Optionally, the one or more bacteria are exposed to, or treated with, urea or methylcarbamate. Optionally, the one or more bacteria are

<img file="MX360847B_D0015.tif" />

<img file="MX360847B_D0016.tif" />

IMPI

ICANO

H.H

<img file="MX360847B_D0017.tif" />

, a mixture of inducing agents comprising urea or methylcarbamate and one or more of asparagine and cobalt. 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 cultured in a medium for a predetermined amount of time to cultivate the bacteria, and the inducing agent could be added at one or more predetermined times to induce the desired enzyme activity in the bacteria. Furthermore, the inducing agent could be added to the culture medium (or to a different mixture that includes previously cultured bacteria) to induce the desired activity in the bacteria after the growth of the bacteria is complete or during a second phase of growth or maintenance.

Although not intended to be limited to a particular mechanism, "inducing bacteria can result in the production or activation (or increased production or increased activity) of one or more enzymes, such as nitrile hydrate, amidase, asparaginase, ACC deaminase, cyanoalanine synthase-like enzyme, alkane monooxygenase, ammonium monoxygenase, methane monooxygenase, toluene dioxygenase, and / or cyanidase, and induction of one or more of these enzymes may play a role in delaying a response to cooling damage in a plant or part of a plant. Nitrile hydratases, amidases, "asparaginases, ACC deaminases, enzymes similar to cyanoalanine synthase, enzymes of the AMO (alkane or ammonium) monooxygenase type, methane monooxygenase, toluene dioxygenase, and cyanidase 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, and each class of enzyme has recognized enzymatic activities.

Methods of inducing enzymatic activity can be accomplished without the requirement to introduce dangerous nitriles, such as acrylonitrile, into the environment. Previously, it was believed that the induction of specific enzymatic activity in certain microorganisms required the addition of chemical inducers. For example, upon induction of nitrile activity it hydrates in

<img file="MX360847B_D0018.tif" />

Mexican IMPI

FROM THE PROPERTY Rhodococcus rhodochrous and Pseudomonas chloroaphis, it was believed de'WSHéfra necessary to supplement with dangerous chemicals, such as the acetonitrile, acrylonitrile, acrylamide, and the like. However, the enzymatic activity in the microorganisms that produce nitrile hydrate can be induced with the use of non-hazardous medium additives, such as amide-containing amino acids and derivatives thereof, and, optionally stabilized with trehalose. Optionally, asparagine, glutamine, or combinations thereof can be used as inducers. Methods of induction and stabilization of enzyme activity in microorganisms are described in US Patent No. 7,531,343 and in US Patent No. 7,531,344, which are incorporated herein by reference.

The disclosed methods of inducing enzyme activity provide for the production and stability of a number of enzymes using modified media, immobilization, and stabilization techniques, as described herein. For example, enzyme activity can be induced and stabilized through the use of media comprising amide-containing amino acids, or derivatives thereof, and, optionally, stabilized with trehalose. In some embodiments, the induction and stabilization methods comprise culturing a nitrile-producing microorganism hydrate in a medium comprising one or more amide-containing amino acids or derivatives thereof, and optionally trehalose. Optionally, methods of inducing nitrile hydrate are disclosed using a medium supplemented with amide-containing amino acids or derivatives thereof, which preferably include asparagine, glutamine, or a combination thereof. Optionally, methods for inducing nitrile hydratase using a nutritionally complete medium supplemented solely with asparagine are disclosed. Optionally, methods for inducing nitrile hydratase using a nutritionally complete medium supplemented with glutamine only are disclosed. Optionally, methods of stabilizing nitrile hydratase using a nutritionally complete medium supplemented with trehalose only are disclosed. More particularly, the induction and stabilization methods comprise cultivating the microorganism in the medium and optionally harvesting the cultured microorganisms or enzymes produced by the microorganisms.

Enzyme induction and stabilization can

IMPI

INDUSTRIAL

<img file="MX360847B_D0019.tif" />

illustrious.

However, while induction methods eliminate the need for hazardous chemicals for induction of enzyme activity, the use of such additional inducers is not excluded. For example, one or more nitriles could be used to aid in the development of specific activity. Media supplemented with succinonitrile and cobalt may be useful for enzyme induction, including, for example, nitrile hydratase, amidase, asparaginase I, ACC deaminase, cyanoalanine synthase-like enzyme, alkane monooxygenase, ammonium monoxygenase, methane monooxygenase, toluene dioxinase , and cyanidase. However, the use of nitriles is not necessary for the induction of enzyme activity. While the use of nitriles and other dangerous chemicals is definitely not preferred, such use is optionally possible.

Stabilization of enzyme activity can be accomplished through immobilization methods, such as fixation, entrapment, and crosslinking, thereby, extending the time during which enzyme activity can be used. Accordingly, in some embodiments, induction methods and methods for delaying a response to cooling damage also at least partially comprise immobilization of the microorganism. Stabilization can be provided by immobilization of the enzymes, enzyme extracts, and / or microorganisms that produce the enzymes or enzyme extracts. For example, enzymes or enzyme extracts extracted from the microorganisms or the induced microorganisms themselves can be immobilized on a substrate as a means of stabilizing the induced activity. Optionally, the microorganisms that produce nitrile hydrate are at least partially immobilized. Optionally, enzymes or microorganisms are at least partially trapped in or located on the surface of a substrate. This allows the presentation of an immobilized material with induced activity (for example, a catalyst) in such a way as to facilitate the reaction of the catalyst with a target material and the recovery of a desired product while simultaneously retaining the catalyst in the reaction medium and in a reactive mode.

Any substrate generally useful for the fixation of enzymes, enzyme extracts,

<img file="MX360847B_D0020.tif" />

ιΜΡΙ

PROPERTY and / or microorganisms can be used. Optionally, the substratofióftT | Wénd of the same. Alginate is a linear copolymer with hingii ^ h? Mopílirngri ™^and β-D-manurqnatn (M) linked in (I-4) and their C-5 OL-guluronate (G) epimer residues, respectively, covalently linked to each other 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 organized blocks. Optionally, calcium alginate is used as the substrate. Calcium alginate can, for example, be crosslinked, such as with polyethyleneimine, to form a hardened calcium alginate substrate. A further description of such immobilization techniques can be found in Bucke, "Cell Cell Immobilization in Calcium Alginate, Methods in Enzymology, vol. 135, Part B (ed. K. Mosbach) pp. 175-189 (1987), which is incorporated herein by reference. The stabilizing effect of immobilization using polyethyleneimine crosslinked calcium alginate is addressed in US Patent Application Serial Number 11 / 695,377, filed April 2, 2007, which is incorporated herein by reference in its entirety.

