Amide hydrolase mutant and application thereof

Abstract

The invention discloses an amide hydrolase mutant and application thereof. The invention provides a mutant protein of amidase CaADH, which is obtained according to any one of the following modes: the 67th amino acid residue G on the amino acid sequence of the amidase CaADH as shown in SEQ ID NO: 1 is mutated into D, or the 67th amino acid residue G is mutated into E, or the 67th amino acid residue G is mutated into L, and the mutant protein has the same function; experiments prove that the amide hydrolase CaADH and the mutant thereof can be used for efficiently degrading OTA and realizing OTA detoxification.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme preparation, and relates to an amide hydrolase mutant and application thereof. BACKGROUND

[0002] Mycotoxins are secondary metabolites produced by some fungi, which can infect grains, nuts and fruits, etc., and pose a threat to humans and animals. Common mycotoxins include aflatoxins, fumonisins, zearalenone (ZEN), deoxynivalenol and ochratoxin, etc.

[0003] Ochratoxin is a kind of secondary metabolite produced by Aspergillus and Penicillium fungi, which has an isocoumarin-phenylalanine complex structure. Among the 19 kinds of similar compounds found so far, ochratoxin A (OTA) is the most toxic and widely distributed. Animal experiments show that it can induce liver cancer, kidney cancer and intestinal tumors. As a highly toxic 2B carcinogen, the threat of ochratoxin A (OTA) to food safety cannot be ignored. Its pollution range is wide, and the toxicity mechanism is complex (involving liver and kidney damage, carcinogenesis and teratogenesis), which needs to be reduced by strict detection and scientific prevention and control to reduce exposure risk.

[0004] At present, the detoxification methods of OTA include physical method, chemical method and biological method. The physical detoxification method includes heat treatment, adsorbent adsorption, etc. Chemical agents usually use alkaline reagents or organic reagents for treatment. The biological method refers to the degradation by microorganisms or enzymatic hydrolysis to achieve detoxification, and is highly regarded for its good specificity, green environmental protection and other characteristics. Amide hydrolase can hydrolyze OTA to generate non-toxic ochratoxin alpha (OTa) and L-phenylalanine, and this hydrolysis pathway is considered to be the most effective detoxification method of OTA. Enzymatic detoxification has shown high efficiency. Although enzymatic detoxification has shown potential for industrial application, there are still significant technical bottlenecks in the prior art: most of the reported OTA hydrolytic enzymes belong to mesophilic enzymes, and their optimum action temperatures mainly distribute in the range of 28-50℃. In the industrial production scene, high-temperature processing environment will cause the enzyme activity to decrease sharply, forcing the production process to increase the cooling treatment link, which significantly increases equipment investment and energy consumption. Therefore, it is urgent to obtain a new enzyme molecular skeleton with heat resistance through gene mining technology, and to construct a high-activity enzyme mutant by using protein engineering technologies such as rational design and directed evolution, so as to promote the industrialization of OTA detoxification technology. SUMMARY

[0005] The technical problem solved by the present application is to find an efficient and heat-resistant OTA detoxification enzyme.

[0006] In order to solve the above technical problems, the present application provides an amide enzyme in a first aspect Caa mutant protein of ADH, which is any one of: A1) the mutant protein comprises the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function; Ca A2) the mutant protein comprises the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to L, and has the same function; A3) the mutant protein has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the protein of any one of A1) - A2) except for the mutated amino acid residue, and has the same function; Ca A4) the mutant protein comprises a sequence of a protein of any one of A1) - A2) with a tag connected to the N-terminus and / or C-terminus of the protein. In some embodiments, the mutant protein of A1) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function. In some embodiments, the mutant protein of A2) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to L, and has the same function.

[0007] Ca In some embodiments, the mutant protein of A1) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function. In some embodiments, the mutant protein of A2) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to L, and has the same function.

[0008] Ca In some embodiments, the mutant protein of A1) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function. In some embodiments, the mutant protein of A2) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to L, and has the same function.

