Group of amide hydrolase mutants capable of efficiently degrading ochratoxin A as well as encoding gene, recombinant vector and application of amide hydrolase mutants

Abstract

The invention discloses a group of amide hydrolase mutants capable of efficiently degrading ochratoxin A as well as a coding gene, a recombinant vector and application of the amide hydrolase mutants, and belongs to the technical field of enzyme engineering. The invention also provides application of the mutant of the group of recombinant amide hydrolase in detoxification of ochratoxin A in beer or corn flour polluted by ochratoxin A. The activity of the mutant on ochratoxin A is obviously improved compared with the activity of the existing recombinant amide hydrolase ADH3; beer or corn flour containing ochratoxin A is treated for 10-15 minutes, the degradation rate of ochratoxin A can reach 100%, and the degradation efficiency is high in the field.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to a group of amidohydrolase mutant proteins for efficiently degrading ochratoxin A and encoding genes, recombinant vectors, recombinant bacteria and applications thereof. Background Art

[0002] Mycotoxins are toxic secondary metabolites produced by dozens of filamentous fungi, which widely contaminate various crops and their products, and seriously threaten the health of humans and livestock. More than 400 mycotoxins have been discovered so far; among them, the most toxic and harmful mycotoxins include aflatoxins (AF), fumonisins (FUM), ochratoxin A (OTA), deoxynivalenol (DON) and zearalenone (ZEN).

[0003] Among them, ochratoxin A (OTA) is mainly found in grains and their products contaminated by Penicillium verrucosum, Aspergillus ochraceus and Aspergillus carbonarius. OTA has a variety of toxic side effects on humans, including hepatotoxicity, nephrotoxicity, immunotoxicity, teratogenicity, mutagenicity, genotoxicity, embryotoxicity, etc. The former National Health and Family Planning Commission and the State Food and Drug Administration jointly issued the National Food Safety Standard (China Food Safety National Standard, 2017 Edition), which stipulates that the legal limit of OTA in alcoholic beverages and cereal products for human consumption is 2μg / kg and 5μg / kg respectively.

[0004] At present, the detoxification methods of OTA include physical, chemical and biological methods. Among them, physical detoxification includes adsorption, ultraviolet light, γ radiation and cold plasma; physical detoxification has disadvantages such as product nutritional loss, high requirements for equipment and instruments, and high application cost. Chemical detoxification methods include ozone treatment, formic acid treatment, citric acid treatment, potassium carbonate treatment, hydrogen peroxide treatment, sodium thiosulfate treatment, etc. However, there are negative effects such as decreased food nutrients, poor palatability, and chemical residues. Biological methods refer to the use of certain microorganisms or enzymes to remove OTA. Due to its advantages of low cost, high efficiency, environmental friendliness, little side effect on nutrients, and applicability to various liquid and solid foods, the biological detoxification research of OTA has received great attention in the past decade. The hydrolysis of the amide bond in the OTA molecule to generate non-toxic ochratoxin α (OTα) and phenylalanine through the hydrolysis of protease A, lipase, carboxypeptidase and amide hydrolase is currently recognized as the most thorough OTA detoxification method. Therefore, obtaining more efficient OTA detoxification enzymes and their encoding genes through gene mining or genetic engineering technology will provide an important biological solution for the efficient detoxification of OTA.

[0005] ADH3 can efficiently hydrolyze OTA to generate non-toxic ochratoxin α (OTα) and phenylalanine. The catalytic activity of ADH3 is 50-30,000 times higher than that of other OTA hydrolases, and has important application prospects. How to further modify ADH3 through genetic engineering technology to further enhance its hydrolysis activity against OTA will greatly enhance the application potential of ADH3 in OTA detoxification. Summary of the invention

[0006] To solve the above problems, the present invention performs protein engineering on ADH3 to obtain mutants ADH3-S88K / I325A and ADH3-S88R / I325A, which have 8 and 9 times higher hydrolysis efficiencies for OTA than ADH3, respectively. Specifically, the present invention is based on the amidohydrolase ADH3 derived from Stenotrophomonas sp.CW117, and through structural analysis, rational design and site-directed mutagenesis of the ADH3 and OTA complex, the 88th serine in the amino acid sequence of ADH3 is mutated into arginine and the 325th isoleucine is mutated into alanine, or the 88th serine in the amino acid sequence of ADH3 is mutated into lysine and the 325th isoleucine is mutated into alanine, which can further enhance its hydrolysis activity for OTA, thereby improving its application value in the industry.