Optionally, the substrate comprises an amide-containing polymer. Any polymer comprising one or more amide groups can be used. Optionally, the substrate comprises a polyacrylamide polymer.

Stabilization can be achieved by crosslinking. For example, the induced microorganisms can be chemically crosslinked to form clumps of cells. Optionally, the induced microorganisms are crosslinked using glutaraldehyde. For example, the microorganisms can be suspended in a mixture of deionized water and glutaraldehyde followed by the addition of polyethyleneimine until maximum flocculation is achieved. Crosslinked microorganisms (usually in the form of particles made up of several cells) can be collected by simple filtration. A further description of such techniques is provided in Lopez-Gallego, et al, J. Biotechnol. 119: 70-75 (2005), which is incorporated herein by reference. In certain embodiments, crosslinking kills or inactivates the microorganism. Accordingly, optionally, the induced microorganisms used in the

IMPI present methods are dead (annihilated) or inactivated, sei ^^ t ^ ede to exhibit catalytic activity. _______________________________

Optionally, the microorganisms, enzymes, and / or enzyme extracts can be encapsulated rather than allowed to remain in classical Brownian motion. Said encapsulation facilitates the collection, retention, and reuse of the microorganisms and usually comprises the fixation of the microorganisms to a substrate. Such binding may also facilitate stabilization of the microorganisms, enzymes, and / or enzyme extracts as described above, or may only be to aid in the easy handling of the induced microorganisms, enzymes, or enzyme extracts.

The microorganisms, enzymes, and / or enzyme extracts can be immobilized by any generally recognized method for immobilization of microorganisms, enzymes, and / or enzyme extracts such as sorption, electrostatic bond, covalent bond, and the like. Generally, microorganisms, enzymes, and / or enzyme extracts are immobilized or trapped on a solid support that aids in the recovery of microorganisms, enzymes, or enzyme extracts from a mixture or solution, such as a detoxification reaction. Suitable solid supports include, but are not limited to, granular activated carbon, compost, wood, or wood products, (e.g., paper, wood chips, wood chips, crushed pallets, or trees), bran (e.g., bran from wheat), metal or metal oxide particles (eg alumina, ruthenium, iron oxide), ion exchange resins, DEAE-cellulose, DEAE-SEPHADEX® polymer, waxy / coating materials (such as those used as a coating for fruits and vegetables and optionally including a microbe control agent such as a fungicide or insecticide), ceramic beads, crosslinked polyacrylamide beads, buckets, beads, or other gel forms, alginate beads, κ-carrageenan cubes, as well as solid particles that can be recovered from aqueous solutions due to their inherent magnetic capacity. The shape of the catalyst is variable (in that the desired dynamic properties of the particular entity are integrated with volume / surface area relationships that influence catalytic activity). Optionally, the induced microorganism is immobilized on alginate beads that have been crosslinked with polyethyleneimine or are

<img file="MX360847B_D0021.tif" />

immobilized on a polyacrylamide type polymer.

In some embodiments, the compositions and the mediation and stabilization further comprise one or more amino acids than otinteneii ainide-e-derivatives thereof. Amide-containing amino acids can, for example, be selected from the group consisting of asparagine, glutamine, derivatives thereof, or combinations thereof. For example, amide-containing amino acids can include natural forms of asparagines, anhydrous asparagine, asparagine monohydrate, natural forms of glutamine, glutamine anhydrous, and / or glutamine monohydrate, each in the form of the L-isomer or the D-isomer.

The concentration of the amide containing amino acids or derivatives thereof in the medium can vary depending on the desired end result of the culture. For example, a culture can be carried out for the purpose of producing microorganisms that have a specific enzymatic activity. Optionally, a culture can be carried out for the purpose of forming and harvesting a specific enzyme from the cultured microorganisms. Optionally, a culture can be carried out for the purpose of forming and harvesting a plurality of enzymes having the same or different activities and functions.

The amount of amide containing amino acids, or derivatives thereof, added to the culture medium or mixture can generally be up to 10,000 parts per million (ppm) (i.e. 1% by weight) based on the total weight of the medium or mix. Induction methods are particularly beneficial, however, in that enzyme activity can be induced by adding even smaller amounts. Optionally, the one or more amide-containing amino acids are present at a concentration of at least 50 ppm. By way of other examples, the concentration of amide containing amino acids or derivatives thereof is in the range of 50 ppm to 5,000 ppm, 100 ppm to 3,000 ppm, 200 ppm to 2,000 ppm, 250 ppm to 1500 ppm, 500 ppm. at 1250 ppm, or 500 ppm at 1000 ppm.

In some modalities, induction methods include the use of trehalose. The concentration of trehalose in the compositions or medium used in the induction methods can be less than 1 gram per liter (g / L). Optionally, the trehalose concentration is in the range of I g / L to 50 g / L, or 1 g / L to 10 g / L. Optionally, the concentration of trehalose in the medium is at least 4 g / L.

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX360847B_D0022.tif" />

Amino acids containing amide or derivatives thereof and / or trehalose are added to a nutritionally complete medium. A suitable nutritionally complete medium is generally a culture medium that can provide a microorganism with the necessary nutrients required for its growth, which at a minimum includes a carbon and / or nitrogen source. A specific example is the commercially available R2A agar medium, which typically consists of agar, yeast extract, protease peptone, casein hydrolyzate, glucose, soluble starch, sodium pyruvate, dipotassium hydrogen phosphate, and magnesium sulfate. Another example of a nutritionally complete liquid medium is Malt Extract and Yeast Extract Agar (YEMEA), which consists of glucose, malt extract, and yeast extract (but specifically excludes agar). Additionally, media of similar composition, but of plant origin can be used for the disclosed methods. Any nutritionally complete media known in the art could be used for the disclosed methods, the above media being only described for exemplary purposes. Such nutritionally complete media may be included in the compositions described herein.

