A thermostable ochratoxin a-degrading enzyme and applications, methods and products

Abstract

The application discloses a heat-stable ochratoxin A (OTA) degradation enzyme and application, method and product, and belongs to the technical field of microorganisms. Rhodothermus marinus After OTA degradation enzyme from thermophilic microorganisms is optimized, the OTA degradation enzyme is expressed in E. coli in a heterologous manner, and an enzyme capable of effectively catalyzing ochratoxin A to degrade into a non-toxic substance is obtained through purification, the specific activity of the enzyme reaches 6666 U / mg, the half-life of the enzyme at 90 DEG C is up to 7.91 min, and the enzyme is superior to all known OTA detoxification enzymes. The technical problem that OTA degradation enzymes in the prior art have poor stability and cannot meet relatively harsh environmental conditions in the industry is successfully solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and specifically relates to a heat-stable ochratoxin A degrading enzyme and application, method and product. BACKGROUND

[0002] For understanding the technical content of the present application:

[0003] Ochratoxin A (OTA) is a mycotoxin produced by some fungi of the Penicillium and Aspergillus genera, which has strong nephrotoxicity, hepatotoxicity, teratogenicity, immunotoxicity and carcinogenicity to humans and animals. OTA widely exists in mold-contaminated cereals, feed, coffee beans, grapes and grape products, posing a threat to food and feed safety. Therefore, it is urgent to develop efficient and sustainable detoxification methods to remove OTA from food and feed.

[0004] Due to the stable chemical structure of OTA, it is difficult to remove the contaminated OTA in products. At present, physical, chemical and biological methods have been used for the research and application of OTA degradation, such as physical methods of heat treatment, irradiation treatment, adsorption, etc.; chemical methods of alkali treatment, oxychloride treatment, etc. However, these methods have problems such as incomplete toxin degradation or secondary pollution in practical application, which is difficult to meet the actual needs of food, feed processing and food safety protection. Compared with physical and chemical detoxification methods, biological enzyme detoxification can utilize the interaction between enzyme and toxin to convert toxic substrates into non-toxic or low-toxic degradation products. Due to its high efficiency, strong specificity, environmental friendliness and other advantages, it has become one of the key research directions for removing OTA.

[0005] However, due to the activity and stability of biological enzymes being easily affected by the environment, most of the reported OTA hydrolases have OTA degradation activity, but due to their poor stability, they cannot meet the relatively harsh environmental conditions (such as high temperature) in industry. Therefore, it is urgent to explore new types of OTA detoxification enzymes that can tolerate extreme conditions.

[0006] Retrieved relevant patent documents:

[0007] The document discloses that the OTA degrading enzyme has good degradation effect on OTA, and the degradation rate of OTA reaches 100% after 1h of reaction at 37℃.

[0008] Retrieved relevant non-patent documents:

[0009] Xu, Nana et al. disclosed in 2025 that a OTA degrading enzyme was obtained from a thermophilic bacterium Thermonema rossianumA novel OTA detoxifying enzyme TrADH was identified, the gene encoding TrADH was cloned into pET32a expression vector, transformed into E. coli BL21 (DE3) for expression, the optimum temperature of the enzyme is 65℃, the activity temperature range is 45-85℃, and 50% activity is retained after 30 minutes of 70℃ treatment.

[0010] The prior art represented by the foregoing documents at least has the following unsolved technical problems or defects:

[0011] Poor thermal stability, and relevant evidence is that the half-life is only 30 min at 70℃.

[0012] In solving the above problems or overcoming the above defects, the present application has encountered the following difficulties and obstacles:

[0013] (1) Mining and screening candidate enzymes with OTA detoxification function and higher thermal stability from protein databases face difficulties such as insufficient data reliability, inaccurate function prediction, and difficulty in obtaining extreme microbial sequences.