[0007] The first object of the present invention is to provide a group of amidohydrolase mutants ADH3-S88K / I325A and ADH3-S88R / I325A that can efficiently degrade ochratoxin A, characterized in that the amino acid sequence of the mutant ADH3-S88K / I325A consists of the amino acid sequence at positions 21 to 427 as shown in SEQ ID NO.4, and the amino acid sequence of the mutant ADH3-S88R / I325A consists of the amino acid sequence at positions 21 to 427 as shown in SEQ ID NO.6.

[0008] The second object of the present invention is to provide a gene encoding the amidohydrolase mutant.

[0009] Furthermore, an amidohydrolase mutant gene consisting of the nucleotide sequence shown at positions 61 to 1284 of the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.5.

[0010] The polynucleotide sequences encoding the amidohydrolase mutants ADH3-S88K / I325A and ADH3-S88R / I325A of the present invention can be obtained by a variety of methods. For example, the polynucleotides can be obtained by PCR amplification as is well known in the art. These techniques include (but are not limited to):

[0011] (1) Using a probe to hybridize with a gene or cDNA library to detect homologous polynucleotide sequences;

[0012] (2) Activity screening of expression libraries to detect cloned polynucleotide fragments with common structural features. The gene library may include an environmental metagenomic library or a clone library constructed from a pure culture strain;

[0013] (3) Access database websites such as NCBI (National Center for Biotechnology Information) and use gene name, gene ID or other related information to search, download or copy the sequence for subsequent use.

[0014] The DNA fragment sequence of the present invention can also be obtained by the following method:

[0015] (1) Separating double-stranded DNA sequences from genomic DNA;

[0016] (2) chemically synthesizing a DNA sequence to obtain a double-stranded DNA of the amidohydrolase;

[0017] (3) Chemical method to extract target DNA sequence from cells.

[0018] The third object of the present invention is to provide a recombinant vector carrying the gene.

[0019] Furthermore, the vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

[0020] In the present invention, the polynucleotide encoding the amidohydrolase mutants ADH3-S88K / I325A and ADH3-S88R / I325A 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.

[0021] Transformation of host cells with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence can be carried out using conventional techniques well known in the art. When the host is a eukaryotic organism such as Pichia pastoris, competent cells that can absorb DNA can be harvested after the exponential growth phase, treated with sorbitol, and transformed with plasmids by electroporation. When the host is other eukaryotic organisms, the following DNA transfection methods can be used: calcium phosphate coprecipitation method, or conventional mechanical methods such as microinjection, liposome packaging, etc.

[0022] Utilizing the polynucleotide sequence of the present invention, expressing or producing the recombinant amidohydrolase by conventional recombinant DNA technology comprises the following steps:

[0023] (1) transforming or transfecting a suitable transformant cell with a polynucleotide encoding a recombinant amide hydrolase of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0024] (2) culturing the transformed cells in a suitable culture medium;

[0025] (3) Isolate and purify proteins from culture medium or cells.

[0026] 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 to promote the expression of the target protein.

[0027] 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 isoelectric precipitation, organic solvent precipitation treatment, protein precipitant treatment (salting out method), density gradient centrifugation, ultrasonic sterilization, ultracentrifugal filtration, 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.

[0028] The fourth object of the present invention is to provide the use of the recombinant amidohydrolase mutants ADH3-S88K / I325A and ADH3-S88R / I325A in degrading OTA.

[0029] Furthermore, the application includes degrading OTA in beer or corn flour.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a group of highly efficient recombinant amide hydrolases ADH3-S88K / I325A and ADH3-S88R / I325A and their encoding genes, and also provides the use of the amide hydrolase mutants ADH3-S88K / I325A and ADH3-S88R / I325A in detoxifying OTA in beer or corn flour contaminated with OTA. The mutants ADH3-S88K / I325A and ADH3-S88R / I325A with improved catalytic activity treat beer or corn flour containing OTA for 10-15 minutes at room temperature, and the degradation rate of OTA reaches 100%, which is highly efficient in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1The chemical reaction equation for the enzyme-catalyzed hydrolysis and detoxification of OTA to generate phenylalanine and ochratoxin α;