Optionally, the disclosed compositions and media may further contain additives. Commonly, the other supplements or nutrients are those useful to aid in increased cell growth, increased cell mass, or accelerated growth. For example, the compositions and media may comprise a carbohydrate source in addition to any carbohydrate source already present in the nutritionally complete medium.

As described above, most media typically contain some carbohydrate content (eg, glucose); however, it may be useful to include an additional source of carbohydrate (eg, maltose or less refined sugars, such as dextrose equivalents that could be dextrose polymers, or any carbohydrate that supports cell growth and induction of desired activity. ). The type of carbohydrate provided in excess may depend on the desired culture result. For example, the addition of carbohydrates, such as maltose or maltodextrin, has been found to provide

IMPI enhanced induction of asparaginase I. Additionally, INDUSTRIAL

<img file="MX360847B_D0023.tif" />

as maltose or maltodextrin, it potentially improves the stability of enzyme activity (eg, nitrile hydratase activity).

In some embodiments, the compositions and media further comprise cobalt. Cobalt or a salt thereof can be added to the mixture or to the media. For example, adding cobalt (eg, cobalt chloride) to the media can be particularly useful in increasing the mass of the enzyme produced by cultured microorganisms. Cobalt or a salt thereof can, for example, be added to the culture medium so that the concentration of cobalt is in an amount of up to 400 ppm. Cobalt can, for example, be present at a concentration of 5 ppm to 400 ppm, 10 ppm to 100 ppm, 10 ppm to 80 ppm, or 10 ppm to 25 ppm.

In some embodiments, the compositions and media further comprise urea. Urea or a salt thereof can be added to the mix or media. Urea or a salt thereof can, for example, be added to the culture medium so that the urea concentration is in an amount of up to 10 g / L. Urea can, for example, be present in a concentration of 5 g / L to 30 g / L, 5 g / L to 20 g / L, 5 g / L to 12 g / L, or 7 g / L to 10 g / L. Optionally, urea is present at a concentration of 7.5 g / L. Optionally, both urea and cobalt are added to the media.

The compositions and media may also include additional components. For example, other suitable components of the medium may include commercial additives, such as cottonseed protein, maltose, maltodextrin, and other commercial carbohydrates. Optionally the medium further comprises maltose or maltodextrin. Maltose or maltodextrin, for example, can be added to the culture medium so that the concentration of maltose or maltodextrin is at least 1 g / L. Optionally, the compositions and media are free of any nitrile-containing compounds. Nitrile compounds were previously required in the culture medium to induce enzymatic activity towards two or more nitrile compounds. The compositions described herein accomplish this through the use of fully safe trehalose and / or amide containing amino acids or derivatives thereof; for the

<img file="MX360847B_D0024.tif" />

IMPI, therefore, the medium can be free of any compound that contains ^ J ^^ jCANo 'NDUSTRÍa?

Enzymatic activity, as used herein, generally refers to the ability of an enzyme to act as a catalyst in a process, such as the conversion of one compound to another compound. Similarly, the desired activity mentioned herein can include the activity of one or more enzymes that are actively expressed by one or more microorganisms. In particular, nitrile hydrate 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. ACC deaminase catalyzes the hydrolysis of 1-aminocyclopropan-l-carboxylate to ammonia and Q-ketobutyrate. Cyanoalanine synthase catalyzes the formation of the non-protein amino acid cyanoalanine from cysteine ​​and cyanide. Cyanidase catalyzes the hydrolysis of cyanide to ammonia and format. Alkane or ammonium monooxygenase (AMO) and methane monooxygenase catalyzes the hydrolysis of ethylene to ethylene epoxide. Toluene dioxygenase can, for example, oxidize ethylene, and is known as an enzyme similar to AMO. The degradation activity of ethylene results in the degradation of the produced ethylene. By degrading ethylene and / or HCN, the plant cannot respond to ethylene in a normal way (i.e., the cascade effects of ethylene signaling are decreased and the plant's ability to respond to an ethylene signal is disrupted) . In the case of cooling damage, with no intention of being limited by theory, the plant will respond to cooling damage with an ethylene explosion, mimicking accelerated maturation / decomposition. By degrading ethylene and / or HCN, the plant does not respond to cooling damage or transient temperature, and normal maturation (i.e., maturation that is not accelerated) can be experienced when returning to the plant at room temperature.

Activity can be referred to in terms of units per mass of enzyme or cells (usually based on the dry weight of cells, eg, units / mg psc). A "unit generally refers to the ability to convert a specific amount of a compound into a different compound under a defined set of conditions as a function of time. Optionally, a unit of nitrile activity hydrates refers to the ability to

IMPI convert 1 pmol of acrylonitrile to its corresponding amide by

INÍT1Tl (TO MEXICANO

<img file="MX360847B_D0025.tif" />

Cells (dry weight) at a pH of 7.0 and at a temperature of 30 ° C. Similarly, 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 at a temperature of 30 ° C. Furthermore, one unit of asparaginase I activity refers to the ability to convert 1 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. Also, one unit of ACC deaminase activity refers to the ability to convert 1 pmol of 1-aminocyclopropan-carboxylate to ammonia and □ -ketobutyrate per minute, per milligram of cells (dry weight) at a pH of 7.0 and a temperature of 30 ° C. Furthermore, one unit of cyanoalanine synthase activity refers to the ability to convert 1 pmol of cysteine ​​and cyanide to cyanoalanine per minute, per milligram of cells (dry weight) at a pH of 7.0 and a temperature of 30 ° C. Furthermore, one unit of cyanidase activity refers to the ability to convert 1 pmol of cyanide to ammonia and format per minute, per milligram of cells (dry weight) at a pH of 7.0 and a temperature of 30 ° C. In addition, an alkane or ammonium monooxygenase (AMO) or methane monooxygenase activity unit refers to the ability to convert 1 pmol of ethylene to ethylene epoxide. Furthermore, a toluene dioxygenase unit refers to the ability to convert 1 pmol of ethylene to ethylene epoxide. Assays to measure nitrile hydratase activity, amidase activity, asparaginase activity, ACC deaminase activity, activity of the enzyme similar to cyanoalanine synthase, activity of alkane or ammonium monooxygenase (AMO) activity, of methane monooxygenase, toluene dioxygenase activity (similar to AMO), and cyanidase activity are known in the art and include, for example, detection of free ammonia. See, eg, Fawcett and Scott, J. Clin. Pathol. 13: 156-9 (1960).