[0014] (2) In the heterologous expression of proteins, enzymes from different microorganisms have great differences in the demand for heterologous expression systems, which will face difficulties such as low protein expression, poor solubility, and activity loss. SUMMARY

[0015] The purpose of the present application is to provide:

[0016] A heat-stable ochratoxin A degrading enzyme and its application, method and product, and related technologies, to solve the technical problems or combinations of the OTA degrading enzyme in the prior art, such as poor thermal stability and difficulty in meeting industrial needs.

[0017] Term explanation:

[0018] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the inventive subject matter belongs. Unless otherwise indicated, all patents, patent applications, publications, and other documents referenced herein are hereby incorporated by reference in their entirety. If there is a plurality of definitions for a term herein, those in this section prevail.

[0019] It should be understood that the foregoing summary and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter of the present application. In the present application, the singular is intended to include the plural unless specifically stated otherwise. It should also be noted that, unless otherwise indicated, "or" and "and / or" are used in the inclusive sense of "and / or." Furthermore, the use of "including" as well as other forms such as "contain," "comprise," and "comprises" is not limiting.

[0020] The definitions of standard chemical terms can be found in the reference "Principles and Techniques of Enzymology (3rd Edition), Higher Education Press, Lin Ying".

[0021] Unless otherwise specified, the methods used are conventional methods within the skill in the art, such as gene cloning techniques (including PCR amplification, vector construction, transformation, etc.), protein purification techniques (including affinity chromatography, SDS-PAGE electrophoresis, etc.), enzyme activity detection methods (including liquid chromatography, mass spectrometry, etc.), and the like.

[0022] Unless a specific definition is provided, the use of each type of commercially available product used herein is in accordance with standard techniques. For example, the use of a kit can be carried out according to the manufacturer's instructions, or in accordance with a manner known in the art or described in the present disclosure. In general, the above-mentioned techniques and methods can be carried out according to conventional methods well known in the art, according to the descriptions in the multiple summary and more specific literatures cited and discussed in the present specification.

[0023] The term "homology" as used herein refers to the degree of similarity between two nucleic acid sequences or amino acid sequences, usually calculated by sequence alignment software (such as BLAST) and expressed as a percentage.

[0024] The term "codon optimization" as used herein refers to the adjustment of the codons of a target gene according to the codon bias of the host cell, so as to improve the expression level of the target gene in the host cell, without changing the encoded amino acid sequence.

[0025] The term "heterologous expression" as used herein refers to the process of introducing a gene from one organism into a different host organism, so that it is transcribed and translated in the host organism to produce the corresponding protein.

[0026] The term "thermal stability" as used herein refers to the ability of an enzyme to maintain its catalytic activity at higher temperature conditions, usually measured by the time (such as half-life) during which the enzyme activity is maintained at a certain percentage at a specific temperature.

[0027] In a first aspect, the present application provides a thermostable ochratoxin A degrading enzyme.

[0028] The thermostable ochratoxin A degrading enzyme is derived from a thermophilic microorganism Rhodothermus marinus (1) the amino acid sequence of the amide hydrolase in Rhodothermus marinus

[0029] MRWSIFLLLLGLAAGVQAQSASGRLLVHCGTVIDPGVSPEPMSERTIIVRGERIEAVQPGFVTPQEGDQVVDLRRAYCLPGLIDMHTHLSMESRKGGYLDRFQMSPALQALRASVYARRTLMAGFTTVRDVGGNEGIDLALRDAINQGWIVGPRMFVAGKSLAIMGGHADPTNGFREDILGIPTEAEGVVDGVASARRGARLAIKRGADVIKITATGGVLSIARDGSSPQFFEDEIRAIVEVARDFGLKVAAHAHGDEGMQRAIRAGVASIEHGTFMSDQTMQMMKEYGVYLVPTITAGRSVADSAKIPGYYVPVVAEKARRIGPVIQETFARAYRAGVPIAFGTDAGVFRHGRNALEFVYMVEAGMPPIEAIKAATYNAADLLGQLDNLGTLEPGKWADVIAVERNPLEDISALQEVSFVMKAGVIYKQNGQPVPQPVR, SEQ ID NO. 1.

