A nitrilase and its application in the synthesis of 2-hydroxy-4-methylthiobutyric acid

Abstract

The present invention discloses a nitrilase and its application in the synthesis of 2-hydroxy-4-methylthiobutyric acid. The amino acid sequence of the nitrilase disclosed in the present invention is as shown in SEQ ID NO:2, and the amino acid sequence of its mutant is as shown in SEQ ID NO:6. The nitrilase of the present invention can efficiently catalyze 2-hydroxy-4-methylthiobutyronitrile to produce 2-hydroxy-4-methylthiobutyric acid. In particular, the substrate conversion rate of the mutant KvNLE-P168M reaches 99%, and the production efficiency of the product 2-hydroxy-4-methylthiobutyric acid is 7.4 kg / kg DCW / h, which has important industrial application value for the green and efficient preparation of methionine hydroxy analogs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and relates to a nitrilase and its mutants derived from Klebsiella variicola, nucleic acid molecules encoding them, vectors and cells containing these nucleic acid molecules, as well as their preparation methods and their applications in the preparation of 2-hydroxy-4-methylthiobutyric acid. Background Art

[0002] 2-Hydroxy-4-methylthiobutyric acid (HMTBA) is a methionine hydroxy analogue. In animals, HMTBA can be converted into methionine and has the same biological potency as an equal amount of methionine. In addition, methionine hydroxy analogues also exhibit some unique properties, such as good antibacterial effects, enhancing the immunity and antioxidant capacity of the animal body, and effectively reducing the damage of stress response. Therefore, HMTBA is widely used as a superior methionine source in the feed additives for poultry and pigs.

[0003] Currently, methionine hydroxy analogues are mainly prepared by the cyanohydrin hydrolysis method. 2-Hydroxy-4-methylthiobutyronitrile (HMBTN) is hydrated to 2-hydroxy-4-methylthiobutyramide (HMTBAm) under the catalysis of sulfuric acid. After dilution with water, it is heated to completely hydrolyze into HMTBA, and then extracted with an organic solvent. This method requires the use of strong acids, the reaction is carried out at a relatively high temperature, the equipment corrosion is serious, and it produces toxic hydrogen cyanide and a large amount of ammonium sulfate or ammonium bisulfate, resulting in serious environmental pollution and difficult product separation.

[0004] Using biocatalysis to carry out the conversion of nitriles has the advantages not only of mild reaction conditions, less environmental pollution and fewer by-products, but also meeting the requirements of atom economy, environmental protection and sustainable development.

[0005] Companies such as Coda and Rhône-Poulenc have all studied the process of preparing 2-hydroxy-4-methylthiobutyric acid by hydrolyzing 2-hydroxy-4-methylthiobutyronitrile catalyzed by nitrilase, and the reaction is shown in formula (I). Li Zongtong et al. screened a strain producing nitrilase and used the whole cells of this strain to convert HMTBN into HMTBA. The substrate concentration was 150 mM, and the cells could only be used once. In summary, there are still problems in the biocatalytic conversion of existing nitrile substances, such as poor tolerance of the enzyme to the substrate and low conversion intensity.

[0006]

[0007] In summary, in the existing biocatalytic process for preparing methionine hydroxy analog using nitrilase, there are still problems such as poor tolerance of the key enzyme nitrilase hydrolysis reaction to substrates and low conversion intensity, which limit its application on an industrial scale. Therefore, it is of great industrial application value to develop a highly efficient nitrilase with strong substrate tolerance through methods such as genomic data mining and prepare methionine hydroxy analog green and efficiently. SUMMARY OF THE INVENTION

[0008] The object of the present invention is to provide a nitrilase and its mutants, nucleic acid molecules encoding them, vectors and cells containing these nucleic acid molecules, as well as their preparation methods and applications for green and efficient hydrolysis preparation of methionine hydroxy analog using them as catalysts, so as to solve the problems of poor tolerance of the existing enzyme to substrates and low conversion intensity in the technology of catalytic synthesis of methionine hydroxy analog by nitrilase.

