Amide hydrolase LlADH capable of efficiently degrading ochratoxin A as well as mutant protein, coding gene, recombinant vector and application of amide hydrolase LlADH

Abstract

The invention discloses an amide hydrolase LlADH capable of efficiently degrading ochratoxin A as well as a mutant protein, a coding gene, a recombinant vector and application of the amide hydrolase LlADH, and belongs to the technical field of enzyme engineering. The invention further provides the optimal temperature and the optimal pH of the amide hydrolase and application of the amide hydrolase in degradation of ochratoxin A. The recombinant amide hydrolase treats corn flour and wheat flour samples containing 1000 [mu] g / kg of ochratoxin A for 10 min under the conditions of 45 DEG C and pH 8.5, and the degradation rate of the ochratoxin A reaches 100%.

Description

Technical Field

[0001] The invention relates to an amide hydrolase L1ADH for efficiently degrading ochratoxin A and a mutant protein, a coding gene, a recombinant vector and application thereof, belonging to the technical field of enzyme engineering. Background Art

[0002] Mycotoxins are a class of toxic metabolites produced by molds, which are commonly found in agricultural products and their products such as wheat, corn, soybeans, peanuts, and fruits that are contaminated by molds. Well-known mycotoxins include aflatoxins (AF), fumonisins (FUM), zearalenone (ZEN), deoxynivalenol (DON), and ochratoxin A (OTA). Mycotoxins have toxic side effects such as suppressing immunity, causing pathological changes and damage to tissues and organs, and causing cancer and teratogenesis. They can be transmitted through the food chain through contaminated food, posing a serious threat to human and livestock health.

[0003] Among them, ochratoxin A (OTA for short) is mainly produced by molds such as Aspergillus or Penicillium. OTA can cause kidney damage, liver toxicity, teratogenicity, carcinogenicity and immunosuppression. At present, the detoxification methods of OTA include physical, chemical and biological methods. Among them, physical detoxification includes water washing, adsorption, cold plasma treatment, etc. The physical detoxification method has the disadvantages of low efficiency, product nutritional loss, and high detoxification cost. Chemical detoxification methods include alkali treatment, ammoniation treatment, ozone treatment, etc., but there are problems such as chemical reagent residues and destruction of food flavor and nutritional value. The use of specific biological enzymes to catalyze the degradation of OTA into low-toxic or even non-toxic products has the advantages of low cost, high safety, and strong specificity. It is the most promising OTA detoxification method.

[0004] Amide hydrolases can specifically hydrolyze OTA to generate non-toxic ochratoxin α (OTα) and phenylalanine (Phe). This detoxification pathway is recognized as the most thorough way to detoxify OTA. How to discover or obtain more efficient OTA detoxification amidohydrolases through genetic engineering technology is crucial to the study of OTA biological detoxification. Summary of the invention

[0005] To solve the above problems, the present invention uses bioinformatics technology to mine and obtain the amidohydrolase LlADH from Lysobacter luteus from the National Center for Biotechnology Information (full name in English: National Center of Biotechnology Information, abbreviated as: NCBI). The present invention first discovered that LlADH can efficiently hydrolyze OTA to generate non-toxic OTα and phenylalanine. Further, through protein structure analysis, rational design and site-directed mutagenesis of LlADH, LlADH was transformed, and it was found that the single-site mutant LlADH / I326A obtained by mutating the isoleucine (I) at the 326th position of the amino acid sequence of LlADH to alanine (A) has higher hydrolysis activity for OTA; in addition, based on the dominant mutation of I326A, the serine (S) at the 89th position in the LlADH / I326A sequence was mutated to tyrosine (Y), phenylalanine (F), tryptophan ( W), aspartic acid (D), glutamic acid (E) or lysine (K), and the obtained double-site mutants LlADH / I326A / S89Y, LlADH / I326A / S89F, LlADH / I326A / S89W, LlADH / I326A / S89D, LlADH / I326A / S89E, and LlADH / I326A / S89K can further improve its detoxification efficiency for OTA, thereby having greater application value in the OTA detoxification industry.