[0030] (2) Nucleic acid sequence encoding amide hydrolase in Rhodothermus marinus

[0031]

[0032] The amino acid sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the amino acid sequences with more than 90% homology with SEQ ID NO. 3.

[0033] The amino acid sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the amino acid sequences with more than 90% homology with SEQ ID NO. 3.

[0034] The amino acid sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the amino acid sequences with more than 90% homology with SEQ ID NO. 3.

[0035] The amino acid sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the amino acid sequences with more than 90% homology with SEQ ID NO. 3.

[0036] The amino acid sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the amino acid sequences with more than 90% homology with SEQ ID NO. 3.

[0037] The amino acid sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the amino acid sequences with more than 90% homology with SEQ ID NO. 3.

[0038] The modification mode of the amino acid sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application includes but is not limited to at least one of phosphorylation, acetylation, methylation, ubiquitination, glycosylation, hydroxylation or oxidation modification; the modification site can be one or more, which can be single modification or multiple modifications at a single site, or multiple sites can be modified at the same time.

[0039] In a second aspect, the application provides a nucleic acid encoding a heat-stable ochratoxin A degrading enzyme

[0040] The technical features of the nucleic acid include nucleotide sequences, modification modes, etc.

[0041] The nucleotide sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the nucleotide sequences with more than 75% homology with SEQ ID NO. 4.

[0042] The nucleotide sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the nucleotide sequences with more than 75% homology with SEQ ID NO. 4.

[0043] The nucleotide sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the nucleotide sequences with more than 75% homology with SEQ ID NO. 4.

[0044] The nucleotide sequence of the heat-resistant ochratoxin A degrading enzyme constructed by the application is selected from the nucleotide sequences with more than 75% homology with SEQ ID NO. 4.

[0045] The nucleotide sequence with homology of more than 75% to SEQ ID NO. 4 comprises SEQ ID NO. 8.

[0046] The modification method of the technical feature nucleotide comprises at least one of fluorination modification, methylation modification, phosphorothioate modification or acetylation modification.

[0047] Based on further solving of the technical problem of the present application, the preferred scheme of the second aspect comprises:

[0048] The first preferred scheme: the sequence of the nucleic acid is the nucleotide sequence shown in SEQ ID NO. 4. This scheme clearly defines the specific nucleotide sequence, which is the basis for realizing efficient expression of the enzyme.

[0049] The second preferred scheme: the nucleic acid comprises modified nucleotides, and the modification method is methylation modification, phosphorothioate modification or acetylation modification. This scheme can improve the stability or expression efficiency of the nucleic acid in the host cell by modifying the nucleic acid, thereby further optimizing the production process of the enzyme.

[0050] In the third aspect, the present application provides an expression vector capable of expressing the ochratoxin A degrading enzyme of the first aspect or containing the nucleic acid of the second aspect.

[0051] The technical feature "expression vector" comprises at least one of pET24a, pET28a, pET32a, pGEX-4T, pMAL-p2x, pMAL-c2X, but is not limited to the above.

[0052] In the fourth aspect, the present application provides a host cell comprising the ochratoxin A degrading enzyme of the first aspect, the nucleic acid of the second aspect or the expression vector of the third aspect.

[0053] The host cell comprises at least one of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Trichonympha, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria and halophilic bacteria, but is not limited to the above. The Escherichia coli comprises at least one of BL21 (DE3), BL21Star (DE3), Rosetta (DE3), Rosetta-gami, Origami (DE3), DH5α, TOP10, MG1655, W3310 and BW25113.

[0054] In the fifth aspect, the present application provides an application of the heat-stable ochratoxin A degrading enzyme and related substances, and the application subject comprises the enzyme of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect and the host cell of the fourth aspect.

[0055] In a sixth aspect, the present application provides a method for degrading OTA, using the ochratoxin A degrading enzyme of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the host cell of the fourth aspect.

[0056] In a seventh aspect, the present application provides a product for degrading OTA, comprising the ochratoxin A degrading enzyme of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the host cell of the fourth aspect.