Generally, any bacterial, fungal, plant, or animal cell capable of producing or being induced to produce nitrile hydratase, amidase, asparaginase, ACC deaminase activity, enzyme activity similar to cyanoalanine synthase, activity of alkane or ammonium monoxygenase (AMO) , methane monooxygenase activity, toluene dioxygenase activity, and cyanidase activity, or any combination thereof, can be used herein. A nitrile

IMPI hydratase, amidase, asparaginase, ACC deaminase, enzyme

<img file="MX360847B_D0026.tif" />

INDUSTRIAL

I alkane or ammonium monooxygenase, methane monoxygenase, toluene dioxygenase, and / or cyanidase can be constitutively produced in a cell from a particular organism (eg, a bacterium, a fungus, a plant cell, or an animal cell) or, alternatively , a cell can produce the desired enzyme or enzymes only after induction with a suitable inducing agent. "Constitutionally" is intended to mean that at least one enzyme disclosed herein is continuously produced or expressed in a particular cell type. Other cell types, however, may need to be induced, as described above, to express nitrite hydratase, amidase, asparaginase, ACC deaminase, enzyme cyanoalanine synthase, alkane or ammonium monooxygenase, methane monooxygenase, toluene dioxygenase, and cyanidase. in a quantity or level of enzymatic activity sufficient to retard a development process of a plant of interest. That is, an enzyme described herein can be produced only (or produced at sufficient levels) after exposure to or treatment with a suitable inducing agent. Such inducing agents are known and described above. For example, the one or more bacteria are treated with an inducing agent, such as asparagine, glutamine, cobalt, urea, trehalose, or any mixture thereof, more particularly a mixture of asparagine, cobalt, and urea. Furthermore, as disclosed in US Patent Nos. 7,531,343 and 7,531,344, which are incorporated herein by reference in their entirety, entitled Induction and Stabilization of Enzyme Activity in Microorganisms, "the activity of asparaginase I can be induced in Rhodococcus rhodochrous DAP 96622 (Gram positive) or Rhodococcus rhodochrous DAP 96253 (Gram positive), in a medium supplemented with amino acids containing amide or derivatives thereof. Other Rhodococcus strains can also be preferentially induced to exhibit enzymatic activity of asparaginase I using amide-containing amino acids or derivatives thereof.

P. chloroaphis (ATCC Deposit No. 43051), which produces asparaginase I activity in the presence of asparagine and ACC deaminase, and B. kletoglutamicum (ATCC Deposit No. 21533), a Gram positive bacterium that has also been shown to produce activity of asparaginase, are also used in the disclosed methods. Fungal cells, such as those from gender

IMPI

Fusarium, plant cells, and animal cells, which express an Nfttfü € DlMolrata $ »'JH r OF PROPERTY

INDUSTRIAL

<img file="MX360847B_D0027.tif" />

An asparaginase can also be used herein, either as whole cells or as a source from which one or more of the above enzymes is isolated.

The amino acid and nucleotide sequences for various nitrile hydratases, amidases, and asparaginases from various organisms are disclosed in publicly available sequence databases. A non-limiting listing of representative nitrile hydratases and aliphatic amidases known in the art is presented below in Tables 1 and 2 and in the sequence listing. The protein value mentioned in Tables 1 and 2 provides an overview of the percent confidence intervals (% Confidence Interval) of identifying isolated proteins based on mass spectroscopy data.

Table 1: Amino Acid Sequence Information for Nitrile Hydratases

Representative

Source organism

Do not give

Identifier

Protein value (% of

Access

Sequence

Rhodococcus sp.

Nocardia sp.

Rhodococcus rhodochrous

Uncultivated bacteria (BD2); nitrile beta subunit

806580 SEQ ID NO: 1

27261874 SEQ ID NO: 2

49058

27657379

SEQ ID NO: 3

SEQ ID NO: 4

Confidence interval)

100%

100%

100%

100% hydratase

Rhodococcus sp.

Rhodococcus rhodochrous

Uncultivated bacteria (SP1);

806581

581528

7657369

SEQ ID NO: 5

SEQ ID NO: 6

SEQ ID NO: 7

100%

100%

100% alpha subunit of nitrile hydratase

<img file="MX360847B_D0028.tif" />

<img file="MX360847B_D0029.tif" />

Table 2:

IMPI

Amino Acid Sequence Information

INDUSTRIAL

Representative

<td>Source organism</td><td>Do not give Access</td><td>Identifier Sequence</td><td>Protein Value (% of 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>SEQIDNO: 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>

Optionally, host cells that have been genetically engineered to express a nitrile hydratase, amidase, asparaginase, ACC deaminase, cyanoalanine synthase-like enzyme, alkane monooxygenase, toluene dioxygenase, and / or cyanidase can be exposed to a plant or part of a plant to prevent or delay a response to cooling damage. Specifically, a polynucleotide encoding a nitrile hydratase, amidase, asparaginase, ACC deaminase, cyanoalanine synthase-like enzyme, alkane or ammonium monooxygenase, methane monooxygenase, or toluene dioxygenase (or multiple polynucleotides each) , amidase, asparaginase, ACC deaminase, cyanoalanine synthase-like enzyme, alkane or ammonium monooxygenase, methane monooxygenase, toluene dioxygenase, or cyanidase) can be introduced by standard molecular biology techniques into a host cell to produce a transgenic cell that expresses one or more of the enzymes. The use of the terms polynucleotide, polynucleotide construction, "nucleotide," or nucleotide construction

IMPI

MfrififeianfleAiW

6e the INDUSTRIAL property which comprises polynucleotides or nucleotides. Those of ordinary skill in the art will recognize that polynucleotides and nucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides described herein encompass all sequence forms including, but not limited to, single-chain forms, double-chain forms, and the like.