[0009] The inventor of the present invention obtained a nitrilase KvNLE from Klebsiella variicola with the strain preservation number DSM 16265 in the German Collection of Microorganisms and Cell Cultures (DSMZ), which can catalyze the hydrolysis of 2-hydroxy-4-methylthiobutyronitrile to prepare 2-hydroxy-4-methylthiobutyric acid. Its amino acid sequence is shown in SEQ ID NO:2, and its nucleotide sequence is shown in SEQ ID NO:1. By further modifying the nitrilase KvNLE by molecular means, a mutant KvNLE-P168M was obtained by replacing proline at position 168 with methionine. Its amino acid sequence is shown in SEQ ID NO:6, and its nucleotide sequence is shown in SEQ ID NO:5. Compared with KvNLE, the production efficiency of 2-hydroxy-4-methylthiobutyric acid by KvNLE-P168M was significantly improved, reaching 7.4 kg / kg DCW / h.

[0010] In the first aspect, the present invention provides a nitrilase KvNLE, whose amino acid sequence is shown in SEQ ID NO:2, and it can catalyze the hydrolysis of 2-hydroxy-4-methylthiobutyronitrile to prepare 2-hydroxy-4-methylthiobutyric acid.

[0011] The above-mentioned nitrilase KvNLE provided by the present invention can be a natural, recombinant or synthetic active polypeptide. Specifically, the active polypeptide can be a product of natural purification, a product of chemical synthesis, or a product produced using recombinant technology from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants).

[0012] In some embodiments, the above-mentioned nitrilase is a recombinant genetically engineered strain constructed by transforming a recombinant vector containing its encoding gene into an Escherichia coli expression host (such as E. coli BL21(DE3)), then culturing the strain, and adding an inducer to induce expression to obtain the nitrilase.

[0013] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned nitrilase, and its nucleotide sequence is as shown in SEQ ID NO:1.

[0014] The above-mentioned nucleic acid molecule provided by the present invention can generally be obtained by using a PCR instrument for amplification or by artificial synthesis.

[0015] In a third aspect, the present invention provides a mutant of the above-mentioned nitrilase, and its amino acid sequence is as shown in SEQ ID NO:6. Compared with the amino acid sequence shown in SEQ ID NO:2, proline at position 168 is mutated to methionine, and its production efficiency of 2-hydroxy-4-methylthiobutyric acid is significantly improved compared with the above-mentioned nitrilase.

[0016] The above-mentioned mutant provided by the present invention can be artificially synthesized, or its encoding gene can be first synthesized and then obtained by biological expression, such as obtaining it by expression from a prokaryote (Escherichia coli) using recombinant technology.

[0017] In some embodiments, the above-mentioned mutant is a recombinant genetically engineered bacterium constructed by transforming a recombinant vector containing its encoding gene into an Escherichia coli expression host (such as E. coli BL21(DE3)), then culturing the strain, and adding an inducer to induce expression to obtain the mutant.

[0018] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned mutant, and its nucleotide sequence is as shown in SEQ ID NO:5.

[0019] The above-mentioned nucleic acid molecule provided by the present invention can generally be obtained by using a PCR instrument for amplification or by artificial synthesis.

[0020] In a fifth aspect, the present invention provides a recombinant vector, which contains any one of the above-mentioned nucleic acid molecules.

[0021] In some embodiments, the above-mentioned recombinant vector is pET26b(+)-KvNLE or pET26b(+)-KvNLE-P168M, which are obtained by replacing the sequence between the BamHI and EcoRI restriction enzyme sites of pET-26b(+) with the nucleic acid molecule encoding the above-mentioned nitrilase or the nucleic acid molecule of the above-mentioned mutant, respectively, and keeping the remaining sequences unchanged.

[0022] In a sixth aspect, the present invention provides a recombinant cell, which contains any one of the above-mentioned recombinant vectors.

[0023] In some embodiments, the recombinant cells are induced to express the above-mentioned nitrilase or mutant.

[0024] In some embodiments, the method for constructing the recombinant cells comprises the following:

[0025] Transform the recombinant vector into an expression host cell, culture it and add an inducer to induce expression to obtain the above-mentioned nitrilase or mutant.