[0009] In some embodiments, the mutant protein of A1) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function. In some embodiments, the mutant protein of A2) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to L, and has the same function.

[0010] In some embodiments, the mutant protein of A1) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function. In some embodiments, the mutant protein of A2) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to L, and has the same function. Ca In some embodiments, the mutant protein of A1) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function. Ca In some embodiments, the mutant protein of A2) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to L, and has the same function. In some embodiments, the mutant protein of A1) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function.

[0011] In some embodiments, the mutant protein of A2) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to L, and has the same function. Ca In some embodiments, the mutant protein of A1) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function. Ca In some embodiments, the mutant protein of A2) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to L, and has the same function. In some embodiments, the mutant protein of A1) is the amidase of SEQ ID NO: 1 with the 67th amino acid residue G mutated to E, and has the same function.

[0012] The mutant protein described above, The amino acid sequence of the mutant protein shown in A1) includes SEQ ID NO: 7. The amino acid sequence of the mutant protein shown in A2) includes SEQ ID NO: 9.

[0013] The mutant protein described above, In some embodiments, the amino acid sequence of the mutant protein shown in A1) can be SEQ ID NO: 7, or can be sequentially connected end to end in the order of SEQ ID NO: 3 1-163, SEQ ID NO: 3 164-170, and SEQ ID NO: 7.

[0014] In some embodiments, the amino acid sequence of the mutant protein shown in A2) can be SEQ ID NO: 9, or can be sequentially connected end to end in the order of SEQ ID NO: 3 1-163, SEQ ID NO: 3 164-170, and SEQ ID NO: 9.

[0015] In a second aspect, the present application provides a biological material related to the mutant protein of the first aspect, which is any one of the following C1) to C4): C1) a nucleic acid molecule encoding the mutant protein of the first aspect; C2) an expression cassette containing the nucleic acid molecule of C1); C3) a recombinant vector containing the nucleic acid molecule of C1), or a recombinant vector containing the expression cassette of C2); C4) a recombinant microorganism containing the nucleic acid molecule of C1), or a recombinant microorganism containing the expression cassette of C2), or a recombinant microorganism containing the recombinant vector of C3).

[0016] In the biological material described above, the nucleic acid molecule of C1) is any one of the following c1) to c4): The nucleotide sequence of the nucleic acid molecule shown in c1) includes SEQ ID NO: 8. The nucleotide sequence of the nucleic acid molecule shown in c2) includes SEQ ID NO: 10. c3) a DNA molecule having 75% or more identity with any one of the nucleotide sequences defined in c1) to c2), and encoding the mutant protein of the first aspect; c4) a DNA molecule hybridizing to any one of the nucleotide sequences defined in c1) to c2) under stringent conditions, and encoding the mutant protein of the first aspect.

[0017] In the above, In some embodiments, the nucleotide sequence of the nucleic acid molecule of c1) can be SEQ ID NO: 8, or can be sequentially connected in a head-to-tail manner from the 1st to the 489th of SEQ ID NO: 4, the 490th to the 510th of SEQ ID NO: 4, and SEQ ID NO: 8.

[0018] In some embodiments, the nucleotide sequence of the nucleic acid molecule of c2) can be SEQ ID NO: 10, or can be sequentially connected in a head-to-tail manner from the 1st to the 489th of SEQ ID NO: 4, the 490th to the 510th of SEQ ID NO: 4, and SEQ ID NO: 10.

[0019] The stringent conditions are hybridization in a solution of 6xSSC, 0.5% SDS at 65°C, and then washing the membrane with 2xSSC, 0.1% SDS and 1xSSC, 0.1% SDS respectively.

[0020] In a third aspect, the present application provides the mutant protein of the first aspect for use in any one of the following: D1) degrading ochratoxin A; D2) hydrolyzing ochratoxin A into ochratoxin alpha; D3) preparing a product for degrading ochratoxin A; D4) detoxifying ochratoxin A; D5) preparing an ochratoxin A detoxifying agent.

