CN108251385B - Ochratoxin degrading enzyme, encoding gene, recombinant vector, cell, additive and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to ochratoxin degrading enzyme, an encoding gene, a recombinant vector, a cell, an additive and application thereof, and more particularly relates to ochratoxin degrading enzyme which has the amino acid sequence shown in SEQ ID NO: 2 or a mutant thereof, a gene encoding the enzyme, a vector and a cell containing the encoding gene, an additive containing the enzyme and/or the cell and/or a fermentation product thereof, the use of the enzyme, the encoding gene, the vector, the cell or the additive in degrading ochratoxin and/or other mycotoxins, and a method for degrading ochratoxin and/or other mycotoxins. The invention has the following advantages: the method is environment-friendly, can efficiently and temporarily degrade ochratoxin, and does not produce any harmful by-product. The enzyme can tolerate high-temperature catalysis up to 80 ℃, has low requirement on pH value and good stability, and can degrade fumonisins and T2 toxins to a certain degree.

Description

Ochratoxin degrading enzyme, encoding gene, recombinant vector, cell, additive and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to ochratoxin degrading enzyme, a coding gene thereof, a recombinant vector and a recombinant cell containing the coding gene, an additive containing the degrading enzyme and/or the recombinant cell and/or a fermentation product thereof, application of the degrading enzyme, the coding gene of the degrading enzyme, the recombinant vector, the recombinant cell or the additive in degrading ochratoxin and/or other mycotoxins, and a method for degrading ochratoxin and/or other mycotoxins.

Background

Mycotoxins are secondary metabolites generated by toxic fungi in the harm process, and mainly comprise aflatoxin, deoxynivalenol (also called vomitoxin, DON), Zearalenone (ZEN), Fumonisin (FUM), ochratoxin, T-2 toxin and the like. And ochratoxin is another mycotoxin which attracts wide attention in the world after aflatoxin. It is an important mycotoxin that contaminates food.

Ochratoxins, a mycotoxin produced by aspergillus ochraceus (aspergillus ochraceus) and penicillium viridum (penicillium viricanum), are classified into two types, a and B, with a high toxicity. The ochratoxin with toxic effect concentration can be produced by using aspergillus ochraceus at 4 deg.C. Animals ingested 1ppm body weight dose of ochratoxin a can be lethal in 5-6 days. Common lesions are tubular epithelial injury and lymphatic necrosis of the intestinal tract. Feeding ration containing 1ppm ochratoxin for 3 months can cause animal polydipsia, frequent micturition, growth retardation and feed utilization rate reduction; kidney damage can be detected for weeks when fed diets as low as 200 ppb. Other clinical symptoms are diarrhea, anorexia and dehydration. Sometimes clinical symptoms are not evident, and in areas where ochratoxin intoxication is endemic, the only observable lesions at slaughter are pale, hard kidneys.

Marquardt et al (1992) have shown that the feed containing OTA in the amount of 0.3-16mg/kg can cause poisoning of livestock and poultry, and the death rate is increased by 2% -58%. Madsen et al (1982) reported that continuous feeding of feed containing OTA at 200. mu.g/kg for 4 months had little effect on pigs, whereas when OTA content was greater than 1400. mu.g/kg, the feed intake and growth rate of pigs were significantly reduced and water intake increased. Huff et al (1974) reported that continuous feeding of feed containing 0.5-1.0mg/kg OTA for 3 weeks had no effect on the weight gain of broiler chickens, while continuous feeding of feed containing 0.5mg/kg OTA for 6 weeks decreased the egg laying performance and feed conversion rate of layer chickens.

Hult et al (1976) reported that Rui Tu stipulates that OTA in a mixed feed for swine and poultry must not exceed 200. mu.g/kg and 1000. mu.g/kg, respectively. Relevant regulations are also being enacted in the united states. Other countries have not seen regulations on the amount of OTA allowed. GB 2761 + 2011 stipulates that the allowable amount of OTA in grains, beans and products thereof is not more than 5 mu g/kg.

At present, for removing mycotoxin, the common methods at home and abroad mainly comprise physical removal, adsorption, chemical treatment and the like. The adsorbent adsorbs a large amount of micronutrients in the feed and food while adsorbing toxins, and the toxins adsorbed by the clay cannot be decomposed, so that secondary pollution is caused.

The biological degradation can efficiently convert the toxin into a non-toxic product, and is environment-friendly and safe. The method has become the treatment technical approach with the most development prospect in the current mycotoxin reduction technology, is safe, environment-friendly and efficient, and is in line with the development trend of energy conservation and emission reduction in China by using modern biotechnology to develop research. Currently, the method for producing ethanol by fermenting grain polluted by mycotoxin as a raw material is one of the most main means, but the toxin is further concentrated in a large amount of byproduct vinasse obtained by fermentation production, and the toxin greatly exceeds the national limit standard and cannot be utilized.

However, the existing biological removal method for ochratoxin has low removal efficiency, long degradation time, or poor thermal stability and acid-base stability, so that a method capable of saving energy, protecting environment and efficiently degrading ochratoxin is urgently needed to be developed.

Disclosure of Invention

The present invention has been made to overcome the above-mentioned drawbacks of the prior art, and an object of the present invention is to provide an enzyme capable of degrading ochratoxin with high efficiency for a short time, and having improved thermal stability and acid and alkali resistance.

In order to achieve the above objects, the present invention provides an ochratoxin-degrading enzyme in a first aspect, wherein the degrading enzyme has an amino acid sequence as shown in the following (a) and/or (b):

(a) SEQ ID NO: 2;

(b) SEQ ID NO: 2, wherein one or more of amino acid residues 11, 89, 179 and 239 in the amino acid sequence shown in the specification still has ochratoxin degradation enzyme activity after substitution, deletion or addition.