[0026] Furthermore, the recombinant vector is any of the above-mentioned recombinant vectors, and the expression host cell is a conventional host cell in the art, as long as it can satisfy that the recombinant vector can stably replicate by itself and the genes carried by it can be effectively expressed. It can be a prokaryotic biological cell or a eukaryotic biological cell, such as Escherichia coli, yeast, etc. Preferably, the expression host of Escherichia coli is E. coli BL21(DE3).

[0027] In some embodiments, the recombinant cells are recombinant bacteria E. coli BL21(DE3) / pET26b(+)-KvNLE and recombinant bacteria E. coli BL21(DE3) / pET26b(+)-KvNLE-P168M. The recombinant cells can be recombinant genetically engineered bacteria. The culture medium used when the recombinant genetically engineered bacteria express nitrilase or its mutant can be a culture medium in the art that can enable the recombinant genetically engineered bacteria to grow and express the nitrilase or its mutant of the present invention, such as LB medium.

[0028] There are no special requirements for the culture method and culture conditions. It is only necessary to ensure the normal growth of the recombinant genetically engineered strain, and induce the expression of nitrilase and its mutant under appropriate temperature conditions. The preferred culture method is: inoculate the recombinant bacteria E. coli BL21(DE3) / pET26b(+)-KvNLE or the recombinant bacteria E. coli BL21(DE3) / pET26b(+)-KvNLE-P168M into a test tube containing LB liquid medium with kanamycin, culture at 37 °C and 220 rpm for 12 hours, transfer it to a 500 mL Erlenmeyer flask containing 100 mL of LB liquid medium with kanamycin according to an inoculation amount of 1-2% (v / v), and place it at 37 °C and 220 rpm for culture. When the OD 600 reaches 0.6 - 0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 100 - 500 μM as an inducer, induce at 16 °C for 16 - 24 hours, then centrifuge the culture solution, collect the cell precipitate, and wash it with physiological saline to obtain the recombinant cells.

[0029] More specifically, the method for constructing the above-mentioned recombinant cells may comprise the following steps:

[0030] (1) Amplification of the gene of nitrilase KvNLE;

[0031] (2) Construction of the recombinant expression plasmid pET-26b(+)-KvNLE;

[0032] (3) Obtaining of the recombinant expression plasmid pET26b(+)-KvNLE-P168M;

[0033] (4) Transformation of the recombinant expression plasmids pET-26b(+)-KvNLE and pET26b(+)-KvNLE-P168M into host cells;

[0034] (5) Screening of positive clone strains on a plate resistant medium.

[0035] In a seventh aspect, the present invention provides a method for preparing a nitrilase or a mutant thereof, comprising:

[0036] Culturing the above-mentioned recombinant cell and adding an inducer for induction to obtain a culture;

[0037] Isolating the above-mentioned nitrilase or a mutant thereof from the culture;

[0038] Wherein, the method for culturing and inducing the recombinant cell and the method for isolating the nitrilase and its mutant from the culture are all conventional methods in the art.

[0039] In an eighth aspect, the present invention provides the use of the above-mentioned nitrilase, the above-mentioned nucleic acid molecule, the above-mentioned mutant, the above-mentioned recombinant vector of any one of the above, the above-mentioned recombinant cell of any one of the above, and / or the nitrilase or a mutant thereof prepared by the above-mentioned method in the preparation of 2-hydroxy-4-methylthiobutyric acid.

[0040] In a ninth aspect, the present invention provides a method for preparing 2-hydroxy-4-methylthiobutyric acid, comprising the following steps: using the above-mentioned nitrilase, the above-mentioned mutant, the above-mentioned recombinant cell of any one of the above, and / or the nitrilase or a mutant thereof prepared by the above-mentioned method as a catalyst to catalyze the hydrolysis reaction of 2-hydroxy-4-methylthiobutyronitrile to prepare 2-hydroxy-4-methylthiobutyric acid.

[0041] In some embodiments, in the above-mentioned method, the temperature of the hydrolysis reaction is 20-40 °C, such as 20 °C, 30 °C, 40 °C, or any value or range between any two of these values, preferably 30 °C; the pH of the hydrolysis reaction is 5-9, such as pH 5, pH 6, pH 7, pH 8, pH 9, or any value or range between any two of these values, preferably pH 8; the reaction feed includes: 2-hydroxy-4-methylthiobutyronitrile, ammonia;

[0042] The final concentration of 2-hydroxy-4-methylthiobutyronitrile is 30 - 250 mM, such as 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, or any value or range between any two of these values, where 250 mM is preferred;

[0043] The addition amount of ammonia is 1.5 - 10 times the equivalent of the theoretical yield of 2-hydroxy-4-methylthiobutyric acid, such as 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 times the equivalent, and 3 times the equivalent is preferred.