Variants and fragments of polynucleotides that encode polypeptides that retain the desired enzyme activity (i.e., activity of nitrile hydratase, amidase, asparaginase, ACC deaminase, enzyme cyanoalanine synthase, alkane or ammonium monooxygenase, methane) may also be used herein. monooxygenase, toluene dioxygenase, or cyanidase). By fragment is meant a portion of the polynucleotide and therefore also encodes a portion 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 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 up to the total number of amino acids present in an enzyme amino acid sequence of full length. Variant is intended to mean considerably similar sequences. Generally, variants of a particular enzyme sequence will be at least 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 respect to the sequence of the reference enzyme, as determined by standard sequence alignment programs. The variant polynucleotides described herein will encode polypeptides with the desired enzyme activity. By way of example, the relationship between two polynucleotides or two polypeptides can be described as identity. The identity between two sequences can be determined using the Needleman-Wunsch algorithm (Needleman and

Wunsch, 1970, J. Mo !. Biol. 48: 443-453) as implemented in

IMPI

Mamatn &amp; taredl

OF INDUSTRIAL PROPERTY

<img file="MX360847B_D0030.tif" />

EMBOSS (EMBOSS: The European Open Software Suite for Molecular Biology, Rice et al, 2000,

Trends Genet. 16: 276-7). The Needle result labeled longest identity is used as the identity percent and is calculated as (Identical Residues (i.e. nucleotides or peptides) x 100) / (Alignment Length - Total Number of Spaces in Alignment).

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 encoding a nitrile hydratase, amidase, asparaginase, ACC deaminase, cyanoalanine synthase, alkane or ammonium monooxygenase, methane monoxygenase, toluene dioxygenase, and / or cyanidase-like enzyme. Optionally, the polynucleotide will be presented in such a way that the sequence can access the interior of a host cell, including its potential insertion into the genome of the host cell. The disclosed methods do not depend on a particular protocol for introducing a sequence into a host cell, only that the polynucleotide accesses the interior of at least one host cell. Methods for introducing polynucleotides into host cells are well known, including, but not limited to, stable transfection methods, transient transfection methods, and virus-regulated methods. Stable transfection is intended to mean that the polynucleotide construct introduced into a host cell integrates into the host 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 is not integrated into the host genome.

Additionally, the nucleotide sequence of nitrile hydratase, amidase, asparaginase, ACC deaminase, cyanoalanine synthase-like enzyme, alkane or ammonium monooxygenase, methane monooxygenase, toluene dioxygenase or cyanidase can be contained in, for example, a plasmid for introduction into the host cell. The usual plasmids of interest include vectors that have cloning sites, origins of replication, and defined selectable markers. The plasmid may further include transcription and translation initiation sequences and transcription and translation terminators. Plasmids can also include

IMPI gene expression cassettes containing at least one sequence^Nt¡S ^ * ^^ | {^^ d

INDUSTRIAL sequences that allow the replication of the cassette in eukaryotes, or prokaryotes, or both, (for

<img file="MX360847B_D0031.tif" />

example, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. 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, Gene 8: 81-97 (1979); Roberts et al, Nature

328: 731-734 (1987); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152 (Academic Press, Inc., San Diego, California) (1989); Sambrook et al, Molecular Cloning: A Laboratory Manual, Vols. 1-3 (2d ed; Coid Spring Harbor Laboratory Press, Plainvlew, New York) (1989); and Ausubel et al, Current Protocols in Molecular Biology, Current Protocols (Greene Publishing Associates, Inc., and John Wiley &amp; Sons, Inc., New York; 1994 Supplement) (1994). Transgenic host cells that express one or more of the enzymes as whole cells or as a biological source from which one or more enzymes can be isolated can be used in the methods described.

Apparatus and vehicles are further provided to prevent or retard a response to cooling damage and to carry out the disclosed methods. In particular embodiments, an apparatus or vehicle for preventing or retarding a response to cooling damage is described herein comprising a catalyst comprising one or more bacteria selected from the group consisting of Rhodococcus spp., Pseudomonas chloroaphis, Brevibacterium ketoglutamicum, and mixtures thereof. Rhodococcus rhodochrous DAP strain 96253, Rhodococcus rhodochrous DAP strain 96622, Rhodococcus erythropolis, or mixtures thereof can be used in certain aspects. The one or more bacteria in an apparatus or vehicle are provided in an amount sufficient to prevent or delay a response to cooling damage, as defined herein above. In other aspects, the catalyst comprises one or more enzymes (i.e., nitrile hydratase, amidase, asparaginase, ACO deaminase, cyanoalanine synthase-like enzyme, alkane or ammonium monooxygenase, methane monooxygenase, toluene dioxygenase, and / or cyanidase) in one amount or level of enzyme activity sufficient to prevent or delay a response to cooling damage. The sources of the enzymes desired for use as a catalyst in the aparSff§ ^ 48 $ &amp; $ ^ herein were also described in detail above. For example, the catalyst may be used in the form of whole cells that produce (or that are induced or genetically modified to produce) one or more of the enzymes disclosed herein or may comprise the enzyme (s) (s) in a isolated, purified, or semi-purified form. Optionally, the apparatus or vehicle delivers a sufficient amount or effective amount of the catalyst into a refrigerated device to prevent or delay the response to cooling damage in the plant or part of the plant.