In a second aspect, the present invention also provides a gene encoding an ochratoxin-degrading enzyme, wherein the gene has a nucleotide sequence encoding an ochratoxin-degrading enzyme as described above.

In a third aspect, the present invention also provides a recombinant vector, wherein the recombinant vector contains the gene as described above.

In a fourth aspect, the present invention also provides a recombinant cell, wherein the recombinant cell contains the gene or the recombinant vector as described above.

In a fifth aspect, the present invention also provides an additive, wherein the additive comprises a degrading enzyme as described above and/or comprises a recombinant cell as described above and/or a fermentation product thereof.

In a sixth aspect, the present invention also provides the use of a degrading enzyme as described above, a gene as described above, a recombinant vector as described above, a recombinant cell as described above and/or a fermentation product thereof or an additive as described above for degrading ochratoxins and/or other mycotoxins.

In a seventh aspect, the present invention also provides a method of degrading ochratoxins and/or other mycotoxins, wherein the method comprises: the degrading enzyme as described above, the recombinant cell as described above and/or its fermentation product or the additive as described above is contacted with the sample to be treated under the conditions of the enzymatic degradation reaction.

The invention has the following advantages: the method is environment-friendly, can efficiently and temporarily degrade ochratoxin, and does not produce any harmful by-product. The ochratoxin degrading enzyme provided by the invention can tolerate high temperature up to 80 ℃, has low requirement on pH value and has good stability. In addition, the ochratoxin degrading enzyme of the invention can degrade fumonisins and T2 toxin to a certain extent.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the present invention provides an ochratoxin-degrading enzyme, wherein the degrading enzyme has an amino acid sequence as shown in (a) and/or (b):

(a) SEQ ID NO: 2;

(b) SEQ ID NO: 2, and one or more of the amino acid residues at 11 th, 89 th, 179 th and 239 th positions in the amino acid sequence shown in the figure 2 still has degrading enzyme activity after being substituted, deleted or added.

The 20 amino acid residues constituting a protein can be classified into four types according to the side chain polarity: 1. non-polar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro); 2. polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), aspartic acid (Asn), glutamine (Gln) and tyrosine (Tyr); 3. positively charged amino acids: arginine (Arg), lysine (Lys), and histidine (His); 4. negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "biochemistry" (second edition) on the book, shengdi, wang spec rock, pages 82-83, higher education press, 1990, 12 months). If the substitution of amino acid residues belonging to the same class, for example, substitution of Arg for Lys or Leu for Ile, occurs in the protein, the role of the residues in the protein domain (e.g., the role of providing positive charge or forming a hydrophobic pocket structure) is not changed, and thus the steric structure of the protein is not affected, and thus the function of the protein can still be achieved. The substitution of an amino acid residue in the same class may occur at any amino acid residue position of the above enzymes.

As mentioned above, the enzymes provided by the present invention may also be modified or mutated to obtain the derived proteins. The "derived protein" of the present invention means a protein having a difference in amino acid sequence from the enzyme having the above amino acid sequence, and may have a difference in modified form which does not affect the sequence, or both. These proteins include natural or induced genetic variants. The induced variants may be obtained by various techniques, such as random mutagenesis by irradiation or mutagenic agents, etc., or by techniques such as site-directed mutagenesis or other known molecular biology techniques. The "derived proteins" also include analogs having residues of natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta-amino acids, gamma-amino acids, etc.).

Modifications (which do not generally alter primary structure, i.e., do not alter amino acid sequence) include: chemically derivatized forms of the protein such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those proteins that result from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility. In the present invention, however, N-and/or O-glycosylation modification at any one of asparagine (Asn), serine (Ser) and threonine (Thr) residues of the degrading enzyme can further improve solubility and thermostability.

For ease of purification, additional modifications of (a) or (b) may also be made using tags commonly used in the art, such as at least one of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc. The label does not influence the activity of the enzyme provided by the invention, and whether the label is added or not can be selected according to requirements in the practical application process.

Preferably, the degrading enzyme has the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

In a second aspect, the present invention also provides a gene encoding ochratoxin-degrading enzyme, wherein the gene has a nucleotide sequence encoding the degrading enzyme.

It is well known in the art that 18 amino acids, of the 20 different amino acids that make up a protein, are each encoded by 2-6 codons, except for Met (ATG) or Trp (TGG), which are each encoded by a single codon (Sambrook et al, molecular cloning, Cold spring harbor laboratory Press, New York, USA, second edition, 1989, see appendix D page 950). That is, due to the degeneracy of genetic code, there is usually more than one codon determining one amino acid, and the substitution of the third nucleotide in the triplet codon will not change the composition of the amino acid, so that the nucleotide sequences of genes encoding the same protein may differ. From the amino acid sequences disclosed in the present invention and the amino acid sequences obtained from the amino acid sequences without the change in the enzyme activity, it is fully possible for those skilled in the art to derive the nucleotide sequences of the genes encoding them, which are obtained by biological methods (e.g., PCR method, mutation method) or chemical synthesis methods, based on the well-known codon tables, and therefore, the partial nucleotide sequences should be included in the scope of the present invention.

Preferably, the gene has the sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: 5.

As described above, the 5 'end and/or 3' end of the nucleotide sequence may be linked to a coding sequence for a tag commonly used in the art (e.g., at least one of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc), respectively.

The nucleotide sequence provided by the invention can be obtained by a Polymerase Chain Reaction (PCR) amplification method, a recombination method or an artificial synthesis method. For example, one skilled in the art can easily obtain templates and primers based on the nucleotide sequences provided by the present invention, and obtain the relevant sequences by PCR amplification.