[0006] The first object of the present invention is to provide an amide hydrolase L1ADH for efficiently degrading ochratoxin A, wherein the amino acid sequence of the amide hydrolase consists of the amino acid sequence shown in SEQ ID NO.2.

[0007] The second object of the present invention is to provide a single-site mutant of a recombinant amide hydrolase that efficiently degrades ochratoxin A; the amino acid sequence of the single-site mutant of the recombinant amide hydrolase consists of the amino acid sequence shown in SEQ ID NO.4, and the amino acid sequence shown in SEQ ID NO.4 is that the isoleucine at position 326 of the sequence shown in SEQ ID NO.2 is mutated to alanine.

[0008] A double-site mutant of a recombinant amide hydrolase for efficiently degrading ochratoxin A, wherein the amino acid sequence of the double-site mutant of the recombinant amide hydrolase consists of an amino acid sequence as shown in SEQ ID NO.6, or SEQ ID NO.8, or SEQ ID NO.10, or SEQ ID NO.12, or SEQ ID NO.14, or SEQ ID NO.16, wherein the amino acid sequence shown in SEQ ID NO.6 is a mutation of the 89th serine in SEQ ID NO.4 to tyrosine, the amino acid sequence shown in SEQ ID NO.8 is a mutation of the 89th serine in SEQ ID NO.4 to phenylalanine, the amino acid sequence shown in SEQ ID NO.10 is a mutation of the 89th serine in SEQ ID NO.4 to tryptophan, the amino acid sequence shown in SEQ ID NO.12 is a mutation of the 89th serine in SEQ ID NO.4 to aspartic acid, the amino acid sequence shown in SEQ ID NO.14 is a mutation of the 89th serine in SEQ ID NO.4 to glutamic acid, and the amino acid sequence shown in SEQ ID NO.16 is a mutation of the 89th serine in SEQ ID NO.4 to glutamic acid. The serine at position 89 in NO.4 mutated to lysine.

[0009] The double-site mutants LlADH / I326A / S89Y, LlADH / I326A / S89F, LlADH / I326A / S89W, LlADH / I326A / S89D, LlADH / I326A / S89E, and LlADH / I326A / S89K have 2-5 times higher hydrolysis activity for OTA than LlADH, thus having greater application prospects for OTA detoxification.

[0010] The gene encoding the above-mentioned amidohydrolase L1ADH consists of the nucleotide sequence shown in SEQ ID NO.1.

[0011] The amino acid sequence encoding the single-site mutant L1ADH / I326A of the above-mentioned recombinant amide hydrolase consists of the nucleotide sequence shown in SEQ ID NO.3.

[0012] The amino acid sequences of the double-site mutants LlADH / I326A / S89Y, LlADH / I326A / S89F, LlADH / I326A / S89W, LlADH / I326A / S89D, LlADH / I326A / S89E, and LlADH / I326A / S89K encoding the above-mentioned recombinant amide hydrolase are composed of the nucleotide sequences shown in SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, and SEQ ID NO.16, respectively.

[0013] The polynucleotide sequence encoding the amidohydrolase of the present invention can be obtained by a variety of methods. These methods include (but are not limited to): (1) using a probe to hybridize with a gene or cDNA library to detect homologous polynucleotide sequences. (2) activity screening of an expression library to detect cloned polynucleotide fragments with common structural characteristics, and the expression library may include an environmental metagenomic library or a clone library constructed from a pure culture strain. The DNA fragment sequence of the present invention can also be obtained by the following methods: (1) isolating a double-stranded DNA sequence from genomic DNA; (2) chemically synthesizing a DNA sequence to obtain the double-stranded DNA of the amidohydrolase.

[0014] The third object of the present invention is to provide a recombinant vector carrying the gene.

[0015] Furthermore, the vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

[0016] In the present invention, the polynucleotide encoding the amidohydrolase or the recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector. "Host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell.

[0017] Transformation of host cells with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence can be carried out by conventional techniques well known in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2 The treatment is well known in the art, and an alternative is MgCl 2 If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation method, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0018] Utilizing the polynucleotide sequence of the present invention, expressing or producing the recombinant amidohydrolase by conventional recombinant DNA technology comprises the following steps:

[0019] (1) transforming or transfecting a suitable host cell with a polynucleotide encoding a recombinant amidohydrolase of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0020] (2) culturing host cells in a suitable culture medium;

[0021] (3) Isolate and purify proteins from culture medium or cells.

