Field

[0001] The present application is directed to enzymes useful for detoxifying mycotoxins. More specifically, the present application is directed to enzymes capable of catalyzing the conversion of trichothecene mycotoxins to products with reduced toxicity.

Background

[0002] Fusarium head blight and ear rot are fungal diseases caused by multiple Fusarium species, including F. graminearum, that affect many crops (such as wheat, barley and corn). These diseases result in considerable economic losses due to reduced yields and the reduction in grain quality due to contamination with mycotoxins such as the trichothecene mycotoxins. The trichothecene mycotoxins are a family of compounds having the following general chemical formula:

[0003] During the course of plant infection, one of the most commonly detected trichothecene mycotoxins, deoxynivalenol (DON), also known as vomitoxin, or by lUPAC name, (3a,7a)-3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one, is secreted by the fungal pathogen and acts as a potent virulence factor, aiding in disease spread.

Deoxynivalenol (DON)

[0004] DON has negative consequences on both human and animal health, causing gastrointestinal issues especially in mono-gastric animals. The acute exposure leads to emesis associated with a total loss of appetite while long-term exposure leads to lack of weight gain, reduced immunity, and increased sensitivity towards diseases. Furthermore, DON is widespread within the cereal industry, with reports showing its presence in 55% of samples tested.

[0005] Current recommendations in the North American economic zone suggest limiting DON in the human food chain to less than 1 ppm DON, while feedlot cattle and poultry should have no more than 10 ppm DON at most (contaminated feed cannot be more than 50% of animals' diet). Due to increased sensitivity, swine cannot be exposed to more than 5 ppm (contaminated feed cannot be more than 20% of their diet). During seasons where the fungal infections are particularly prevalent, levels of DON in grains can exceed 20 ppm, putting pressure on animal feed producers to meet these recommendation levels. Contaminated grain lots must be sold at deeply discounted rates for alternative uses such as the ethanol industry. Due to the large economic losses, DON mitigation strategies have been heavily explored in the past three decades. Several attempts to reduce DON levels through chemical means have been demonstrated but have been deemed impractical due to the associated costs or simply to their detrimental effect on the final grain quality.

[0006] The detoxification of DON by various species of microbes has been demonstrated using a wide variety of microorganisms. Earlier reported mechanisms of DON microbial detoxification have focused on de-epoxidation, transformation of the C3 carbon (or attached groups), or the complete mineralization of DON. Under the reported conditions, multiple strains have been shown to de-epoxidize DON under anaerobic conditions, but the exact mechanism(s) by which these species accomplish this detoxification are unknown. While very limited numbers of studies were able to demonstrate the complete mineralization of DON, many were able to show that targeting the hydroxyl group on the C3 carbon is a common strategy to detoxify DON. Among the reported modifications of C3 groups are acetylation, oxidation, epimerization and glycosylation, each of which have been demonstrated to reduce DON toxicity.

[0007] Recently, the bacterium Devosia mutans was reported to epimerize DON to 3-ep; ^' -DON enzymatically, eliminating or significantly reducing its biotoxicity in many cell lines and experimental animals (He, J.W., et al (2015), Toxicology of 3-ep;- deoxynivalenol, a deoxynivalenol-transformation product by Devosia mutans 17-2-E- 8, Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 84: 250-259; He, J.W.et al (2015), An epimer of deoxynivalenol: purification and structure identification of 3-ep;- deoxynivalenol, Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment 32: 1523-1530; He, J.W., et al (2016), Bacterial epimerization as a route for deoxynivalenol detoxification: the influence of growth and environmental conditions, Frontiers in Microbiology 7: 572).

0008] This epimerization was shown to be a two-step process, as seen below.

DON 3-oxo-DON 3-ep/-DON

[0009] The first step involves oxidation of the 3a (R) hydroxy group of DON to a carbonyl group in 3-oxo-DON (also known as 3-keto-DON). The carbonyl group of 3-oxo-DON is subsequently reduced in a second step with high stereoselectivity to form 3-ep/ ^' -DON, which contains a 3-hydroxy group having a stereochemical configuration (S) opposite to that found in DON (Hassan, Y. I. , et al (2017). The enzymatic epimerization of deoxynivalenol by Devosia mutans proceeds through the formation of a 3-keto-DON intermediate, Scientific Reports 7: 6929). Both the 3-oxo- DON intermediate and the final 3-ep/ ^' -DON stereoisomer have been reported to have a reduced toxicity compared to that of DON itself. 3-ep; ^' -DON is reported to have at least 50-fold less toxicity than DON itself.

[0010] International patent application publication WO 2016/154640 and related US patent application publication US 2018/008001 1 describe alcohol dehydrogenases capable of transforming the 3-hydroxyl group of trichothecene mycotoxins to a 3-oxo group.

[001 1 ] It is desirable to provide isolated enzymes capable of catalyzing the conversion of deoxynivalenol to less toxic products, such as 3-ep; ^' -DON.

Embodiments of such isolated enzymes may advantageously be used to

decontaminate animal feed, by methods including but not limited to direct application of the isolated enzymes to the contaminated feed, or by expression of the enzymes by transgenic microorganisms (including but not limited to lactic acid bacteria or yeasts). In addition, plants genetically modified to express embodiments of such enzymes may advantageously be useful to detoxify DON produced by Fusarium infection of such plants in situ.

Summary

[0012] In one aspect, the present application provides a method for decontaminating a biological material contaminated with a trichothecene mycotoxin, wherein the trichothecene mycotoxin bears a 3-hydroxy group having a first stereochemical configuration, the method comprising exposing the biological material to an oxidizing enzyme or to an organism expressing the oxidizing enzyme, wherein the oxidizing enzyme has activity to oxidize the 3-hydroxy group having the first stereochemical configuration to a 3-oxo group, thereby to convert the trichothecene mycotoxin to an oxidized trichothecene product.

[0013] In at least one embodiment, the method further comprises subsequently or concurrently exposing the biological material to a reducing enzyme or to an organism expressing the reducing enzyme, wherein the reducing enzyme has activity to reduce the 3-oxo group of the oxidized trichothecene product to a 3-hydroxy group having a second stereochemical configuration different from the first stereochemical configuration, thereby to convert the oxidized trichothecene product to an epimerized trichothecene product.

[0014] In at least one embodiment, the epimerized trichothecene product has a toxicity lower than the toxicity of the trichothecene mycotoxin. In at least one embodiment, the oxidized trichothecene product has a toxicity lower than the toxicity of the trichothecene mycotoxin. In at least one embodiment, the trichothecene mycotoxin is selected from deoxynivalenol and 15-acetyldeoxynivalenol.

[0015] In another aspect, the present application provides an isolated oxidizing enzyme having activity to oxidize a trichothecene mycotoxin comprising a 3-hydroxy group to form an oxidized trichothecene product comprising a 3-oxo group, wherein the enzyme has a sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7 and variants thereof. In at least one embodiment, the isolated oxidizing enzyme has activity to convert deoxynivalenol to 3-oxo-deoxynivalenol. In at least one embodiment, the isolated oxidizing enzyme has activity to convert 15-acetyl- deoxynivalenol to 3-oxo-15-acetyldeoxynivalenol.

[0016] A further aspect of the present application provides an isolated reducing enzyme having activity to reduce an oxidized trichothecene product comprising a 3-oxo group to form an epimerized trichothecene product bearing a 3-hydroxy group, wherein the 3-hydroxy group of the epimerized trichothecene product has a second stereochemical configuration different from a first stereochemical configuration of a 3-hydroxy group in a trichothecene mycotoxin. In at least one embodiment, the isolated reducing enzyme has activity to convert 3-oxo-deoxynivalenol to 3-ep; ^' - deoxynivalenol. In at least one embodiment, the isolated reducing enzyme has activity to convert 3-oxo- 15-acetyldeoxynivalenol to 3-ep/ ^' - 15-acetyldeoxynivalenol. In at least one embodiment, the isolated reducing enzyme has an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:1 1 and variants thereof. [0017] According to a further aspect of the present application, there is provided a polynucleotide encoding at least one of an oxidizing enzyme as described herein and a reducing enzyme as described herein. In at least one embodiment, the

polynucleotide encodes an oxidizing enzyme as described herein. In at least one embodiment, the polynucleotide encodes a reducing enzyme as described herein.

[0018] In another aspect, the present application provides a vector comprising a polynucleotide encoding at least one of an oxidizing enzyme as described herein and a reducing enzyme as described herein. Yet another aspect of the present invention provides a transgenic organism expressing at least one of an oxidizing enzyme and a reducing enzyme as described herein. In at least one embodiment, the transgenic organism is a plant. In at least one embodiment, the transgenic organism is a microorganism.

Brief Description of the Drawings

[0019] Further features of the present invention will become apparent from the following written description and the accompanying figures, in which:

[0020] Figure 1A is a bar graph showing the concentration of 3-oxo-deoxynivalenol (3-oxo-DON) after 2 hours and 60 hours in reaction mixtures containing

deoxynivalenol (DON) and partially purified Devosia mutans DepA (DmDepA) in the presence and absence of pyrroloquinoline quinone (PQQ) and Ca ²⁺ ;

[0021] Figure 1 B is a bar graph showing the relative concentration of 3-oxo-DON in reaction mixtures containing DON and DmDepA expressed in Escherichia coli in the absence (Apo-DmDepA) and presence (PQQ-DmDepA) of PQQ;

[0022] Figure 1 C is a bar graph showing relative concentrations of DON and 3-oxo- DON present in reaction mixtures containing DON, PQQ and phenazine methosulfate in the presence and absence of DmDepA;

[0023] Figure 2A is a bar graph showing relative concentrations of DON and 3-oxo- DON present in reaction mixtures containing DON, CaC , phenazine methosulfate, and Devosia yakushimensis DepA (DyDepA) expressed and purified in E. coli in the absence (Apo-Enzyme) or presence (Holo-Enzyme) of PQQ. Reactions were carried out in the absence or presence (APO-PQQ, Holo-PQQ) of PQQ;

[0024] Figure 2B is a bar graph showing the concentration of 3-oxo-deoxynivalenol (3-oxo-DON) in reaction mixtures containing DON, PQQ, phenazine methosulfate and CaCb in addition to no enzyme (Negative control), DyDepA or lyophilized DyDepA; [0025] Figure 2C is a bar graph showing the relative concentration of 3-oxo-DON in reaction mixtures containing DON, PQQ and CaCI ₂ in addition to no enzyme, DyDepA and no cofactor or DyDepA and various possible cofactors;

[0026] Figure 3A is a bar graph showing the relative activity of Devosia mutans DepB (DmDepB) expressed in and purified from E. coli in various buffers;

[0027] Figure 3B is a graph showing the concentration of 3-ep/ ^' -deoxynivalenol (3-ep/ ^' -DON) in reaction mixtures containing 3-oxo-DON, NADPH and DmDepB at various pH values;

[0028] Figure 3C is a bar graph showing the concentration of 3-ep/ ^' -DON in reaction mixtures containing 3-oxo-DON and DmDepB in the presence of NADH or NADPH;

[0029] Figure 3D is a bar graph showing the effect of heat treatment on the stability of DmDepB;

[0030] Figure 3E is a bar graph showing the effect of temperature on the activity of DmDepB;

[0031 ] Figure 3F is a bar graph showing the relative concentration of 3-ep/ ^' -DON in reaction mixtures containing 3-oxo-DON and NADPH in the absence or presence of DmDepB or lyophilized DmDepB;

[0032] Figure 4 is a bar graph showing the concentration of 3-ep; ^' -DON in reaction mixtures containing 3-oxo-DON and NADPH in the absence or presence of Rhizobium leguminosarum DepB (R/DepB);

[0033] Figure 5 is a bar graph showing relative concentrations of DON, 3-oxo-DON and 3-ep; ^' -DON produced from a reaction mixture containing DyDepA, DON, phenazine methosulfate, PQQ and CaCI ₂ which is allowed to react for 2.75 h, then incubated with DmDepB and NADPH overnight; and

[0034] Figure 6 is a bar graph showing relative concentrations of

15-acetyldeoxynivalenol (15-AcDON) and 3-oxo-15-acetyldeoxynivalenol (3-oxo-15- AcDON) in reaction mixtures containing 15-AcDON, PQQ, phenazine methosulfate and CaCI ₂ in the presence or absence of DyDepA.

