CN111394333B

CN111394333B - Ochratoxin detoxification enzyme, and coding gene, recombinant vector and application thereof

Info

Publication number: CN111394333B
Application number: CN201910004498.1A
Authority: CN
Inventors: 周育; 王旭; 陈楠; 杜郑君
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2019-01-03
Filing date: 2019-01-03
Publication date: 2021-05-18
Anticipated expiration: 2039-01-03
Also published as: CN111394333A

Abstract

The invention discloses ochratoxin detoxification enzyme, and a coding gene, a recombinant vector and application thereof, and belongs to the technical field of enzyme engineering. The invention provides ochratoxin detoxification enzyme and a coding gene thereof, and also provides the optimum pH value of the ochratoxin detoxification enzyme and application of the ochratoxin detoxification enzyme in degradation of ochratoxin and biological detoxification of ochratoxin in foods. The ochratoxin detoxification enzyme treats ochratoxin A in a buffer system with pH 7.3 at 37 ℃ for 2min, and the degradation rate of the ochratoxin A reaches 100%, which is unprecedented in the field.

Description

Ochratoxin detoxification enzyme, and coding gene, recombinant vector and application thereof

Technical Field

The invention relates to ochratoxin detoxification enzyme, and a coding gene, a recombinant vector and application thereof, and belongs to the technical field of enzyme engineering.

Background

The aspergillus mycotoxins are mainly secondary metabolites produced by aspergillus strains producing toxin such as aspergillus flavus, aspergillus parasiticus, aspergillus ochraceus and the like, more than twenty structural analogs have been separated and identified, wherein the aflatoxin B1(AFB1) and the aspergillus ochraceus A (OTA) have the highest toxicity, and the aflatoxins are most widely polluted in agricultural production and food industry. In 1993, the World Health Organization (WHO) cancer research institute classified aflatoxins as class I carcinogens and ochratoxins as class II B carcinogens. Studies have shown that ochratoxins are the major cause of human balm's disease and have become the focus of growing public health and scientific attention due to their strong nephrotoxicity, as well as possible carcinogenesis, teratogenicity and mutagenicity. Contamination of cereal and oil crops with aspergillus mycotoxins has become a global problem, in animal husbandry, aspergillus mycotoxins can harm animal health by contaminating feed, resulting in low animal husbandry productivity, causing severe economic losses, and affecting human health by contaminating food either directly or indirectly (animal product transmission). With the gradual deepening of people's understanding of the harmfulness of the aspergillus mycotoxins, research on the detection, pollution prevention and control and detoxification technology of the mycotoxins is continuously carried out, and the research is relevant to the quality of life, health and economic benefits of people.

The detoxification method of ochratoxin commonly adopted at present comprises the following steps: physical adsorption and chemical decomposition methods can effectively reduce or degrade the pollution concentration of ochratoxin in food and feed, but the detoxification methods still have the problems of high cost, difficult thorough detoxification, sensory changes of the food and feed caused by the detoxification methods in the treatment process, and loss of nutrient components.

Biological detoxification is considered to be one of the most promising methods for removing ochratoxin A pollution in recent years due to the advantages of low cost, safety, high efficiency and the like, biological detoxification enzymes and genes thereof are screened, the biological functions of the detoxification enzymes are perfected, critical biological materials and matching technologies are accumulated for biological detoxification methods, and the research on the biological detoxification technology in China is effectively promoted. Ochratoxin detoxification enzymes are a general term for enzymes that degrade ochratoxin, and most of them belong to the class of hydrolases. Widely present in fungi, plants, insects and bacteria, and have been a research hotspot in the fields of biological, chemical and environmental science since the discovery. However, the existing enzyme for degrading ochratoxin has low degradation efficiency or is not suitable for industrial application, and no enzyme which has high degradation rate of ochratoxin and is suitable for industrial production exists at present.

Disclosure of Invention

In order to solve the problems, the invention provides the ochratoxin detoxification enzyme derived from the lysobacter strain of xanthomonas, which realizes heterologous high expression in a prokaryotic expression system, is soluble protein, is easy to purify in the later period and has low commercial production cost.