[0021] In a fourth aspect, the present application provides the biomaterial of the second aspect for use in any one of the following: D1) degrading ochratoxin A; D2) hydrolyzing ochratoxin A into ochratoxin alpha; D3) preparing a product for degrading ochratoxin A; D4) detoxifying ochratoxin A; D5) preparing an ochratoxin A detoxifying agent.

[0022] The detoxifying agent is a biological detoxifying agent.

[0023] In a fifth aspect, the present application provides a method for degrading ochratoxin A, comprising the step of degrading ochratoxin A with the mutant protein of the first aspect, so as to degrade ochratoxin A.

[0024] In the above method, the temperature for degrading ochratoxin A is 25-80°C.

[0025] Further, the temperature for degrading ochratoxin A is 65°C.

[0026] In a sixth aspect, the present application provides a product for degrading ochratoxin A, which comprises the mutant protein of the first aspect or the biological material of the second aspect.

[0027] The product described above is an enzyme preparation or ochratoxin A detoxification agent.

[0028] Experiments of the present application prove that the present application finds an amide hydrolase Ca ADH and its mutants can efficiently degrade OTA, achieving OTA detoxification. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a wild-type recombinant amidase Ca HPLC analysis results of ADH degrading OTA.

[0030] Figure 2 is a recombinant amidase Ca Comparison of relative enzyme activities of ADH and its mutants degrading OTA, WT represents wild Ca ADH, G67D, G67E, G67L represent mutants. DETAILED DESCRIPTION

[0031] The present application will be further described in detail below in conjunction with the specific embodiments, and the examples given are only for the purpose of illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0032] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0033] In the following examples, unless otherwise specified, the quantitative tests were set up in triplicate, and the results were averaged.

[0034] In the following examples, the test materials and reagents are as follows: genes and vectors: E. coli expression vector pET32a(+) (69015-3, Novagen, USA) and E. coli strain BL21 (DE3) (Beijing Quanshi Gold Biotechnology Co., Ltd.); enzymes and other biochemical reagents; ochratoxin (OTA, CAS No: 303-47-9); IPTG; culture medium; E. coli culture medium LB.

[0035] Example 1, preparation of recombinant amidase Ca ADH and mutants 1. Expression of amide hydrolase Ca Construction of vectors for ADH and mutant genes 1) Wild type Ca Construction of ADH recombinant plasmid The objective gene is derived from amide hydrolase of the warm fungi domain Ca ADH. Amide hydrolase Ca The amino acid sequence of ADH is shown as SEQ ID NO: 1, which encodes amide hydrolase Ca The nucleotide sequence of the gene of ADH is shown as SEQ ID NO: 2.

[0036] Expression of amide hydrolase Ca Recombinant vector pET32a-Tev-ADH encoding gene of ADH Ca ADH is a TEV- Ca ADH gene (490-1725 of SEQ ID NO: 4, i.e. SEQ ID NO: 2 shown amide hydrolase with TEV enzyme cutting site (amino acid sequence ENLYFQG) at N terminal Ca The gene of ADH replaces the fragment between the BamHI and HindIII restriction enzyme cutting sites of plasmid pET32a(+) to make TEV- Ca The ADH gene is downstream of T7 promoter and lac operator and is regulated by them, and the obtained vector. The TrxA tag, TEV enzyme cutting site and Ca ADH gene in the vector are co-expressed to obtain recombinant protein TrxA-TEV- Ca ADH.

[0037] Recombinant protein TrxA-TEV- Ca The amino acid sequence of ADH is SEQ ID NO: 3, wherein SEQ ID NO: 3 is TrxA tag, SEQ ID NO: 3 is TEV enzyme cutting site, and SEQ ID NO: 3 is Ca ADH protein.