Once the nucleotide sequence of interest is obtained, the amino acid sequence of interest can be obtained in large quantities by recombinant methods. The nucleotide sequence obtained is usually cloned into a vector and transferred into a host, and the relevant nucleotide sequence is isolated from the propagated host cells by a conventional method.

In a third aspect, the present invention also provides a recombinant vector, wherein the recombinant vector contains the above gene.

In the present invention, the recombinant vector may contain the above-mentioned gene provided by the present invention. As the "vector" used in the recombinant vector, various vectors known in the art, such as various commercially available plasmids, cosmids, phages, retroviruses and the like can be used, and plasmids are preferably used as the vector in the present invention. The recombinant vector can be constructed by digesting a linear plasmid with various endonucleases having a cleavage site at the multiple cloning site of the vector (for example, Sal I, BamH I, EcoR I and the like can be used for pUC 18; Nde I, Nhe I, EcoR I, BamH H, HindIII and the like can be used for pPICZ alpha A; and BamH I, HindIII, Nde I, Xho I and the like can be used for PET30 a) and ligating the linear plasmid with a gene fragment cleaved with the same endonuclease to obtain a recombinant plasmid. The invention preferably adopts Nde I and Xho I double enzyme digestion PET30a vector and gene fragment connected with the vector, and the recombinant vector is constructed by ligase connection.

In a fourth aspect, the present invention also provides a recombinant cell, wherein the recombinant cell contains the gene or the recombinant vector.

In the present invention, the cell may be a cell containing the gene of the present invention, or a recombinant cell obtained by transforming, transducing or transfecting the above recombinant vector into a host cell by a method conventional in the art, such as calcium chloride method, chemical transformation or electroporation transformation, preferably electroporation transformation.

In a preferred embodiment of the invention, the cell is a spore. It will be understood that the term "spore" as used herein refers to a fungal or bacterial spore, endospore or exospore.

The cell according to the invention may be any suitable cell. More preferably, any suitable bacterial, fungal or plant cell. Even more preferably, the cell is selected from escherichia coli, Streptomyces (Streptomyces), Hansenula (Hansenula), Trichoderma (Trichoderma), in particular Trichoderma reesei (t. reesei), Bacillus (Bacillus), Lactobacillus (Lactobacillus), aspergillus (in particular aspergillus niger), a plant cell and/or a spore of Bacillus, Trichoderma or aspergillus.

In a fifth aspect, the present invention also provides an additive, wherein the additive comprises the degrading enzyme provided by the present invention, and/or comprises the recombinant cell and/or a fermentation product thereof.

In a preferred case, the additive has the degrading enzyme provided by the present invention as an active ingredient. In the additive, the content of the degrading enzyme is 0.001-10g/kg, preferably 0.01-8g/kg, more preferably 0.1-5 g/kg. The additive may further contain a solvent (e.g., a protein protecting agent such as glycerol, a saccharide, and a protease inhibitor), an agonist, and the like, which are known to those skilled in the art.

According to the invention, the feed and the additive also contain a physiologically acceptable carrier, wherein the physiologically acceptable carrier is selected from at least one of the following substances: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat bran or wheat component, rice or rice bran, sucrose, starch, Na₂SO₄Talc and PVA and mixtures thereof.

It will be apparent to those skilled in the art that these additives may be added to feed and/or grain and oil material contaminated with mycotoxins to reduce the level of toxins present.

The feed material of the invention may comprise: a) cereals, for example, small grain cereals (such as wheat, barley, rye, oats, and combinations thereof) and/or large grain cereals such as maize or sorghum; b) by-products from cereals, such as corn gluten meal, distillers dried grains with solubles (DDGS), wheat bran, wheat middlings, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) ensiling the feed; d) proteins from the following sources: such as soy, sunflower, peanut, lupin, pea, broad bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; e) oils and fats obtained from plant and animal sources; f) minerals and vitamins.

The grain and oil of the present invention refers to the general term of grains and oil materials such as grains, beans, etc. and the finished products and semi-finished products thereof, especially to the products which can be eaten by human beings. For example, the grain oil may be a grain oil product that is edible to humans and is common in the art, and specifically, the grain oil may include at least one of grains and agricultural byproducts thereof, oil and fat products, wines, milks and products thereof, and the like.

It will be apparent to the skilled person that the feed or additive according to the invention may further comprise other components such as stabilisers and/or extenders and/or enzymes.

Wherein the enzyme may be selected from, but is not limited to: aflatoxin detoxication enzyme, ochratoxin lactonase, fumonisin carboxyesterase, fumonisin aminotransferase, aminopolyol amine oxidase, deoxynivalenol epoxide hydrolase, carboxypeptidase, Aspergillus niger aspartic protease PEPAa, PEPAb, PEPAc and PEPAd, elastase, aminopeptidase, pepsin or pepsin-like protease, trypsin or trypsin-like protease, bacterial protease, enzyme involved in starch metabolism, fiber degradation, lipid metabolism, protein or enzyme involved in glycogen metabolism, amylase, arabinase, arabinofuranosidase, catalase, cellulase, chitinase, rennin, cutinase, deoxyribonuclease, epizyme, esterase, galactosidase, isomerase, glucanase, endoglucanase, glucoamylase, glucose oxidase, glucosidases, including beta-glucosidase, glucuronidase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, lipolytic enzyme, laccase, lyase, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectin depolymerase, pectin methylesterase, pectinolytic enzyme, peroxidase, phenoloxidase, phytase, polygalacturonase, protease, rhamnogalacturonase, ribonuclease, african sweet fruit element, transferase, transporter, transglutaminase, xylanase, hexose oxidase, acid phosphatase, and combinations thereof.

