Amide hydrolase PwADH / DM for degrading ochratoxin A as well as coding gene, recombinant vector and application of amide hydrolase PwADH / DM

Abstract

The invention discloses amide hydrolase PwADH / DM for degrading ochratoxin A as well as a coding gene, a recombinant vector and application of the amide hydrolase PwADH / DM, and belongs to the technical field of enzyme engineering. The amide hydrolase is an amide hydrolase protein of a two-site mutant obtained by further modification on the basis of the existing amide hydrolase capable of efficiently degrading ochratoxin A. The invention provides application of the amide hydrolase in hydrolysis of OTA and detoxification of beer and nuts polluted by OTA. Compared with the existing amide hydrolase PwADH, the amide hydrolase has the advantages that the hydrolysis and detoxification efficiency of the amide hydrolase on OTA is obviously improved, and the amide hydrolase is used for treating beer and almond powder samples polluted by ochratoxin A for 10-15 minutes at room temperature, so that the degradation rate of the ochratoxin A can reach 100%.

Description

Technical Field

[0001] The present invention relates to an improved high-efficiency ochratoxin A (OTA) hydrolase, in particular to the catalytic characteristics and applications of amide hydrolase optimized by protein engineering, and belongs to the technical fields of enzyme engineering, molecular biology and food safety. Background Art

[0002] Mycotoxins are secondary metabolites produced by more than 200 kinds of fungi and are widely present in various crops and food raw materials contaminated by molds. Common mycotoxins include aflatoxins, zearalenone, trichothecenes, deoxynivalenol, T-2 toxin, ochratoxin, fumonisin, patulin, citrinin, and ergot alkaloids, etc. These toxins can cause a series of clinical and subclinical symptoms, such as decreased appetite, intestinal damage, vaccine failure, liver overload, immune function suppression, etc., posing a long-term threat to the health of animals and humans. Mycotoxins have extremely high stability and are not easily degraded during the storage, transportation, processing, and cooking of food. Therefore, they can exist in grains and their related products for a long time and are transmitted through the food chain, causing a continuous threat to food safety.

[0003] Ochratoxin A (OTA) is a secondary metabolite mainly produced by molds such as Aspergillus ochraceus and Penicillium viridicatum, and has toxic side effects such as carcinogenicity, teratogenicity, immunotoxicity, and nephrotoxicity. Long-term exposure to OTA may cause serious health problems, such as renal tubular damage, immunosuppression, and apoptosis. OTA contamination not only endangers human health but also seriously affects the growth and production performance of animals. Although there are currently studies on the degradation and detoxification of OTA by physical methods (such as heating, freezing, ultraviolet irradiation) and chemical methods (such as ozone treatment, acid-base treatment, etc.); however, these methods have certain limitations. For example, physical methods often lead to problems such as loss of food nutrition, high requirements for detoxification equipment, and high application costs; while chemical methods may produce harmful substances, damage the flavor and nutrition of food, and have poor specificity. Using biological enzymes to specifically degrade OTA has the advantages of high specificity, good safety, and small loss of food nutrition, and is the most promising OTA detoxification strategy.

[0004] Amide hydrolase is a type of hydrolase that can specifically hydrolyze OTA to produce non-toxic ochratoxin α (OTα) and phenylalanine, and this catalytic reaction is currently recognized as the most efficient OTA detoxification method. In order to achieve more efficient degradation and detoxification of OTA, it is urgent to obtain a more efficient OTA hydrolysis and detoxification enzyme through gene mining or molecular modification technology to meet the needs of industrial applications. Summary of the Invention

[0005] To solve the above problems, the present invention is based on the highly efficient OTA hydrolase PwADH discovered previously (described in the invention "An Amide Hydrolase for Degrading Ochratoxin A Efficiently, Its Coding Gene, Recombinant Vector and Application", patent number: ZL202410194281.2). The amino acid sequence of PwADH is the amino acid sequence at positions 21 to 428 shown in SEQ ID NO.2; the nucleotide coding sequence corresponding to the amino acid sequence of PwADH is the nucleotide sequence at positions 61 to 1296 shown in SEQ ID NO.1. Two amino acid sites of the PwADH protein were modified to obtain a double mutant protein PwADH / DM with a 1.9-fold higher OTA hydrolysis and detoxification efficiency than PwADH.