Detailed Description

[0035] In one aspect, the present invention provides a method for decontaminating a biological material contaminated with a trichothecene mycotoxin. As used herein, the term "decontaminating" or "decontamination" when used in relation to a biological material contaminated with a trichothecene mycotoxin is intended to mean reducing the toxicity of the material which is attributable to the presence of the trichothecene mycotoxin. In at least one embodiment, the toxicity of the material which is attributable to the trichothecene mycotoxin is reduced by reducing the amount and/or concentration of the trichothecene mycotoxin contaminating the biological material. As used herein, the term "contaminating", "contaminated" or "contamination" when used in relation to a biological material contaminated with a trichothecene mycotoxin is intended to mean that the biological material contains the trichothecene mycotoxin in an amount or concentration that renders the biological material more toxic to an animal than the biological material would be in the absence of the trichothecene mycotoxin. The trichothecene mycotoxin may be found within the biological material, on the surface of the biological material or otherwise associated with the biological material such that an animal ingesting or otherwise coming into contact with the biological material will ingest or otherwise come into contact with, or is at risk of ingesting or otherwise coming into contact with, the trichothecene mycotoxin.

[0036] In at least one embodiment, the biological material is plant material which is or has been exposed to or infected by a fungus which produces the trichothecene mycotoxin. Such fungi are well known and include but are not limited to species of Fusarium. In at least one embodiment, the plant material is a food crop, including but not limited to grains, cereals, wheat, oats, barley, corn, buckwheat, rice, sorghum, millet, rye, triticale and other food crops well known in the art, or a food or feed product derived from such a food crop, including but not limited to malt, silage, distiller's dried grains with solubles (DDGS) and other such products.

[0037] In at least one embodiment, the plant material is used for food by an animal. In at least one embodiment, the animal is a human or an animal whose meat, milk, eggs or other products may be used for food by a human. Such animals include but are not limited to ruminants (including but not limited to cattle, oxen, bison, buffalo, yaks and other bovines, and sheep, rams, ewes, lambs, wethers, goats, bucks, does, kids and other caprines), swine (including but not limited to pigs, hogs, boars, sows, gilts, barrows and other swine), poultry (including but not limited to chickens, ducks, geese, turkeys and other poultry), rabbits, and fish (including but not limited to salmon, trout, tilapia, halibut and other fish raised for food in aquaculture). In at least one embodiment, the animal is a companion animal or a pet, including but not limited to dogs, cats, birds, rodents, rabbits, fish and horses.

[0038] In at least one embodiment, the decontamination comprises converting the trichothecene mycotoxin into one or more products which have a lower toxicity to an animal than the trichothecene mycotoxin itself. In at least one embodiment, the trichothecene mycotoxin comprises a 3-hydroxy group having a first stereochemical configuration. Such trichothecene mycotoxins include but are not limited to deoxynivalenol (DON), 15-acetyldeoxynivalenol, T-2 toxin, HT-2 toxin, nivalenol, fusarenone-X, 15-acetoxyscirpenol, 4,15-diacetoxyscirpenol, T-2 tetraol, deacetylneosolaniol, acetylneosolaniol, sporotrichiol, and derivatives thereof, including but not limited to glycosylate, sulfonate or sulfate derivatives thereof. In at least one embodiment, the trichothecene mycotoxin is selected from deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-AcDON).

DON 15-AcDON

[0039] In at least one embodiment, the first stereochemical configuration is the stereochemical configuration found in naturally-occurring trichothecene mycotoxins. In at least one embodiment, the first stereochemical configuration is a. In at least one embodiment, the decontamination comprises oxidizing the 3-hydroxy group having a first stereochemical configuration to a 3-oxo group and/or epimerizing the 3-hydroxy group having a first stereochemical configuration to a 3-hydroxy group having a second stereochemical configuration which is different from the first stereochemical configuration.

[0040] It has now been found that a biochemical pathway discovered in the bacterium Devosia mutans, herein referred to as the DON EDjmerization (Dep) pathway, contains at least two putative enzymes. The first enzyme, herein designated as DepA, has activity to catalyze the oxidation of DON to 3-oxo-DON, as shown below:

[0041] The second enzyme, herein designated as DepB, has activity to catalyze the reduction of 3-oxo-DON to 3-ep; ^' -DON, as shown below:

[0042] Thus, in at least one embodiment, the method comprises a first step of exposing the biological material to an oxidizing enzyme or to an organism expressing the oxidizing enzyme, wherein the oxidizing enzyme has activity to oxidize the 3-hydroxy group of a trichothecene mycotoxin having a first stereochemical configuration to a 3-oxo group, thereby to convert the trichothecene mycotoxin to an oxidized trichothecene product.

[0043] In at least one embodiment, the method comprises a second step of exposing the biological material to a reducing enzyme or to an organism expressing the reducing enzyme, wherein the reducing enzyme has activity to reduce the 3-oxo group to a 3-hydroxy group having a second stereochemical configuration different from the first stereochemical configuration, thereby to convert the oxidized trichothecene product to an epimerized trichothecene product.

[0044] The biological material can be exposed to the reducing enzyme subsequently to and/or concurrently with exposure of the biological material to the oxidizing enzyme. As used herein in the context of exposure of biological material to an oxidizing enzyme and/or a reducing enzyme as described herein, the term

"subsequent" or "subsequently" is intended to mean that the biological material is first exposed to the oxidizing enzyme in the absence of the reducing enzyme, and exposure of the biological material to the reducing enzyme is delayed and takes place only after the biological material has been exposed to the oxidizing enzyme for a period of time. Subsequent exposure of the biological material to the reducing enzyme can take place either in the presence of the oxidizing enzyme, or in the absence of the oxidizing enzyme. In this way, the trichothecene mycotoxin contaminating the biological material can be at least partially converted to the oxidized trichothecene product before exposure to the reducing enzyme occurs.

[0045] As used herein in the context of exposure of biological material to an oxidizing enzyme and/or a reducing enzyme as described herein, the term "concurrent" or "concurrently" is intended to mean that the biological material is exposed to the oxidizing enzyme in the presence of the reducing enzyme. In this way, the oxidized trichothecene product can be converted to the epimerized trichothecene product by the action of the reducing enzyme as the oxidized trichothecene product is being produced from the trichothecene mycotoxin contaminating the biological material by the action of the oxidizing enzyme. Thus, in at least one embodiment, exposure of the biological material to the reducing enzyme can be both concurrent with (in the presence of) and subsequent to (at a delayed time with respect to) exposure to the oxidizing enzyme.

[0046] The present application also provides an isolated oxidizing enzyme having activity to oxidize a trichothecene mycotoxin comprising a 3-hydroxy group to form an oxidized trichothecene product comprising a 3-oxo group. In at least one

embodiment, the isolated oxidizing enzyme has activity to convert deoxynivalenol to 3-oxo-deoxynivalenol. In at least one embodiment, the isolated oxidizing enzyme has activity to convert 15-acetyldeoxynivalenol to 3-oxo-15-acetyldeoxynivalenol. In at least one embodiment, the isolated oxidizing enzyme is DepA or a variant thereof.

[0047] In at least one embodiment, the DepA is isolated from a species of Devosia. In at least one embodiment, the species of Devosia is Devosia mutans strain

17-2-E-8. In at least one such embodiment, the DepA has an amino acid sequence selected from SEQ ID NO:1 , SEQ ID NO:2, and variants thereof.

[0048] In at least one embodiment, the species of Devosia is Devosia

yakushimensis. In at least one such embodiment, the DepA has an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:5, and variants thereof.

[0049] In at least one embodiment, the species of Devosia is Devosia

epidermidihirudinis. In at least one such embodiment, the DepA has an amino acid sequence selected from SEQ ID NO:7 and variants thereof.

[0050] In addition, the present application provides an isolated reducing enzyme having activity to reduce an oxidized trichothecene product comprising a 3-oxo group to form an epimerized trichothecene product bearing a 3-hydroxy group, wherein the 3-hydroxy group of the epimerized trichothecene product has a second

stereochemical configuration different from a first stereochemical configuration of a 3-hydroxy group in a trichothecene mycotoxin. In at least one embodiment, the isolated reducing enzyme has activity to convert 3-oxo-deoxynivalenol to 3-ep; ^' - deoxynivalenol. In at least one embodiment, the isolated reducing enzyme has activity to convert 3-oxo- 15-acetyldeoxynivalenol to 3-ep/ ^' - 15-acetyldeoxynivalenol. In at least one embodiment, the reducing enzyme is DepB or a variant thereof.

[0051] In at least one embodiment, the DepB is isolated from a bacterial species. In at least one embodiment, the bacterial species is Devosia mutans strain 17-2-E-8. In at least one such embodiment, the DepB has an amino acid sequence selected from SEQ ID NO:9 and variants thereof. In at least one embodiment, the bacterial species is Rhizobium leguminosarum. In at least one such embodiment, the DepB has an amino acid sequence selected from SEQ ID NO: 1 1 and variants thereof.

[0052] As used herein, the term "variant" when used in reference to an enzyme is intended to refer to an enzyme which completely or partially retains the enzymatic activity of a reference enzyme to which the variant is being compared, but which differs in its amino acid sequence from the sequence of the reference enzyme by one or more amino acid residues. The differences between the sequence of the variant and the sequence of the reference enzyme can include substitution of one or more amino acid residues with different amino acid residues, insertion of additional amino acid residues or deletion of amino acid residues. In certain embodiments, a variant can differ from a reference enzyme by conservative substitution of one or more amino acid residues with replacement amino acid residues which may have similar properties, including but not limited to charge, size and hydrophilicity, to the amino acid residues which the new residues replace. In certain embodiments, variants may completely or partially retain the enzymatic activity of the reference enzyme. In at least one embodiment, the reference enzyme is an oxidizing enzyme or a reducing enzyme as described herein.

[0053] In at least one embodiment, the sequence of a variant can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% identity to the sequence of a reference enzyme. As used herein, the term "percent identity" or "% identity" when used in reference to the sequence of a polypeptide or a polynucleotide is intended to mean the percentage of the total number of amino acid or nucleotide residues, respectively, in the sequence which are identical to those at the corresponding position of a reference polypeptide or polynucleotide sequence. In at least one embodiment, when the length of the variant sequence and the length of the reference sequence are not identical, percent identity can be calculated based on the total number of residues in the variant sequence or based on the total number or residues in the reference sequence. Percent identity can be measured by various local or global sequence alignment algorithms well known in the art, including but not limited to the Smith-Waterman algorithm and the Needleman-Wunsch algorithm. Tools using these or other suitable algorithms include but are not limited to BLAST (Basic Local Alignment Search Tool) and other such tools well known in the art.

[0054] In at least one embodiment, the DepA has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% identity to SEQ ID NO: 1 . In at least one embodiment, the DepA has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% identity to SEQ ID NO:2. In at least one embodiment, the DepA has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% identity to SEQ ID NO:4. In at least one embodiment, the DepA has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% identity to SEQ ID NO:5. In at least one embodiment, the DepA has at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% identity to SEQ ID NO:7.