[0038] Recombinant protein TrxA-TEV- Ca The nucleotide sequence of the coding gene of ADH is SEQ ID NO: 4, wherein SEQ ID NO: 4 is TrxA tag encoding nucleic acid, SEQ ID NO: 4 is TEV enzyme cutting site encoding nucleic acid, and SEQ ID NO: 4 is Ca The coding nucleic acid of ADH protein.

[0039] 2) Expression CaRecombinant plasmid of ADH mutant By studying wild type Ca ADH protein structure, the amino acids near the active region involved in substrate interaction were selected for site-directed mutagenesis to increase the activity of the enzyme on substrate OTA. That is, the present application uses structure analysis and site-directed mutagenesis technology to mutate Ca ADH to obtain Ca ADH mutant, changes the original OTA hydrolase activity problem, improves Ca ADH activity of OTA degradation. Through research, the following three mutants are determined: Ca ADH mutant G67D, G67E and G67L.

[0040] Ca ADH mutant G67D is to mutate the glycine at position 67 of wild type Ca ADH shown in SEQ ID NO: 1 to aspartic acid, and the amino acid residues at other positions remain unchanged. Ca The amino acid sequence of ADH mutant G67D is SEQ ID NO: 5, and the nucleotide sequence of its encoding gene is SEQ ID NO: 6; Ca ADH mutant G67E is to mutate the glycine at position 67 of wild type Ca ADH shown in SEQ ID NO: 1 to glutamic acid, and the amino acid residues at other positions remain unchanged. Ca The amino acid sequence of ADH mutant G67E is SEQ ID NO: 7, and the nucleotide sequence of its encoding gene is SEQ ID NO: 8.

[0041] Ca ADH mutant G67L is to mutate the glycine at position 324 of wild type Ca ADH shown in SEQ ID NO: 1 to leucine, and the amino acid residues at other positions remain unchanged. Ca The amino acid sequence of ADH mutant G67L is SEQ ID NO: 9, and the nucleotide sequence of its encoding gene is SEQ ID NO: 10.

[0042] The above recombinant vector is prepared as follows: using site-directed mutagenesis technology, taking pET32a-Tev- Ca ADH plasmid as the template, and using the primers shown in Table 1 for PCR, respectively, to obtain each CaPlasmids of ADH mutants; then add restriction enzyme DpnI to react at 37 ℃ to remove the original template. The purified reaction product is transformed into E. coli competent cells, and primary screening is performed with antibiotics, and DNA sequencing is performed to determine the successfully mutated genes, obtaining expression of each Ca Plasmids of ADH mutants, named pET32a-Tev- Ca ADH-G67D, pET32a-Tev- Ca ADH-G67E, and pET32a-Tev- Ca ADH-G67L.

[0043] Table 1 is the primer required for mutation

[0044] Recombinant vector pET32a-Tev- Ca ADH-G67D expressed recombinant protein TrxA-Tev- Ca ADH-G67D, the recombinant protein TrxA-Tev- Ca The amino acid sequence of ADH-G67D is SEQ ID NO: 3 1-163, SEQ ID NO: 3 164-170, and SEQ ID NO: 5 in turn end to end; the recombinant protein TrxA-Tev- Ca The nucleotide sequence of the coding gene of ADH-G67D is SEQ ID NO: 4 1-489, SEQ ID NO: 4 490-510, and SEQ ID NO: 6 in turn end to end; Recombinant vector pET32a-Tev- Ca ADH-G67E expressed recombinant protein TrxA-Tev- Ca ADH-G67E, the recombinant protein TrxA-Tev- Ca The amino acid sequence of ADH-G67E is SEQ ID NO: 3 1-163, SEQ ID NO: 3 164-170, and SEQ ID NO: 7 in turn end to end; the recombinant protein TrxA-Tev- Ca The nucleotide sequence of the coding gene of ADH-G67E is SEQ ID NO: 4 1-489, SEQ ID NO: 4 490-510, and SEQ ID NO: 8 in turn end to end; Recombinant vector pET32a-Tev- Ca ADH-G67L expressed recombinant protein TrxA-Tev- CaADH-G67L, the recombinant protein TrxA-Tev- Ca The amino acid sequence of ADH-G67L is sequentially connected in order from the first to the last of SEQ ID NO: 3 1-163, SEQ ID NO: 3 164-170 and SEQ ID NO: 9; the recombinant protein TrxA-Tev- Ca The nucleotide sequence of the coding gene of ADH-G67L is sequentially connected in order from the first to the last of SEQ ID NO: 4 1-489, SEQ ID NO: 4 490-510 and SEQ ID NO: 10.