[0044] In some embodiments, in any of the above-described methods, the buffer used in the hydrolysis reaction is a conventional buffer in the art, such as Tris-HCl buffer, and its concentration is preferably 100 mM.

[0045] In some embodiments, in any of the above-described methods, it includes using any of the above-described recombinant cells to catalyze the hydrolysis reaction of 2-hydroxy-4-methylthiobutyronitrile to prepare 2-hydroxy-4-methylthiobutyric acid;

[0046] Specifically, the frozen dry cells or freeze-dried powder of the above-described recombinant cells can be used as a catalyst for whole-cell catalysis to produce 2-hydroxy-4-methylthiobutyric acid, and the amount of the frozen dry cells or freeze-dried powder used is 0.01 - 0.05 g / g of 2-hydroxy-4-methylthiobutyronitrile, preferably 0.03 g / g of 2-hydroxy-4-methylthiobutyronitrile. Among them, the freeze-dried powder is obtained by crushing any of the above-described recombinant cells to obtain a cell lysate, and then freeze-drying the cell lysate. The frozen dry cells are directly freeze-dried from the recombinant cells without crushing.

[0047] It should be understood that the nitrilase KvNLE or its mutant KvNLE-P168M of the present invention can be used in the form of whole cells of an engineered bacterium, or in the form of a crude enzyme without purification, or in the form of a partially purified or fully purified enzyme. The nitrilase KvNLE or its mutant KvNLE-P168M of the present invention can also be prepared into a catalyst in the form of an immobilized enzyme or immobilized cells using immobilization techniques known in the art.

[0048] In some embodiments, in any of the above-described methods, the hydrolysis reaction is carried out in a stirred or oscillating environment, for example, reacting under stirring at 100 - 500 rpm.

[0049] The beneficial effects of the present invention are as follows:

[0050] The nitrilase provided by the present invention can efficiently catalyze the conversion of 2-hydroxy-4-methylthiobutyronitrile to 2-hydroxy-4-methylthiobutyric acid. In particular, the substrate conversion rate of the mutant KvNLE-P168M reaches 99%, and the production efficiency of 2-hydroxy-4-methylthiobutyric acid is as high as 7.4 kg / kg DCW / h, which has important industrial application value for the green and efficient preparation of methionine hydroxy analogs. Detailed implementation manners

[0051] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional operations in the art or experimental methods recommended by the manufacturers of reagent kits and instrument equipment. The reagents and biological materials used in the embodiments can be obtained from commercial sources unless otherwise specified.

[0052] Klebsiella variicola is derived from the German Collection of Microorganisms and Cell Cultures (DSMZ), and the strain preservation number is DSM 16265.

[0053] pET-26b(+) is a product of Novagen, and the product catalog number is 69862-3CN.

[0054] Example 1: Obtaining the gene sequence of nitrilase KvNLE from Klebsiella variicola DSM 16265

[0055] 1. Extract the genomic DNA of Klebsiella variicola DSM 16265.

[0056] 2. Using the genomic DNA of Klebsiella variicola DSM 16265 as a template and KvNLE-FP and KvNLE-RP as primers for PCR amplification to obtain a fragment containing the nitrilase gene. The nucleotide sequence of the nitrilase gene is shown in SEQ ID NO:1, and the amino acid sequence of the encoded nitrilase KvNLE is shown in SEQ ID NO:2.

[0057] KvNLE-FP: 5’-CGCGGATCCATGATGAACAAACCG-3’ (SEQ ID NO:3);

[0058] KvNLE-RP: 5’-CCGGAATTCCTACTCCTCCTCTTC-3’ (SEQ ID NO:4).