[0006] Specifically, the present invention is based on the highly efficient OTA hydrolase PwADH discovered previously. Further, through protein structure and amino acid sequence analysis, rational design and site-directed mutagenesis techniques of PwADH, it was found that glycine at position 88 in the amino acid sequence of PwADH was mutated to tyrosine, and isoleucine at position 325 was mutated to alanine, obtaining the double mutant protein PwADH / G88Y / I325A (abbreviated as PwADH / DM). This double mutant protein PwADH / DM has higher OTA hydrolysis activity than PwADH, and the mutant PwADH / DM has broader application value in OTA detoxification in the field of food safety.

[0007] The first object of the present invention is to provide an amide hydrolase PwADH / DM for degrading ochratoxin A. The amino acid sequence of the amide hydrolase consists of the amino acid sequence at positions 21 to 428 shown in SEQ ID NO.4. The amino acid sequence shown in SEQ ID NO.4 is obtained by mutating glycine at position 88 and isoleucine at position 325 in SEQ ID NO.2 to tyrosine and alanine, respectively.

[0008] The second object of the present invention is to provide a coding gene encoding the amide hydrolase PwADH / DM.

[0009] Furthermore, an amide hydrolase PwADH / DM gene consisting of the nucleotide sequence at positions 61 to 1296 of the nucleotide sequence shown in SEQ ID NO.3.

[0010] The polynucleotide sequence encoding the amide hydrolase PwADH / DM of the present invention can be obtained by various methods. For example, polynucleotides can be obtained by PCR amplification well-known in the art. These techniques include (but are not limited to):

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

[0012] (2) Active screening of the expression library to detect the polynucleotide fragments of the clones with common structural features. The gene library may include an environmental metagenomic library or a clone library constructed from a pure cultured strain.

[0013] (3) Enter database websites such as NCBI (National Center for Biotechnology Information), and use gene names, gene IDs or other relevant 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 methods:

[0015] (1) Isolate the double-stranded DNA sequence from genomic DNA;

[0016] (2) Chemically synthesize the DNA sequence to obtain the double-stranded DNA of the amide hydrolase;

[0017] (3) Extract the target DNA sequence from cells by chemical methods.

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

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

[0020] In the present invention, the polynucleotide encoding the amide hydrolase PwADH / DM or the recombinant vector containing the polynucleotide can be transformed or transfected into a host cell to constitute a genetically engineered host cell containing the nucleotide 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, etc.

[0021] Transforming the host cell with the DNA sequence of the present invention or the recombinant vector containing the said DNA sequence can be carried out by conventional techniques well-known to those skilled in the art. When the host is a eukaryote such as Pichia pastoris, the competent cells capable of absorbing DNA can be harvested after the exponential growth phase, treated by the sorbitol method, and the plasmid transformation can be carried out by electroporation. When the host is other eukaryotes, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, liposome packaging, etc.

[0022] Using the polynucleotide sequence of the present invention to express or produce the recombinant amide hydrolase by conventional recombinant DNA techniques, including the following steps:

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

[0024] (2) Culture the transformant cells in a suitable medium;

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

[0026] In step (2), according to the host cell used, the culture medium used in the culture can be selected from various conventional culture media. The culture is carried out under conditions suitable for the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method, and the cells are cultured for a further period of time to promote the expression of the target protein.

[0027] In step (3), the recombinant enzyme can be coated inside the cell, or 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 point precipitation, organic solvent precipitation treatment, protein precipitant treatment (salting-out method), density gradient centrifugation, sonication for cell disruption, ultrafiltration by 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.

[0028] The fourth object of the present invention is to provide the application of the recombinant amide hydrolase PwADH / DM in degrading OTA. Further, the application includes degrading OTA in beer and nuts.

