A group of amidohydrolase mutants that efficiently degrade ochratoxin A, their encoding genes, recombinant vectors, and applications

Abstract

This invention discloses a group of highly efficient amide hydrolase mutants for degrading ochratoxin A, their encoding genes, recombinant vectors, and applications, belonging to the field of enzyme engineering technology. This invention also provides the application of these recombinant amide hydrolase mutants in the detoxification of ochratoxin A from beer or corn flour contaminated with ochratoxin A. The activity of these mutants against ochratoxin A is significantly improved compared to the existing recombinant amide hydrolase ADH3. After treating beer or corn flour containing ochratoxin A for 10-15 minutes, the degradation rate of ochratoxin A reaches 100%, which is highly efficient in this field.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a group of mutant amide hydrolases that efficiently degrade ochratoxin A, their encoding genes, recombinant vectors, recombinant bacteria, and their applications. Background Technology

[0002] Mycotoxins are toxic secondary metabolites produced by dozens of filamentous fungi, widely contaminating various crops and their products, and seriously threatening human and livestock health. More than 400 mycotoxins have been identified to date; among them, the most toxic and widely harmful mycotoxins include aflatoxin (AF), fumonisin (FUM), ochratoxin A (OTA), deoxynivalenol (DON), and zearalenone (ZEN).

[0003] Ochratoxin A (OTA) is mainly found in grains and their products contaminated with Penicillium verruciformis, Aspergillus ochraceus, and Aspergillus carbonisata. OTA has various toxic side effects on humans, including hepatotoxicity, nephrotoxicity, immunotoxicity, teratogenicity, mutagenicity, genotoxicity, and embryotoxicity.

[0004] Currently, OTA detoxification methods include physical, chemical, and biological methods. Physical detoxification methods include adsorption, ultraviolet radiation, gamma radiation, and cold plasma; however, physical detoxification methods have disadvantages such as product nutrient loss, high equipment requirements, and high application costs. Chemical detoxification methods include ozone treatment, formic acid treatment, citric acid treatment, potassium carbonate treatment, hydrogen peroxide treatment, and sodium thiosulfate treatment. However, these methods have negative impacts such as decreased nutritional content, poor palatability, and chemical residues. Biological methods refer to the use of certain microorganisms or enzymes to remove OTA. Due to their advantages of low cost, high efficiency, environmental friendliness, minimal side effects on nutrients, and applicability to various liquid and solid foods, biological detoxification research on OTA has received considerable attention in the past decade. The most thorough OTA detoxification method currently recognized is the hydrolysis of the amide bonds within the OTA molecule through the action of proteinase A, lipase, carboxypeptidase, and amide hydrolase, producing non-toxic ochratoxin α (OTα) and phenylalanine. 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] Amide hydrolase ADH3 can efficiently hydrolyze OTA to produce non-toxic ochratoxin α (OTα) and phenylalanine. ADH3's catalytic activity is 50-30,000 times higher than other OTA hydrolases, showing significant application potential. Further genetic engineering of ADH3 to enhance its OTA hydrolytic activity will greatly improve its application potential in OTA detoxification. Summary of the Invention

[0006] To address the aforementioned issues, this invention employs protein engineering to modify ADH3, obtaining mutants ADH3-S88K / I325A and ADH3-S88R / I325A, which exhibit OTA hydrolysis efficiencies 8-fold and 9-fold higher than ADH3, respectively. Specifically, based on the amide hydrolase ADH3 derived from Stenotrophomonas sp. CW117, this invention utilizes structural analysis, rational design, and site-directed mutagenesis of the ADH3-OTA complex. By mutating serine at position 88 of the ADH3 amino acid sequence to arginine and isoleucine at position 325 to alanine, or by mutating serine at position 88 to lysine and isoleucine at position 325 to alanine, its OTA hydrolysis activity can be further enhanced, thereby increasing its industrial application value.

[0007] The first objective of this invention is to provide a set of highly efficient amide hydrolase mutants ADH3-S88K / I325A and ADH3-S88R / I325A for degrading ochratoxin A, characterized in that the amino acid sequence of the mutant ADH3-S88K / I325A consists of the amino acid sequence from position 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 from position 21 to 427 as shown in SEQ ID NO.6.

[0008] A second objective of this invention is to provide a coding gene that encodes the amide hydrolase mutant.