[0055] In at least one embodiment, the DepB has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% identity to SEQ ID NO:9. In at least one embodiment, the DepB has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 99.9% identity to SEQ ID NO: 1 1.

[0056] Another aspect of the present application provides a polynucleotide encoding an oxidizing enzyme or a reducing enzyme as described herein. In at least one embodiment, the polynucleotide is messenger RNA (mRNA) having a sequence which can be translated to generate the oxidizing enzyme or reducing enzyme. In at least one embodiment, the polynucleotide is DNA, at least one strand of which can be transcribed to produce mRNA which in turn can be translated to generate the oxidizing enzyme or reducing enzyme. In at least one embodiment, the DNA can be expressed by a biochemical system, including but not limited to a cell or a cell-free protein expression system, as well known in the art, to produce the oxidizing enzyme or reducing enzyme. In at least one such embodiment, the DNA can be incorporated into a vector configured for expression of the DNA in a host cell, as well known in the art.

[0057] In at least one embodiment, the polynucleotide can include a variant polynucleotide sequence which hybridizes to a polynucleotide comprising a sequence encoding an oxidizing enzyme or a reducing enzyme as described herein under at least moderately stringent conditions. By "at least moderately stringent hybridization conditions" it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrid, is determined by the melting temperature (T _m ), which in sodium-containing buffers is a function of the sodium ion concentration ([Na ⁺ ]) and temperature (T _m = 81.5°C - 16.6 (Logi ₀ [Na ⁺ ]) + 0.41 (%(G+C) - 600/I), where %G+C is the percentage of cytosine and guanine nucleotides in the nucleic acid and I is the length of the nucleic acid in base pairs, or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule, a 1 % mismatch may be assumed to result in about a 1 °C decrease in T _m . For example, if nucleic acid molecules are sought that have a >95% identity, the final wash temperature may be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions.

[0058] In some embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate (SSC)/5x Denhardt's solution/1 .0% sodium dodecylsulfate (SDS) at T _m - 5°C based on the above equation, followed by a wash of 0.2x SSC/0.1 % SDS at 60°C. Moderately stringent

hybridization conditions include a washing step in 3x SSC at 42°C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001 .

[0059] Therefore, in at least one embodiment, the DepA is encoded by a

polynucleotide having a sequence selected from SEQ ID NO:3 or variants thereof. In at least one embodiment, the DepA is encoded by a polynucleotide having a sequence selected from SEQ ID NO:6 or variants thereof. In at least one

embodiment, the DepA is encoded by a polynucleotide having a sequence selected from SEQ ID NO:8 or variants thereof.

[0060] In at least one embodiment, the DepB is encoded by a polynucleotide having a sequence selected from SEQ ID NO: 10 or variants thereof. In at least one embodiment, the DepB is encoded by a polynucleotide having a sequence selected from SEQ ID NO: 12 or variants thereof.

[0061 ] In at least one embodiment, the isolated oxidizing enzyme or isolated reducing enzyme can be isolated from an organism or microorganism naturally expressing the oxidizing enzyme or reducing enzyme, or from a culture thereof. In at least one embodiment, the isolated oxidizing enzyme or isolated reducing enzyme can be recombinantly produced by expression in a suitable host cell of a vector comprising a polynucleotide having a sequence encoding one or more of the oxidizing enzyme or reducing enzyme. In addition, the oxidizing enzyme or reducing enzyme can be at least partially purified after isolation or recombinant production. Suitable vectors and host cells, including but not limited to prokaryotic and eukaryotic host cells adapted for the production of recombinant proteins, and methods of isolating or recombinantly producing such proteins, including methods of at least partial purification of such proteins, are well known in the art or can be readily identified and carried out by the skilled person in light of the teaching herein with no more than routine experimental effort. In at least one embodiment, the host cell is a plant cell. In at least one embodiment, the host cell is a microorganism.

[0062] Yet another aspect of the present invention provides a transgenic organism expressing at least one of an oxidizing enzyme and a reducing enzyme as described herein. In at least one embodiment, the transgenic organism is a microorganism. Such microorganisms can be useful in embodiments of a method as described herein in which the biological material is exposed to an organism expressing the oxidizing enzyme or the reducing enzyme or both. Alternatively, such microorganisms can be useful in the recombinant production of one or more of the oxidizing enzyme or the reducing enzyme on a commercial scale.

[0063] In at least one embodiment, the transgenic organism is a plant. In at least one such embodiment, the transgenic plant can be an agriculturally relevant crop plant which is advantageously resistant to contamination with trichothecene mycotoxins that may be produced during exposure to or infection by fungal species producing such trichothecene mycotoxins.

[0064] As used herein, the terms "about" or "approximately" as applied to a numerical value or range of values are intended to mean that the recited values can vary within an acceptable degree of error for the quantity measured given the nature or precision of the measurements, such that the variation is considered in the art as equivalent to the recited values and provides the same function or result. For example, the degree of error can be indicated by the number of significant figures provided for the measurement, as is understood in the art, and includes but is not limited to a variation of ±1 in the most precise significant figure reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" can mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

[0065] As used herein, the term "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is "substantially" aligned would mean that the object is either completely aligned or nearly completely aligned. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.

[0066] The use of "substantially" is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a composition that is

"substantially free of particles would either completely lack particles, or so nearly completely lack particles that the effect would be the same as if it completely lacked particles. In other words, a composition that is "substantially free of an ingredient or element may still actually contain such item as long as there is no measurable effect thereof.

EXAMPLES

[0067] Other features of the present invention will become apparent from the following non-limiting examples which illustrate, by way of example, the principles of the invention. Unless otherwise indicated, all reactions are carried out at room temperature.

Example 1 : DepA from Devosia mutans 17-2-E-8 (DmDepA)

Measurement of DepA activity

[0068] One part test sample is combined with four parts buffer (Tris, pH 7.5) with a final concentration of deoxynivalenol (DON) at 100 μg ^■ mL ^~1 (ppm). After an overnight incubation, the reaction is subjected to HPLC (high performance liquid

chromatography). Samples and standards (20 μΙ_) are separated by a Proteo Phenomenex column (Jupiter™, 4 μηι, 250 mm ^χ 4.6 mm) attached to a Shimadzu uHPLC system (Mandel Scientific Company, Guelph, ON, Canada). An 1 1 minute isocratic elution consisting of 12% acetonitrile in water is used to detect DON followed by a column wash of 80 percent acetonitrile in water for 3 minutes and a re- equilibration of the column at 12% acetonitrile in water for 6 minutes. 3-oxo- deoxynivalenol (3-oxo-DON) is eluted using a 15 minute isocratic elution of 23% acetonitrile in water followed by a column wash of 90 percent acetonitrile in water for 3 minutes and a re-equilibration of the column at 23% acetonitrile in water for 6 minutes. DON and 3-oxo-DON are detected by monitoring the absorbance at 218 nm and quantified by comparing to a known DON or 3-oxo-DON standard (TripleBond, Guelph, Canada).

Partial Purification

[0069] A frozen glycerol stock of Devosia mutans 17-2-E-8 was used to inoculate LB medium (lysogeny broth or Luria-Bertani medium) containing 34 μg ^■ mL ^~1 of kanamycin, and the inoculated media was incubated for two weeks at 28°C with shaking at 200 RPM. The resulting starter culture was used to inoculate four litres of LB medium and the cultures were grown for 6 days, centrifuged to pellet the cells and the pellets were stored at -20°C. Frozen pellets were thawed and cells were resuspended in 50 mM Tris, pH 8.0 with 150 mM NaCI and were lysed by extensive sonication. The supernatant was centrifuged and filtered through a 0.45 μηι syringe filter, and Halt™ Protease Inhibitor Cocktail (ThermoFisher) without EDTA was added to provide a lysate.

[0070] The clarified lysate was tested for DepA activity as described above.

Compared to control reactions, in which no 3-oxo-DON was detected, the treated samples showed a peak corresponding to 3-oxo-DON and a corresponding decrease in the amount of DON.

[0071] A series of protein purification steps were undertaken to isolate the responsible enzyme. After ammonium sulfate precipitation of the lysate (Duong-Ly, K.C., and Gabelli, S.B. (2014) Salting out of proteins using ammonium sulfate precipitation, Methods in enzymology 541 : 85-94), most of the DepA activity was found among the proteins that precipitated between 50-70% of saturating conditions. This fraction was then dialyzed to remove excess ammonium sulfate and heated at 60°C to precipitate less stable proteins.

[0072] The protein solution was then subjected to anion exchange chromatography (AKTAprime™ plus with a HiTrap™ Q FF column (GE Healthcare)). Fractions (and flow through) were assessed for DepA activity and fractions containing high DepA activity were pooled and concentrated by ultrafiltration using an Amicon™ stirred cell with an Ultracel™ 10 kDa ultrafiltration disc (Millipore). Analysis of fractions by SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis) revealed a band of approximately 62 kDa, the intensity of which was positively correlated with DepA activity.

[0073] The pooled fractions were then subjected to size exclusion column chromatography performed using a HiPrep™ 16/60 Sephacryl™ S-200 HR size exclusion column. The resulting fractions were again assessed for DepA activity and active fractions were pooled and concentrated. SDS-PAGE was performed and the abundance of the band representing one or more proteins of approximately 62 kDa had a positive correlation with DepA activity. DepA activity in the partially purified samples was found to be stable for at least one month at 4°C and after a heat treatment of 60°C.

Initial Cofactor Analysis

[0074] Cofactors such as NADH, NADPH, NAD ⁺ , NADP ⁺ , ATP and PQQ

(pyrroloquinoline quinone), were individually added to DepA activity assay reactions, and the only cofactor found to increase DepA activity was PQQ. In addition, it was found that addition of EDTA to the protein solution abolished DepA activity. The EDTA was removed by dialysis and 20 μΙ_ of the resulting crude extract was incubated with 50 μg ^■ mL ^~1 or 100 μg ^■ mL ^"1 DON and 1 mM metal, in Tris buffer pH 7.5 in a final volume of 100 μΙ_, in the absence and in the presence of 100 μΜ PQQ. Buffers and water used to dissolve substrates in this experiment were treated with Chelex 100 (Sigma). As seen from the results presented in Figure 1A, addition of Ca ²⁺ in the absence of exogenous PQQ showed no detectable activity after 2 hours, and only minimal activity was seen after ~60 hours incubation. The addition of exogenous PQQ in the absence of exogenous Ca ²⁺ increased the amount of 3-oxo- DON formed after ~60 hours, and coaddition of PQQ and Ca ²⁺ increased the amount of 3-oxo-DON formed after both 2 and ~60 hours. Addition of Ca ²⁺ , and to a lesser extent, Mg ²⁺ and Mn ²⁺ _, in the presence of PQQ appeared to restore activity in overnight reactions, as shown in Table 1 . Percent activity is determined relative to the highest activity observed.

Table 1 : Metal specificity of partially purified DmDepA

[0075] These results indicate that DmDepA is a PQQ- and metal-dependent enzyme. Without being bound by theory, it is hypothesised that the metal stabilizes the binding of PQQ, as removal of the metal by EDTA treatment appeared to allow a substantial amount of PQQ to dissociate from the enzyme. It is possible that a small amount of Ca ²⁺ remained bound to the enzyme following the EDTA treatment, accounting for the increased activity observed when PQQ was added in the absence of additional exogenous Ca ²⁺ . Further, because PQQ is known to bind strongly to some PQQ- dependent dehydrogenases, it is believed that the partially purified enzyme is in the form of the holoenzyme including tightly bound PQQ, and therefore retained activity in the absence of exogenously added PQQ, even after repeated treatment by dialysis and column chromatography.