[0029] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a recombinant amide hydrolase PwADH / DM and its coding gene, and also provides the application of this amide hydrolase in hydrolyzing OTA and detoxifying beer and nuts contaminated with OTA. The amide hydrolase PwADH / DM reaches a degradation rate of 100% for OTA after treating beer and almond powder contaminated with OTA at room temperature for 10 - 15 minutes, and this degradation efficiency is extremely high in the art. Description of the Drawings

[0030] Figure 1 Chemical reaction equation for the detoxification of OTA by enzymatic hydrolysis to produce phenylalanine and ochratoxin α;

[0031] Figure 2 SDS-PAGE protein electrophoresis of purified recombinant amide hydrolase PwADH and mutant PwADH / DM with enhanced hydrolysis activity;

[0032] Figure 3 High performance liquid chromatography of the reaction products of purified recombinant amide hydrolase PwADH and mutant PwADH / DM with enhanced hydrolysis activity in hydrolyzing OTA;

[0033] Figure 4Comparison of the relative enzyme activities of PwADH and PwADH / DM in hydrolyzing OTA;

[0034] Figure 5 Comparison of the hydrolysis effects of the recombinant amidase PwADH and the mutant PwADH / DM with enhanced hydrolysis activity on OTA in beer;

[0035] Figure 6 Comparison of the hydrolysis effects of the recombinant amidase PwADH and the mutant PwADH / DM with enhanced hydrolysis activity on OTA in almond powder;

[0036] Figure 7 Alignment diagram of the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.4. Specific implementation mode

[0037] The present invention will be 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 be able to implement it, but the specific embodiments cited do not limit the present invention.

[0038] Example 1: Construction of the amidase PwADH / DM mutant

[0039] The amidase PwADH derived from Pseudoxanthomonas wuyuanensis was obtained by mining through bioinformatics technology from the National Center of Biotechnology Information (English full name: National Center of Biotechnology Information, abbreviated: NCBI). The nucleotide sequence of PwADH is shown in SEQ ID NO.1, and the amino acid sequence of the PwADH amidase encoded by it is shown in SEQ ID NO.2 (such as Figure 7 Sequence1 in), containing 428 amino acids (hereinafter, the description of the amino acid sequences at all specific positions of PwADH and PwADH / DM refers to the amino acid numbers corresponding to them in the full-length PwADH); among them, the amino acids at positions 1-20 in the amino acid sequence shown in SEQ ID NO.2 are the signal peptides for protein expression. In order to obtain a mature PwADH protein without signal peptides, we handed over the nucleotide sequence encoding the amino acid sequence at positions 21-428 of PwADH to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis and cloned it into the Escherichia coli expression vector pET46EK to obtain the recombinant plasmid pET46EK-PwADH.

[0040] In addition, through the structural analysis of the PwADH protein structure and amino acid sequence, glycine at the 88th position of the full-length amino acid sequence of PwADH was mutated to tyrosine, and isoleucine at the 325th position was mutated to alanine, to construct a double-site mutant PwADH / G88Y / I325A (hereinafter simply referred to as PwADH / DM) with enhanced OTA hydrolysis activity. The mutant PwADH / DM has the amino acid sequence from the 21st to the 428th position as shown in SEQ ID NO.4 (such as Sequence2 in Figure 7 ), and the gene encoding PwADH / DM has the nucleotide sequence from the 61st to the 1296th position as shown in SEQ ID NO.3.

[0041] The nucleotide sequence of the mature double-site mutant PwADH / DM without a signal peptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the synthesized gene was ligated to the expression vector pET46EK using the restriction enzymes NcoI and XhoI, and finally the recombinant plasmid pET46EK-PwADH / DM was obtained.

[0042] In this example, the amide hydrolase gene PwADH was obtained by total gene synthesis with reference to the method described in ZL201910004511.3. The nucleotide sequence of PwADH is as shown in SEQ ID NO.3, and the amino acid sequence of the PwADH amide hydrolase it encodes is as shown in SEQ ID NO.4.

[0043] Example 2: Expression and purification of PwADH protein and double-site mutant protein in Escherichia coli

[0044] The constructed recombinant plasmids pET46EK-PwADH and pET46EK-PwADH / DM were respectively transformed into Escherichia coli BL21(DE3) competent cells, and the strains were screened on LB plates containing 100 μg / mL ampicillin. Pipette 1.0 mL of the seed bacterial solution and inoculate it into fresh 5 mL of LB, and culture it overnight with shaking at 180 rpm at 37 °C, and then scale it up to 200 mL of LB medium for culture. Finally, it was scaled up to 10 L of LB medium. When the OD 600 value reached 0.6 to 0.8, the culture temperature was reduced to 16 °C, and the concentration of IPTG was added to 0.3 mM to induce the large expression of the protein. After 16 h of protein induction, the bacterial solution was centrifuged at 6000 rpm for 10 min to collect Escherichia coli cells.