A vehicle for compositions to prevent or retard a response to cooling damage can, for example, be selected from the group consisting of paper, DEAC, cellulose, waxes, glutaraldehyde, and granular activated carbon. By way of an example, the catalyst can be incorporated into / on a paper or plastic label (eg, sticker) that is placed on a fruit and adheres to the fruit to prevent or delay a response to cooling damage. The sticker may include an adhesive layer that has a surface that adheres to the fruit and a paper layer that includes the catalyst and that adheres to the surface opposite the surface that adheres to the fruit. The sticker can be provided in a backing layer. The sticker can be designed to release the catalyst to release the catalyst in a continuous (for example, a constant release) or non-continuous form (for example, a first-time release followed by a second-time release, etc.) . Optionally, the sticker can be designed to change colors upon catalyst release. Optionally, the sticker can be designed to determine fruit or vegetable damage based on the color of the sticker. As an example, the sticker could change color (for example, from yellow to green) when the fruit or vegetable has been damaged, as determined by the ability of the catalyst to determine or evaluate the quality of the fruit or vegetable. The sticker has the advantage that the sticker can be removed before consumption of the fruit or vegetable, which prevents the consumer from ingesting the catalyst and eliminates the need to wash the catalyst. Additionally, the sticker provides a defined catalyst charge on the fruit

<img file="MX360847B_D0032.tif" />

IMPI or vegetable. By way of another example, the catalyst is incorporated into the industrial product can coat the desired product. By way of another example, the catalyst may be incorporated into a post-harvest protection additive (eg, Ull pessiula). In another example, the catalyst may be incorporated into a material (eg, tissue paper, plastic cup, or other wrapper) designed to contain the fruit or vegetable such that the fruit or vegetable is nested within the material. infused with the catalyst.

The apparatuses for preventing or retarding a response to cooling damage disclosed herein can be supplied in various suitable formats and may be suitable for single or multiple uses (eg, rechargeable). Additionally, the devices or vehicles disclosed herein find utility in domestic and commercial settings. For example, such devices or vehicles can be integrated into domestic or commercial refrigerators, included in trains, trucks, etc., to transport the fruit, vegetable or flower over long distances, or be used as independent cabinets for the storage or transport of such vegetable products. Illustrative, non-limiting apparatus are described hereinafter and illustrated in Figures 5-8.

In particular embodiments, the catalyst is provided 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 bacteria (or enzymes) to retard or accelerate the plant development process is preserved. For example, the catalyst can be immobilized on a matrix comprising alginate (eg, calcium alginate), carrageenan, DEAE-cellulose, or polyacrylamide. Other such matrices are well known in the art and may further be crosslinked with any appropriate crosslinking agent, including but not limited to glutaraldehyde and / or polyethyleneimine, to increase the mechanical strength of the catalyst matrix. In one aspect, the catalyst is immobilized on a glutaraldehyde crosslinked DEAE-cellulose matrix. The catalyst, particularly the catalyst in immobilized form, can furthermore 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 catalyst replacement, or allows air flow through the catalyst.

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL MONEY

<img file="MX360847B_D0033.tif" />

In one embodiment, the matrix comprises alginate, or salts thereof. Alginate is a linear copolymer with homopolymeric blocks of β-D-manuronate (M) linked in (I-4) and its epimer residues of C-5 □ -L-guluronate (G), respectively, covalently linked to each other 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 organized blocks. In one embodiment, calcium alginate is used as a substrate, more particularly calcium alginate that has been crosslinked, such as with polyethyleneimine, to form a hardened calcium alginate substrate. A further description of such immobilization techniques can be found in Bucke (1987) "Ce // Immobilization in Calcium Alginate in Methods in Enzymology, Vol. 135 (B) (Academic Press, Inc., San Diego, California; Mosbach, ed .), which is incorporated herein by reference. An illustrative method of immobilization using polyethyleneimine crosslinked calcium alginate is also described in Example 5 below. In another embodiment, the matrix comprises an amide-containing polymer. Any polymer comprising one or more amide groups can be used. In one embodiment, the substrate comprises a polyacrylamide polymer.

Greater mechanical strength of an immobilized catalyst matrix can be achieved by crosslinking. For example, cells can be chemically crosslinked to form clumps of cells. In one embodiment, the collected cells are crosslinked 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. Crosslinked cells (usually in the form of particles made up of several cells) can be collected by simple filtration. A further description of such techniques is provided in Lopez-Gallego et al. (2005) J. Biotechnol. 119: 70-75, which is incorporated herein by reference in its entirety.

In certain aspects, the immobilized catalyst or one or more modular catalyst elements are placed on, placed on, or adhered to a physical structure. The physical structure includes, but is not limited to, a film, foil, liner, box, pouch, bag, or slotted camera capable of accommodating one or more niodtMSII ^^ gKgg [t8diz ^^^^ gS ^ pas modalities , the physical structure includes a suitable container to transport or store the fruit, vegetables, or flowers. The physical structure may further comprise more than one individual structure, whereby all the individual structures are connected to a central catalyst or modular catalyst element. A physical structure previously described herein may optionally be cooled by external means or comprise a refrigeration unit within the physical structure itself.

In an apparatus disclosed herein, elements may optionally be included to monitor the effectiveness of the catalyst to prevent or delay a response to cooling damage (for example, to assess when the catalyst or catalyst module should be replaced) or to measure or control air flow, humidity / moisture content, and carbon dioxide levels. Any apparatus for preventing or retarding a response to cooling damage may further comprise one or more elements to allow air flow to or through the catalyst or modular catalyst element. The skilled person will readily visualize other possible modifications to the apparatus described herein to monitor and control atmospheric conditions (eg, air flow, humidity, and carbon dioxide levels) of the catalyst, the modular catalyst element, or the physical structure. Conditions such as temperature, atmospheric composition (eg, relative humidity, O levels₂ and CO₂, physical stress, light, chemical stress, radiation, water stress, growth regulators, and pathogen attacks play an important role in respiration rates and considerably impact the shelf life of fruits, vegetables, flowers, and other products. related to plants. 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 normally in the range of about 0 ° to about 20 ° C with levels of O₂ and CO₂ within the approximate ranges of 1-10% and 0-20%, respectively. A relative humidity of approximately 50% to approximately 100%, particularly 85% to approximately, is generally recommended for the storage of fruits, vegetables, and related plant products.

<img file="MX360847B_D0034.tif" />

relationship

95%, more particularly from about 90% to approxÍJ ^^^^^ ^w DELArROTIWAC _ _ _

IhlDUSTUAL significant between respiration rate and shelf life of plant products, control of the above factors is important to retard eHet ^ dhyfl ^^ tlIgñW ^ ódifcfós · Therefore, a carbon dioxide scavenger can be provided in the apparatus to reduce the carbon dioxide content.