The first object of the present invention is to provide an ochratoxin detoxification enzyme, the amino acid sequence of which is shown in (a) or (b):

(a) an amino acid sequence shown as SEQ ID NO. 1;

(b) and (b) an amino acid sequence which has 95% or more homology with the amino acid sequence shown in (a) and has ochratoxin detoxification enzyme activity.

Further, the ochratoxin detoxification enzyme includes any peptide protein fragment containing the amino acid sequence shown in SEQ ID NO.1 or its variant, such as conservative variant, bioactive fragment or derivative thereof, as long as the peptide protein fragment or peptide protein variant has a homology of more than 95% with the amino acid sequence, and the peptide protein fragment or peptide protein variant is within the protection scope of the present invention. Particular such alterations may include deletions, insertions or substitutions of amino acids in the amino acid sequence; where conservative changes to a variant are made, the substituted amino acid has similar structural or chemical properties as the original amino acid, e.g., replacement of isoleucine with leucine, and the mutant may also have non-conservative changes, e.g., replacement of glycine with tryptophan. The fragment, derivative or analogue of the polypeptide protein of the present invention refers to a peptide protein that substantially retains the same biological function or activity as the ochratoxin detoxification enzyme of the present invention, and may be: (I) one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and the substituted amino acid may or may not be encoded by the genetic code; (II) one or more of the amino acid residues is substituted with another group; (III) the mature peptide protein is fused to another compound (such as a compound that extends the half-life of the peptide protein, e.g., polyethylene glycol); (IV) peptide protein sequences formed by fusing additional amino acid sequences to the mature peptide protein (e.g., sequences used to purify the peptide protein or proprotein sequence).

Further, the ochratoxin detoxification enzymes may be recombinant, natural or synthetic, pure natural purified products, or chemically synthesized products, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). The peptide proteins of the invention may be glycosylated depending on the host used in the recombinant production scheme. The ochratoxin detoxification enzymes of the invention may or may not also include the initial methionine residue.

It is a second object of the present invention to provide a gene encoding the ochratoxin detoxification enzyme.

Furthermore, the gene has a nucleotide sequence shown in SEQ ID NO.2, or a nucleotide sequence which has more than 70% of homology with the nucleotide shown in SEQ ID NO. 2.

Due to the specificity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.2 is within the scope of the present invention as long as it has more than 70% homology with the polynucleotide. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Variants of the polynucleotide include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, without substantially altering the function of the peptide protein encoded thereby.

Furthermore, polynucleotides which hybridize to the polynucleotide shown in SEQ ID No.2 (having at least 50% homology, preferably 70% homology) are also within the scope of the present invention, in particular polynucleotides which hybridize under stringent conditions to the nucleotides described herein. The "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2SSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum, 0.1% Ficoll, 42 deg.C; or (3) hybridization occurs only when the homology between two sequences is at least 95% or more, preferably 97% or more. And, the peptide protein encoded by the hybridizable polynucleotide hybridizes to SEQ ID NO: the peptide protein shown in 1 has the same biological functions and activities.

The polynucleotide sequence encoding the ochratoxin detoxification enzyme of the invention can be obtained in a number of ways. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include (but are not limited to): (1) hybridization of probes to gene or cDNA libraries to detect homologous polynucleotide sequences and (2) activity screening of expression libraries, which may include environmental metagenomic libraries or libraries of clones created from a pure culture of the strain, to detect cloned polynucleotide fragments having common structural features. The DNA fragment sequences of the present invention can also be obtained by the following methods: (1) isolating double-stranded DNA sequences from genomic DNA; (2) chemically synthesizing a DNA sequence to obtain double-stranded DNA of the ochratoxin detoxification enzyme.

The genes of the present invention can be screened from these cNDA libraries by conventional methods. These methods include (but are not limited to):

(1) DNA-DNA or DNA-RNA hybridization; (2) the appearance or loss of function of a marker gene; (3) the protein product of gene expression is detected by measuring biological activity. The above methods can be used singly or in combination of multiple methods.