[0009] Further, the amide hydrolase mutant gene is composed 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 amide hydrolase mutants ADH3-S88K / I325A and ADH3-S88R / I325A described in this invention can be obtained by a variety of methods. For example, polynucleotides can be obtained by PCR amplification, a technique well known in the art. These techniques include (but are not limited to):

[0011] (1) Use probes to hybridize with genes or cDNA libraries to detect homologous polynucleotide sequences;

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

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

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

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

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

[0017] (3) Extracting target DNA sequences from cells using chemical methods.

[0018] A third objective of this invention is to provide a recombinant vector carrying the gene.

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

[0020] In this invention, polynucleotides encoding the amylase mutants ADH3-S88K / I325A and ADH3-S88R / I325A, or recombinant vectors containing such polynucleotides, can be transformed or introduced into host cells to constitute genetically engineered host cells containing such nucleotides or recombinant vectors. "Host cells" refer to prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells.

[0021] Transformation of host cells using the DNA sequence described in this invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known in the art. When the host is a eukaryote such as Pichia pastoris, competent cells capable of absorbing DNA can be harvested after the exponential growth phase, treated with sorbitol, and transformed with plasmids via electroporation. When the host is another eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation, or conventional mechanical methods such as microinjection, liposome packaging, etc.

[0022] Using the polynucleotide sequence of the present invention, expressing or producing recombinant amide hydrolases via conventional recombinant DNA technology includes the following steps:

[0023] (1) Transform or transfect suitable transformant cells with the polynucleotide of the recombinant amide hydrolase encoded by the present invention, or with a recombinant expression vector containing the polynucleotide.

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

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

[0026] In step (2), the culture medium used in the culture can be any conventional culture medium, depending on the host cells used. Culture is carried out under conditions suitable for the host cells. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method, and the cells are cultured for a further period to promote the expression of the target protein.

[0027] In step (3), the recombinase may be encapsulated intracellularly, expressed on the cell membrane, or secreted extracellularly. The recombinant protein is separated and purified using various separation methods based on 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, protein precipitant treatment (salting out), density gradient centrifugation, ultrasonic disruption, ultracentrifugation 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 thereof.

[0028] A fourth objective of this invention is to provide the application of the recombinant amide hydrolase mutants ADH3-S88K / I325A and ADH3-S88R / I325A in the degradation of OTA.

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

[0030] Compared to existing technologies, the advantages of this invention are as follows: This invention provides a set of highly efficient recombinant amide hydrolases ADH3-S88K / I325A and ADH3-S88R / I325A and their encoding genes, and also provides the application of these amide hydrolases mutants ADH3-S88K / I325A and ADH3-S88R / I325A in the detoxification of OTA from OTA-contaminated beer or corn flour. The mutants ADH3-S88K / I325A and ADH3-S88R / I325A with enhanced catalytic activity achieve a 100% degradation rate of OTA in beer or corn flour containing OTA after 10-15 minutes at room temperature. This degradation efficiency is highly efficient in the field. Attached Figure Description

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

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

[0033] Figure 3High-performance liquid chromatography (HPLC) chromatograms of the OTA degradation reaction products of purified recombinant amide hydrolase ADH3 and modified mutants ADH3-S88K / I325A and ADH3-S88R / I325A;

[0034] Figure 4 Comparison of relative enzyme activities for OTA degradation by ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A;

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

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

[0037] Figure 7 This is a comparison diagram of the amino acid sequences shown in SEQ ID NO.4 and SEQ ID NO.6. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0039] Example 1: Synthesis of the ADH3 gene for amide hydrolase and construction of its mutants

[0040] The Stenotrophomonas sp. CW117, deposited on June 14, 2015, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China), has the accession number CCTCC NO: M2015371. Classified as *Stereotrophomonas* strain CW117, its heterologously expressed amidase gene, ADH3, is a highly efficient OTA hydrolysis and detoxification enzyme. The nucleotide sequence of ADH3 is shown in SEQ ID NO.1, demonstrating significant application development potential. The full-length amino acid sequence of ADH3 contains 427 amino acids (the following descriptions of all amino acid sequences in ADH3 and its mutants refer to their corresponding sequences within the full-length amino acid sequence of ADH3). The full-length amino acid sequence of the ADH3 protein is shown in SEQ ID NO.2. The first 20 amino acids in sequence NO.2 are the signal peptide for ADH3 protein expression. To obtain a mature ADH3 protein without the signal peptide, the nucleotide sequence encoding amino acids 21 to 427 of ADH3 was given to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis, following the method described in ZL201910004511.3. Then, the synthesized ADH3 gene was ligated into the E. coli expression vector pET46EK using restriction endonucleases NcoI and XhoI to obtain the recombinant plasmid pET46EK-ADH3.