In particular modalities, it pro air permeable catalyst apparatuses provided to prevent or retard a response to cooling damage comprising multiple layers. For example, as shown in Figure 5, 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, Brevibacteríum ketoglutamicum, and mixtures thereof) or enzymes (a nitrite hydratase, amidase, asparaginase, ACC deaminase, enzyme similar to cyanoalanine synthase, alkane or ammonium monooxygenase, methane monooxygenase, toluene dioxygenase, cyanidase, and mixtures thereof), wherein the one or more bacteria or enzymes are provided in an amount sufficient to prevent or delay a response to cooling damage, 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 normally allow air flow to the catalyst layer 16 although, in some embodiments, it may be advantageous to have an outer layer that is not air permeable, for example if the apparatus It 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 can be provided in reusable or non-reusable sachets or bags. In one embodiment, the catalyst layer (16) comprises Rhodococcus spp. Cells, particularly the Rhodococcus rhodochrous DAP strain 96253, the Rhodococcus rhodochrous DAP strain 96622, Rhodococcus erythropolis, or mixtures thereof. Bacterial cells used as a catalyst in an apparatus disclosed herein can be induced with one or more inducing agents (eg, asparagine, glutamine, cobalt, urea, or a mixture thereof), as described in detail above.

IMPI

MEXICAN INSTITUTE. - ... MEXICAN INSTITUTE

Figures 6A-6C illustrate alternative devices for prevenfri ^ R ^^^ r

<img file="MX360847B_D0035.tif" />

: a al

<img file="MX360847B_D0036.tif" />

cooling damage. These devices comprise multiple layers, where one or more of the layers are removable. As shown in Figure 6A, the apparatus may include an air permeable structural layer (22) and a catalyst layer (24). The removable layers 26 and / or 28 can be arranged along the structural layer 22 and / or the catalyst layer 24 and are normally intended to be removed before using or activating the catalyst. In certain aspects, removal of removable layers 26 and 28 exposes an adhesive that facilitates placement or coupling of the catalyst structure to a separate physical structure. Figure 6B illustrates an alternative embodiment where the apparatus (30) includes two air permeable structural layers (32) and (34), an intermediate catalyst layer (36) and a removable layer (38). Figure 6C illustrates yet another embodiment where apparatus 40 includes two air permeable structural layers 42 and 44, an intermediate catalyst layer 46, and two removable layers 48 and 50.

Figures 7A and 7B illustrate an alternative embodiment (60) where the catalyst is attached to the interior of a container such as a cardboard box. As shown in Figure 7A, one side (62) of the container includes a catalyst layer (64) attached thereto by use of an adhesive layer (66). A peel film 68 may be provided adjacent to the catalyst layer 64 to protect the catalyst layer from exposure to the environment. The peeled film (68) can be removed to activate the catalyst in the catalyst layer (64) by exposing the catalyst to a portion of the plant provided in the container so as to prevent or delay an unwanted response to damage by cooling.

Figure 7B illustrates a catalyst structure (70) before the catalyst structure is attached to the interior of the container as shown in Figure 7B. In addition to the catalyst layer (64), the adhesive layer (66), and the release film (68), the catalyst structure (70) includes an additional release film (72). The peel-off film (72), like the peel-off film (68), protects the catalyst structure (70) when it is packed, transported, or stored. The release film (72) can be removed to expose the adhesive layer (66) to allow the catalyst structure (70) to attach to the interior of the container

TMPI in the form illustrated in Figure 3A. ^{A x} * ^to MEXICAN INSTITUTE

FROM PROnWAD

Figure 8 illustrates a catalyst structure (80) that includes slots (

<img file="MX360847B_D0037.tif" />

to receive a catalyst module (for example module (86)). □^,ri^,FÚÜCl! UTIt3^rtyiSTK'a (Wr (857 '5?) is air permeable and can be easily inserted into or removed from slot 84. Consequently, catalyst module 86 can be easily replaced if a new catalyst module is desired for their use in catalyst structure 80. Catalyst module 86 includes a catalyst as described herein and which is preferably immobilized in a matrix. The catalyst structure (80) can include opposing air-permeable surfaces (88) and (90), such as mesh grids, to allow air flow through the catalyst module (86). The catalyst structure 80 may, in alternative embodiments, include only one air permeable surface, two non-opposing air permeable surfaces, or more than two air permeable surfaces as would be understood by a person skilled in the art. Although Figure 8 includes two slots 82 and 84 for receiving a catalyst module (for example module 86), a person skilled in the art would understand that catalyst structure 80 could include one or more slots to receive a module. The catalyst structure (80) can be arranged within a container used to transport part of a plant, such as fruit or flowers, or can be attached to a container, for example, through the use of an adhesive layer as described at the moment.

The person skilled in the art will further recognize that any of the methods, apparatus, physical structures, compositions, or vehicles disclosed herein may be combined with other known methods, apparatus, physical structures, compositions, and vehicles to retard or accelerate the process. of plant development, particularly those processes generally associated with ethylene biosynthesis (eg, response to cooling damage). Furthermore, as described above, an increased production of ethylene has also been observed during attack on plants or parts of a plant by pathogenic organisms. Accordingly, the methods and apparatus disclosed herein may find further use in improving the plant's response to pathogens.

Materials, compositions, and components are disclosed that can be used for,

<img file="MX360847B_D0038.tif" />

<img file="MX360847B_D0039.tif" />

Pl can be used in combination with, can be used in pr Ot LA MONtOAD

INDUSTRIAL ___ of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, materials are disclosed so long as a specific reference to each of the various individual and collective combinations and permutations of these compounds is not explicitly disclosed. , each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and various modifications that can be made to various molecules including the method are discussed, each and every combination and permutations of the method, and the modifications that are possible are specifically contemplated unless otherwise stated. specifically state otherwise. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Accordingly, if there is a variety of additional steps that can be carried out, it is understood that each of these additional steps can be carried out with any specific method steps or combination of method steps of the disclosed methods, and that each of such combinations or subsets of combinations is specifically contemplated and should be considered as disclosed.

The publications cited herein and the material for which they are cited are specifically incorporated by reference in their entirety.