[0059] Example 2: Construction of the nitrilase KvNLE expression strain

[0060] 1. The fragment containing the nitrilase gene obtained in Example 1 was digested with BamHI and EcoRI to obtain a gene fragment; pET-26b(+) was digested with BamHI and EcoRI to obtain a vector fragment; the gene fragment and the vector fragment were ligated to obtain a recombinant expression plasmid, which was named pET26b(+)-KvNLE. This plasmid was sent for sequencing, and the result was consistent with the expectation.

[0061] 2. The recombinant expression plasmid pET26b(+)-KvNLE obtained in step 1 was transformed into the expression host E. coli BL21(DE3) by chemical transformation, and spread on an LB solid medium containing 50 μg / mL kanamycin for screening to obtain the recombinant strain E. coli BL21(DE3) / pET26b(+)-KvNLE expressing nitrilase KvNLE.

[0062] Example 3: Obtaining the gene sequence of the mutant KvNLE-P168M

[0063] Using the recombinant expression plasmid pET-26b(+)-KvNLE obtained in Example 2 as a template, whole plasmid PCR was performed with KvNLE-P168M-FP and KvNLE-P168M-RP as primers to introduce site-directed mutagenesis, and the mutant recombinant expression plasmid pET26b(+)-KvNLE-P168M was obtained. Among them, the nucleotide sequence of the nitrilase mutant is shown in SEQ ID NO:5, and the amino acid sequence of the nitrilase mutant KvNLE-P168M encoded by it is shown in SEQ ID NO:6.

[0064] KvNLE-P168M-FP: 5’-TGGGAGCATCTGCAGATGCTGTCCCGTTATGCG-3’ (SEQ ID NO:7);

[0065] KvNLE-P168M-RP: 5’-CGCATAACGGGACAGCATCTGCAGATGCTCCCA-3’ (SEQ ID NO:8).

[0066] Example 4: Construction of the mutant KvNLE-P168M expression strain

[0067] The mutant recombinant expression plasmid pET26b(+)-KvNLE-P168M obtained in Example 3 was transformed into the expression host E. coli BL21(DE3) by chemical transformation, and spread on an LB solid medium containing 50 μg / mL kanamycin for screening to obtain the recombinant bacterium E. coli BL21(DE3) / pET26b(+)-KvNLE-P168M expressing the mutant KvNLE-P168M.

[0068] Example 5: Preparation of Enzyme

[0069] The recombinant bacterium E. coli BL21(DE3) / pET26b(+)-KvNLE obtained in Example 2 and the recombinant bacterium E. coli BL21(DE3) / pET26b(+)-KvNLE-P168M obtained in Example 4 were respectively inoculated into an LB test tube medium containing 50 μg / mL kanamycin and cultured at 37 °C and 220 rpm for 12 hours. Then, they were transferred to a 100 mL LB shake flask medium containing 50 μg / mL kanamycin at an inoculation amount of 1% (v / v) and cultured at 37 °C and 220 rpm until OD 600 = 0.6 - 0.8. IPTG with a final concentration of 300 μM was added, and the cells were induced to culture at 16 °C for 20 hours. The induced culture broth was centrifuged at 9000 rpm for 10 minutes to collect the cell precipitate, which was washed with physiological saline to obtain resting cells. The resting cells were directly freeze-dried to obtain freeze-dried cells, or freeze-dried to obtain freeze-dried powder after ultrasonic disruption, and stored at 4 °C.

[0070] Example 6: Preparation of 2-Hydroxy-4-methylthiobutyric Acid by Enzyme Catalysis

[0071] The original enzyme KvNLE and the mutant enzyme KvNLE-P168M prepared in Example 5 were used as biocatalysts in the preparation reaction of 2-hydroxy-4-methylthiobutyric acid.

[0072] 200 mL reaction system: 250 mM substrate 2-hydroxy-4-methylthiobutyronitrile, 0.2 g freeze-dried cells (equivalent to 0.03 g / g 2-hydroxy-4-methylthiobutyronitrile), ammonia was added dropwise in the form of ammonia water with a final concentration of 3 times the equivalent of the theoretical yield of 2-hydroxy-4-methylthiobutyric acid, the buffer was 100 mM Tris-HCl buffer, and the reaction was stirred at 30 °C and pH 8.0 at 200 rpm for 5 hours.