Protein sequencing and expression of DmDepA

[0076] Aliquots of two fractions showing high DepA activity from the size exclusion chromatography step described above were subsequently sent for protein sequencing at the Advanced Analysis Centre at the University of Guelph (Guelph, Ontario, Canada). Sequencing results showed that these two fractions contained 70 and 83 proteins, respectively, 51 and 57 of which, respectively, contained at least 2 unique peptide sequences. An analysis of the protein sequencing results identified seven target proteins as being possible candidates for having PQQ-dependent dehydrogenase activity. The subcellular localization of the proteins was predicted using PSORTb 3.0 (Yu, N.Y. et al. (2010) PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes, Bioinformatics (Oxford, England) 26: 1608-1615). Signal sequences, as predicted by SignalP 4.0 (Petersen, T.N., et al (201 1) SignalP 4.0: discriminating signal peptides from transmembrane regions, Nature Methods 8: 785-786) were removed prior to synthesis of the gene.

[0077] The DNA sequences of the candidate genes were codon-optimized for overexpression in Escherichia coli, synthesized and cloned into the bacterial expression vector pET28a. Each of the candidate genes in pET28a was transformed into E. coli BL21 , which was propagated at 37°C in 500 mL of LB media containing 34 mg- L ¹ kanamycin, shaking at 175 rpm. When the cultures reached an optical density of approximately 0.7, 150 μΜ of isopropyl β-d-l-thiogalactopyranoside (IPTG) was added to initiate protein expression. The cultures were incubated overnight at 15°C and harvested by centrifugation at 8000 g for 10 minutes and frozen at -20°C. Each pellet was thawed and resuspended in 5 mL of 50 mM Tris, pH 8.0, 150 mM NaCI. The cells were then lysed by sonication using the following parameters: 30% amplitude with a cycle of 30 seconds on, 40 seconds off for 8 cycles. The lysate was centrifuged at 7142 g for 30 minutes to remove the insoluble pellet and filtered through a 0.45 μηι filter. An aliquot of this was set aside to test for DepA activity. [0078] The clarified lysate (approximately 5 ml.) was then mixed with an equal volume of 50 mM sodium phosphate with 300 mM NaCI and 30 mM imidazole, pH 8.0 (wash buffer) and incubated with 300 μΙ_ of HisPur™ Ni-NTA (nickel-nitrilotriacetic acid) agarose resin (ThermoFisher) for one hour. The mixture was then briefly centrifuged and the supernatant was removed from the tubes. The beads were resuspended in 1 mL of wash buffer and transferred to 1.5 mL tubes. The tubes were briefly spun, supernatant removed and resuspended with 1 mL of wash buffer. This was repeated for a total of 5 washes. The proteins which bound to the beads were eluted with 1 mL of 50 mM sodium phosphate with 300 mM NaCI and 250 mM imidazole, pH 8.0 and the beads removed by centrifugation.

[0079] The expressed proteins were then each tested for DepA activity by separately incubating crude lysate and/or purified protein with DON in the presence of PQQ and CaCI ₂ . The only candidate protein having the ability to transform DON to 3-oxo-DON was the protein identified as fig|6666666.163324.CDS.378 (GenBank accession number KFL25551.1), herein designated DmDepA.

[0080] The native amino acid sequence of DmDepA is:

MKLECLRQNWGLALSTALIASLSAPAFAQHADGAAAETAAPGQSAIENFQPVTAED

LAGGNAANWPILRGNYQGWGYTQLDQINKDNVGQLQLAWARTMEPGSNEGSAIA

YNGWFLGNANDWQAIDGKTGNLIWEYRRKLPPASKFINSLGAAKRSIALFGDKVY FVSWDNFWALDAKTGKLAWETNRGQGVEEGVSNSSGPIWDGWIAGSTCQYSG FGCYVTGTDAESGEELWRNTFIPRPGEEGDDTWGGAPYENRWMTGAWGQITYDP ELDLVYYGSTGAGPASEVQRGTEGGTLAGTNTRFAVKPKTGEWWKHQTLPRDN WDSECTFEMMWSTTVNPDAGADGMMSVGANVPRGETRKVLTGVPCKTGVAWQ FDAETGDYFWSKATVEQNSIASIDDKGLVTVNEDMILKEPGKDYNYCPTFLGGRDW PSAGYLPKSNLYVIPLSNACYDLKARTTEATPADVYNTDSTVKLAPGKTNMGRVDAI DVATGATKWSFETEAALYDPVMTTAGDLVFVGSTDRMFRALDAETGKEVWSTRLP GAISGYTTSYSIDGRQYVAWAGGSLGTGFFKAAVPGVDAVQGGNGIYVFALPEAK

(SEQ ID NO:1)

[0081] The amino acid sequence of DmDepA modified for expression in E. coli is: MHADGAAAETAAPGQSAIENFQPVTAEDLAGGNAANWPILRGNYQGWGYTQLDQI NKDNVGQLQLAWARTMEPGSNEGSAIAYNGWFLGNANDWQAIDGKTGNLIWEY RRKLPPASKFINSLGAAKRSIALFGDKVYFVSWDNFWALDAKTGKLAWETNRGQG VEEGVSNSSGPIWDGWIAGSTCQYSGFGCYVTGTDAESGEELWRNTFIPRPGEE GDDTWGGAPYENRWMTGAWGQITYDPELDLVYYGSTGAGPASEVQRGTEGGTL AGTNTRFAVKPKTGEWWKHQTLPRDNWDSECTFEMMWSTTVNPDAGADGMMS VGANVPRGETRKVLTGVPCKTGVAWQFDAETGDYFWSKATVEQNSIASIDDKGLV TVNEDMILKEPGKDYNYCPTFLGGRDWPSAGYLPKSNLYVIPLSNACYDLKARTTE ATPADVYNTDSTVKLAPGKTNMGRVDAIDVATGATKWSFETEAALYDPVMTTAGDL VFVGSTDRMFRALDAETGKEVWSTRLPGAISGYTTSYSIDGRQYVAWAGGSLGTG FFKAAVPGVDAVQGGNGIYVFALPEAK (SEQ ID NO:2)

[0082] The codon-optimized nucleotide sequence of the DmDepA gene modified for overexpression in E. coli is:

ATGCACGCGGATGGTGCGGCGGCGGAAACCGCGGCGCCGGGCCAGAGCGCG ATCGAGAACTTTCAACCGGTTACCGCGGAAGATCTGGCGGGTGGCAACGCGGC GAACTGGCCGATCCTGCGTGGTAACTACCAGGGTTGGGGCTATACCCAGCTGG ACCAAATTAACAAGGATAACGTTGGTCAGCTGCAGCTGGCGTGGGCGCGTACG ATGGAGCCGGGTAGCAACGAAGGCAGCGCGATTGCGTACAACGGTGTGGTTTT TCTGGGCAACGCGAACGACGTGGTTCAAGCGATCGATGGTAAAACCGGCAACC TGATTTGGGAATATCGTCGTAAGCTGCCGCCGGCGAGCAAATTCATCAACAGCC TGGGTGCGGCGAAGCGTAGCATTGCGCTGTTCGGCGACAAAGTGTACTTTGTT AGCTGGGACAACTTCGTGGTTGCGCTGGATGCGAAGACCGGTAAACTGGCGTG GGAAACCAACCGTGGTCAGGGCGTTGAGGAAGGTGTGAGCAACAGCAGCGGC CCGATCGTGGTTGACGGTGTGGTTATTGCGGGCAGCACCTGCCAATACAGCGG TTTTGGCTGCTATGTTACCGGTACCGATGCGGAAAGCGGCGAGGAACTGTGGC GTAACACCTTCATCCCGCGTCCGGGCGAGGAAGGCGATGATACCTGGGGTGGC GCGCCGTACGAGAACCGTTGGATGACCGGTGCGTGGGGCCAGATTACCTATGA CCCGGAACTGGATCTGGTTTACTATGGTAGCACCGGTGCGGGTCCGGCGAGCG AGGTGCAACGTGGTACCGAAGGTGGCACCCTGGCGGGTACCAACACCCGTTTT GCGGTTAAGCCGAAAACCGGTGAGGTGGTTTGGAAGCACCAGACCCTGCCGCG TGACAACTGGGATAGCGAGTGCACCTTCGAAATGATGGTGGTTAGCACCACCGT GAACCCGGATGCGGGTGCGGATGGCATGATGAGCGTTGGTGCGAACGTGCCG CGTGGCGAAACCCGTAAGGTTCTGACCGGTGTGCCGTGCAAAACCGGCGTTGC GTGGCAGTTTGACGCGGAAACCGGTGATTACTTCTGGAGCAAGGCGACCGTGG AACAAAACAGCATCGCGAGCATTGACGATAAAGGTCTGGTGACCGTTAACGAGG ACATGATCCTGAAGGAACCGGGCAAAGATTACAACTATTGCCCGACCTTTCTGG GTGGCCGTGACTGGCCGAGCGCGGGTTACCTGCCGAAGAGCAACCTGTATGTT ATTCCGCTGAGCAACGCGTGCTACGATCTGAAAGCGCGTACCACCGAGGCGAC CCCGGCGGACGTTTATAACACCGATAGCACCGTGAAGCTGGCGCCGGGTAAAA CCAACATGGGCCGTGTTGACGCGATCGATGTGGCGACCGGTGCGACCAAGTG GAGCTTCGAAACCGAAGCGGCGCTGTACGACCCGGTGATGACCACCGCGGGT GATCTGGTGTTTGTTGGCAGCACCGACCGTATGTTCCGTGCGCTGGATGCGGA AACCGGTAAAGAAGTTTGGAGCACCCGTCTGCCGGGTGCGATCAGCGGCTACA CCACCAGCTATAGCATTGATGGTCGTCAGTATGTTGCGGTGGTTGCGGGTGGC AGCCTGGGTACCGGCTTCTTTAAAGCGGCGGTGCCGGGTGTTGATGCGGTGCA AGGTGGCAACGGCATCTATGTGTTCGCGCTGCCGGAAGCGAAATGA (SEQ ID NO:3)

[0083] DmDepA heterologously expressed in E. coli (15 μg) was incubated with 100 μς- ηιΙ. ^"1 deoxynivalenol (DON), in Tris buffer pH 7.5 in the presence or absence of 100 μΜ PQQ. As seen from the results presented in Figure 1 B, DmDepA heterologously expressed in E. coli (apo-DmDepA) is not active to convert DON to 3- oxo-DON in the absence of added PQQ. However, in the presence of exogenously added PQQ (PQQ-DmDepA), the expressed DmDepA is capable of transforming DON to 3-oxo-DON. Without being bound by theory, it is believed that because E. coli does not produce PQQ, DmDepA is expressed in E. coli as the apoenzyme, which is inactive in the absence of the cofactor PQQ. Furthermore, as seen from the results presented in Figure 1 C, incubation of 100 μg ^■ mL ^~1 DON, 40 μΜ phenazine (to regenerate reduced PQQ), and 100 μΜ PQQ in Tris buffer at pH 7.5 overnight at room temperature in the presence of DmDepA allowed complete conversion of DON to 3-oxo-DON, while in the absence (control) of DmDepA, no conversion was observed. Under these conditions, DmDepA was found to have a feat (s ^~1 ) of 4.18 ± 0.12, a Km app of 32 ± 4 μΜ and a catalytic efficiency (feat/Km) of 13.0 χ 10 ⁴ IV - s ^-1 . [0084] It is notable that the use of genomic library screening techniques to attempt to identify DmDepA from the Devosia mutans 17-2-E-8 genome sequence by expressing the candidate gene in common expression systems, such as E. coli and yeast, which do not produce PQQ natively, would have produced the inactive apoenzyme, and the function of DmDepA may not have been recognized in the absence of the realization that the relatively uncommon cofactor PQQ was required for activity. In contrast, the present method which combines protein purification from the native source of the enzyme with standard biochemical techniques, allowed isolation and partial purification of the holoenzyme containing tightly bound PQQ cofactor. Thus, the holoenzyme DmDepA could be readily identified under these conditions by its catalytic activity.