[0022] In step (2), the culture medium used in the culture can be selected from various conventional culture media according to the host cells used. The culture is carried out under conditions suitable for the host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method, and the cells are cultured for a period of time.

[0023] In step (3), the recombinant enzyme can be encapsulated in the cell, expressed on the cell membrane, or secreted outside the cell. The recombinant protein is separated 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, osmotic sterilization, ultrasonic treatment, ultracentrifugation, affinity chromatography, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography and various other liquid chromatography techniques and combinations of these methods.

[0024] The fourth object of the present invention is to provide the use of the amidohydrolase L1ADH, the single-site mutant of the recombinant amidohydrolase, and the double-site mutant of the recombinant amidohydrolase in degrading OTA. Further, the use includes degrading OTA in corn flour or wheat flour.

[0025] The fifth object of the present invention is to provide an enzyme preparation comprising the amidohydrolase L1ADH, a single-site mutant of the recombinant amidohydrolase, and a double-site mutant of the recombinant amidohydrolase.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention provides a recombinant amide hydrolase L1ADH and its mutant protein, and also provides the optimum temperature and pH of the amide hydrolase, and its application in detoxification of ochratoxin A in hydrolyzed corn flour or wheat flour.

[0028] 2. The recombinant amide hydrolase with double-site mutant provided by the present invention has a 2-5-fold improvement in the detoxification activity of ochratoxin A compared to the original recombinant amide hydrolase L1ADH, and has a greater application prospect for OTA detoxification.

[0029] 3. The recombinant amide hydrolase of the double-site mutant was used to treat corn flour or wheat flour containing 1000 μg OTA / kg at 45°C and pH 8.5 for 10 minutes, and the hydrolysis and detoxification efficiency of OTA reached 100%, which is highly efficient in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The chemical reaction equation for the enzymatic hydrolysis of OTA to generate phenylalanine and ochratoxin α;

[0031] Figure 2 SDS-PAGE protein electrophoresis of purified recombinant amidohydrolase L1ADH and mutants L1ADH / I326A, L1ADH / I326A / S89Y, L1ADH / I326A / S89F, L1ADH / I326A / S89W, L1ADH / I326A / S89D, L1ADH / I326A / S89E, and L1ADH / I326A / S89K;

[0032] Figure 3 It is a comparative diagram of the relative enzyme activities of L1ADH and its mutants L1ADH / I326A, L1ADH / I326A / S89Y, L1ADH / I326A / S89F, L1ADH / I326A / S89W, L1ADH / I326A / S89D, L1ADH / I326A / S89E, and L1ADH / I326A / S89K in degrading OTA;

[0033] Figure 4 The HPLC chromatograms of the reaction products of OTA degradation by the purified recombinant amide hydrolase L1ADH and mutants L1ADH / I326A, L1ADH / I326A / S89Y, L1ADH / I326A / S89F, L1ADH / I326A / S89W, L1ADH / I326A / S89D, L1ADH / I326A / S89E, and L1ADH / I326A / S89KA;

[0034] Figure 5 The degradation effects of recombinant amide hydrolase L1ADH and the most active mutant L1ADH / I326A / S89Y on OTA in corn flour were compared.

[0035] Figure 6 The degradation effects of recombinant amide hydrolase L1ADH and the most active mutant L1ADH / I326A / S89Y on OTA in wheat flour were compared.

[0036] Figure 7 This is a diagram showing the results of enzyme activity detection of recombinant amide hydrolase L1ADH under different conditions;

[0037] Figure 8 is a comparison diagram of the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.6;

[0038] Fig. 9 is a comparison diagram of the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.8;

[0039] Fig.10is a comparison diagram of the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.10;

[0040] Fig.11 is a comparison diagram of the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.12;

[0041] Fig.12 is a comparison diagram of the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.14;

[0042] Fig.13 This is a comparison diagram of the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.16;. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0044] Example 1: Synthesis of the amidohydrolase L1ADH gene and construction of its mutants.