[0057] For the technical solution involving: SEQ ID NO. 3 and the amino acid sequence with more than 90% homology to SEQ ID NO. 3.

[0058] On the basis of SEQ ID NO. 3 and the amino acid sequence with more than 90% homology to SEQ ID NO. 3, the same type of amino acid residue substitution occurs, such as Leu replacing Ile, etc. The three-dimensional conformation of the degrading enzyme protein has not been substantially changed, so the protein still has the function of degrading ochratoxin. The substitution of the same amino acid described herein can occur at any position of the degrading enzyme.

[0059] As the same inventive concept as the present application, there is a modification of the amino acid residues based on SEQ ID NO. 3, including acetylation, glycosylation, phosphorylation, etc. The modification of the degrading enzyme generally does not change the amino acid sequence structure, so the protein still has the function of degrading ochratoxin.

[0060] The present application has at least the following beneficial effects:

[0061] 1. Compared with the prior art, the ochratoxin A degrading enzyme provided by the present application performs excellently in terms of thermal stability. The related enzyme in the prior art has a half-life of only 30 minutes at 70℃, while the enzyme of the present application still retains 78% relative enzyme activity after 4 hours of heat incubation at 70℃, and the half-life is 7.91 minutes at 90℃, which can meet the needs of harsh environmental conditions such as high temperature in industrial production.

[0062] 2. The specific activity of the ochratoxin A degrading enzyme provided by the present application reaches 6666 U / mg, which is superior to all known OTA detoxification enzymes (U = the amount of enzyme required to catalyze 1 μg of OTA to product per minute). BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 The test results of the degradation rate of ochratoxin A.

[0064] Figure 2 The chromatogram of the ochratoxin A degradation test.

[0065] Figure 3 Mass spectrum for ochratoxin A degradation test.

[0066] Figure 4 Results of ochratoxin A degrading enzyme optimum temperature experiment.

[0067] Figure 5 Results of ochratoxin A degrading enzyme optimum pH experiment.

[0068] Figure 6 Results of ochratoxin A degrading enzyme heat stability experiment at 90°C.

[0069] Figure 7 Results of ochratoxin A degrading enzyme (ADH6-OTase-M) long-term heat stability experiment at 70°C.

[0070] Figure 8 Results of ochratoxin A degrading enzyme (ADH6-OTase-M) low pH stability experiment. DETAILED DESCRIPTION

[0071] The following non-limiting examples can enable those of ordinary skill in the art to more fully understand the present application, but are not intended to limit the present application in any way. The following merely illustrates the scope of the application claimed and those skilled in the art can make many changes and modifications to the application disclosed without departing from the scope of the application claimed.

[0072] The present application is further described in the following specific examples. The various instruments, devices, equipment, reagents, products, etc. used in the examples of the present application are obtained through conventional commercial sources unless otherwise specified.

[0073] Example 1

[0074] The present application provides an enzyme having amide hydrolase activity with 429 amino acid residues, and the amino acid sequence is shown in SEQ ID NO. 3. The protein has a molecular weight of 46.3 kDa, and a theoretical isoelectric point of 6.54.

[0075] According to the codon bias of the E. coli engineering strain, the nucleotide sequence encoding the degradation enzyme of SEQ ID NO. 3 is obtained by screening using Geneart and other codon optimization software, and is specifically shown in SEQ ID NO. 4.

[0076] SEQ ID NO. 3:

[0077] MQSASGRLLVHCGTVIDPGVSPEPMSERTIIVRGERIEAVQPGFVTPQEGDQVVDLRRAYCLPGLIDMHTHLSMESRKGGYLDRFQMSPALQALRASVYARRTLMAGFTTVRDVGGNEGIDLALRDAINQGWIVGPRMFVAGKSLAIMGGHADPTNGFREDILGIPTEAEGVVDGVASARRGARLAIKRGADVIKITATGGVLSIARDGSSPQFFEDEIRAIVEVARDFGLKVAAHAHGDEGMQRAIRAGVASIEHGTFMSDQTMQMMKEYGVYLVPTITAGRSVADSAKIPGYYVPVVAEKARRIGPVIQETFARAYRAGVPIAFGTDAGVFRHGRNALEFVYMVEAGMPPIEAIKAATYNAADLLGQLDNLGTLEPGKWADVIAVERNPLEDISALQEVSFVMKAGVIYKQNGQPVPQPVRHHHHHH.