The polynucleotide sequences of the gene of the present invention or various DNA fragments obtained as described above can be determined by the dideoxy chain termination method according to a conventional method. Such polynucleotide sequencing may also be performed using commercial sequencing kits and the like. Sequencing was repeated to obtain a full-length cDNA sequence. Sometimes it is necessary to sequence the cDNA of multiple clones to splice the full-length cDNA sequence.

The third purpose of the invention is to provide a recombinant vector carrying the gene.

Further, the vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

In the present invention, the nucleotide sequence encoding ochratoxin detoxification enzyme may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention. "vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Vectors suitable for use in the present invention also include, but are not limited to: expression vectors based on the T7 promoter for expression in bacteria; pcDNA3.1 vector expressed in mammalian cells and baculovirus-derived vector expressed in insect cells. In general, any plasmid or vector can be used to construct recombinant expression vectors, preferably the pET vector series as well as other prokaryotic expression vector series, so long as it is replicable and stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation regulatory elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding ochratoxin detoxification enzymes and appropriate transcription/translation regulatory elements. These methods include in vitro recombinant DNA sequences, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct the synthesis of mRNA. Representative examples of such promoters are: lac or trp promoter of E.coli; the PL promoter of a bacteriophage; eukaryotic promoters include CMV early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. The insertion of enhancer sequences into vectors will enhance transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase gene transcription. Examples include the SV40 enhancer at the late side of the replication origin at 100 to 270 bp, the polyoma enhancer at the late side of the replication origin, and adenovirus enhancers.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein for eukaryotic cell culture, or kanamycin or ampicillin for E.coli, and the like.

The fourth purpose of the invention is to provide a recombinant bacterium for expressing the ochratoxin detoxification enzyme.

Further, bacterial, fungal, plant, insect or animal cells are used as host cells.

In the present invention, a polynucleotide encoding ochratoxin detoxification enzyme or a recombinant vector containing the polynucleotide may be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the nucleotide or recombinant vector. "host cell" refers to prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells such as salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells such as Drosophila S2 or Sf 9; animal cells such as CHO, COS or Bowes melanoma cells.

Transformation of a host cell with a DNA sequence according to the invention or a recombinant vector containing said DNA sequence may be carried out by conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase and treated by the CaCl2 method, as is well known in the art, alternatively with MgCl 2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, or conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The recombinant ochratoxin detoxification enzymes can be expressed or produced by conventional recombinant DNA techniques using the polynucleotide sequences of the invention. Generally, the following steps are performed:

(1) transforming or transfecting a suitable host cell with the polynucleotide encoding ochratoxin detoxification enzyme of the invention, or with a recombinant expression vector containing the polynucleotide;

(2) culturing the host cell in a suitable medium;

(3) separating and purifying protein from culture medium or cell.

In step (2), the medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is carried out under conditions suitable for the host cell. After the host cells have been grown at the appropriate cell density, the selected promoter is induced by suitable means and the cells are cultured for an additional period of time.

In step (3), the recombinase may be encapsulated inside the cell, expressed on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting-out method), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, affinity chromatography, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.

The fifth purpose of the invention is to provide the application of the ochratoxin detoxification enzyme in degradation of ochratoxin A.

Further, the application comprises degrading ochratoxin A in oil, grains or tea.

It is a sixth object of the present invention to provide an enzyme preparation comprising the ochratoxin detoxification enzyme.

The invention has the beneficial effects that:

the invention provides ochratoxin detoxification enzyme and a coding gene thereof, and also provides the optimum pH value of the ochratoxin detoxification enzyme and application of the ochratoxin detoxification enzyme in degradation of ochratoxin and biological detoxification of ochratoxin in foods. The ochratoxin detoxification enzyme treats ochratoxin A in a buffer system with pH 7.3 at 37 ℃ for 2min, and the degradation rate of the ochratoxin A reaches 100%, which is unprecedented in the field.