[0045] The amide hydrolase L1ADH gene from Lysobacter luteus was obtained by mining from the National Center of Biotechnology Information (NCBI) of the United States through bioinformatics technology, and the amino acid sequence of the L1ADH amide hydrolase encoded by it is shown in SEQ ID NO. 2. By structural analysis of the amino acid sequence of the amide hydrolase L1ADH, the isoleucine at position 326 of the amino acid sequence shown in SEQ ID NO. 2 was mutated to alanine, and the amino acid sequence of the mutant L1ADH / I326A was obtained as shown in SEQ ID NO. 4.

[0046] On the basis of the L1ADH / I326A dominant mutation, the serine (S) at position 89 of the amino acid sequence shown in SEQ ID NO.4 was mutated to tyrosine (Y), or phenylalanine (F), or tryptophan (W), or aspartic acid (D), or glutamic acid (E), or lysine (K), and a total of 6 double-site mutants were further obtained. The amino acid sequences of the 6 double-site mutants and the nucleotide coding sequences encoding the 6 double-site mutants are as follows:

[0047] The amino acid sequence of the L1ADH amidohydrolase is SEQ ID NO.2 ( Figure 8Sequence 2 in the sequence, the nucleotide coding sequence encoding L1ADH is shown in SEQ ID NO.1;

[0048] The amino acid sequence of the mutant L1ADH / I326A is SEQ ID NO.4, and the nucleotide coding sequence encoding L1ADH / I326A / S89Y is shown in SEQ ID NO.3;

[0049] The amino acid sequence of the mutant L1ADH / I326A / S89Y is SEQ ID NO.6 ( Figure 8 Sequence 6 in the present invention), the nucleotide coding sequence encoding L1ADH / I326A / S89Y is shown in SEQ ID NO.5;

[0050] The amino acid sequence of the mutant L1ADH / I326A / S89F is SEQ ID NO.8 ( Fig. 9 Sequence 8 in ), the nucleotide coding sequence encoding L1ADH / I326A / S89F is shown in SEQ ID NO.7;

[0051] The amino acid sequence of the mutant L1ADH / I326A / S89W is SEQ ID NO.10 ( Fig.10 Sequence 10 in the sequence, the nucleotide coding sequence encoding L1ADH / I326A / S89W is shown in SEQ ID NO.9;

[0052] The amino acid sequence of the mutant L1ADH / I326A / S89D is SEQ ID NO.12 ( Fig.11 Sequence 12 in the present invention), the nucleotide coding sequence encoding L1ADH / I326A / S89D is shown in SEQ ID NO.11;

[0053] The amino acid sequence of the mutant L1ADH / I326A / S89E is SEQ ID NO.14 ( Fig.12 Sequence 14 in the present invention), the nucleotide coding sequence encoding L1ADH / I326A / S89E is shown in SEQ ID NO. 13;

[0054] The amino acid sequence of the mutant L1ADH / I326A / S89K is SEQ ID NO.16 ( Fig.13 Sequence 16 in the sequence, and the nucleotide coding sequence encoding L1ADH / I326A / S89K is shown in SEQ ID NO.15.

[0055] The above SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13 and SEQ ID The nucleotide sequence shown in NO.15 was submitted to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis and cloned into the Escherichia coli expression vector pET46EK to obtain recombinant plasmids pET46EK-LlADH, pET46EK-LlADH / I326A, pET46EK-LlADH / I326A / S89Y, pET46EK-LlADH / I326A / S89F, pET46EK-LlADH / I326A / S89W, pET46EK-LlADH / I326A / S89D, pET46EK-LlADH / I326A / S89E and pET46EK-LlADH / I326A / S89K.

[0056] Example 2: Expression and purification of L1ADH and mutant proteins in E. coli.