[0032] Figure 2 The SDS-PAGE protein electrophoresis of purified recombinant amide hydrolase ADH3, improved mutants ADH3-S88K / I325A and ADH3-S88R / I325A;

[0033] Figure 3 The HPLC chromatograms of the reaction products of OTA degradation by purified recombinant amide hydrolase ADH3 and improved mutants ADH3-S88K / I325A and ADH3-S88R / I325A;

[0034] Figure 4 The relative enzymatic activities of ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A in degrading OTA are compared;

[0035] Figure 5 To compare the degradation effects of recombinant amide hydrolase ADH3, improved mutants ADH3-S88K / I325A and ADH3-S88R / I325A on OTA in beer;

[0036] Figure 6 To compare the degradation effects of recombinant amide hydrolase ADH3, improved mutants ADH3-S88K / I325A and ADH3-S88R / I325A on OTA in corn flour;

[0037] Figure 7 It is a comparison diagram of the amino acid sequences shown in SEQ ID NO.4 and SEQ ID NO.6. DETAILED DESCRIPTION

[0038] 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.

[0039] Example 1: Synthesis of ADH3 amidohydrolase gene and construction of its mutants

[0040] The Stenotrophomonas sp. CW117 deposited in China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, zip code 430072) on June 14, 2015 has a deposit number of CCTCC NO: M2015371; the classification name is: Stenotrophomonas sp. CW117 strain, according to which the amidase gene heterologously expressed amide hydrolase ADH3 is a highly efficient OTA hydrolysis detoxification enzyme, the nucleotide sequence of ADH3 is shown in SEQ ID NO.1, and has great application development potential; the full-length amino acid sequence of ADH3 contains 427 amino acids (the following description of all amino acid sequences in ADH3 and its mutants refers to the corresponding sequence numbers in the full-length amino acid sequence of ADH3); the full-length amino acid sequence of ADH3 protein is shown in SEQ ID NO.2; wherein, SEQ ID Amino acids 1-20 in the NO.2 sequence are the signal peptide for the expression of ADH3 protein. In order to obtain a mature ADH3 protein without a signal peptide, the nucleotide sequence encoding the amino acid sequence from positions 21 to 427 of ADH3 was submitted to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis with reference to the method described in ZL201910004511.3. The synthesized ADH3 gene was then connected to the Escherichia coli expression vector pET46EK using restriction endonucleases NcoI and XhoI to obtain the recombinant plasmid pET46EK-ADH3.

[0041] According to the protein structure analysis of the amino acid sequence of the amidohydrolase ADH3, the serine at position 88 of the sequence shown in SEQ ID NO.2 is mutated into arginine, and the isoleucine at position 325 is mutated into alanine to obtain a double-site mutant ADH3-S88R / I325A, or the serine at position 88 of the sequence shown in SEQ ID NO.2 is mutated into lysine and the isoleucine at position 325 is mutated into alanine to obtain a double-site mutant ADH3-S88K / I325A. The amino acid sequence of the obtained mutant ADH3-S88K / I325A is the amino acid sequence at positions 21 to 427 as shown in SEQ ID NO.4 (as shown in Figure 7 The amino acid sequence of the mutant ADH3-S88R / I325A is the amino acid sequence at positions 21 to 427 as shown in SEQ ID NO.6 (as Figure 7 Sequence2 in ).

[0042] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.4 is the 61st to 1284th nucleotide sequence shown in SEQ ID NO.3; the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.6 is the 61st to 1284th nucleotide sequence shown in SEQ ID NO.5; the nucleotide sequences of mutants ADH3-S88K / I325A and ADH3-S88R / I325A were submitted to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis, and the synthesized genes were connected to the recombinant vector pET46EK using restriction endonucleases NcoI and XhoI to obtain recombinant plasmids pET46EK-ADH3-S88K / I325A and pET46EK-ADH3-S88R / I325A.

[0043] Example 2: Expression and purification of ADH3, mutant proteins ADH3-S88K / I325A and ADH3-S88R / I325A in Escherichia coli.