Preferably, the method of preparing the additive according to the invention comprises a mixing step comprising mixing at least one of the degrading enzymes, recombinant cells and fermentation products thereof as described above, optionally together with at least one physiologically acceptable carrier, solvent, agonist, stabilizer, bulking agent or enzyme.

It will be apparent to the skilled person that as a preventive step, additives may be added to any feed and/or grain and oil material.

For example, when the additive is used in the feed for farmed animals, the additive may further comprise at least one of Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, enterococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp.

For example, when the additive is used for silage or cattle feed, the additive further contains at least one of propionibacterium propionicum, lactobacillus buchneri and lactobacillus paracasei.

For example, when the additive is used in the feed of meat poultry, growing-finishing pigs and aquaculture animals, the feed or additive also contains bacillus coagulans and/or brevibacillus laterosporus.

Based on the above description, the invention also provides a grain oil or feed, wherein the grain oil or feed contains the additive.

In a sixth aspect, the present invention also provides the use of a degrading enzyme as described above, a gene as described above, a recombinant vector as described above, a recombinant cell as described above and/or a fermentation product thereof or an additive as described above for degrading ochratoxins and/or other mycotoxins.

According to the invention, the other mycotoxins are preferably fumonisins and T2 toxin.

In a seventh aspect, the present invention also provides a method of degrading ochratoxins and/or other mycotoxins, wherein the method comprises: the degrading enzyme as described above, the recombinant cell as described above and/or its fermentation product or the additive as described above is contacted with the sample to be treated under the conditions of the enzymatic degradation reaction.

According to the present invention, the conditions of the contacting may include: the temperature is 20-80 ℃, and the pH value is 2-9; preferably, the temperature is 30-80 ℃, and the pH value is 4-8; more preferably, the temperature is 30-70 ℃ and the pH is 6-8.

In the present invention, the time of the enzymatic degradation reaction may be 5 to 60min, preferably 10 to 30 min. According to the invention, the other mycotoxins are preferably fumonisins and T2 toxin.

According to the invention, the sample to be treated can be grain oil and/or feed. The grain and/or feed has been described in detail above and will not be described further.

The present invention will be described in detail below by way of examples.

In the following examples and comparative examples, ochratoxin a, fumonisin FB1 and T2 toxin standards were purchased from sigma;

detecting fumonisins FB1 by high performance liquid chromatography according to a method in GB 5009.240-2016 standard, detecting vomitoxins by a method in GB/T30956-2014 standard, and detecting T2 toxins by a method in GB/T23501-2009 standard;

the toxin degradation rate = (mass of toxin in sample before reaction-mass of toxin in sample after reaction)/mass of toxin in sample before reaction × 100%.

Example 1

This example illustrates the degrading enzymes provided by the present invention, and their preparation and use.

(1) Obtaining of genes

The following nucleotide fragments were synthesized by an artificial chemical synthesis method (entrusted to treasure bioengineering (Dalian) Co., Ltd., the same shall apply hereinafter); in SEQ ID NO: 1, adding Nde I enzyme cutting site after the 5 'end initiation codon ATG, and adding Xho I enzyme cutting site and termination codon TAG at the 3' end.

(2) Construction of recombinant plasmid

The PET30a plasmid (His-tagged, available from Invitrogen USA) was double digested with restriction enzymes Nde I and Xho I (available from NEB) in a 37 ℃ water bath for 4h, with the following restriction enzyme systems (50. mu.L):

and (4) carrying out agarose gel electrophoresis on the enzyme digestion product, and then purifying and recovering. Then, ligation was performed using T4 ligase (purchased from Takara) for 4 hours at room temperature to obtain a recombinant plasmid. The ligation system (10. mu.L) used was as follows:

(3) obtaining of recombinant strains

DH 5. alpha. competent cells (purchased from Takara) were electroporated on a Bio-Rad Gene Pulse electrotransfer apparatus using the recombinant plasmid obtained in step (2) under the following conditions: the voltage 1500V, the capacitance 25. mu.F, the resistance 200. omega. and the conversion time vary from one DNA sample to another and are automatically given by the instrument, generally in the range of 3.5-4 s. Then, the transformed cells were spread on an LB solid plate containing kanamycin for screening, inverted culture was performed at 37 ℃ for 2 days, and positive colonies were picked and inoculated in an LB liquid medium to obtain a recombinant strain.

Then, the plasmid was extracted by a method known in the art, and sequencing was performed by Taobao bioengineering (Dalian) Co., Ltd. to show that the above gene was successfully transformed into E.coli, indicating that the recombinant strain of the present invention was successfully constructed.

(4) Preparation of enzymes

The obtained recombinant strain was inoculated into LB liquid medium (beef extract 5g, peptone 10g, sodium chloride 5g, water supplemented to 1000mL, sterilized at 115 ℃ for 20min, pH 7), cultured at 37 ℃ for 3 days to OD₆₀₀The value is 2-6, the cells are collected by centrifugation and washed with phosphate buffer (PBS, 135mM NaCl, 2.7mM KCl, 1.5mM KH)₂PO₄，8mM K₂HPO₄pH 7.2) suspension, ultrasonic disruptionAnd centrifuging to obtain supernatant to obtain crude enzyme solution.