[0057] The eight recombinant plasmids constructed in Example 1 were transformed into competent cells of E. coli Shuffle T7E.coil, and strains were selected on LB plates containing 100 μg / mL ampicillin. 1.5 mL of seed culture was inoculated into 6 mL of fresh LB, and cultured overnight at 37°C with shaking at 230 rpm / min, and then expanded to 100 mL of LB medium. Finally, the culture was expanded to 5 L of LB medium. At the OD of the bacteria, 600 When the value reaches 0.8, the culture temperature is lowered to 16°C, and IPTG is added to a concentration of 0.2mM to induce the massive expression of the protein. After another 16h of protein induction, the bacterial solution is centrifuged at 6000rpm for 6min to collect the E. coli cells. Then the cells are resuspended with a buffer (20mM Tris-HCl, pH 8.5), and the cells are broken by a cell crusher, and then centrifuged at 17000rpm for 45min, and the supernatant is collected for the next step of purification. Then the target protein is eluted using a nickel ion chromatography column in sequence using a fast protein liquid chromatography instrument, and then the target protein is dialyzed in 5L (20mM Tris-HCl, pH 8.5 at 4°C overnight, and then purified by an ion column. Finally, the purified target protein is concentrated in 20Mm Tris-HCl, pH8.5 buffer and stored at -80°C. LlADH and its mutant proteins are expressed and purified according to the above steps.

[0058] Figure 2 The SDS-PAGE protein electrophoresis diagram of the purified recombinant amide hydrolase L1ADH and mutant protein.

[0059] Example 3: Determination of OTA hydrolysis activity.

[0060] In order to analyze the difference in the hydrolysis activity of L1ADH and mutants towards OTA, this example further determined the hydrolysis activity of recombinant amide hydrolase L1ADH, mutants L1ADH / I326A, L1ADH / I326A / S89Y, L1ADH / I326A / S89F, L1ADH / I326A / S89W, L1ADH / I326A / S89D, L1ADH / I326A / S89E and L1ADH / I326A / S89K towards OTA.

[0061] The activity test method of OTA hydrolase is as follows:

[0062] The mixture (100 μL) of each reaction was in a mixture including 200 μg / mL OTA, 10 μL enzyme (10 μg / mL), and pH 8.5. After mixing, it was placed in a shaking metal bath at 45°C and 800 rpm for 20 min. Each reaction was performed in 3 parallels and repeated 3 times. After the reaction, an equal volume of acetonitrile was added to terminate the enzyme reaction, and then centrifuged at 12000 rpm for 10 min, and the supernatant reaction solution was filtered through a 0.22 μm filter membrane. Then, each group of reactions was determined and analyzed by high performance liquid chromatography (HPLC, Shimadzu SPD-M20A), and the analytical column was InertSustain C18 column (4.6×250 mm, 5 μm), mobile phase A: water, mobile phase B: 95% acetonitrile + 5% acetic acid, flow rate 1 mL / min, detection wavelength 330 nm, elution time 20 min, and elution concentration 50%.

[0063] Among them, the HPLC detection results of the degradation products of OTA by LlADH and LlADH / I326A / S89Y are as follows: Figure 3 As shown in the figure, the peaks are eluted at retention time 4.6min and retention time 12.3min respectively. Among them, the peak time of retention time 4.6min is consistent with the product standard OTα, so the product corresponding to the chromatographic peak at retention time 4.6min is OTα; while the peak time of retention time 12.3min is consistent with the substrate standard OTA, so the product corresponding to the chromatographic peak at retention time 12.3min is OTA.

[0064] Furthermore, by comparing the peak areas of OTα generated by LlADH and each mutant after hydrolyzing OTA, the difference in OTA hydrolysis activity between LlADH and each mutant was calculated. Figure 4As shown, the hydrolysis activities of LlADH / I326A, LlADH / I326A / S89Y, LlADH / I326A / S89F, LlADH / I326A / S89W, LlADH / I326A / S89D, LlADH / I326A / S89E and LlADH / I326A / S89K towards OTA were 1.76, 5.12, 1.98, 3.48, 2.43, 2.99 and 2.01 times that of LlADH, respectively.

[0065] Among them, the double mutant LlADH / I326A / S89Y had the highest activity; therefore, we further used the mutant protein LlADH / I326A / S89Y as a representative to test its application in the hydrolysis and detoxification of OTA-contaminated grains.