Drawings

FIG. 1 shows the electrophoresis of the Haematitum toxin detoxification enzyme SDS-PAGE protein expressed by pronucleus;

FIG. 2 shows the effect of ochratoxin detoxification enzyme on the degradation of ochratoxin A in food products (wheat flour);

FIG. 3 shows the effect of ochratoxin detoxification enzyme on the degradation of ochratoxin A in Fuzhuan tea.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The method for measuring the activity of ochratoxin detoxification enzyme comprises the following steps:

the transformant E.coli BL21(DE)3 containing pGEX-4T-1/adh239 expression vector is expressed under the optimal condition, 10 mu L of the supernatant of the expression bacterial liquid after disruption is placed in a 2mL centrifuge tube, 490 mu L of ochratoxin A standard buffer solution (the pH is 7.3 by using 1. multidot. PBS) is added, the final concentration of ochratoxin A is about 40 mu g/L, the reaction is carried out for 2min, and 1.5mL of acetonitrile is added into the reaction system to stop the reaction. The residual amount of OTA is detected by using high performance liquid chromatography, and experimental results show that after reacting for 2min, the recombinase crude enzyme solution can completely degrade substrates in a reaction system. The blank control group is the supernatant of E.coli BL21(DE)3 containing pGEX-4T-1 empty plasmid after crushing, and the crude enzyme solution has no ochratoxin A degradation activity after crushing the control group.

Example 1: synthesis and cloning of ochratoxin detoxification enzyme gene cDNA

The strain is derived from a flavomonads bacterium lysobacter strain obtained in the early stage of a laboratory, an open reading frame nucleotide sequence of ochratoxin A hydrolase gene is obtained through analysis by gene sequence determination, and a primer upstream primer (adh239-F) (SEQ ID NO.3) of a complete coding reading frame is designed and amplified: 5' -ATCTGGTTCCGCGTGGATCCATGACCGTCCGCC TGGTCCG-3'; downstream primer (adh239-R) (SEQ ID NO. 4): 5' -TCACGATGCGGCCGCTCGAGTCATGGCGACG TCCCGGGC-3', restriction enzyme sites (BamHI and XhoI sites are added according to the invention, underlined, and italic sequence is homologous sequence at downstream end of pGEX-4T-1 vector) are introduced on upstream and downstream primers, respectively. Underlined, and italicized sequence is pGEX-4T-1 vector downstream end homologous sequence), and the ochratoxin detoxification enzyme gene encoding the amino acid shown in SEQ ID No.1 is obtained by an in vitro amplification technology, and the sequence of the ochratoxin detoxification enzyme gene is shown in SEQ ID No. 2. Under the premise of ensuring correct reading frame, the expression recombinant plasmid pGEX-4T-1/adh239 is constructed and then transferred into E.coil BL21(DE 3). The theoretical isoelectric point of the ADH239 enzyme is 6.10 and the theoretical molecule is obtained by sequence analysisThe amount was 46.1 kDa.

Example 2: heterologous expression of ochratoxin detoxification enzyme genes

The E.coil BL21(DE3)/pGEX-4T-1/adh239 transformant obtained in example 1 was shake-cultured overnight in 100mL of LB liquid medium containing 100. mu.g/mL of ampicillin. 1.0mL of the seed cell suspension was inoculated into 100mL of a fresh LB liquid medium (containing 100. mu.g/mL of ampicillin), and shake-cultured at 37 ℃ at 180 r/min. Bacteria solution OD₆₀₀When the concentration reaches 0.6, 0.2mmol/mL IPTG is added to induce expression for 4h at 16 ℃. The mixture was subjected to refrigerated centrifugation at 7000r/min for 10min, and the supernatant was discarded. The cells were suspended in 10mL of 1 XPhosphate buffer solution (PBS), and disrupted by ultrasonication under ice bath conditions. Centrifugation and inclusion body renaturation of the pellet were carried out. SDS-PAGE shows that the enzyme is present in both the supernatant and the pellet (inclusion body) after the disruption of the cells, and the apparent molecular weight (including GST tag 26kDa) is about 72kDa (FIG. 1), which corresponds to the theoretical molecular weight.