[0085] DmDepA expressed in E. coli retains significant activity after lyophilization. 100 μΙ_ aliquots of purified DmDepA expressed in E. coli were frozen at -80°C, lyophilized overnight and left at room temperature for 3 days. The lyophilized sample was resuspended in 100 μΙ_ of water, and allowed to react at a concentration of 50 μg ^■ mL ^"1 with 100 μg ^■ mL ^"1 DON, 100 μΜ PQQ, 40 μΜ phenazine methosulfate, and 1 mM CaC in Tris pH 7.5. Reactions (3 replicates) were stopped after 10 minutes and the concentration of 3-oxo-DON in the reaction mixture was measured. The lyophilized DmDepA retained significant activity to convert DON to 3-oxo-DON (approximately 90% compared to the activity of the unlyophilized and freshly thawed DmDepA).

Example 2: DepA from Devosia yakushimensis (DyDepA)

Partial Purification

[0086] DyDepA was partially purified from a culture of Devosia yakushimensis grown under the conditions described in Example 1 for culture of D. mutans 17-2-E-8, using methods similar to those described in Example 1. Thus, the pellet obtained from precipitation of the cell lysate with 50%-70% ammonium sulfate was resuspended and separated by anion exchange chromatography (AKTAprime™ plus with a HiTrap™ Q FF column (GE Healthcare)). Fractions containing high DepA activity were pooled and concentrated by ultrafiltration using an Amicon™ stirred cell with an Ultracel™ 10 kDa ultrafiltration disc (Millipore), then were further purified by hydrophobic interaction chromatography (HIC) using a HiPrep™ Phenyl FF (high sub) 16/60 column. Protein sequencing and expression of DyDepA

[0087] The fraction found to have the highest DepA activity was subjected to protein sequencing and was found to contain 85 proteins. Analysis of the protein sequencing results identified a candidate PQQ-dependent glucose dehydrogenase sequence, which was modified for expression in E. coli by removal of predicted signal sequences, codon-optimization of the resulting DNA sequence, gene synthesis and cloning into pET28a as described in Example 1. The pET28a construct was transformed into E. coli BL21 and expressed as described in Example 1 , except that two 1 L cultures were grown at a time and the pellets pooled. Cells were lysed under the same conditions except 15 sonication cycles were used. The clarified lysate was mixed with an equal volume of wash buffer and incubated with one mL of Ni-NTA agarose for an hour at 4°C. The mixture was then poured into a Flex-Column™ (Kimble Chase Life Science, Vineland, NJ, USA) and washed with 40 mL of wash buffer. The enzyme was eluted with eight mL of elution buffer. The buffer was changed to 50 mM Tris, 150 mM NaCI, pH 8.0 by repeated dilutions in the stirred cell, followed by concentration. The purified enzyme (herein designated DyDepA) was then aliquoted and stored at -80°C.

[0088] The native amino acid sequence of DyDepA is:

MKSKISVLLASVAVLGLASHAYGQVDISALAPVTDEVLANPEPGDWPSYGRTIENYR YSPLDQITKDNVGQLTLVWARAMEPGNMEAAPIEYQGVLFTGNPGDWQAIDAATG QLIWEYRRTLPDKALLNTLGEAKRGIALYEDKIYMATWDNFIVALDAKTGQVAWETD RGGGTDLVSNTTGPIVANGVWAGSTCQYSEFGCYVTGHDAATGEELWRNSFIPN KGEEGDDTWGNSEFDQRWMTGAWGQMTYDPTLDLVYYGSTGAGPASETQRGTE GGTMFGTNTRFAVKPKTGEIVWRHQVLPRDNWDQECTYEMVPVDIDVGPSADMD GLLAIGQNASGTKRVLTGVPCKTGILWQFDAETGDFIYARSTVEQNLISSVDDTGLV TVNEAAMPLVPDVAVHMCPTFLGGRDWSPTAFNPDSKVMFVPLTNMCGDLTALDQ EPTGLDVYNTEMAYKMPEGVTDAGRIDAIDWTGKTVWSYTQQMPQYSPIVATAG GLIFTGGADRKLKAIDQETGELVWSTTLASRASGHPITYEANGRQFIAIPAGGPGFAT NLITASGATDDAVSGSNTLYVFALPETK (SEQ ID NO:4)

[0089] SEQ ID NO:4 has 57% identity and 69% similarity to SEQ ID NO: 1.

[0090] The amino acid sequence of DyDepA modified for expression in E. coli is:

MQVDISALAPVTDEVLANPEPGDWPSYGRTIENYRYSPLDQITKDNVGQLTLVWAR AMEPGNMEAAPIEYQGVLFTGNPGDWQAIDAATGQLIWEYRRTLPDKALLNTLGE AKRGIALYEDKIYMATWDNFIVALDAKTGQVAWETDRGGGTDLVSNTTGPIVANGV WAGSTCQYSEFGCYVTGHDAATGEELWRNSFIPNKGEEGDDTWGNSEFDQRWM TGAWGQMTYDPTLDLVYYGSTGAGPASETQRGTEGGTMFGTNTRFAVKPKTGEIV WRHQVLPRDNWDQECTYEMVPVDIDVGPSADMDGLLAIGQNASGTKRVLTGVPC KTGILWQFDAETGDFIYARSTVEQNLISSVDDTGLVTVNEAAMPLVPDVAVHMCPTF LGGRDWSPTAFNPDSKVMFVPLTNMCGDLTALDQEPTGLDVYNTEMAYKMPEGV TDAGRIDAIDWTGKTVWSYTQQMPQYSPIVATAGGLIFTGGADRKLKAIDQETGEL VWSTTLASRASGHPITYEANGRQFIAIPAGGPGFATNLITASGATDDAVSGSNTLYV FALPETK (SEQ ID NO:5) [0091] The codon-optimized nucleotide sequence of the DyDepA gene modified for overexpression in E. coli is:

ATGCAGGTTGACATCAGCGCGCTGGCGCCGGTTACCGATGAAGTGCTGGCGAA CCCGGAGCCGGGTGACTGGCCGAGCTACGGCCGTACCATCGAAAACTACCGTT ATAGCCCGCTGGACCAGATTACCAAGGATAACGTTGGTCAACTGACCCTGGTGT GGGCGCGTGCGATGGAGCCGGGTAACATGGAAGCGGCGCCGATTGAGTACCA AGGCGTGCTGTTCACCGGCAACCCGGGTGACGTGGTTCAGGCGATCGATGCG GCGACCGGTCAACTGATTTGGGAGTATCGTCGTACCCTGCCGGACAAGGCGCT GCTGAACACCCTGGGCGAAGCGAAGCGTGGTATCGCGCTGTACGAGGATAAAA TTTATATGGCGACCTGGGACAACTTCATCGTTGCGCTGGATGCGAAAACCGGTC AGGTGGCGTGGGAAACCGACCGTGGTGGCGGTACCGATCTGGTTAGCAACACC ACCGGTCCGATTGTGGCGAACGGTGTGGTTGTGGCGGGTAGCACCTGCCAATA CAGCGAGTTTGGCTGCTATGTTACCGGTCACGACGCGGCGACCGGCGAGGAAC TGTGGCGTAACAGCTTCATCCCGAACAAAGGCGAGGAAGGTGACGATACCTGG GGCAACAGCGAATTTGATCAGCGTTGGATGACCGGCGCGTGGGGTCAAATGAC CTACGACCCGACCCTGGATCTGGTTTACTATGGTAGCACCGGTGCGGGTCCGG CGAGCGAAACCCAGCGTGGTACCGAGGGCGGTACCATGTTCGGCACCAACACC CGTTTTGCGGTGAAGCCGAAAACCGGCGAGATCGTTTGGCGTCACCAGGTGCT GCCGCGTGACAACTGGGATCAAGAGTGCACCTACGAAATGGTTCCGGTGGACA TCGATGTTGGTCCGAGCGCGGACATGGATGGCCTGCTGGCGATTGGTCAAAAC GCGAGCGGCACCAAGCGTGTTCTGACCGGTGTGCCGTGCAAAACCGGCATTCT GTGGCAGTTCGACGCGGAAACCGGTGATTTTATCTATGCGCGTAGCACCGTTGA GCAAAACCTGATTAGCAGCGTGGACGATACCGGCCTGGTTACCGTGAACGAAG CGGCGATGCCGCTGGTGCCGGACGTTGCGGTGCACATGTGCCCGACCTTTCTG GGCGGTCGTGACTGGAGCCCGACCGCGTTCAACCCGGATAGCAAGGTTATGTT TGTGCCGCTGACCAACATGTGCGGTGACCTGACCGCGCTGGATCAGGAGCCGA CCGGCCTGGACGTTTACAACACCGAAATGGCGTATAAGATGCCGGAGGGTGTG ACCGATGCGGGTCGTATCGACGCGATTGATGTTGTGACCGGTAAAACCGTTTG GAGCTACACCCAGCAAATGCCGCAGTATAGCCCGATCGTGGCGACCGCGGGC GGTCTGATTTTCACCGGCGGTGCGGACCGTAAGCTGAAAGCGATCGATCAAGA AACCGGTGAACTGGTGTGGAGCACCACCCTGGCGAGCCGTGCGAGCGGTCAC CCGATTACCTACGAAGCGAACGGCCGTCAGTTCATCGCGATTCCGGCGGGCGG TCCGGGTTTTGCGACCAACCTGATCACCGCGAGCGGTGCGACCGACGATGCGG TTAGCGGCAGCAACACCCTGTATGTGTTTGCGCTGCCGGAAACCAAATAA (SEQ ID NO:6)

Characterization of DyDepA

[0092] DyDepA, both partially purified from Devosia yakushimensis and expressed in and purified from E. coli, was confirmed, using methods similar to those described in Example 1 , to have activity to completely convert DON to 3-oxo-DON in the presence of PQQ and Ca ²⁺ . Figure 2A shows the results of experiments in which DyDepA (0.425 μg ^■ mL ^"1 ) expressed in E. coli is reacted with 100 μg ^■ mL ^~1 DON, 40 μΜ phenazine methosulfate and 1 mM CaCI ₂ in Tris buffer pH 7.5, in the absence or in the presence of 100 μΜ PQQ. As seen from the results presented in Figure 2A, DyDepA expressed and purified in the absence of PQQ (Apo-Enzyme) lacks activity to convert DON to 3-oxo-DON in the absence of exogenously added PQQ, but when the enzyme is expressed and purified in the presence of PQQ (100 μΜ PQQ was added prior to the final dilution and subsequent concentration) (Holo-Enzyme), or allowed to react with DON in the presence of exogenously added PQQ (Apo- Enzyme-PQQ; Holo-Enzyme-PQQ), complete conversion to 3-oxo-DON occurs.