[0041] Based on protein structure analysis of the amino acid sequence of the amide hydrolase ADH3, the 88th serine in the sequence shown in SEQ ID NO.2 was mutated to arginine, and the 325th isoleucine was mutated to alanine, resulting in the double-site mutant ADH3-S88R / I325A. Alternatively, the 88th serine in the sequence shown in SEQ ID NO.2 was mutated to lysine, and the 325th isoleucine was mutated to alanine, resulting in the double-site mutant ADH3-S88K / I325A. The amino acid sequence of the obtained mutant ADH3-S88K / I325A is as shown in SEQ ID NO.4, amino acid sequences from positions 21 to 427 (e.g., as shown in SEQ ID NO.4). Figure 7 Sequence 1), the amino acid sequence of the mutant ADH3-S88R / I325A is the amino acid sequence from position 21 to 427 as shown in SEQ ID NO. 6 (e.g. Figure 7 Sequence 2 in the middle.

[0042] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.4 is the nucleotide sequence from position 61 to 1284 as shown in SEQ ID NO.3; the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.6 is the nucleotide sequence from position 61 to 1284 as shown in SEQ ID NO.5; the nucleotide sequences of the mutants ADH3-S88K / I325A and ADH3-S88R / I325A were given to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis, and the synthesized gene was ligated into the recombinant vector pET46EK using restriction endonucleases NcoI and XhoI to obtain the 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 *E. coli* BL21(DE3) competent cells, and strains were screened on LB agar plates containing 100 μg / mL ampicillin. 1.5 mL of seed culture was inoculated into 8 mL of fresh LB agar and cultured overnight at 37°C with shaking at 220 rpm / min. The culture was then scaled up to 100 mL of LB agar. Finally, the culture was scaled up to 8 L of LB agar. The OD of the bacterial cells... 600 When the pH reached 0.6, the culture temperature was lowered to 16℃, and IPTG was added to a concentration of 0.3 mM to induce high protein expression. After 20 hours of protein expression, the bacterial culture was centrifuged at 5000 rpm for 5 min to collect E. coli cells. The cells were then resuspended in buffer (20 mM Tris-HCl, pH 8.0), and lysed using a French cell disruptor. The cells were then centrifuged at 15000 rpm for 50 min, and the supernatant was collected for further purification.

[0045] ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A proteins without the 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 2SDS-PAGE gel images of purified recombinant amidohydrolases ADH3, ADH3-S88K / I325A and ADH3-S88R / I325A obtained from Escherichia coli.

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

[0048] To compare the differences in the activities of recombinant amide hydrolases ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A, this example further determined the hydrolytic activity of recombinant amide hydrolases ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A on OTA.

[0049] The activity assay method for OTA hydrolase is as follows:

[0050] Each reaction mixture (100 μL) was placed in a 20 mM Tris-HCl, pH 8.0 buffer solution, containing 200 μg / mL OTA substrate and 10 μL enzyme (10 μg / mL). After mixing, the mixture was placed in a shaking metal bath at 40°C and 800 rpm for 20 min. Each reaction was performed in triplicate, with three replicates. The enzyme reaction was terminated by adding an equal volume of acetonitrile, followed by centrifugation at 12000 rpm for 10 min. The supernatant was then filtered through a 0.22 μm organic nylon membrane. Subsequently, each reaction was analyzed by high-performance liquid chromatography (HPLC, Shimadzu SPD-M20A) using an InertSustain C14 column. 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 high-performance liquid chromatography (HPLC) results 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, peaks were observed at retention times of 4.9 min and 12.6 min. The peak time at 4.9 min was consistent with that of the product standard OTα; therefore, the substance with a retention time of 4.9 min is OTα. The peak time at 12.6 min was consistent with that of the substrate standard OTA. Therefore, the substance with a retention time of 12.6 min is OTA. Next, the activity differences were calculated by comparing the peak areas of OTα, the hydrolysis products of recombinant amide hydrolases ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A.