The crude enzyme solution was placed on ice, and the ground ammonium sulfate powder was slowly added thereto with stirring until saturation with ammonium sulfate. Standing at 4 deg.C for about 24h, centrifuging at 12000r/min for 50min, discarding supernatant, and dissolving precipitate with small amount of PBS (pH 7.2). The PBS-solubilized pellet was dialyzed, ammonium sulfate removed, and resuspended in buffer (pH 7.4, 50mM NaCl, containing 10mM imidazole). According to the fact that expressed recombinase contains His labels, an Ni column is used for affinity chromatography purification, after the Ni column is balanced by 1mL/min, the resuspended crude enzyme liquid is directly loaded at the flow rate of 0.5 mL/min; continuing to use 1mL/min buffer (pH 7.4, 50mM NaCl containing 10mM imidazole) to elute unadsorbed or adsorbed non-specific hybrid protein; the target protein was collected by elution with a buffer (pH 7.4, 50mM NaCl, 500mM imidazole) to obtain a purified enzyme solution.

(5) Effect of temperature and pH on enzyme Activity

Taking a proper amount of the enzyme solution obtained in the step (4) and a proper amount of ochratoxin A standard substance, and preparing by using normal saline so that the concentration of the enzyme in the obtained mixed solution is 100ng/ml, and the concentration of the ochratoxin A is 1000 ppb. The reaction temperature is 20 ℃, 37 ℃, 60 ℃ and 80 ℃ (pH value 7), the reaction pH value is 2, 3, 5, 7 and 9 (temperature 37 ℃), 20 microliter reaction product is subjected to high performance liquid chromatography to detect ochratoxin A residue after 30min reaction, and the degradation rate is calculated. The results are shown in tables 1 and 2.

Example 2

This example illustrates the degrading enzymes provided by the present invention, and their preparation and use.

A degrading enzyme was prepared according to the method of example 1, and the effect of temperature and pH on the enzyme activity was measured, except that the enzyme activity was measured using SEQ ID NO: 3 substitution of SEQ ID NO: 1.

example 3

This example illustrates the degrading enzymes provided by the present invention, and their preparation and use.

A degrading enzyme was prepared according to the method of example 1, and the effect of temperature and pH on the enzyme activity was measured, except that the enzyme activity was measured using SEQ ID NO: 4 substitution of SEQ ID NO: 1.

example 4

This example illustrates the degrading enzymes provided by the present invention, and their preparation and use.

A degrading enzyme was prepared according to the method of example 1, and the effect of temperature and pH on the enzyme activity was measured, except that the enzyme activity was measured using SEQ ID NO: 5 substitution of SEQ ID NO: 1.

comparative example 1

This comparative example is a blank control

A degrading enzyme was prepared according to the method of example 1, except that a plasmid of the empty vector PET30a, which was not transfected, was used instead of the recombinant plasmid used in example 1.

Comparative example 2

This example illustrates the degrading enzymes provided by the present invention, and their preparation and use.

A degrading enzyme was prepared according to the method of example 1, and the effect of temperature and pH on the enzyme activity was measured, except that the enzyme activity was measured using SEQ ID NO: 6 (amidase sequence from CN 103209597A) in place of SEQ ID NO: 1.

TABLE 1

TABLE 2

The results of the above examples show that the ochratoxin degrading enzyme provided by the invention can efficiently degrade ochratoxin A, has high stability, and is suitable for industrial production. In addition, the ochratoxin degradation enzyme provided by the invention also has the characteristics of acid resistance and high temperature resistance, so that the application range of the ochratoxin degradation enzyme is further expanded.

In addition, the experimental results show that SEQ ID NO: 1 (SEQ ID NO: 3-5), and in addition, experiments prove that the ochratoxin degradation enzyme provided by the invention has the degradation efficiency of 90% of the ochratoxin A on ochratoxin B, and the degradation efficiency of fumonisin and T2 toxin is 80% and 75% of the ochratoxin A respectively.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

SEQUENCE LISTING

<110> Zhongliang Nutrition and health research institute Co., Ltd

COFCO Ltd.

<120> ochratoxin-degrading enzyme, encoding gene, recombinant vector, cell, additive and application thereof

<130> I40391COF

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 1443

<212> DNA

<213> Artificial

<220>

<223> The sequence is synthesized.

<400> 1

atggtccgcc gaattgcttc agctacacct cgcgtgcaat cgcccatgtc gccattgggc 60

acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120

atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180

gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240

ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300

cccgtgctca tgcctggttt gtgggattgc gacatgcatt ttggcgggga tgacgattat 360

tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420

cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480

gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcattcg 540

tctggcgctg cactcagtca gacagctgga cacggcgata tcttcgctct tccagcaggc 600

gaagtactgg ggagttatgg agtaatgaac ccacgccctg ggtactgggg ggcagggccg 660

ctatgtatcg ccgatggcgt agaggaggtc cgacgagcag tgaggttgca gatcatgcgc 720

ggtgcaaagg ttatcaaagt gatggcctct gggggtgtca tgtcgcgaga cgataatccc 780

aactttgcac agttctctcc agaagaactg aaggtgatag tggaagaggc ggctcgacag 840

aaccggatcg tttctgcaca tgtgcatggc aaggcgggga ttatggctgc tatcaaagca 900

ggctgcaaga gtctggagca tgtgtcttat gctgacgagg aggtctggga gctcatgaaa 960

gagaagggaa ttttgtatgt ggccacacgc tcggttattg aaatctttct ggctagtaat 1020

ggagaggggt tggtgaaaga gtcgtgggcc aagttgcagg cccttgccga ttcgcatttg 1080

aaagcttatc agggagctat taaggcgggt gttaccattg cgttgggaac ggataccgcc 1140

cccggtggtc ctaccgcact tgagttgcag tttgccgtcg agagaggagg tatgacgccg 1200

ttggaggcca tcaaagccgc aactgcgaac gctcccctgt cagttggtcc acaagcaccg 1260

ttgacgggtc agcttcgcga ggggtatgag gcagatgtga ttgcgttgga ggagaatcca 1320

ttggaggaca tcaaagtctt tcaggagccg aaggcagtta cccacgtctg gaagggaggg 1380

aaactgttca aaggtccagg tattggtccg tggggagaag atgcacgtaa tccttttctg 1440

tag 1443

<210> 2

<211> 480

<212> PRT

<213> Artificial

<220>

<223> The sequence is synthesized.