[0078] The sequence of the enzyme gene in the experiment (SEQ ID NO. 4) was synthesized by Shanghai Biosynth and cloned into the pUC57 vector. The amplification primer pair containing the homologous region (upstream primer: 5'-TTTAACTTTAAGAAGGAGATATACAATGCAGAGCGCAAGCGGTCG-3', SEQ ID NO. 5; downstream primer: 5'-GTCGACGGAGCTCGAATTCGTTAATGATGATGGTGATGGTGACGCACCGG-3', SEQ ID NO. 6) was used for PCR amplification of the target gene SEQ ID NO. 4. The amide hydrolase coding gene (named ADH6-OTase) was connected to the expression vector pET-24a(+) by Gibson connection technology to construct the recombinant expression vector pET-24a(+)-ADH6-OTase. The recombinant vector was transformed into E. coli TOP10. After antibiotic screening and sequencing verification, the positive recombinant vector was obtained and extracted, and then transformed into the expression host E. coli BL21(DE3). Finally, the host cell BL21(DE3) containing the recombinant expression vector pET-24a(+)-ADH6-OTase was obtained.

[0079] SEQ ID NO. 4:

[0080]

[0081] The obtained positive transformants were picked to single colonies in 6 mL (containing a final concentration of 100 μg / mL kanamycin) LB liquid medium at 37°C, 200 r / min in a shaker overnight to obtain seed liquid. 1.0 mL of seed liquid was added to fresh sterile 50 mL (containing a final concentration of 100 μg / mL kanamycin) LB liquid medium, and cultured at 37°C, 200 r / min until OD600=0.6, then 0.4 mM IPTG was added to induce expression for 16 h. The bacterial liquid was collected at 4°C, 5000 x g centrifuged for 10 min, and the supernatant was discarded. The cells were resuspended with buffer, broken by ultrasonic wave disrupter, centrifuged at 4°C, 5000 x g for 10 min, and the supernatant was collected, filtered with a 0.22 μM inorganic filter membrane, and the obtained enzyme was named ADH6-OTase.

[0082] Example 2

[0083] The difference from Example 1 is that the amino acid sequence is shown as SEQ ID NO. 7, the nucleotide sequence is shown as SEQ ID NO. 8, and the mutated enzyme is named ADH6-OTase-M.

[0084] SEQ ID NO. 7:

[0085] MQSASGRLLVHCGTVIDPGVSPEPMSERTIIVRGERIEAVQPGFVTPQEGDQVVDLRRAYCLPGLIDMHTHLSMESRKGGYLDRFQMSPALQALRASVYARRTLMAGFTTVRDVGGNEGIDLALRDAINQGWIVGPRMFVAGKSLAIMGGHADPTNGFREDILGIPTEAEGVVDGVASARKGARLAIKRGADVIKITATGGVLSIARDGSSPQFFEDEIRAIVEVARDFGLKVAAHAHGDEGMQRAIRAGVASIEHGTFMSDQTMQMMKEYGVYLVPTITAGRSVADSAKIPGYYVPVVAEKARRIGPVIQETFARAYRAGVPIAFGTDAGVFRHGRNALEFVYMVEAGMPPIEAIKAATYNAADLLGQLDNLGTLEPGKWADVIAVERNPLEDISALQEVSFVMKAGVIYKQNGQPVPQPVRHHHHHH.