[0045] 2. Expression of amide hydrolase Ca Recombinant bacteria of ADH coding gene The above-mentioned expression of amide hydrolase Ca Recombinant vectors pET32a-TEV of ADH and each mutant Ca ADH, pET32a-Tev- Ca ADH-G67D, pET32a-Tev- Ca ADH-G67E and pET32a-Tev- Ca ADH-G67L is transformed into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli BL21 (DE3) / pET-32a-TEV- Ca ADH, BL21 (DE3) / pET32a-Tev- Ca ADH-G67D, BL21 (DE3) / pET32a-Tev- Ca ADH-G67E, BL21 (DE3) / pET32a-Tev- Ca ADH-G67L.

[0046] The following BL21 (DE3) / pET-32a-TEV- Ca ADH is called recombinant Escherichia coli Ca ADH (WT), BL21 (DE3) / pET32a-Tev- Ca ADH-G67D is called recombinant Escherichia coli G67D, BL21 (DE3) / pET32a-Tev- Ca ADH-G67E is called recombinant Escherichia coli G67E, BL21 (DE3) / pET32a-Tev- Ca ADH-G67L is called recombinant Escherichia coli G67L.

[0047] 3. Amide hydrolase Ca Expression and purification of ADH and its mutants Take the above-constructed recombinant Escherichia coli Ca ADH, G67D, G67E, and G67L were each inoculated into 100 mL of LB medium and cultured at 37 °C with shaking at 220 rpm for 12 h. Then, they were transferred at a 1% ratio to 5 L of LB medium and cultured at 37 °C with shaking at 220 rpm for approximately 6 h (OD600≈0.6). Next, IPTG was added to a final concentration of 0.2 mM, and the cells were induced at 16 °C with shaking at 220 rpm for 20 h. The cells were then collected by centrifugation. The bacterial cells were resuspended in a balanced buffer (25 mM Tris, 300 mM NaCl, 10% glycerol, pH 7.5). The cells were lysed using a low-temperature, high-pressure continuous flow cell disruptor (4 °C, 1000 bar, disruption for 30 minutes, JN-30c low-temperature, high-pressure continuous flow cell disruptor, Guangzhou Juneng Nanobiotechnology Co., Ltd.) to obtain fragmented bacterial cells.

[0048] Centrifuge 15,000 g of the broken bacterial cell fragments for 1 hour to remove the precipitate and collect the supernatant.

[0049] To obtain high-purity enzyme protein, the supernatant was eluted using a fast protein liquid chromatography (FPLC) system and a nickel ion affinity chromatography column (buffer A: 25 mM Tris, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 7.5; buffer B: 25 mM Tris, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 7.5, solvent: water). 80% of buffer B was collected to obtain the target protein. 200 µL of LTEV protease (Zeye Biotechnology, ZY130873, 300U) was added to the target protein for enzymatic digestion to remove the TrxA tag on the vector. Simultaneously, the target protein was dialyzed in 5 L of dialysis buffer (25 mM Tris, 300 mM NaCl, 10% glycerol, pH 7.5, solvent: water) overnight at 4 °C. The target protein, after enzyme digestion, was passed through a nickel affinity chromatography column once more. The streamlet protein without the TrxA tag was collected. The purified target protein was dialyzed against buffer (25 mM Tris, 300 mM NaCl, 10% glycerol, pH 7.5, balance water), concentrated, collected, and stored at -80 °C to obtain the target protein. Ca ADH solution, Ca ADH mutant G67D solution, Ca ADH mutant G67E solution, CaADH mutant G67L solution (solvent is a buffer containing 25 mM Tris, 300 mM NaCl, 10% glycerol at pH 7.5).