[0073] After the reaction was completed, 1 mL of the reaction solution was taken and centrifuged to remove the biocatalyst. The supernatant was analyzed by high-performance liquid chromatography to detect the concentration of 2-hydroxy-4-methylthiobutyric acid. After calculation, when the original enzyme KvNLE was used as the biocatalyst, the conversion rate of the substrate 2-hydroxy-4-methylthiobutyronitrile (conversion rate = (initial amount of reactant - remaining amount of reactant) / initial amount of reactant × 100%) was 21%, and the production efficiency of the product 2-hydroxy-4-methylthiobutyric acid (production efficiency = amount of product formed in the reaction / (reaction time * amount of catalyst)) was 1.6 kg / kg DCW / h. In contrast, when the mutant enzyme KvNLE-P168M was used as the biocatalyst, the conversion rate of the substrate 2-hydroxy-4-methylthiobutyronitrile reached 99%, and the production efficiency of the product 2-hydroxy-4-methylthiobutyric acid reached 7.4 kg / kg DCW / h.

[0074] The high-performance liquid chromatography detection method was as follows:

[0075] An Agilent high-performance liquid chromatograph was used, with a WatersSpherisorb S5 ODS2 chromatographic column (specification 4.6×150 mm); mobile phase: 0.1 M phosphoric acid: acetonitrile = 9:1 (volume ratio); flow rate: 1.0 mL / min; detection wavelength: 210 nm; temperature: 40°C; all samples were centrifuged at 10,000 rpm and filtered through a 0.22 μm organic filter membrane, and the sample injection volume was: 10 μL.

[0076] The peak elution time of the 2-hydroxy-4-methylthiobutyronitrile standard was 6.6 min, and the peak elution time of the 2-hydroxy-4-methylthiobutyric acid standard was 3.6 min.

[0077] The peak elution time of 2-hydroxy-4-methylthiobutyronitrile in the reaction solution was 6.6 min, and the peak elution time of 2-hydroxy-4-methylthiobutyric acid was 3.6 min.

[0078] As mentioned above, only the preferred specific embodiments of the present invention are described, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