[0086] SEQ ID NO. 8:

[0087]

[0088] Detection Example 1 Test of ochratoxin A degradation

[0089] The enzyme solution purified in Example 1 and Example 2 was diluted to 0.1 μg / mL with Tris-HCl buffer as the experimental group, and the same volume of Tris-HCl buffer without enzyme was used as the control group. The OTA standard substance stock solution (final reaction concentration of 10 μg / mL) was added, and incubated at 80℃ for 5 min and 15 min, respectively. After incubation, an equal volume of acetonitrile was added, and after vigorous shaking, it was filtered through a 0.22 μm organic filter membrane. The OTA content after reaction was detected based on the national standard (GB5009.96-2016) liquid chromatography method, and the degradation rate was calculated as (A1-A2) / A1x100%, where A1 was the chromatographic peak area of the control group OTA, and A2 was the chromatographic peak area of the experimental group OTA. According to the degradation rate of the enzyme, the degradation rate was as high as 99.7% after 15 min of incubation (see Figure 1 ), and the specific activity of the enzyme was 6666 U / mg, which was better than all known OTA detoxification enzymes (U defined as the amount of enzyme required to catalyze 1 μg of OTA to product per minute), indicating that the enzyme had good OTA degradation effect. The degradation product was identified by ultra-high performance liquid chromatography tandem time-of-flight mass spectrometry. The mass spectrometry conditions were as follows: negative ion mode, ion source temperature 350℃, capillary voltage 4000V, dry gas flow rate 11L / min, sheath gas temperature 250℃, sheath gas flow rate 11L / min, atomization gas pressure 55psi, and scanning range 100~500m / z.

[0090] Through the analysis of the product of OTA degradation by the degradation enzyme, it was found that with the extension of the incubation time of the enzyme, there was a significant increase in the absorption peak of the compound at a retention time of 0.853 min (see Figure 2 ), which was identified by mass spectrometry as OTa Figure 3 ), and it was speculated to be the OTA degradation product. Based on this, the degradation pathway of the degradation enzyme to OTA was as follows:

[0091]

[0092] Detection Example 2 Optimum temperature experiment

[0093] Take the enzyme solution after purification of Example 1 and Example 2, dilute to 0.05 μg / mL with Tris-HCl buffer as the experimental group, take the same volume of Tris-HCl buffer without enzyme as the control group, respectively add OTA standard substance stock solution (the final reaction concentration is 10 μg / mL), incubate at different temperatures (37℃, 45℃, 55℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃) for 15 min, after incubation, add the same volume of acetonitrile, after vigorous shaking, pass through 0.22 μm organic filter membrane, based on the national standard (GB5009.96-2016) liquid chromatography method to detect the content of OTA after reaction, calculate the degradation rate according to the OTA content = (A1-A2) / A1*100%, wherein A1 is the chromatographic peak area of OTA in the control group, and A2 is the chromatographic peak area of OTA in the experimental group.

[0094] According to the OTA degradation rate results (see Figure 4 ), when the incubation temperature is 80℃, the degradation activity of ADH6-OTase and ADH6-OTase-M provided by the application is high, and the OTA degradation activity is good at 70-90℃.

[0095] Optimum pH experiment of detection example 3

[0096] Take the enzyme solution after purification of Example 1 and Example 2, dilute to 0.05 μg / mL with different pH (4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11) Britton-Robinson buffer as the experimental group, take the same volume of Britton-Robinson buffer without enzyme as the control group, respectively add OTA standard substance stock solution (the final reaction concentration is 10 μg / mL), incubate at 80℃ for 15 min, after incubation, add the same volume of acetonitrile, after vigorous shaking, pass through 0.22 μm organic filter membrane, based on the national standard (GB5009.96-2016) liquid chromatography method to detect the content of OTA after reaction, calculate the degradation rate according to the OTA content = (A1-A2) / A1*100%, wherein A1 is the chromatographic peak area of OTA in the control group, and A2 is the chromatographic peak area of OTA in the experimental group.

[0097] According to the OTA degradation rate results (see Figure 5 ), when the buffer pH is 9.0, the OTA degradation activity of ADH6-OTase and ADH6-OTase-M provided by the application is the highest, and the OTA degradation activity is good at pH 7.5-10.0.