<400> 2

Met Val Arg Arg Ile Ala Ser Ala Thr Pro Arg Val Gln Ser Pro Met

1 5 10 15

Ser Pro Leu Gly Thr Thr Tyr Cys Val Arg Pro Asn Pro Val Ser Leu

20 25 30

Asn Leu Gln Arg Arg Pro Leu Val Ile Ala Ser Thr Asp Glu Ala Lys

35 40 45

Val Thr Ile Ile Tyr Ala Gly Leu Leu Ile Pro Gly Asp Gly Glu Pro

50 55 60

Leu Arg Asn Ala Ala Leu Val Ile Ser Asp Lys Ile Ile Ala Phe Val

65 70 75 80

Gly Ser Glu Ala Asp Ile Pro Lys Asp Tyr Leu Arg Ser Thr Gln Ser

85 90 95

Thr His Arg Val Pro Val Leu Met Pro Gly Leu Trp Asp Cys Asp Met

100 105 110

His Phe Gly Gly Asp Asp Asp Tyr Tyr Asn Asp Tyr Thr Ser Gly Leu

115 120 125

Ala Thr His Pro Ala Ser Ser Gly Ala Arg Leu Ala Arg Gly Cys Trp

130 135 140

Glu Ala Leu Gln Asn Gly Tyr Thr Ser Tyr Arg Asp Leu Ala Gly Tyr

145 150 155 160

Gly Cys Glu Val Ala Lys Ala Ile Asn Asp Gly Thr Ile Val Gly Pro

165 170 175

Asn Val His Ser Ser Gly Ala Ala Leu Ser Gln Thr Ala Gly His Gly

180 185 190

Asp Ile Phe Ala Leu Pro Ala Gly Glu Val Leu Gly Ser Tyr Gly Val

195 200 205

Met Asn Pro Arg Pro Gly Tyr Trp Gly Ala Gly Pro Leu Cys Ile Ala

210 215 220

Asp Gly Val Glu Glu Val Arg Arg Ala Val Arg Leu Gln Ile Met Arg

225 230 235 240

Gly Ala Lys Val Ile Lys Val Met Ala Ser Gly Gly Val Met Ser Arg

245 250 255

Asp Asp Asn Pro Asn Phe Ala Gln Phe Ser Pro Glu Glu Leu Lys Val

260 265 270

Ile Val Glu Glu Ala Ala Arg Gln Asn Arg Ile Val Ser Ala His Val

275 280 285

His Gly Lys Ala Gly Ile Met Ala Ala Ile Lys Ala Gly Cys Lys Ser

290 295 300

Leu Glu His Val Ser Tyr Ala Asp Glu Glu Val Trp Glu Leu Met Lys

305 310 315 320

Glu Lys Gly Ile Leu Tyr Val Ala Thr Arg Ser Val Ile Glu Ile Phe

325 330 335

Leu Ala Ser Asn Gly Glu Gly Leu Val Lys Glu Ser Trp Ala Lys Leu

340 345 350

Gln Ala Leu Ala Asp Ser His Leu Lys Ala Tyr Gln Gly Ala Ile Lys

355 360 365

Ala Gly Val Thr Ile Ala Leu Gly Thr Asp Thr Ala Pro Gly Gly Pro

370 375 380

Thr Ala Leu Glu Leu Gln Phe Ala Val Glu Arg Gly Gly Met Thr Pro

385 390 395 400

Leu Glu Ala Ile Lys Ala Ala Thr Ala Asn Ala Pro Leu Ser Val Gly

405 410 415

Pro Gln Ala Pro Leu Thr Gly Gln Leu Arg Glu Gly Tyr Glu Ala Asp

420 425 430

Val Ile Ala Leu Glu Glu Asn Pro Leu Glu Asp Ile Lys Val Phe Gln

435 440 445

Glu Pro Lys Ala Val Thr His Val Trp Lys Gly Gly Lys Leu Phe Lys

450 455 460

Gly Pro Gly Ile Gly Pro Trp Gly Glu Asp Ala Arg Asn Pro Phe Leu

465 470 475 480

<210> 3

<211> 1443

<212> DNA

<213> Artificial

<220>

<223> The sequence is synthesized.