[0044] The constructed recombinant plasmids pET46EK-ADH3, pET46EK-ADH3-S88K / I325A and pET46EK-ADH3-S88R / I325A were transformed into competent E. coli BL21 (DE3) cells, and strains were screened on LB plates containing 100 μg / mL ampicillin. 1.5 mL of seed culture was inoculated into 8 mL of fresh LB, cultured overnight at 37°C with shaking at 220 rpm / min, and then expanded to 100 mL of LB medium. Finally, it was expanded to 8 L of LB medium. At the OD of the bacteria, 600 When the value reached 0.6, the culture temperature was lowered to 16°C, and IPTG was added to a concentration of 0.3 mM to induce the massive expression of the protein. After 20 hours of protein expression, the bacterial solution was centrifuged at 5000 rpm for 5 minutes to collect the E. coli cells. The cells were then resuspended in a buffer (20 mM Tris-HCl, pH 8.0), broken with a French cell disruptor, and centrifuged at 15000 rpm for 50 minutes, and the supernatant was collected for the next step of purification.

[0045] ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A proteins without signal peptide were purified by nickel column affinity chromatography and ion exchange chromatography, and concentrated in 20 mM Tris-HCl, pH 8 buffer, and stored at -80°C.

[0046] Figure 2The SDS-PAGE protein electrophoresis gel image of the purified recombinant amide hydrolase ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A expressed in Escherichia coli.

[0047] Example 3: Determination of OTA hydrolysis activity.

[0048] In order to compare the differences in the activities of recombinant amide hydrolases ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A, this example further determined the activities of recombinant amide hydrolases ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A in hydrolyzing OTA.

[0049] The activity test method of OTA hydrolase is as follows:

[0050] The mixture (100 μL) of each reaction was in 20 mM Tris-HCl, pH 8.0 buffer, including substrate 200 μg / mL OTA and 10 μL enzyme (10 μg / mL). After mixing, it was placed in a shaking metal bath at 40°C and 800 rpm for 20 min. Each reaction was done 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. The supernatant reaction solution was filtered through a 0.22 μm organic nylon filter membrane; then each group of reactions was tested and analyzed by high performance liquid chromatography (HPLC, Shimadzu SPD-M20A), and the analytical column was InertSustain C 18 column (4.6×250mm, 5μm), mobile phase A: H2O, mobile phase B: 95% acetonitrile + 5% glacial acetic acid, flow rate 1mL / min, detection wavelength 330nm, elution time 20min, elution concentration constant at 50%.

[0051] The results of HPLC detection of the degradation products of OTA by recombinant amide hydrolases ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A are as follows: Figure 3 As shown, the peaks are eluted at retention time 4.9min and retention time 12.6min respectively. Among them, the peak time of retention time 4.9min is consistent with the product standard OTα, so the substance with retention time 4.9min is OTα; while the peak time of retention time 12.6min is consistent with the substrate standard OTA. Therefore, the substance with retention time 12.6min is OTA. Then, the activity difference was calculated by comparing the peak areas of the hydrolysis products OTα of recombinant amide hydrolase ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A.

[0052] from Figure 3 The data showed that the products of OTA hydrolysis by ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A were OTα and phenylalanine, and OTα could be detected. Figure 4 In the present invention, we regard the peak area value of the hydrolysis product OTα of ADH3 as 100%, and compare the peak areas of the hydrolysis products OTα of ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A as the relative enzyme activity. Figure 4 As shown, the activities of ADH3-S88K / I325A and ADH3-S88R / I325A against OTA were 8-fold and 9-fold higher than those of ADH3, respectively.

[0053] Therefore, the mutant proteins ADH3-S88K / I325A and ADH3-S88R / I325A have higher application value in the field of OTA detoxification.

[0054] Example 4: Application of recombinant amide hydrolases ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A in detoxification of OTA-contaminated beer.

[0055] The OTA standard stock solution was mixed with beer to prepare OTA-contaminated beer (the final concentration was 500 μg OTA per liter of beer). The purified recombinant amide hydrolases ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A were added to the OTA-contaminated beer samples, respectively. The OTA degradation reaction was carried out at 37°C, and samples were taken at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min. Three replicates were set for each degradation experiment. At the same time, the OTA-contaminated beer mixture without the addition of recombinant amide hydrolases ADH3, ADH3-S88K / I325AA or DH3 / S88R / I325A was used as a negative blank control. The same amount of samples were taken each time in the treatment group and the blank control group, and the extraction, purification and analysis were carried out according to the OTA standard detection method.