[0066] Example 4: Application of recombinant amide hydrolase L1ADH and double mutant L1ADH / I326A / S89Y in detoxification of OTA-contaminated corn flour.

[0067] The OTA standard stock solution was mixed with corn flour to prepare corn flour contaminated with OTA (the final concentration was 1000 μg OTA per kg corn flour). The purified recombinant amide hydrolase LlADH and double mutant LlADH / I326A / S89Y were mixed with the OTA contaminated corn flour respectively. The prepared samples were reacted at 45°C and pH 8.5, and samples were taken at 0min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min and 50min. Three replicates were set for each degradation experiment. At the same time, the OTA contaminated corn flour sample without the addition of recombinant amide hydrolase LlADH or double mutant LlADH / I326A / S89Y was used as a negative control. The sample size of the treatment group and the blank control group was the same each time, and the extraction, purification and analysis were carried out according to the OTA standard detection method.

[0068] The degradation rate of OTA by recombinant amide hydrolase was measured according to the toxin extraction and purification detection method described in ZL201910004498.1. The results are as follows Figure 5 As shown, in the negative control group without enzyme addition, OTA existed stably and was not degraded within 0-50 min; while the recombinant amidohydrolase LlADH and LlADH / I326A / S89Y completely degraded OTA in the sample within 50 min and 10 min, respectively, and the degradation efficiency reached 100%; the degradation efficiency of LlADH / I326A / S89Y for OTA was significantly better than that of LlADH, which is consistent with the relative enzyme activity determination results of LlADH and LlADH / I326A / S89Y for OTA in Example 3.

[0069] Example 5: Application of recombinant amide hydrolase L1ADH and double mutant L1ADH / I326A / S89Y in detoxification of OTA-contaminated wheat flour.

[0070] The OTA standard stock solution was mixed with corn flour to prepare wheat flour contaminated with OTA (the final concentration was 1000 μg OTA per kg corn flour). The purified recombinant amide hydrolase LlADH and double mutant LlADH / I326A / S89Y were mixed with OTA-contaminated corn flour respectively, and reacted at pH 8.5 and 45°C. Samples were taken at 0min, 5min, 10min, 15min, 20min, 25min, 30min, 35min and 40min, and 3 replicates were set for each degradation experiment. At the same time, OTA-contaminated corn flour samples without the addition of recombinant amide hydrolase LlADH or double mutant LlADH / I326A / S89Y were used as negative controls. The same amount of samples were taken each time in the treatment group and the blank control group, and extraction, purification and analysis were performed according to the OTA standard detection method.

[0071] The degradation rate of OTA by recombinant amide hydrolase was measured according to the OTA extraction and purification detection method described in ZL201910004498.1. The results are as follows Figure 6 As shown, in the negative control group without enzyme addition, OTA was stably present and not degraded within 0-40 min; while the recombinant amidohydrolase LlADH and LlADH / I326A / S89Y completely degraded OTA in the sample within 40 min and 10 min, respectively, with a degradation efficiency of 100%; the degradation efficiency of LlADH / I326A / S89Y for OTA was significantly better than that of LlADH, which is consistent with the relative enzyme activity determination results of LlADH and LlADH / I326A / S89Y for OTA in Example 3.

[0072] Example 6: Optimization of enzyme catalytic reaction conditions of amidohydrolase L1ADH

[0073] Referring to the activity test method of OTA hydrolase shown in Example 3, the optimal temperature of LlADH was tested, and the reaction temperatures were set at 10°C, 20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 60°C, 70°C, and 80°C, respectively, and 3 replicates were set for each degradation experiment.

[0074] Referring to the activity test method of OTA hydrolase shown in Example 3, the optimal pH of LlADH was tested, and the pH of the reaction solution was set to 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0, respectively, and 3 replicates were set for each degradation experiment.

[0075] Referring to the activity test method of OTA hydrolase shown in Example 3, the effect of metal ions on L1ADH was tested. 10 mM metal ions (CaCl 2 ,MgCl 2 ,CoCl 2 ,MnCl 2 ,NiCl 2 ,FeCl 2 ,CuCl 2 and ZnCl 2 ), and each degradation experiment was repeated 3 times.