<400> 3

atggtccgcc gaattgcttc agctacacct cacgtgcaat cgcccatgtc gccattgggc 60

acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120

atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180

gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240

ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300

cccgtgctca tgcctggttt gtgggattgc gacatgcatt ttggcgggga tgacgattat 360

tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420

cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480

gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcattcg 540

tctggcgctg cactcagtca gacagctgga cacggcgata tcttcgctct tccagcaggc 600

gaagtactgg ggagttatgg agtaatgaac ccacgccctg ggtactgggg ggcagggccg 660

ctatgtatcg ccgatggcgt agaggaggtc cgacgagcag tgaggttgca gatctttcgc 720

ggtgcaaagg ttatcaaagt gatggcctct gggggtgtca tgtcgcgaga cgataatccc 780

aactttgcac agttctctcc agaagaactg aaggtgatag tggaagaggc ggctcgacag 840

aaccggatcg tttctgcaca tgtgcatggc aaggcgggga ttatggctgc tatcaaagca 900

ggctgcaaga gtctggagca tgtgtcttat gctgacgagg aggtctggga gctcatgaaa 960

gagaagggaa ttttgtatgt ggccacacgc tcggttattg aaatctttct ggctagtaat 1020

ggagaggggt tggtgaaaga gtcgtgggcc aagttgcagg cccttgccga ttcgcatttg 1080

aaagcttatc agggagctat taaggcgggt gttaccattg cgttgggaac ggataccgcc 1140

cccggtggtc ctaccgcact tgagttgcag tttgccgtcg agagaggagg tatgacgccg 1200

ttggaggcca tcaaagccgc aactgcgaac gctcccctgt cagttggtcc acaagcaccg 1260

ttgacgggtc agcttcgcga ggggtatgag gcagatgtga ttgcgttgga ggagaatcca 1320

ttggaggaca tcaaagtctt tcaggagccg aaggcagtta cccacgtctg gaagggaggg 1380

aaactgttca aaggtccagg tattggtccg tggggagaag atgcacgtaa tccttttctg 1440

tag 1443

<210> 4

<211> 1440

<212> DNA

<213> Artificial

<220>

<223> The sequence is synthesized.

<400> 4

atggtccgcc gaattgcttc agctacacct gtgcaatcgc ccatgtcgcc attgggcaca 60

acatactgcg tccgtcctaa tcctgtttca ctgaatcttc aaagaagacc tctcgtgatc 120

gcatcaacag acgaggccaa ggtcactata atatatgccg gactattaat ccctggcgac 180

ggagaacctc tgcgcaatgc tgccctagtc atcagcgata agatcatcgc gttcgttgga 240

tccgaagccg acatccctaa ggactacctc cggtccacgc agtctactca tcgtgtcccc 300

gtgctcatgc ctggtttgtg ggattgcgac atgcattttg gcggggatga cgattattac 360

aacgattata catctggtct ggccactcat ccagcatcat caggtgctcg actagcccgt 420

ggttgctggg aagcattgca gaatgggtat acatcctacc gcgacctagc cggatacggg 480

tgcgaggtcg caaaggcgat caatgatggc actatcgttg gtccaaacgt gcattcgtct 540

ggcgctgcac tcagtcagac agctggacac ggcgatatct tcgctcttcc agcaggcgaa 600

gtactgggga gttatggagt aatgaaccca cgccctgggt actggggggc agggccgcta 660

tgtatcgccg atggcgtaga ggaggtccga cgagcagtga ggttgcagat ctttcgcggt 720

gcaaaggtta tcaaagtgat ggcctctggg ggtgtcatgt cgcgagacga taatcccaac 780

tttgcacagt tctctccaga agaactgaag gtgatagtgg aagaggcggc tcgacagaac 840

cggatcgttt ctgcacatgt gcatggcaag gcggggatta tggctgctat caaagcaggc 900

tgcaagagtc tggagcatgt gtcttatgct gacgaggagg tctgggagct catgaaagag 960

aagggaattt tgtatgtggc cacacgctcg gttattgaaa tctttctggc tagtaatgga 1020

gaggggttgg tgaaagagtc gtgggccaag ttgcaggccc ttgccgattc gcatttgaaa 1080

gcttatcagg gagctattaa ggcgggtgtt accattgcgt tgggaacgga taccgccccc 1140

ggtggtccta ccgcacttga gttgcagttt gccgtcgaga gaggaggtat gacgccgttg 1200

gaggccatca aagccgcaac tgcgaacgct cccctgtcag ttggtccaca agcaccgttg 1260

acgggtcagc ttcgcgaggg gtatgaggca gatgtgattg cgttggagga gaatccattg 1320

gaggacatca aagtctttca ggagccgaag gcagttaccc acgtctggaa gggagggaaa 1380

ctgttcaaag gtccaggtat tggtccgtgg ggagaagatg cacgtaatcc ttttctgtag 1440

<210> 5

<211> 1446

<212> DNA

<213> Artificial

<220>

<223> The sequence is synthesized.

<400> 5

atggtccgcc gaattgcttc agctacacct cgcgtgcaat cgcccatgtc gccattgggc 60

acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120

atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180

gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240

ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300

cccgtgctca tgcctggttt gtgggattgc gacatgcatt ttggcgggga tgacgattat 360

tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420

cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480

gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcatcat 540

tcgtctggcg ctgcactcag tcagacagct ggacacggcg atatcttcgc tcttccagca 600

ggcgaagtac tggggagtta tggagtaatg aacccacgcc ctgggtactg gggggcaggg 660

ccgctatgta tcgccgatgg cgtagaggag gtccgacgag cagtgaggtt gcagatcatg 720

cgcggtgcaa aggttatcaa agtgatggcc tctgggggtg tcatgtcgcg agacgataat 780

cccaactttg cacagttctc tccagaagaa ctgaaggtga tagtggaaga ggcggctcga 840

cagaaccgga tcgtttctgc acatgtgcat ggcaaggcgg ggattatggc tgctatcaaa 900

gcaggctgca agagtctgga gcatgtgtct tatgctgacg aggaggtctg ggagctcatg 960

aaagagaagg gaattttgta tgtggccaca cgctcggtta ttgaaatctt tctggctagt 1020

aatggagagg ggttggtgaa agagtcgtgg gccaagttgc aggcccttgc cgattcgcat 1080

ttgaaagctt atcagggagc tattaaggcg ggtgttacca ttgcgttggg aacggatacc 1140

gcccccggtg gtcctaccgc acttgagttg cagtttgccg tcgagagagg aggtatgacg 1200

ccgttggagg ccatcaaagc cgcaactgcg aacgctcccc tgtcagttgg tccacaagca 1260

ccgttgacgg gtcagcttcg cgaggggtat gaggcagatg tgattgcgtt ggaggagaat 1320

ccattggagg acatcaaagt ctttcaggag ccgaaggcag ttacccacgt ctggaaggga 1380

gggaaactgt tcaaaggtcc aggtattggt ccgtggggag aagatgcacg taatcctttt 1440

ctgtag 1446

<210> 6

<211> 1443

<212> DNA

<213> Artificial

<220>

<223> The sequence is synthesized.