Example 3: application of heterologous recombinant ochratoxin detoxification enzyme in ochratoxin degradation

1. Experimental Material

The enzyme preparation is crude enzyme solution of a broken supernatant after expression of the pGEX-4T-1/adh239 transformant, and other reagents are analytically pure chemical reagents.

2. Experimental methods

10 mu L of supernatant after disruption of the pGEX-4T-1/adh239 transformant expression was put into a 2mL centrifuge tube, 490 mu L of ochratoxin A standard buffer (pH 7.3 using 1 × PBS) was added, the final concentration of ochratoxin A was about 40 mu g/L, the reaction was carried out for 2min, and 1.5mL of acetonitrile was added to the reaction system to stop the reaction. The residual amount of OTA was measured by HPLC, and the experimental results showed that after 2min of reaction, the broken supernatant degraded all OTA.

Example 4: application of heterologous recombinant ochratoxin detoxification enzyme in detoxification of ochratoxin-polluted grains

1. Experimental Material

The enzyme preparation is crude enzyme solution of broken supernatant after pGEX-4T-1/adh239 transformant expression, and the experimental material is wheat flour. All other reagents are analytical pure chemical reagents.

2. Experimental methods

Adding a proper amount of OTA standard stock solution into 50mL of phosphate buffer solution, uniformly mixing, immediately pouring into 500g of crushed wheat flour sample, uniformly stirring until the final concentration of OTA reaches 80.0 mu g/kg, and airing in a cool and ventilated place for later use. 200mL of enzyme preparation is uniformly mixed with 200g of prepared toxin-contaminated wheat flour, the prepared sample is cultured at 37 ℃, samples are taken at 0h, 1h, 3h and 6h, and 3 times of degradation experiments are set for each degradation experiment. Meanwhile, 200mL of fresh buffer solution and 200g of prepared toxin-contaminated wheat flour are mixed uniformly to serve as negative control treatment. The treated group and the blank control group were sampled 20g each time, immediately extracted, purified and analyzed according to the ochratoxin standard detection method.

And (5) extracting and purifying toxins. Accurately weighing 10g of cultured sample into a 150mL triangular flask with a plug, adding 1g of NaCl and 20mL of methanol-water mixed solution (volume ratio is 4:1), shaking and extracting at normal temperature for 30min, taking down the plug, filtering in a clean cup by using a cano filter paper (or centrifuging at 3000r/min at normal temperature for 5min), accurately transferring 5.0mL of supernatant, adding 20mL of pure water to dilute and mix uniformly, filtering by using glass fiber filter paper for 1-2 times until the filtrate is clarified, and performing immunoaffinity column purification operation.

Accurately 10.0mL (representing 1.0g of sample) of the clarified filtrate was removed and injected into a glass syringe, the pressure was adjusted to allow the solution to slowly pass through the immunoaffinity column at a flow rate of 1-2 drops/second until a portion of the air passed through the column, the column was rinsed with 10mL of pure water (repeated once), all the effluent was discarded and a portion of the air was passed through the column. Accurately adding 1.5mL of methanol for elution at the flow rate of 1 drop/second, collecting the eluent in a glass sample bottle, and carrying out HPLC-FLD detection.

The dynamic degradation curve of ochratoxin detoxification enzyme on aflatoxin is shown in figure 2, and the experimental result shows that the 3-hour degradation rate of the ochratoxin detoxification enzyme-treated toxin-contaminated wheat flour (with the final concentration of 40 mug/L) is 100%. The ochratoxin detoxification enzyme has high-efficiency degradation capability on ochratoxin A in wheat flour.

Example 5: application of heterologous recombinant ochratoxin detoxification enzyme in detoxification of ochratoxin-polluted dark green tea

1. Experimental Material

The enzyme preparation is crude enzyme liquid of a supernatant fluid after the disruption of the expression of the pGEX-4T-1/adh239 transformant, and the experimental material is Fuzhuan tea. All other reagents are analytical pure chemical reagents.