[0098] Temperature stability experiment of detection example 4

[0099] Take the enzyme solution after purification in Example 1 and Example 2, dilute to 0.05 μg / mL with Tris-HCl buffer, set different heat incubation times (0 min, 3 min, 6 min, 9 min, 12 min, 15 min, 18 min) respectively, take 0 min heat incubation time as the control group, carry out heat incubation before enzyme reaction at 90℃, after heat incubation, add OTA standard substance stock solution (final concentration is 10 μg / mL) respectively, incubate at 80℃ for 15 min, after incubation, add equal volume of acetonitrile, after violent oscillation, pass through 0.22 μm organic filter membrane, detect the content of OTA after reaction based on national standard (GB5009.96-2016) liquid chromatography. According to the content of OTA, calculate the relative enzyme activity = (R1 / R2) x 100%, wherein R1 is the degradation rate of the experimental group, and R2 is the degradation rate of the control group.

[0100] According to the relative enzyme activity results (see Figure 6 ), the ochratoxin A degrading enzyme mutant ADH6-OTase-M provided by the application has a half-life of 7.91 min at 90℃, which is increased by 3.61 min compared with ADH6-OTase, and the thermal stability data graph of ADH6-OTase-M is shown in Figure 7 It can be seen that ADH6-OTase-M still retains 78% of the relative enzyme activity after 4 hours of heat incubation at 70℃.

[0101] Detection Example 5 Low pH stability experiment

[0102] Take the enzyme solution after purification in Example 1 and Example 2, dilute to 7.0 μg / mL with pH 2.0 HCl-KCl buffer, set different acid incubation times (2 min, 4 min, 6 min, 8 min, 10 min, 12 min) respectively, take 0 min acid incubation time as the control group, after acid incubation, dilute to 0.05 μg / mL with Tris-HCl buffer, add OTA standard substance stock solution (final concentration is 10 μg / mL), incubate at 80℃ for 15 min, after incubation, add equal volume of acetonitrile, after violent oscillation, pass through 0.22 μm organic filter membrane, detect the content of OTA after reaction based on national standard (GB5009.96-2016) liquid chromatography. According to the content of OTA, calculate the relative enzyme activity = (R1 / R2) x 100%, wherein R1 is the degradation rate of the experimental group, and R2 is the degradation rate of the control group.

[0103] According to the relative enzyme activity results (see Figure 8 ), the ochratoxin A degrading enzyme mutant ADH6-OTase-M provided by the application has a relative enzyme activity of 54.4% after 8 min of incubation at pH 2.

[0104] Finally, it should be noted that the above is only to illustrate the technical solutions of the present application, and is not a limitation on the scope of protection of the present application, and simple modifications or equivalent replacements of the technical solutions of the present application by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A thermostable ochratoxin A degrading enzyme, characterized in that, The amino acid sequence of the ochratoxin A degrading enzyme is shown as SEQ ID NO. 3 or SEQ ID NO.

7.

2. A nucleic acid, characterized in that, The nucleic acid encodes the ochratoxin A degrading enzyme of claim 1.

3. The nucleic acid of claim 2, wherein, The nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 4 or has more than 75% homology with the sequence shown as SEQ ID NO.

4.

4. The nucleic acid of claim 3, wherein, The nucleotide sequence having more than 75% homology with SEQ ID NO. 4 is shown as SEQ ID NO.

8.

5. An expression vector, characterized by, The expression vector can express the ochratoxin A degrading enzyme of claim 1 or the expression vector contains the nucleic acid of any one of claims 2-4.

6. A host cell, characterized in that, The host cell contains the ochratoxin A degrading enzyme of claim 1, the nucleic acid of any one of claims 2-4 or the expression vector of claim 5.

7. Use of the ochratoxin A degrading enzyme of claim 1, the nucleic acid of any one of claims 2-4, the expression vector of claim 5 or the host cell of claim 6 in OTA degradation.

8. A method of degrading OTA, characterized by, Degradation using the ochratoxin A degrading enzyme of claim 1, the nucleic acid of any one of claims 2-4, the expression vector of claim 5 or the host cell of claim 6.

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