[0056] The degradation rate of OTA by recombinant amide hydrolase was measured according to the OTA extraction and purification detection method described in GB 5009.96-2016.

[0057] The results are as follows Figure 5As shown, in the negative control group without enzyme addition, OTA existed stably and was not degraded within 0-30 min; while the recombinant amide hydrolases ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A completely degraded OTA in the sample within 60 min, 15 min and 15 min, respectively, and the degradation efficiency reached 100%; the degradation efficiency of ADH3-S88K / I325A and ADH3-S88R / I325A for OTA was significantly better than that of ADH3, which is consistent with the relative enzyme activity determination of ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A in Example 3.

[0058] When ADH3-S88K / I325A and ADH3-S88R / I325A were used to degrade higher OTA content in beer, the degradation rate was faster.

[0059] Example 5: Application of recombinant amidohydrolase in detoxification of OTA-contaminated corn flour

[0060] The OTA standard stock solution was mixed with the crushed corn flour to prepare the OTA-contaminated corn flour sample (the final concentration was 500 μg OTA per kg corn flour), and the purified recombinant amide hydrolase ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A were added to the OTA-contaminated corn flour sample, respectively, and cultured at 37°C. Samples were taken at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min, and 3 replicates were set for each degradation experiment. At the same time, the OTA-contaminated corn flour mixture without the addition of recombinant amide hydrolase ADH3, ADH3-S88K / I325AA or DH3 / S88R / I325A was used as a negative blank control. The same amount of samples were taken each time in the treatment group and the blank control group, and the extraction, purification and analysis were carried out according to the OTA standard detection method.

[0061] The degradation rate of OTA by recombinant amide hydrolase was measured according to the OTA extraction and purification detection method described in GB 5009.96-2016.

[0062] The results are as follows Figure 6As shown, in the negative control group without enzyme addition, OTA existed stably and was not degraded within 0-30 min; ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A completely degraded OTA in the sample in 40 min, 10 min and 10 min, respectively, and the degradation efficiency reached 100%; the degradation efficiency of ADH3-S88K / I325A and ADH3-S88R / I325A for OTA was significantly better than that of ADH3, which is consistent with the relative enzyme activity determination of ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A in Example 3.

[0063] When ADH3-S88K / I325A and ADH3-S88R / I325A were used to degrade OTA with higher content in grain, the degradation rate was faster.

[0064] 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. A group of amidohydrolase mutants that efficiently degrade ochratoxin A, characterized in that: The amino acid sequence of the amidohydrolase mutant consists of the amino acid sequence at positions 21 to 427 as shown in SEQ ID NO.4 or SEQ ID NO.6, wherein the amino acid sequence shown in SEQ ID NO.4 is that the serine at position 88 in SEQ ID NO.2 is mutated into lysine and the isoleucine at position 325 is mutated into alanine, and the amino acid sequence shown in SEQ ID NO.6 is that the serine at position 88 in SEQ ID NO.2 is mutated into arginine and the isoleucine at position 325 is mutated into alanine.

2. A gene encoding the amidohydrolase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that An amidohydrolase mutant gene consisting of the nucleotide sequence shown at positions 61 to 1284 of the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.

5.

4. A recombinant expression vector carrying the amidohydrolase mutant gene structure according to claim 3.

5. A method for producing the amidohydrolase mutant according to claim 1, comprising at least the following steps: (1) a step of expressing a polypeptide from at least one gene according to any one of claims 2 to 3; and (2) a step of collecting the polypeptides in step (1).

6. Use of the amidohydrolase mutant according to claim 1 in degrading ochratoxin A, characterized in that: The amino acid sequence of the amidohydrolase mutant consists of the amino acid sequence at positions 21 to 427 shown in SEQ ID NO.4 or SEQ ID NO.6, and the application is for purposes other than disease diagnosis and treatment.

7. The use according to claim 6, characterized in that: The recombinant amidohydrolase mutant can effectively remove ochratoxin A from food, including beer and corn flour.

8. A method for efficiently removing ochratoxin A from food, characterized in that: Includes at least the following steps: (A) mixing an aqueous solution containing the amidohydrolase mutant according to claim 6 with a food containing ochratoxin A; (B) reacting the mixture at a pH of 5 to 9.8 and a temperature not exceeding 80° C. until ochratoxin A in the mixture is detected to be below a threshold value.

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