2. Experimental methods

Adding a proper amount of OTA standard stock solution into 50mL of phosphate buffer solution, uniformly mixing, immediately pouring into 500g of crushed tea powder sample, uniformly stirring until the final concentration of OTA reaches 80.0 mu g/kg, and airing in a cool and ventilated place for later use. And (3) uniformly mixing 200mL of enzyme preparation with 200g of prepared toxin-contaminated tea powder, culturing the prepared sample at 37 ℃, and sampling at 0h, 1h, 3h and 6h, wherein 3 times of degradation experiments are set. Meanwhile, 200mL of fresh buffer and 200g of prepared toxin-contaminated tea powder were mixed uniformly to serve as a negative control. The treated group and the blank control group were sampled 20g each time, immediately extracted, purified and analyzed according to the ochratoxin standard detection method.

A dynamic degradation curve of ochratoxin detoxification enzyme to ochratoxin in tea powder is shown in figure 3, and an experimental result shows that the degradation rate of the ochratoxin detoxification enzyme-treated tea powder (with the final concentration of 40 mu g/L) polluted by the toxin in 6 hours is 100%. The ochratoxin detoxification enzyme has high-efficiency degradation capability on ochratoxin A in wheat flour.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> agriculture university of Anhui

<120> ochratoxin detoxification enzyme, and coding gene, recombinant vector and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 438

<212> PRT

<213> (Artificial sequence)