[0093] DyDepA expressed in E. coli retains significant activity after lyophilization. 100 μΙ_ aliquots of purified DyDepA expressed in E. coli were frozen at -80°C, lyophilized overnight and left at room temperature for 3 days. The lyophilized sample was resuspended in 100 μΙ_ of water, and allowed to react at a concentration of 50 μg ^■ mL ^"1 with 100 μg ^■ mL ^"1 DON, 100 μΜ PQQ, 40 μΜ phenazine methosulfate, and 1 mM CaCI ₂ in Tris pH 7.5. Reactions (3 replicates) were stopped after 10 minutes and the concentration of 3-oxo-DON in the reaction mixture was measured. As seen from the results presented in Figure 2B, the lyophilized DyDepA (DyDepA- Lyophilized) retained significant activity to convert DON to 3-oxo-DON (approximately 74% compared to the activity of the unlyophilized and freshly thawed enzyme (DyDepA)).

[0094] The ability of various redox cofactors to regenerate the oxidized form of PQQ during the oxidation of DON to 3-oxo-DON was investigated. DyDepA

(0.425 μg ^■ mL ^"1 ) was allowed to react overnight with 100 μg ^■ mL ^~1 DON, 100 μΜ PQQ, and 1 mM CaCI ₂ , in Tris buffer pH 7.5 in the presence of various redox cofactors provided at a concentration of 40 μΜ. The cofactors tested were phenazine methosulfate, Coenzyme Q1 , Λ/,Λ/,Λ/',Λ/'-tetramethyl-p-phenylenediamine (TMPD), dichlorophenolindophenol (DCIP), potassium ferricyanide, glutathione (GSH), cytochrome C and ascorbic acid. As can be seen in Figure 2C, phenazine methosulfate was the most efficient cofactor tested, while TMPD and DCIP also showed effectiveness.

Example 3: DepA from Devosia epidermidihirudinis (DeDepA)

Expression of DeDepA

[0095] Analysis of the genome of Devosia epidermidihirudinis identified a candidate PQQ-dependent glucose dehydrogenase sequence, which was modified for expression in E. coli by codon-optimization, gene synthesis and cloning into pET28a as described in Example 1 . The pET28a construct was transformed into E. coli BL21 and expressed, and the resulting protein (herein designated DeDepA) was purified by Ni-NTA chromatography.

[0096] The native amino acid sequence of DeDepA is:

M KKRTS I LLASVAM LG MGSTAFAQVD I N ALPAVTDAI LAN PDAG DWPSYG RD ITN YR FSPLDQVNKDNVGQLTLAWARALEPGNLQSAPLEFGGVLFTAAPGDWQAMDAAT GQLIWEYRRQLPDRATLNSLGENKRGIALYEDKIYVATWDNFIVALDAKTGQVAWE SDRGGGADLISNTTGPIVANGVWAGSTCQFSEFGCYVTGHDAATGEELWRNNFIP KKGEEGDDTWGDSTEDQRWMTGAWGQMTYDPELDLVYYGSTGAGPAAEFQRNT VGGTLFGSNTRFAVKPKTGEIVWRHQVLPRDNWDQECTYEMVPVDIDSAPAADME G LLALGTAAPG KKRVLTG VPCKTG VMWQFDAQTG EFI YARDTVQQTLI ESVDNTG L VTVNEAAIPTEVDVATPMCPTYLGGRDWSPTAFNPTSKVMFVPLTNMCADVTVLDQ EPTGLDVYNTELTYKMPEGVTDAGRIDAINVETGKTLWSWTQQTPQYASITATAGG LIFTGGADRRFKAIDQETGELVWSVTLGSRATGHPISYEVDGRQYIAIPAGGPGYAT DLITASGSTVDWSGSNMLYVFALPEQKK (SEQ ID N0:7)

[0097] The DeDepA sequence (SEQ ID N0:7) has 81 % identity and 90% similarity to the DyDepA sequence (SEQ ID NO:4) and has 55% identity and 69% similarity to the DmDepA sequence (SEQ ID NO: 1).

[0098] The codon-optimized nucleotide sequence of the DeDepA gene modified for overexpression in E. coli is:

ATGAAGAAACGTACTTCAATCCTGCTCGCATCCGTTGCGATGCTGGGCATGGGT AG CACTG CCTTTG CACAG GTTG ACATCAATGCACTG CCTGC AGTG ACTG ATG CC ATCCTGGCAAATCCAGACGCTGGTGACTGGCCATCCTATGGCCGTGACATCAC CAACTATCGCTTCAGCCCGCTCGATCAGGTCAATAAAGACAACGTCGGCCAGCT GACCCTGGCTTGGGCCCGCGCCCTTGAGCCCGGCAACCTGCAGTCCGCACCA CTCGAGTTCGGTGGCGTGCTGTTCACCGCCGCTCCAGGCGACGTCGTGCAGG CCATGGACGCTGCGACCGGCCAGCTGATCTGGGAATACCGTCGTCAGTTGCCA GACCGCGCAACGCTCAACTCGCTCGGCGAGAACAAGCGCGGTATCGCTCTGTA TGAAGACAAGATCTACGTCGCGACCTGGGACAACTTCATCGTTGCCCTCGACGC CAAGACCGGTCAGGTCGCTTGGGAAAGCGATCGTGGCGGCGGTGCCGACCTC ATCTCCAACACCACCGGTCCAATCGTGGCCAATGGCGTTGTGGTTGCCGGTTC CACCTGCCAGTTCTCCGAGTTCGGTTGCTATGTAACCGGCCACGACGCTGCGA CCGGTGAAGAACTCTGGCGCAACAACTTCATCCCCAAGAAGGGTGAAGAAGGC GACGACACCTGGGGTGACTCGACTGAAGACCAGCGCTGGATGACCGGCGCAT GGGGTCAGATGACCTATGATCCAGAACTCGACCTGGTGTACTACGGCTCGACC GGTGCTGGCCCAGCTGCTGAATTCCAGCGCAACACCGTTGGCGGCACCCTGTT CGGTTCCAACACCCGCTTTGCTGTGAAGCCAAAGACCGGTGAAATCGTCTGGC GCCACCAGGTTCTGCCACGCGACAACTGGGATCAGGAATGCACCTATGAAATG GTGCCAGTGGATATCGACTCCGCTCCTGCTGCCGACATGGAAGGCCTGCTGGC CCTCGGCACCGCCGCTCCAGGCAAGAAGCGCGTCCTCACCGGCGTGCCTTGC AAGACCGGCGTGATGTGGCAGTTCGACGCCCAGACCGGCGAGTTCATCTATGC TCGTGACACCGTTCAGCAGACGCTGATCGAAAGCGTCGACAACACCGGCCTCG TGACCGTCAACGAAGCGGCCATCCCGACCGAAGTCGACGTCGCAACGCCAATG TGCCCGACCTACCTCGGTGGTCGTGACTGGTCGCCAACCGCGTTCAACCCAAC CAGCAAGGTGATGTTCGTCCCACTGACCAACATGTGCGCCGACGTGACCGTGC TGGACCAGGAGCCAACCGGCCTCGACGTCTACAACACCGAACTCACCTACAAG ATGCCAGAAGGCGTGACCGACGCTGGCCGTATCGACGCAATCAATGTCGAAAC CG G CAAG ACCCTGTG GAG CTGGACGCAG CAG ACCCCACAGTACG CTTCG ATCA CCGCAACCGCAGGCGGCCTGATCTTCACCGGTGGTGCTGATCGTCGCTTCAAG GCAATCGACCAGGAAACCGGCGAGCTGGTTTGGTCCGTTACCCTTGGTTCGCG CGCTACCGGCCACCCAATCAGCTACGAAGTTGATGGTCGCCAGTACATCGCGA TCCCAGCTGGTGGCCCAGGCTACGCAACCGACCTGATCACCGCTTCGGGCTCG ACCGTTGACGTCGTCTCCGGCAGCAACATGCTCTACGTCTTCGCTCTGCCAGAG CAGAAGAAGTAA (SEQ ID NO:8)

Characterization of DeDepA

[0099] DeDepA was confirmed, using methods similar to those described in Example 1 , to have activity to completely convert DON to 3-oxo-DON in the presence of PQQ and Ca ²⁺ . Example 4: DepB from Devosia mutans 17-2-E-8 (DmDepB)

Measurement of DepB activity by HPLC

[0100] Protein solution (20 μΙ_) is added to a solution with a final concentration of 3-oxo-DON of 100 μς-ηιΙ. ^"1 in Tris buffer pH 8.0. After incubation for about an hour and/or overnight at room temperature, the concentration of 3-ep/ ^' -DON is measured by HPLC. Samples and standards (20 μΙ_) are separated by a Proteo Phenomenex column (Jupiter™, 4 μηι, 250 mm χ 4.6 mm) attached to a Shimadzu uHPLC system (Mandel Scientific Company, Guelph, ON, Canada). An 1 1 minute isocratic elution consisting of 12% acetonitrile in water is used to detect DON and 3-ep/ ^' -DON followed by a column wash of 80 percent acetonitrile in water for 3 minutes and a re- equilibration of the column at 12% acetonitrile in water for 6 minutes. 3-oxo-DON is eluted using a 15 minute isocratic elution consisting of 23% acetonitrile in water followed by a column wash of 90 percent acetonitrile in water for 3 minutes and a re- equilibration of the column at 23% acetonitrile in water for 6 minutes. DON, 3-oxo- DON and 3-ep; ^' -DON are detected by monitoring the absorbance at 218 nm and quantified by comparing to a known standard (He, J.W. et al (2015) An epimer of deoxynivalenol: purification and structure identification of 3-ep; ^' -deoxynivalenol, Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment 32: 1523-1530).

Partial Purification

[0101] A cell lysate of Devosia mutans 17-2-E-8 was prepared under conditions similar to those described in Example 1 . Specifically, a culture of D. mutans 17-2-E-8 was grown for two weeks at 28°C in LB medium supplemented with 34 μg ^■ mL ^~1 of kanamycin while shaking at 200 RPM. This starter culture was used to inoculate 4 L of LB medium which was incubated for six days at 28°C while shaking at 150 RPM. The culture was then centrifuged at 8000 g for 30 minutes to pellet the cells. The cell pellets were then frozen at -20°C.

[0102] Frozen pellets were thawed and cells resuspended in 50 mM Tris, pH 8.0 with 150 mM NaCI. The cells were then lysed by sonication using a QSONICA™ Q500 sonicator (Qsonica LLC, Newtown, CT, USA) while on ice. Sonication parameters were as follows: 30% amplitude, 30 seconds on and 50 seconds off for 20 cycles. The cells were allowed to cool for 10 minutes on ice before being subjected to another 20 cycles of sonication. The lysed cells were then centrifuged at 7142 g for 30 minutes to remove the insoluble fraction. The supernatant was filtered through a 0.45 μηι syringe filter and Halt™ Protease Inhibitor Cocktail, (Thermo Fisher, Mississauga, Canada) without EDTA, was added. [0103] The lysate was tested for DepB activity as described above. Compared to a control reaction where no 3-ep; ^' -DON was detected, the sample had a peak appear corresponding to the 3-ep; ^' -DON standard and a corresponding decrease in the amount of DON.

[0104] DmDepB was partially purified from the lysate using methods similar to those described in Example 1 . The active fraction of the lysate was salted out using ammonium sulfate precipitation. Briefly, the ammonium sulfate concentration was brought to 30% of saturation and insoluble proteins were removed by centrifugation at 7142 g for 20 minutes. The ammonium sulfate concentration was brought to 50% of saturation and the insoluble fraction was collected by centrifugation at 7142 g for 20 minutes. This was repeated for 70% saturation. Each of the above fractions was subjected to dialysis using Slide-A-Lyzer™ mini dialysis device 3.5K molecular weight cut-off (MWCO) (ThermoFisher), to remove the excess salt, and tested for DepB activity as described above. The active fraction was then subjected to a heat treatment at 60°C for 15 minutes. The insoluble protein was removed by

centrifugation at 7142 g.