EXAMPLES

Example 1: Method to retard the response to cooling damage in peaches.

Rhodococcus sp DAP 96253 cultures were started from glycerol standard solutions stored at -80 ° C by transferring 1 milliliter (ml) of the glycerol standard to 250 ml of nutrient broth. The culture was incubated at 30 ° C with shaking at 150 revolutions per minute (rpm) for 2 days. Nutrient agar plates were inoculated and incubated for 2 days at 30 ° C; cells from these plates were scraped and used as an inoculum for YEMEA plates supplemented with glucose and the following additives: cobalt, urea, and asparagine. Plates

IMPI

YEMEA were incubated for one week at 30 ° C. The cells ^ fuete ^^ jS ^ fabrics ^^ T ^ Sfeces of YEMEA and were weighed (5-10 grams (g) of wet packed weight), a sample was taken from the cells and the activities of nitrile hydratase were determined ( NHase), amidase and ACC deaminase.

Rhodococco cells (5 g-10 g wet packed weight) were suspended in 10 ml of 50 millimolar (mM) phosphate buffer and transferred to a Petri dish which was placed in a brown paper bag containing 6 peaches they had been stored at 4 ° C for 3 weeks. The bags were closed and left at room temperature for 7 days. This experiment was repeated three times.

The peach samples (10 g) were taken after exposure and transferred to 10 ml of water in a 50 ml tube. The samples were crushed on ice and spun for 10 minutes at 4,000 rpm. 1 ml samples were taken and transferred to microcentrifuge tubes and centrifuged for 10 minutes at 13,000 rpm. The samples were diluted 1: 100, followed by a 1:10 dilution.

A glucose standard solution (1 mg / ml) was prepared and a lOOug / ml standard solution was prepared from the standard solution and used as a standard.

Anthrone reactions were carried out on the samples, standard, and negative control in glass test tubes adding 5 ml of the antrone reagent (200 milligrams (mg) of antrone dissolved in 100 ml of 75% sulfuric acid) for a sample of 1 mi. The solution was mixed and placed in a water bath at 100 ° C for 3.5 minutes. The tubes were allowed to cool and the absorbance at 625 nm was read. The enzymatic activities for rhodococco cells are provided in Table 3.

Table 3: Enzymatic activities of rhodococco cells used in the experiments.

<td>Media</td><td>NHase (units / mg psc)</td><td>Amidase (units / mg psc)</td><td>ACC deaminase (units / mg psc)</td>

<td>G-</td><td> 2</td><td> 0</td><td> 0</td>

<td>GCo</td><td> 81</td><td> 0</td><td> 0</td>

<td>GU</td><td> 26</td><td> 20</td><td> 5</td>

MEXICAN TUTO OF INDUSTRIAL PROPERTY

<img file="MX360847B_D0040.tif" />

<td>GCoU</td><td> 70</td><td> 4</td>

<td>GCoU</td><td>⁶⁰</td><td> 10</td>

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

G: Glucose; Co: Cobalt; U: Urea; Asn: Asparagine

The data demonstrated that the rhodococco catalyst was effective in delaying fruit ripening after the fruit had been stored in the cold for an extended period (Figures 1-4). The catalyst was also able to prevent cooling damage to the fruit. The properties of the peaches are provided in Table 4.

Table 4: Monitoring of the ripening of control peaches and treated with catalyst

<td></td><td colspan="3">Initial measurements</td><td colspan="4">After 7 days</td>

<td></td><td>Brix after 3 weeks at 4 ° C</td><td>PH</td><td>Degree of hardness</td><td>Brix</td><td>Carbohydrate content (mg / ml)</td><td>pH</td><td>Degree of hardness / Comments</td>

<td>Control</td><td> 15.5</td><td> 4</td><td> ++++</td><td> 18</td><td> 167</td><td> 4</td><td> +</td>

<td>G</td><td> 15.5</td><td> 4</td><td> ++++</td><td> 16</td><td> 146</td><td> 4</td><td>Several peaches exhibited some degree of discoloration and fungal growth</td>

<td>GCo</td><td> 15.5</td><td> 4</td><td> ++++</td><td> 17</td><td> 136</td><td> 4</td><td>+++ Two peaches had a slight wrinkled appearance with a brown stain</td>

<img file="MX360847B_D0041.tif" />

<img file="MX360847B_D0042.tif" />

IMPI

MEXICAN INSTITUTE

M PROPERTY

<td>GU</td><td> 15.5</td><td> 4</td><td> ++++</td><td> 17</td><td> 122</td><td> 4</td><td>---- INPUSTMAt ------ 1 +++ A peach</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>TJTéésntóTiscbwracterr '..... with little fungal growth</td>

<td>GCoU</td><td> 15.5</td><td> 4</td><td> ++++</td><td> 17</td><td> 127</td><td> 4</td><td> +++</td>

<td>GCoU Asn</td><td> 15.5</td><td> 4</td><td> ++++</td><td> 17</td><td> 132</td><td> 4</td><td> +++</td>

G: Glucose; Co: Cobato; U: Urea; Asn: Asparagine

Example 2: Method to retard the response to cooling damage in bananas.

Bananas were placed in the refrigerator at 15 ° C for 4 weeks. After 4 weeks, the bananas were removed from the refrigerator and kept at 25-27 ° C in closed containers with humidity control. A select number of bananas are removed from the container and sprayed with catalyst or wrapped in catalyst impregnated paper. Once exposed to the catalyst, the bananas are placed back in the container and signs of response to cooling damage are observed. The same procedure can be performed with catalyst treatment before and after exposure to cold temperatures.

Example 3: Method to delay a response to cooling damage in soybean plant.

Soy bean plants are grown in pots to a defined size with a defined number of leaves. Soybean plants are divided into two groups. The first group is the control group, and the second group is the catalyst sprayed leaf group. Plants are exposed to temperatures of 4 ° C for 12 hours. After exposure to cold temperatures, plants are returned to room temperature (25-27 ° C). Plants are observed to identify signs of response to cooling damage. The same procedure can be done with catalyst treatment before and after exposure to cold temperatures.

IMPI

<img file="MX360847B_D0043.tif" />