<400> 1

Met Thr Val Arg Leu Val Arg Thr Ala Val Cys Thr Ala Leu Ala Leu

1 5 10 15

Ser Cys Leu Ser Phe Ala Ser His Ala Ala Ser Gly Ser Thr Ala Leu

20 25 30

His Cys Gly Gln Leu Phe Asp Ser Arg Ser Gly Asp Val Leu Gly Pro

35 40 45

His Thr Val Leu Ile Arg Asp Asp Arg Ile Ala Ala Val Thr Ala Gly

50 55 60

His Ala Glu Val Pro Gly Thr Pro Gly Ile Asp Leu Ser Gly His Thr

65 70 75 80

Cys Thr Pro Gly Trp Thr Asp Leu His Val His Leu Gly Ser Gln Ser

85 90 95

Ser Pro Asp Ser Tyr Ser Glu Gly Phe Arg Leu Asp Asp Val Asp Tyr

100 105 110

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

115 120 125

Thr Ser Val Arg Asp Leu Gly Gly Glu Val Ser Pro His Leu Arg Asp

130 135 140

Ala Ile Asn Gln Gly Leu Val Asp Gly Pro Arg Ile Trp Ala Ala Gly

145 150 155 160

Lys Ser Ile Ala Thr Thr Gly Gly His Ala Asp Pro Thr Asn Gly Tyr

165 170 175

Asn Ser Ser Leu Ser His Leu Leu Gly Pro Pro Gly Pro Thr Glu Gly

180 185 190

Val Ile Asn Ser Val Ala Asp Ala Arg Gln Ala Val Arg Gln Arg Tyr

195 200 205

Lys Asp Gly Ser Asp Val Ile Lys Ile Thr Ala Thr Gly Gly Val Leu

210 215 220

Ser Tyr Ala Arg Ser Gly Asp Ala Pro Gln Phe Arg Val Asp Glu Ile

225 230 235 240

Gln Ala Ile Val Asp Thr Ala Lys Asp Tyr Gly Tyr His Val Ala Ala

245 250 255

His Ala His Gly Glu Glu Gly Met Arg Arg Ala Val Glu Ala Gly Val

260 265 270

Thr Ser Ile Glu His Gly Thr Tyr Met Ser Asp Glu Val Met Ala Leu

275 280 285

Met Lys Arg Lys Gly Thr Trp Tyr Val Pro Thr Val Tyr Ala Gly Arg

290 295 300

Phe Val Ala Asp Lys Ala Lys Ile Asp Gly Tyr Phe Pro Asp Val Val

305 310 315 320

Arg Pro Lys Ala Ala Arg Ile Gly Glu Leu Ile Gln Ala Thr Ala Ala

325 330 335

Arg Ala Tyr Lys Ala Gly Val Lys Met Ala Phe Gly Thr Asp Met Gly

340 345 350

Val Gly Pro His Gly Asp Asn Ala Arg Glu Phe Val Tyr Leu Val Glu

355 360 365

Ala Gly Val Pro Ala Ala Val Ala Leu Gln Met Ala Thr Ile Arg Ala

370 375 380

Ala Glu Val Leu Gly Val Asp Asp Gln Gly Val Ile Ala Pro Gly Gln

385 390 395 400

Arg Ala Asp Ile Val Ala Val Lys Gly Asn Pro Leu Glu Asp Ile Asn

405 410 415

Arg Val Leu Asp Val Ala Phe Val Met Lys Asp Gly Val Val Tyr Ser

420 425 430

Gln Pro Gly Thr Ser Pro

435

<210> 2

<211> 1317

<212> DNA

<213> (Artificial sequence)

<400> 2

atgaccgtcc gcctggtccg taccgccgtt tgcaccgccc tcgccctttc ctgcctgtcc 60

ttcgccagcc acgccgccag cggatcgacg gccctgcact gcggccaact gttcgacagc 120

cgcagcggcg acgtgctcgg cccgcacacc gtgctgatcc gcgacgaccg gatcgccgcg 180

gtcaccgccg gccacgccga ggtgccgggc acgccgggca tcgatctgtc cgggcacacc 240

tgcacaccgg gctggaccga cctgcacgtg cacctgggca gccagtcgag tccggacagc 300

tactccgaag gcttccgcct ggacgacgtc gattacgcgt tccgctcggt gggctacgcg 360

aagaaaaccc tgctggccgg cttcaccagc gtgcgcgacc tcggcggcga agtcagcccg 420

cacctgcgcg acgcgatcaa ccaggggctg gtggacgggc cgcggatctg ggcggccggc 480

aaatccatcg ccaccaccgg tggccacgcc gatccgacca acggctacaa ctccagcctg 540

tcgcacctgc tcggcccgcc cggcccgacc gagggcgtga tcaactcggt cgccgacgcc 600

cgccaggccg tgcgccagcg gtacaaagac ggcagcgacg tgatcaagat caccgcgacc 660

ggcggcgtgc tgtcgtatgc caggtcaggc gatgccccgc aattccgggt ggacgagatc 720

caggccatcg tcgataccgc aaaggactac ggctaccacg tcgccgccca cgcccacggc 780

gaggagggca tgcgccgcgc ggtggaggcc ggcgtgacca gcatcgaaca cggcacctac 840

atgagcgacg aggtgatggc gctgatgaag cgcaagggca cctggtacgt cccgaccgtc 900

tacgccggcc gcttcgtcgc cgacaaggcg aagatcgacg gctacttccc cgacgtggtg 960

cgcccgaaag cggccaggat cggcgagttg atccaggcca cagccgcgcg cgcctacaag 1020

gccggcgtga agatggcctt cggcaccgac atgggcgtcg gcccgcacgg cgacaacgca 1080

cgcgagttcg tctacctggt cgaagccggt gtcccggccg ccgtcgccct gcagatggcc 1140

acgatccgcg ccgccgaggt cctcggcgtc gacgatcagg gcgtgatcgc gcccggccag 1200

cgcgccgaca tcgtggcggt caagggcaat ccgctggaag acatcaaccg cgtgctcgac 1260

gtcgcgttcg tgatgaagga cggcgtggtc tacagccagc ccgggacgtc gccatga 1317

<210> 3

<211> 40

<212> DNA

<213> (Artificial sequence)

<400> 3

atctggttcc gcgtggatcc atgaccgtcc gcctggtccg 40

<210> 4

<211> 39

<212> DNA

<213> (Artificial sequence)

<400> 4

tcacgatgcg gccgctcgag tcatggcgac gtcccgggc 39

Claims

1. An application of ochratoxin detoxification enzyme in degradation of ochratoxin A is characterized in that the amino acid sequence of the ochratoxin detoxification enzyme is shown as SEQ ID No. 1.

2. The use according to claim 1, which comprises degrading ochratoxin a in cereals or tea.