[0045] The cells were resuspended in buffer (20 mM Tris-HCl, pH 8.0), lysed using a cell disruptor, and then the supernatant was collected by centrifugation at 17,000 rpm for 40 min. The target protein was purified using a nickel ion affinity chromatography column, and the eluted target protein was dialyzed overnight in buffer (20 mM Tris-HCl, pH 8.0) at 4 °C. The dialyzed protein was further purified by an ion exchange chromatography column and concentrated into buffer of 20 mM Tris-HCl pH 8.0, and the final protein was stored at -80 °C. Figure 2 SDS-PAGE electrophoresis patterns of recombinant amide hydrolase PwADH protein and double-site mutant PwADH / DM expressed and purified from Escherichia coli.

[0046] Example 3: OTA hydrolysis activity assay:

[0047] To compare the differences in OTA hydrolysis activities between PwADH and PwADH / DM, the activities of PwADH and PwADH / DM in hydrolyzing OTA were further determined in this example.

[0048] The activity test method of OTA hydrolase is described as follows:

[0049] The mixture (100 μL) of each reaction was in buffer of 20 mM Tris-HCl, pH 8.0, 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 reaction for 20 min. Each reaction was performed in triplicate and the reaction was repeated 3 times. After the reaction, an equal volume of acetonitrile was added to terminate the enzyme reaction, and then centrifuged at 12,000 rpm for 10 min. The supernatant reaction solution was filtered through a 0.22 μm organic nylon syringe filter; then the product of each group of reactions was determined and analyzed by high performance liquid chromatography (HPLC, Shimadzu SPD-M20A), and its analytical column was InertSustain C 18 column (4.6×250 mm, 5 μm), mobile phase A: H 2 O, mobile phase B: 95% acetonitrile + 5% glacial acetic acid, flow rate 1 mL / min, detection wavelength 330 nm, elution time 20 min, elution concentration constant at 50%. The high performance liquid chromatography detection results of the degradation products of OTA by PwADH and PwADH / DM are as Figure 3As shown in the figure, peaks appeared at a retention time of 4.9 min and a retention time of 12.6 min, respectively. Among them, the peak time at a retention time of 4.9 min was consistent with that of the product standard OTα. Therefore, the substance at a retention time of 4.9 min was OTα; while the peak time at a retention time of 12.6 min was consistent with that of the substrate standard OTA. Therefore, the substance at a retention time of 12.6 min was OTA. Then, the activity difference was calculated by comparing the peak areas of the hydrolysis products OTα of PwADH and PwADH / DM.

[0050] From Figure 3 The data showed that the hydrolysis products of OTA by PwADH and PwADH / DM were OTα and phenylalanine, respectively, and OTα could be detected. Therefore, as Figure 4 In the present invention, the peak area value of the hydrolysis product OTα of PwADH was regarded as 100%, and the peak areas of the hydrolysis products OTα of PwADH and PwADH / DM were compared as the relative enzyme activity. The results of the OTA degradation activity determination were as Figure 4 shown. The activity of PwADH / DM was 1.9 times that of PwADH. Therefore, the mutant protein PwADH / DM had higher application value in the OTA degradation and detoxification industry.

[0051] Example 4: Application of recombinant amidohydrolase in detoxifying OTA-contaminated beer

[0052] An OTA standard stock solution was mixed with beer to prepare an OTA-contaminated beer sample (containing 400 μg OTA per liter of the beer sample). After the recombinant plasmids (pET46EK-PwADH and pET46EK-PwADH / DM) were expressed respectively, the bacteria were lysed, and the supernatant of the bacterial cell lysate containing the recombinant amidohydrolase was collected. The purified PwADH and PwADH / DM proteins were obtained from the supernatant. The purified PwADH and PwADH / DM proteins were respectively mixed evenly with the OTA-contaminated beer sample. The prepared samples were subjected to OTA detoxification tests at 25 °C, and samples were taken at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min. Each degradation experiment was set with 3 replicates. At the same time, the OTA-contaminated beer sample without enzyme treatment was used as a negative blank control. The sampling amounts of the enzyme-treated group and the non-enzyme-treated blank control group were the same each time, and extraction, purification, and analysis were carried out according to the OTA standard detection method.