[0052] from Figure 3 Data shows that the products of OTA hydrolysis by ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A are OTα and phenylalanine, and OTα can be detected. Therefore, as... Figure 4 In this invention, we take the peak area of ​​OTα, the hydrolysis product of ADH3, as 100%, and compare the peak areas of OTα, the hydrolysis products of ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A, as the relative enzyme activity. The OTA degradation activity assay results are as follows: Figure 4 As shown, ADH3-S88K / I325A and ADH3-S88R / I325A exhibited 8-fold and 9-fold higher activity for OTA compared to 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 the detoxification of OTA-contaminated beer.

[0055] OTA-contaminated beer (final concentration of 500 μg OTA per liter of beer) was prepared by mixing OTA standard stock solution with beer. Purified recombinant amide hydrolases ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A were added to the OTA-contaminated beer samples, respectively. OTA degradation reactions were carried out at 37℃, with samples taken at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, with three replicates for each degradation experiment. Simultaneously, a mixture of OTA-contaminated beer without the addition of recombinant amide hydrolases ADH3, ADH3-S88K / I325AA, or DH3 / S88R / I325A served as a negative control. The same sample size was used for both the treatment and control groups, and extraction, purification, and analysis were performed according to standard OTA detection methods.

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

[0057] The results are as follows Figure 5As shown, in the negative control group without enzymes, OTA remained stable and did not degrade within 0-30 min. In contrast, recombinant amide hydrolases ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A completely degraded OTA in the samples at 60 min, 15 min, and 15 min, respectively, with a degradation efficiency of 100%. The degradation efficiency of OTA by ADH3-S88K / I325A and ADH3-S88R / I325A 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 using ADH3-S88K / I325A and ADH3-S88R / I325A to degrade higher levels of OTA in beer, the degradation rate is faster.

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

[0060] OTA standard stock solution was mixed with pulverized corn flour to prepare OTA-contaminated corn flour samples (final concentration: 500 μg OTA per kilogram of corn flour). Purified recombinant amide hydrolases ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A were added to the OTA-contaminated corn flour samples, respectively. The samples were incubated at 37°C, and samples were taken at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, with three replicates for each degradation experiment. Simultaneously, a mixture of OTA-contaminated corn flour without the addition of recombinant amide hydrolases ADH3, ADH3-S88K / I325AA, or DH3 / S88R / I325A served as a negative control. The same sample size was used for both the treatment and control groups, and extraction, purification, and analysis were performed according to the standard OTA detection method.

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

[0062] The results are as follows Figure 6As shown, in the negative control group without enzyme, OTA remained stable and did not degrade within 0-30 min. ADH3, ADH3-S88K / I325A, and ADH3-S88R / I325A completely degraded OTA in the sample at 40 min, 10 min, and 10 min, respectively, with a degradation efficiency of 100%. The degradation efficiency of OTA by ADH3-S88K / I325A and ADH3-S88R / I325A 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 using ADH3-S88K / I325A and ADH3-S88R / I325A to degrade OTA content in grains at higher levels, the degradation rate is faster.

[0064] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A group of highly efficient amide hydrolase mutants for degrading ochratoxin A, characterized in that, The amino acid sequence of the amide hydrolase mutant consists of amino acid sequences from positions 21 to 427 as shown in SEQ ID NO. 4 or SEQ ID NO.

6. The amino acid sequence shown in SEQ ID NO. 4 is formed by mutating serine at position 88 of SEQ ID NO. 2 to lysine and isoleucine at position 325 to alanine. The amino acid sequence shown in SEQ ID NO. 6 is formed by mutating serine at position 88 of SEQ ID NO. 2 to arginine and isoleucine at position 325 to alanine.

2. A gene encoding an amide hydrolase mutant as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that, A mutant gene of amide hydrolase consisting of nucleotide sequences from 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 amide hydrolase mutant gene as described in claim 3.

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

6. The application of the amide hydrolase mutant according to claim 1 in the degradation of ochratoxin A, characterized in that, The amino acid sequence of the amide hydrolase mutant consists of amino acid sequences from position 21 to 427 as 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 application according to claim 6, characterized in that, The amide hydrolase mutant can efficiently 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 amide hydrolase mutant of claim 6 with a food containing ochratoxin A; (B) React 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 the threshold.

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