[0076] from Figure 7 According to the data of A, LlADH has a high OTA degradation activity (>70%) in the range of 40-50°C; Figure 7 From the data of B, LIADH has a high OTA degradation activity (>80%) in the pH range of 7.5-8.5. Figure 7 According to the data of C, Mn 2+ 、Ni 2+ , Ca 2+ Mg 2+ 、Co 2+ All of them have an inhibitory effect on the hydrolysis activity of L1ADH. 2+ , Cu 2+ 、Zn 2+ It has a strong inhibitory effect on the hydrolysis activity of LlADH.

[0077] Double mutants and single mutants generally do not change the enzymatic properties, so the enzyme catalytic reaction conditions of the recombinant amidohydrolase LlADH and the double mutant LlADH / I326A / S70Y are the same.

[0078] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. An amide hydrolase L1ADH for efficiently degrading ochratoxin A, characterized in that: The amino acid sequence of the amidohydrolase consists of the amino acid sequence shown in SEQ ID NO.

2.

2. A single-site mutant of a recombinant amide hydrolase that efficiently degrades ochratoxin A, characterized in that: The amino acid sequence of the single-site mutant of the recombinant amidohydrolase consists of the amino acid sequence shown in SEQ ID NO.4, and the amino acid sequence shown in SEQ ID NO.4 is that the isoleucine at position 326 of the sequence shown in SEQ ID NO.2 is mutated to alanine.

3. A double-site mutant of a recombinant amide hydrolase that efficiently degrades ochratoxin A, characterized in that: The amino acid sequence of the double-site mutant of the recombinant amidohydrolase consists of the amino acid sequence shown in SEQ ID NO.6, or SEQ ID NO.8, or SEQ ID NO.10, or SEQ ID NO.12, or SEQ ID NO.14, or SEQ ID NO.16, wherein the amino acid sequence shown in SEQ ID NO.6 is a mutation of the 89th serine in SEQ ID NO.4 to tyrosine, the amino acid sequence shown in SEQ ID NO.8 is a mutation of the 89th serine in SEQ ID NO.4 to phenylalanine, the amino acid sequence shown in SEQ ID NO.10 is a mutation of the 89th serine in SEQ ID NO.4 to tryptophan, the amino acid sequence shown in SEQ ID NO.12 is a mutation of the 89th serine in SEQ ID NO.4 to aspartic acid, the amino acid sequence shown in SEQ ID NO.14 is a mutation of the 89th serine in SEQ ID NO.4 to glutamic acid, and the amino acid sequence shown in SEQ ID NO.16 is a mutation of the 89th serine in SEQ ID NO.4 to lysine.

4. A gene encoding the amidohydrolase L1ADH according to claim 1, characterized in that: The gene of amide hydrolase L1ADH composed of the nucleotide sequence shown in SEQ ID NO.

1.

5. A gene encoding a single-site mutant of the recombinant amidohydrolase according to claim 2, characterized in that: A gene of a single-site mutant of a recombinant amidohydrolase consisting of the nucleotide sequence shown in SEQ ID NO.

3.

6. A gene encoding a double-site mutant of the recombinant amidohydrolase according to claim 3, characterized in that: A gene of a double-site mutant of a recombinant amidohydrolase consisting of the nucleotide sequence shown in SEQ ID NO.5, or SEQ ID NO.7, or SEQ ID NO.9, or SEQ ID NO.11, or SEQ ID NO.13, or SEQ ID NO.

15.

7. A recombinant expression vector carrying the gene of the amidohydrolase L1ADH according to claim 4, the gene of the single-site mutant of the recombinant amidohydrolase according to claim 5, or the gene of the double-site mutant of the recombinant amidohydrolase according to claim 6.

8. Use of the amidohydrolase L1ADH according to claim 1, the single-site mutant of the recombinant amidohydrolase according to claim 2, or the double-site mutant of the recombinant amidohydrolase according to claim 3 in detoxifying agricultural products contaminated by ochratoxin A.

9. The use according to claim 8, characterized in that: The agricultural products include corn flour and wheat flour.

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