[0050] Detection of amide hydrolase by SDS-PAGE Ca ADH, Ca ADH mutant G67D, G67E and G67L protein size, all results are 44 kD, consistent with the expected.

[0051] Ca ADH protein solution, Ca ADH mutant G67D solution, Ca ADH mutant G67E solution and Ca The concentration of ADH mutant G67L protein solution is 5 mg / ml.

[0052] The target protein obtained Ca ADH, Ca ADH mutant G67D, G67E and G67L were used for OTA degradation activity test.

[0053] Example 2, amide hydrolase Ca ADH and its mutants degrade OTA OTA was dissolved in chromatographically pure acetonitrile to prepare a stock solution of 1 mg / ml (denoted as OTA solution), and the reaction system was constructed as follows: 188 ul Tris-HCl solution (50 mM, pH 7.0), 2 ul OTA solution (final concentration 10 ug / ml), 10 ul different amide hydrolase solutions (protein amount 0.5 ug), to obtain the detection reaction system (total volume 200 ul). Ca ADH corresponds to the system diagram denoted as Ca ADH or WT, Ca ADH mutant G67D corresponds to the system diagram denoted as G67D, Ca ADH mutant G67E corresponds to the system diagram denoted as G67E, Ca ADH mutant G67L corresponds to the system diagram denoted as G67L.

[0054] The above different amide hydrolase solutions are respectively prepared in Example 1 Ca ADH protein solution, Ca ADH mutant G67D solution, Ca ADH mutant G67E solution or Ca ADH mutant G67L solution.

[0055] No amide hydrolase was added CaADH as a control reaction system (CK / OTA in the figure).

[0056] The above detection reaction system was set up in 3 replicates. After reaction at 65 °C, 1000 rpm for 3 min, the reaction was terminated by adding three times the volume of acetonitrile, centrifuged at 12000 g for 5 min, and filtered through a 0.22 μm organic phase filter. The degradation of ochratoxin A in the above reaction system was analyzed by high performance liquid chromatography (HPLC).

[0057] The liquid chromatography was Shimadzu LC-20AT high performance liquid chromatography analysis system, and the chromatographic separation column was Ultimate® XB-C18 column (4.6 x 250 mm, 5 μm). The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was acetonitrile. The isocratic gradient elution conditions were as follows: 50% B elution for 20 min. The fluorescence detector was used for detection, the excitation wavelength was 333 nm, the emission wavelength was 460 nm, and the flow rate was 1 ml / min.

[0058] The retention time of OTA standard (CAS No: 303-47-9, Shanghai Yuan Ye Biological Technology Co., Ltd.) was about 11.9 min.

[0059] The retention time of ochratoxin a standard (OTa, CAS No: 19165-63-0, Shanghai Yuan Ye Biological Technology Co., Ltd.) was about 8.0 min.

[0060] The HPLC analysis results are shown in Figure 1, Ca In the ADH detection reaction system, the peak produced at a retention time of about 11.9 min was consistent with the retention time of the OTA standard, i.e. ochratoxin A. The peak produced at a retention time of about 8 min was consistent with the retention time of the OTa standard, i.e. ochratoxin a.

[0061] The degradation rate was calculated by comparing the peak area of residual OTA in the control system and different reaction systems.

[0062] Degradation rate = 〔(initial OTA peak area - residual OTA peak area) / initial OTA peak area〕 x 100% The wild type ADH was used as a control. Ca The degradation rate of ADH was taken as 100%, Ca The degradation rates of ADH mutants G67D, G67E, and G67L were compared with that of the wild type ADH, Ca and were taken as the relative enzyme activity.