[0053] According to the OTA extraction, purification, and detection method described in GB 5009.96-2016, the degradation rate of the recombinant amidohydrolase to OTA was measured. The results were as Figure 5As shown, in the negative control group without enzyme, OTA stably existed in beer and was not degraded within 0 - 30 min. However, the recombinant amidohydrolases PwADH and PwADH / DM completely degraded OTA in the samples within 30 min and 15 min respectively, with a degradation efficiency reaching 100%. The degradation efficiency of PwADH / DM for OTA was significantly better than that of PwADH, which was consistent with the relative enzyme activity determination results of PwADH and PwADH / DM in Example 3.

[0054] Example 5: Application of Recombinant Amidohydrolase in Detoxifying Almonds Contaminated with OTA

[0055] Mix the OTA standard stock solution with the ground almond powder to prepare an almond powder sample contaminated with OTA (containing 400 μg OTA per kilogram of this almond powder sample). After expressing the recombinant plasmids (pET46EK - PwADH and pET46EK - PwADH / DM) respectively and lysing the bacteria, collect the supernatant of the bacterial cell lysate containing the recombinant amidohydrolase, and obtain the purified PwADH and PwADH / DM proteins from the supernatant. Mix the purified PwADH and PwADH / DM proteins evenly with the almond powder sample contaminated with OTA, incubate the prepared samples at 25 °C, and take samples at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min. Each degradation experiment is set with 3 replicates. Use the OTA - contaminated almond powder mixture without enzyme treatment as the negative blank control. The sampling amount of the enzyme - added treatment group and the non - enzyme - added treatment blank control group is the same each time, and extraction, purification, and analysis are carried out according to the OTA standard detection method.

[0056] Measure the degradation rate of the recombinant amidohydrolase for OTA according to the OTA extraction, purification, and detection method recorded in GB 5009.96 - 2016. The results are as Figure 6 shown. In the negative blank control group without enzyme, OTA stably existed in the almond powder solution and was not degraded within 0 - 30 min. However, the recombinant amidohydrolases PwADH and PwADH / DM completely degraded OTA in the samples within 30 min and 15 min respectively, with a degradation efficiency reaching 100%. The degradation efficiency of PwADH / DM for OTA was significantly better than that of PwADH, which was consistent with the relative enzyme activity determination of PwADH and PwADH / DM in Example 3.

[0057] The above - mentioned examples are only preferred examples given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. An ochratoxin A degrading amidohydrolase PwADH / DM, characterized in that: The amino acid sequence of the amidohydrolase consists of the amino acid sequence at positions 21 to 428 as shown in SEQ ID NO.4, wherein the amino acid sequence shown in SEQ ID NO.4 is obtained by mutating the glycine at position 88 into tyrosine and the isoleucine at position 325 into alanine in SEQ ID NO.

2.

2. A gene encoding the amidohydrolase PwADH / DM according to claim 1.

3. The coding gene according to claim 2, characterized in that The amidohydrolase PwADH / DM gene consists of the nucleotide sequence at positions 61 to 1296 of the nucleotide sequence shown in SEQ ID NO.

3. 4 . A recombinant expression vector carrying the gene encoding the amidohydrolase PwADH / DM according to claim 3 .

5. A method for producing the recombinant amidohydrolase PwADH / DM with high detoxification activity according to claim 1, comprising 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 ochratoxin A degrading amidohydrolase PwADH / DM according to claim 1 in degrading ochratoxin A, characterized in that: The amino acid sequence of the amidohydrolase PwADH / DM consists of the amino acid sequence at positions 21 to 428 shown in SEQ ID NO.4, and the application is for purposes other than disease diagnosis and treatment.

7. The use according to claim 6, characterized in that: The amidohydrolase PwADH / DM can effectively remove ochratoxin A from foods, including beer and almond powder.

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 according to claim 6 with a food containing ochratoxin A; (B) reacting the mixture at a pH of 5 to 10 and a temperature not exceeding 80° C. until ochratoxin A in the mixture is detected to be below a threshold value.

9. The method for efficiently removing ochratoxin A from food according to claim 8, characterized in that: The food products include beer and almond meal.

Images