<400> 6

atggtccgcc gaattgcttc agctacacct cgcgtgcaat cgcccatgtc gccattgggc 60

acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120

atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180

gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240

ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300

cccgtgctca tgcctggttt gtgggattgc cacatgcatt ttggcgggga tgacgattat 360

tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420

cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480

gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcattcg 540

tctggcgctg cactcagtca gacagctgga cacggcgata tcttcgctct tccagcaggc 600

gaagtactgg ggagttatgg agtaatgaac ccacgccctg ggtactgggg ggcagggccg 660

ctatgtatcg ccgatggcgt agaggaggtc cgacgagcag tgaggttgca gatcatgcgc 720

ggtgcaaagg ttatcaaagt gatggcctct gggggtgtca tgtcgcgaga cgataatccc 780

aactttgcac agttctctcc agaagaactg aaggtgatag tggaagaggc ggctcgacag 840

aaccggatcg tttctgcaca tgtgcatggc aaggcgggga ttatggctgc tatcaaagca 900

ggctgcaaga gtctggagca tgtgtcttat gctgacgagg aggtctggga gctcatgaaa 960

gagaagggaa ttttgtatgt ggccacacgc tcggttattg aaatctttct ggctagtaat 1020

ggagaggggt tggtgaaaga gtcgtgggcc aagttgcagg cccttgccga ttcgcatttg 1080

aaagcttatc agggagctat taaggcgggt gttaccattg cgttgggaac ggataccgcc 1140

cccggtggtc ctaccgcact tgagttgcag tttgccgtcg agagaggagg tatgacgccg 1200

ttggaggcca tcaaagccgc aactgcgaac gctcccctgt cagttggtcc acaagcaccg 1260

ttgacgggtc agcttcgcga ggggtatgag gcagatgtga ttgcgttgga ggagaatcca 1320

ttggaggaca tcaaagtctt tcaggagccg aaggcagtta cccacgtctg gaagggaggg 1380

aaactgttca aaggtccagg tattggtccg tggggagaag atgcacgtaa tccttttctg 1440

tag 1443

Claims (16)

1. An ochratoxin-degrading enzyme, characterised in that the amino acid sequence of the degrading enzyme is SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3, and (b) 3, and an amino acid sequence encoded by the nucleotide sequence shown in the figure.

2. A gene encoding ochratoxin-degrading enzyme, characterized in that the nucleotide sequence of the gene is the nucleotide sequence encoding ochratoxin-degrading enzyme according to claim 1.

3. The encoding gene of claim 2, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO: 1 or SEQ ID NO: 3, respectively.

4. A recombinant vector comprising the gene of claim 2 or 3.

5. A recombinant cell comprising the gene of claim 2 or 3 or the recombinant vector of claim 4;

the recombinant cell is not a plant cell.

6. The recombinant cell of claim 5, wherein the recombinant cell is selected from one or more of Escherichia coli, Streptomyces, Hansenula, Trichoderma, Bacillus, Lactobacillus, and Aspergillus.

7. An additive comprising the degrading enzyme of claim 1 and/or comprising the recombinant cell of claim 5.

8. The supplement of claim 7, wherein the supplement further comprises one or more of bacillus licheniformis, bacillus subtilis, bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, enterococcus lactis, lactobacillus acidophilus, lactobacillus casei, lactobacillus delbrueckii subsp lactis, lactobacillus plantarum, pediococcus acidilactici, pediococcus pentosaceus, candida utilis, bifidobacterium infantis, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, bifidobacterium animalis, aspergillus oryzae, bacillus lentus, bacillus pumilus, lactobacillus cellobiosus, lactobacillus fermentum, and lactobacillus delbrueckii subsp bulgaricus.

9. The supplement of claim 8, wherein the supplement is for silage or cattle, the supplement further comprising at least one of propionibacterium propionicum, lactobacillus buchneri, and lactobacillus paracasei; or

The additive is used for feed of meat poultry, growing-finishing pigs and aquaculture animals, and also contains bacillus coagulans and/or brevibacillus laterosporus.

10. The supplement according to claim 7, wherein the supplement further comprises at least one physiologically acceptable carrier; the physiologically acceptable carrier is selected from maltodextrin, limestone, cyclodextrin, wheat bran, rice or rice bran, sucrose, starch, Na₂SO₄One or more of talc and PVA.

11. The supplement of claim 10, wherein the supplement further comprises one or more additional enzymes.

12. The additive of claim 11, wherein the one or more additional enzymes are selected from one or more of oxidoreductases, transferases, hydrolases, lyases, and isomerases.

13. The additive according to any one of claims 7-12, wherein the additive contains the degrading enzyme of claim 1 at a level of 0.001-10g/kg additive.

14. Use of the degrading enzyme of claim 1, the gene of claim 2 or 3, the recombinant vector of claim 4, the recombinant cell of claim 5 or 6, or the additive of any one of claims 7 to 13 for degrading ochratoxins.

15. A method of degrading ochratoxins, which method comprises: contacting the degrading enzyme of claim 1, the recombinant cell of claim 5 or 6, or the additive of any one of claims 7-13 with a sample to be treated under conditions of an enzymatic degradation reaction.

16. The method of claim 15, wherein the sample to be treated is grain oil and/or feed.