[0105] The dialyzed active fraction obtained after ammonium sulfate precipitation and heat treatment was subjected to anion exchange column chromatography (AKTAprime™ plus with a HiTrap™ Q FF column (GE Healthcare)) and monitored at 280 nm. The sample was loaded onto the column which had been equilibrated with 20 mM Tris (Buffer A). The column was then rinsed with 8 mL of Buffer A and proteins were eluted by a linear gradient from 0% Buffer B (20 mM Tris, 1 M NaCI) to 100% over 40 mL. Fractions containing the highest activity were pooled,

concentrated by ultrafiltration using an Amicon™ stirred cell with an Ultracel™ 10 kDa ultrafiltration disc (Millipore). The sample was then loaded onto a HiPrep™ 16/60 Sephacryl S-200 HR size exclusion column and eluted over 140 minutes at 1 mL per minute with 20 mM Tris and 300 mL NaCI. Fractions were tested for DepB activity and the most active fractions were pooled and concentrated by ultrafiltration.

Analysis of fractions by SDS-PAGE revealed a band of approximately 38 kDa, the intensity of which was positively correlated with DepB activity. DepB activity in the partially purified samples was found to be stable for at least one month at 4°C and after a heat treatment of 60°C.

Protein sequencing and expression of DmDepB

[0106] Pooled active fractions from the size exclusion chromatography step described above were sent for protein sequencing at the Advanced Analysis Centre at the University of Guelph (Guelph, Ontario, Canada). Sequencing results showed that the pooled active fractions contained 101 proteins, 70 of which contained at least 2 unique peptide sequences. An analysis of the protein sequencing results identified six potential candidate proteins predicted to use NAD(P)H as a cofactor and having a molecular weight of approximately 38 kDa. The subcellular localization of the proteins was predicted using PSORTb 3.0 as described in Example 1 , and all candidates were predicted to be cytosolic.

[0107] The DNA sequences of the candidate genes were codon-optimized for overexpression in E. coli, synthesized and cloned into the bacterial expression vector pET28a using Ndel and BamHI restriction sites to produce proteins with N-terminal polyhistidine tags. Each of the candidate genes in pET28a was transformed separately into E. coli BL21 . E. coli BL21 containing each construct was propagated at 37°C in 500 mL of LB medium supplemented with 34 mg- L ¹ kanamycin while shaking at 175 rpm. When the cultures reached an optical density of approximately 0.7, 150 μΜ of isopropyl β-D-l -thiogalactopyranoside (IPTG) was added to initiate protein expression. The cultures were incubated overnight at 18°C and harvested by centrifugation at 8000 g for 10 minutes and frozen at -20°C. Each pellet was thawed and resuspended in 5 mL of 50 mM Tris, pH 8.0, 150 mM NaCI. The cells were then lysed by sonication using the following parameters: 30% amplitude with a cycle of 30 seconds on, 40 seconds off for 8 cycles. Lysates were centrifuged at 7142 g for 30 minutes to remove the insoluble pellet and filtered through a 0.45 μηι filter.

[0108] The clarified lysates (approximately 5 mL) were then mixed with an equal volume of 50 mM sodium phosphate with 300 mM NaCI and 20 mM imidazole, pH 8.0 (wash buffer) and incubated with 300 μί of HisPur™ Ni-NTA resin

(ThermoFisher) for one hour. Each mixture was then briefly centrifuged and the supernatant was removed from the tubes. The beads were resuspended in 1 mL of wash buffer and transferred to 1 .5 mL tubes. The tubes were briefly spun, supernatant removed and resuspended with 1 mL of wash buffer. This was repeated for a total of 5 washes. The proteins which bound to the beads were eluted with 1 mL of 50 mM sodium phosphate with 300 mM NaCI and 250 mM imidazole (elution buffer), pH 8.0 and the beads removed by centrifugation.

[0109] The expressed proteins were then each tested for DepB activity by separately incubating purified protein (20 μί) with a mixture containing 400 μΜ NADPH and 100 μg ^■ mL ^"1 3-oxo-DON in 50 mM Tris, pH 8.0. The reduction in absorbance at 340 nm was monitored using an Ultrospec™ 3100 pro UV/Vis Spectrophotometer (GE Healthcare/Amersham Biosciences). Activity was confirmed by HPLC analysis as described above. [01 10] The only candidate protein found to have the ability to transform 3-oxo-DON to 3-ep; ^' -DON was the protein identified as fig |6666666.163324. CDS.25 (GenBank accession number KFL28068.1), herein designated DmDepB. DmDepB was subsequently expressed in E. coli and purified by a procedure with the following modifications from that described above. Two 1 L cultures were grown

simultaneously and the pellets pooled. 15 sonication cycles were used to lyse cells. The clarified lysate was mixed with an equal volume of wash buffer and incubated with one mL of Ni-NTA agarose for an hour at 4°C. The mixture was then poured into a Flex-Column™ (Kimble Chase Life Science, Vineland, NJ, USA) and washed with 40 mL of wash buffer. DmDepB was eluted with eight mL of elution buffer. The buffer was changed by repeated dilutions in the stirred cell to 50 mM Tris, 150 mM NaCI, pH 8.0. Aliquots of the purified enzyme were stored at -80°C.

[01 1 1] The native amino acid sequence of DmDepB is:

MEYRKLGNSGTWTSYCLGTMTFGQETDEATSHLIMDDYIKAGGNFIDTANVYSAG VSEEIVGRWLKARPSEARQVWATKGRFPMGAGPNDLGLSRTNLNRALNDSLRRL GVEQIDLYQMHAWDAVTPIEETLRFLDDAVSAGKIAYYGFSNYLGWQVTKAVHVAR ANHWTAPVTLQPQYNLLVRDIEHEIVPACQDAAMGLLPWSPLGGGWLAGKYQRDV MPSGATRLGENPNRGMESYGPRNAQERTWQIIDMVAEIAKERGVSAAQVALAWW ARPAVTAVILGARTREQLADNLGAVAVTLSTEEMERLNRVSAPAMADYPYGERGVS QRHRKMDGGR (SEQ ID NO:9)

[01 12] The codon-optimized nucleotide sequence of the DmDepB gene modified for overexpression in E. coli is:

ATGGAGTACCGTAAGCTGGGCAACAGCGGTACCGTGGTTACCAGCTATTGCCT GGGCACCATGACCTTCGGTCAGGAAACCGACGAAGCGACCAGCCACCTGATCA TGGACGATTACATTAAAGCGGGTGGCAACTTTATCGATACCGCGAACGTTTATA GCGCGGGTGTGAGCGAGGAAATTGTTGGCCGTTGGCTGAAGGCGCGTCCGAG CGAGGCGCGTCAAGTGGTTGTGGCGACCAAAGGTCGTTTCCCGATGGGCGCG GGTCCGAACGACCTGGGCCTGAGCCGTACCAACCTGAACCGTGCGCTGAACGA TAGCCTGCGTCGTCTGGGTGTGGAACAGATCGACCTGTACCAAATGCACGCGT GGGATGCGGTTACCCCGATTGAGGAAACCCTGCGTTTCCTGGACGATGCGGTG AG CG CG G GCAAG ATCG CGTACTATG GTTTTAG CAACTACCTG GG CTG G CAG GT TACCAAAGCGGTTCACGTGGCGCGTGCGAACCACTGGACCGCGCCGGTGACC CTGCAGCCGCAATATAACCTGCTGGTGCGTGACATCGAGCACGAAATTGTTCCG GCGTGCCAGGATGCGGCGATGGGTCTGCTGCCGTGGAGCCCGCTGGGTGGCG GTTGGCTGGCGGGCAAGTACCAACGTGACGTTATGCCGAGCGGTGCGACCCGT CTGGGCGAGAACCCGAACCGTGGCATGGAAAGCTATGGTCCGCGTAACGCGCA GGAGCGTACCTGGCAAATCATTGATATGGTGGCGGAGATCGCGAAAGAACGTG GTGTGAGCGCGGCGCAGGTTGCGCTGGCGTGGGTTGTGGCGCGTCCGGCGGT TACCGCGGTGATTCTGGGTGCGCGTACCCGTGAACAACTGGCGGACAACCTGG GTGCGGTTGCGGTGACCCTGAGCACCGAGGAAATGGAGCGTCTGAACCGTGTT AGCGCGCCGGCGATGGCGGACTACCCGTATGGCGAACGTGGTGTGAGCCAAC GTCACCGTAAGATGGATGGCGGTCGTTAA (SEQ ID NO:10) Activity of DmDepB

[01 13] DmDepB is active to completely convert 3-oxo-DON to 3-ep/ ^' -DON under the conditions for measurement of DepB activity by HPLC described above, as evidenced by the data in Table 2.

Table 2: Concentration of 3-oxo-DON and 3-ep/-DON after treatment with DmDe B.

*As s, the exact amounts may vary from those reported (i.e. a reported amount of 10 mg in a bottle may in fact be anywhere from 9-1 1 mg unless annotated as an analytical standard).

Buffer and pH specificity

[01 14] The activity of DmDepB was tested in several common buffers (Tris

(fr/ ^' s(hydroxymethyl)aminomethane), HEPES (4-(2-hydroxyethyl)-1-piperazine- ethanesulfonic acid), ammonium bicarbonate, sodium phosphate and MOPS (3-(/V- morpholino)propanesulfonic acid)). Reaction mixtures contained 50 mM of buffer, 100 μg ^■ mL- ¹ 3-oxo-DON, 400 μΜ NADPH, and 7 μg DmDepB. Each reaction was allowed to proceed for 10 minutes before quenching with acidified methanol and measuring conversion of 3-oxo-DON to 3-ep/ ^' -DON. As seen from the results presented in Figure 3A, there was no significant difference in the relative activity of DmDepB between the buffers, with the exception of ammonium bicarbonate, in which activity was found to be significantly lower than in the HEPES buffer.

[01 15] In addition, the activity of DmDepB was assessed at pH values from 5-9. Reaction mixtures contained 50 mM of a three component buffer (0.1 M Tris, 0.05 M acetic acid, and 0.05 M 2-(N-morpholino)ethanesulfonic acid) at various pH values, 100 μg ^■ mL ^■1 3-oxo-DON, 400 μΜ NADPH, and 4.7 μg DmDepB. The reaction was allowed to proceed for 15 minutes before quenching with acidified methanol and measuring conversion of 3-oxo-DON to 3-ep/ ^' -DON. As seen from the results presented in Figure 3B, the highest conversion was observed at a pH of 7.5.

However, appreciable activity was observed at a relatively broad range of pH values around neutrality. Cofactor specificity

[01 16] DmDepB was allowed to react with 3-oxo-DON (100 μς- ηιΙ. ^"1 ) in 50 mM Tris buffer at pH 7 in the presence or absence of NADPH (20 mM) or of a commercial regeneration solution (NADPH Regeneration System (V9510), Promega) which uses enzymes to regenerate NADPH. As seen from the results presented in Table 3, in the absence of NADPH, no conversion of 3-oxo-DON to 3-ep/ ^' -DON was observed for DmDepB. In addition, the presence of NADPH is sufficient for activity of DmDepB.

Table 3: NADPH usage by DmDepB.

*As the standards used to determine the concentrations are not analytical standards, the exact amounts may vary from those reported (i.e. a reported amount of 10 mg in a bottle may in fact be anywhere from 9-1 1 mg unless annotated as an analytical standard).