[0063] The detection results are shown in Ca , and CaThe ADH mutants G67E and G67L showed higher OTA degradation activity than the wild type. Ca ADH protein (WT), in which Ca The relative enzyme activities of ADH mutants G67E and G67L are approximately equal to those of the wild type. Ca The activity of ADH was 1.9 and 1.5 times that of the wild type, while the activity of the mutant G67D was slightly lower than that of the wild type, indicating that... Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca ADH mutants G67E and G67L have great application value.

[0064] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A mutant protein of amidase CaADH, which is any one of the following: The mutant protein shown in A1) includes the amidase shown in SEQ ID NO:

1. A1) the mutant protein has an amino acid sequence comprising SEQ ID NO: 7; A2) the mutant protein has an amino acid sequence comprising SEQ ID NO:

9. ADH is a protein with the same function whose amino acid residue G is mutated to E at amino acid residue 67 on its amino acid sequence. A2) mutant proteins shown in SEQ ID NO: 1 include an amylase A3) the mutant protein has an amino acid sequence which is more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identical to the amino acid sequence of any one of A1) - A2), and has the same function; and A4) the mutant protein comprises a tag linked to the N-terminus or / and C-terminus of any one of A1) - A2). ADH amino acid sequence at position 67 amino acid residue G is mutated to L, and a protein having the same function; 2. The mutant protein of claim 1, wherein: A1) the mutant protein has an amino acid sequence comprising SEQ ID NO: 7; A2) the mutant protein has an amino acid sequence comprising SEQ ID NO:

9.

3. The mutant protein of claim 1 or 2, wherein: The amidoase A1) the mutant protein has an amino acid sequence comprising SEQ ID NO: 7; A2) the mutant protein has an amino acid sequence comprising SEQ ID NO:

9. The mutant protein of ADH has higher activity than the amidoase shown in SEQ ID NO: 1 4. A biological material related to the mutant protein of any one of claims 1-3, which is any one of the following C1) to C4): ADH. C1) a nucleic acid molecule encoding the mutant protein of any one of claims 1-3; C2) an expression cassette comprising the nucleic acid molecule of C1); C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2); and C4) a recombinant microorganism comprising the nucleic acid molecule of C1), or a recombinant microorganism comprising the expression cassette of C2), or a recombinant microorganism comprising the recombinant vector of C3). C1) the nucleic acid molecule is any one of the following c1) - c4): c1) the nucleotide sequence of the nucleic acid molecule comprises SEQ ID NO: 8; c2) the nucleotide sequence of the nucleic acid molecule comprises SEQ ID NO: 10; c3) a DNA molecule having 75% or more identity to any one of the nucleotide sequences defined in c1) - c2), and encoding the mutant protein of any one of claims 1-3; and c4) a DNA molecule which hybridizes to any one of the nucleotide sequences defined in c1) - c2) under stringent conditions, and encoding the mutant protein of any one of claims 1-3.

6. Use of the mutant protein of any one of claims 1-3 in any one of the following: D1) degrading patulin A; D2) hydrolyzing patulin A into patulin a; D3) preparing a product for degrading patulin A; D4) detoxifying patulin A; and D5) preparing a patulin A detoxification agent.

7. Use of the biological material of claim 4 or 5 in any one of the following: D1) degrading patulin A; D2) hydrolyzing patulin A into patulin a; D3) preparing a product for degrading patulin A; D4) detoxifying patulin A; and D5) preparing a patulin A detoxification agent.

8. A method for degrading patulin A, comprising the step of degrading patulin A with the mutant protein of any one of claims 1-3, thereby degrading patulin A.

5. The biomaterial of claim 4, wherein:

9. The method of claim 8, wherein: the temperature for degrading patulin A is 25-80°C. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 10. A product for degrading patulin, comprising the mutant protein of any one of claims 1-3 or the biological material of claim 4 or 5.

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