[01 17] The ability of DmDepB to use NADH as a cofactor was also investigated. Reaction mixtures contain 50 mM of Tris pH 7.5, 100 μg ^■ mL ^"1 3-oxo-DON, 400 μΜ NADPH or NADH, NADP ⁺ or NAD ⁺ and 4.7 μg DmDepB. The reaction is allowed to proceed for 15 minutes or overnight before quenching with acidified methanol.

Reactions were carried out in triplicate and the amount of 3-ep/ ^' -DON produced was assessed by HPLC as described above. As seen from the results presented in Figure 3C, after a 15 minute incubation, only reactions containing NADPH contained 3-ep/ ^' - DON, although a small amount of 3-ep/ ^' -DON was detected after the NADH reaction was left overnight (approximately 16 hours). Thus, catalysis is approximately 5000 times less efficient with NADH than with NADPH. Reactions with NAD ⁺ or NADP ⁺ failed to produce 3-ep/ ^' -DON.

Stability

[01 18] The stability of DmDepB, and the activity of the enzyme to catalyze the conversion of 3-oxo-DON to 3-ep; ^' -DON were investigated over a range of temperatures. Aliquots of DmDepB were incubated at a specific temperature between 30°C and 70°C for one hour and cooled on ice. Heat treated DmDepB and untreated DmDepB (maintained at 4°C) were tested for activity at room temperature (23°C ± 2°C). In addition, the activity of DmDepB was measured at temperatures from 20°C to 60°C (5°C intervals). In each case, reaction mixtures for measurement of activity contained 100 g- mL ^" 3-oxo-DON, 400 μΜ NADPH and 5 μg DmDepB in 50 mM of Tris buffer pH 7.5. Reactions were allowed to proceed for 7.5 and 15 minutes before quenching by addition of acidified methanol. Reactions were carried out in triplicate and the amount of 3-ep; ^' -DON produced was assessed by HPLC.

[01 19] As seen from the results presented in Figure 3D, DmDepB remained stable and maintained its activity when incubated for one hour at temperatures from 30°C to 55°C. However, incubation at 60°C reduced activity and incubation at temperatures of 65°C and higher abolished activity. Furthermore, as seen from the results presented in Figure 3E, the activity of DmDepB was highest at 30°C and 35°C, and undetectable at 60°C.

[0120] In separate experiments, frozen aliquots of DmDepB were lyophilized and stored at room temperature for one week. Lyophilized samples were resuspended in water and assessed for activity at 25°C, compared to the activity of DmDepB samples which had been freshly removed from storage at -80°C and thawed on ice. In each case, reaction mixtures for measurement of activity contained 100 μg ^■ mL ^"1 3-oxo-DON, 400 μΜ NADPH and 4.7 μg DmDepB in 50 mM of Tris buffer pH 7.5. Reactions were allowed to proceed for 12 minutes before quenching by addition of acidified methanol. Reactions were carried out in triplicate and the amount of 3-ep; ^' - DON produced was assessed by HPLC. As seen from the results presented in Figure 3F, there was no significant change in the activity of DmDepB after lyophilization.

Example 5: DepB from Rhizobium leguminosarum (R/DepB)

[0121] Analysis of the genome of Rhizobium leguminosarum identified a gene having a sequence similar to that of DmDepB. The gene was expressed in E. coli and purified using methods similar to those described in Example 4 to provide an active protein, herein designated R/DepB.

[0122] The native amino acid sequence of R/DepB is:

MDYRKLGPSGTWTAYCLGTMTFGAEADEAASHKLLDDYFAWGGNFIDTADVYSA GKSEEIIGRWLKARPTEARQAIVATKGRFPMGNGPNDIGLSRRHLSQALDDSLRRL GLEQIDLYQMHAWDALTPIEETLRFLDDAVSSGKIGYYGFSNYVGWHIAKASEIAKA RGYTRPVTLQPQYNLLMRDIELEIVAACQDAGMGLLPWSPLGGGWLTGKYKRDEM PTGATRLGENPNRGGESYAPRNAQERTWAIIGTVEEIAKARGVSMAQVALAWTAA RPAITSVILGARTPEQLADNLGAMKVELSGEEMARLNEVSAPQPLDYPYGKGGINQ RHRKIEGGR (SEQ ID NO:1 1)

[0123] SEQ ID NO: 1 1 has 78% identity to SEQ ID NO:9.

[0124] The codon-optimized nucleotide sequence of the RDepB gene modified for overexpression in E. coli is: ATGGACTATCGTAAACTGGGCCCGAGCGGCACCGTTGTGACCGCGTATTGCCT GGGTACCATGACCTTTGGTGCGGAAGCGGACGAGGCGGCGAGCCACAAGCTG CTG G ACG ATTACTTCG CGTGG G GTG G CAACTTTATCG ACACCG CG G ATGTGTAT AGCGCGGGCAAGAGCGAGGAAATCATTGGCCGTTGGCTGAAAGCGCGTCCGA CCGAGGCGCGTCAGGCGATTGTTGCGACCAAAGGTCGTTTCCCGATGGGCAAC GGTCCGAACGACATTGGCCTGAGCCGTCGTCACCTGAGCCAAGCGCTGGACGA TAGCCTGCGTCGTCTGGGTCTGGAACAGATCGACCTGTACCAAATGCACGCGT GGGATGCGCTGACCCCGATTGAGGAAACCCTGCGTTTCCTGGACGATGCTGTG AG CAGCGG CAAG ATTG GTTACTATG G CTTTAG CAACTATGTTGGTTG G CACATC GCGAAGGCGAGCGAGATTGCGAAAGCGCGTGGCTACACCCGTCCGGTGACCC TGCAGCCGCAATATAACCTGCTGATGCGTGACATCGAGCTGGAAATTGTTGCGG CGTGCCAGGATGCGGGTATGGGCCTGCTGCCGTGGAGCCCGCTGGGTGGCGG TTGGCTGACCGGCAAGTACAAACGTGATGAAATGCCGACCGGTGCGACCCGTC TGGGCGAGAACCCGAACCGTGGCGGTGAAAGCTATGCGCCGCGTAACGCGCA GGAACGTACCTGGGCGATCATTGGTACCGTGGAGGAAATTGCGAAGGCGCGTG GCGTGAGCATGGCGCAAGTTGCGCTGGCGTGGACCGCGGCGCGTCCGGCGAT CACCAGCGTTATTCTGGGTGCGCGTACCCCGGAACAGCTGGCGGACAACCTGG GTGCGATGAAAGTGGAGCTGAGCGGCGAGGAAATGGCGCGTCTGAACGAAGTT AGCGCGCCGCAACCGCTGGATTACCCGTATGGCAAAGGCGGCATTAACCAACG TCATCGTAAGATTGAAGGCGGTCGCTAA (SEQ ID NO:12)

[0125] The activity of R/DepB to convert 3-oxo-DON to 3-ep/ ^' -DON was measured in a reaction mixture containing 400 μΜ NADPH, 100 g-rnL ^" 3-oxo-DON and 3.1 μg of R/DepB. The amount of 3-ep/ ^' -DON produced was assessed by HPLC, as described in Example 4. As seen from the results presented in Figure 4, R/DepB expressed heterologously in E. coli is active to convert 3-oxo-DON to 3-ep/ ^' -DON, both in the crude cell lysate and when purified.

Example 6: Conversion of deoxynivalenol (DON) to 3-ep/-deoxynivalenol (3-ep/- DON

[0126] A reaction mixture containing 34 μg ^■ mL ^"1 DyDepA, 100 μg ^■ mL ^"1 DON, PQQ in a quantity sufficient to saturate the enzyme, 4 μΜ phenazine methosulfate and 1 mM CaCI ₂ in Tris buffer at pH 7.5 was incubated at room temperature for 2.75 hours. 30 μg ^■ mL ^"1 DmDepB and 1 mM NADPH was added to the mixture and incubation was continued overnight (approximately 18 hours). As seen from the results presented in Figure 5, the final reaction mixture contained about 33% 3-oxo-DON and about 67% 3-ep; ^' -DON. No traces of DON were found in the final mixture.

Example 7: Epimerization of 15-acetyldeoxynivalenol (15-AcDON)

[0127] It is considered that other trichothecene mycotoxins in addition to

deoxynivalenol may act as substrates for epimerization in the DepA/B pathway. Thus, the activity of DepA and Dep B enzymes was investigated when

15-acetyldeoxynivalenol (15-AcDON) was used as a substrate, as shown below.

15-AcDON 3-0X0-15- AcDON 3-ep;-15-AcDON

Transformation of 15- cDON to 3-oxo-15- AcDON by DyDepA

[0128] DyDepA (Example 2) was allowed to react overnight with 15-AcDON in a reaction mixture containing 100 μg ^■ mL ^~1 15-AcDON, 100 μΜ PQQ, 40 μΜ phenazine methosulfate, 1 mM CaC and 50 μg ^■ mL ^~1 DyDepA in Tris buffer pH 7.5. Because an HPLC standard is not commercially available for 3-oxo-15-AcDON, progress of the reaction was monitored by LCMS/MS, using a Vanquish™ UHPLC system coupled to a Q Exactive™ Orbitrap™ mass spectrometer (ThermoFisher Scientific). The column used was a ZORBAX Eclipse Plus C18 Rapid Resolution HD 2.1x100 mm 1.8 micron column, eluted with mobile phases A (H ₂ 0 containing 0.1 % formic acid) and B (acetonitrile containing 0.1 % formic acid) at a flow rate 0.300 mL min ¹ . The percentage of B is increased from 5% to 35% over 5 minutes, maintained at 35% for 2 minutes, increased to 70% over 2.5 minutes, increased to 90% over 0.5 minutes, maintained at 90% for 2 minutes, decreased to 5% over 1 minute and maintained at 5% for 6 minutes.

[0129] A compound eluting at a retention time of 7.963 minutes was identified as 3-OXO-15-AcDON by its mass spectral data. 3-oxo-15-AcDON shows a molecular ion (M+H) at the expected mass of 337.12 Da and a fragmentation pattern similar to that of 3-oxo-DON, but having some peaks shifted by an additional mass of 42 Da, attributable to the additional acetyl group in 3-oxo- 15- AcDON. Quantification of 3-OXO-15-AcDON was carried out using the transition from 337.12 to137.0594 at a collision energy of 30 eV. As seen from the results presented in Figure 6, 15-AcDON was quantitatively converted to 3-oxo- 15-AcDON.

Transformation of 3-oxo- 15- AcDON to 3-epi- 15- AcDON by DmDepB

[0130] A reaction mixture containing 100 μg ^■ mL ^"1 15-AcDON, 100 μΜ PQQ, 40 μΜ phenazine methosulfate, 1 mM CaCh and 50 μg ^■ mL ^~1 DyDepA in Tris buffer pH 7.5 was allowed to react for 3 hours and an aliquot was removed. 50 μg ^■ mL ^~1 DmDepB (Example 4) and 1 mM NADPH were added to the reaction mixture and incubation was continued overnight. Again, because HPLC standards are not commercially available for 3-oxo-15-AcDON or 3-ep; ^' -15-AcDON, progress of the reaction was monitored by LCMS/MS as described above. A compound eluting at a retention time of 5.563 minutes was identified as 3-ep/ ^' -15-AcDON by its mass spectral data. By comparison, 15-AcDON, which has the same mass, elutes at a retention time of 5.910 minutes under the same conditions. The compound showed a molecular ion (M+H) at the expected mass of 339.14 Da and a fragmentation pattern similar to that of 15-AcDON, but showing fragment peaks at different intensities.

[0131] The embodiments described herein are intended to be illustrative of the present compositions and methods and are not intended to limit the scope of the present invention. Various modifications and changes consistent with the description as a whole and which are readily apparent to the person of skill in the art are intended to be included. The appended claims should not be limited by the specific embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.