[0079] Sequence

[0080] SEQ ID NO:1

[0081] ATGATGAACAAACCGGTTACCGTTGCCTGCGTTCAGGCCGCCCCCGTTTTTATGGATCT

[0082] TGAAGGCACCATCGACAAAACAGTCACCCTCATCTCTGAAGCCGCCCAGAAAGGTGC

[0083] GGAACTCATCGCTTTTCCGGAGACCTGGATACCCGGTTATCCGTGGTTCTTATGGCTTA

[0084] ACTCGCCCGCAACAAATATGCCCCTGGTTTATCAGTATCATCAGAACTCTCTGGTGCTG

[0085] GACAGTGCCCAGGCGAAGCGAATTGCGGATGCAGCGCAGCAAAATAATATCGTTGTC

[0086] GTTCTGGGCTTCAGCGAGCGCGATCACGGGAGTCTCTACATCTCACAGTGGCTGATTG

[0087] GCAGCAACGGGGAAACCATTGGCATCCGGCGCAAGCTTAAGGCCACACACGTGGAG

[0088] CGTACGCTGTTCGGTGAAAGCGACGGTTCCTCTCTGACTACCTGGGAGACACCTCTG

[0089] GGGAACGTTGGAGCACTCTGCTGCTGGGAGCATCTGCAGCCGCTGTCCCGTTATGCGA

[0090] TGTATTCCCAGCATGAAGAGATACATATTGCCGCCTGGCCCAGCTTCAGTCTTTACACC

[0091] AGCGCAACGGCCGCGCTGGGTCCCGAGGTCAACACGGCGGCTTCACGCCTGTATGCC

[0092] GCAGAGGGTCAGTGCTTCGTGATAGCCCCGTGCGCCGTGGTTTCTGATGAGATGATTG

[0093] ATTTCCTTTGTCCTGACGACGACCGGAGAGCGTTACTCAGTGCCGGGGGGGGACATG

[0094] CCCGAATTTACGGCCCGGACGGAAGAGAACTCGTCACACCTCTCGGGGAAAATGAGG

[0095] AAGGACTGCTTATCGCTGAGCTTGACTCTTCTGCGATTACCTTTGCCAAACTGGCGGC

[0096] TGACCCGGTAGGCCACTATTCACGCCCTGACGTGACACGCCTCCTTTTCAATCCTTCA

[0097] GTCAACAAGACTGTGATTAAACGTCATTCGCCTCCTGAGCTAATTGCCGAACAGGCTG

[0098] CTGCTGAAGAAGAGGAGGAGTAG

[0099] SEQ ID NO:2

[0100] MMNKPVTVACVQAAPVFMDLEGTIDKTVTLISEAAQKGAELIAFPETWIPGYPWFLWLN

[0101] SPATNMPLVYQYHQNSLVLDSAQAKRIADAAQQNNIVVVLGFSERDHGSLYISQWLIGSN

[0102] GETIGIRRKLKATHVERTLFGESDGSSLTTWETPLGNVGALCCWEHLQPLSRYAMYSQHE

[0103] EIHIAAWPSFSLYTSATAALGPEVNTAASRLYAAEGQCFVIAPCAVVSDEMIDFLCPDDDR

[0104] RALLSAGGGHARIYGPDGRELVTPLGENEEGLLIAELDSSAITFAKLAADPVGHYSRPDV

[0105] TRLLFNPSVNKTVIKRHSPPELIAEQAAAEEEEE

[0106] SEQ ID NO:5

[0107] ATGATGAACAAACCGGTTACCGTTGCCTGCGTTCAGGCCGCCCCCGTTTTTATGGATCT

[0108] TGAAGGCACCATCGACAAAACAGTCACCCTCATCTCTGAAGCCGCCCAGAAAGGTGC

[0109] GGAACTCATCGCTTTTCCGGAGACCTGGATACCCGGTTATCCGTGGTTCTTATGGCTTA

[0110] ACTCGCCCGCAACAAATATGCCCCTGGTTTATCAGTATCATCAGAACTCTCTGGTGCTG

[0111] GACAGTGCCCAGGCGAAGCGAATTGCGGATGCAGCGCAGCAAAATAATATCGTTGTC

[0112] GTTCTGGGCTTCAGCGAGCGCGATCACGGGAGTCTCTACATCTCACAGTGGCTGATTG

[0113] GCAGCAACGGGGAAACCATTGGCATCCGGCGCAAGCTTAAGGCCACACACGTGGAG

[0114] CGTACGCTGTTCGGTGAAAGCGACGGTTCCTCTCTGACTACCTGGGAGACACCTCTG

[0115] GGGAACGTTGGAGCACTCTGCTGCTGGGAGCATCTGCAGATGCTGTCCCGTTATGCGA

[0116] TGTATTCCCAGCATGAAGAGATACATATTGCCGCCTGGCCCAGCTTCAGTCTTTACACC

[0117] AGCGCAACGGCCGCGCTGGGTCCCGAGGTCAACACGGCGGCTTCACGCCTGTATGCC

[0118] GCAGAGGGTCAGTGCTTCGTGATAGCCCCGTGCGCCGTGGTTTCTGATGAGATGATTG

[0119] ATTTCCTTTGTCCTGACGACGACCGGAGAGCGTTACTCAGTGCCGGGGGGGGACATG

[0120] CCCGAATTTACGGCCCGGACGGAAGAGAACTCGTCACACCTCTCGGGGAAAATGAGG

[0121] AAGGACTGCTTATCGCTGAGCTTGACTCTTCTGCGATTACCTTTGCCAAACTGGCGGC

[0122] TGACCCGGTAGGCCACTATTCACGCCCTGACGTGACACGCCTCCTTTTCAATCCTTCA

[0123] GTCAACAAGACTGTGATTAAACGTCATTCGCCTCCTGAGCTAATTGCCGAACAGGCTG

[0124] CTGCTGAAGAAGAGGAGGAGTAG

[0125] SEQ ID NO:6

[0126] MMNKPVTVACVQAAPVFMDLEGTIDKTVTLISEAAQKGAELIAFPETWIPGYPWFLWLN

[0127] SPATNMPLVYQYHQNSLVLDSAQAKRIADAAQQNNIVVVLGFSERDHGSLYISQWLIGSN

[0128] GETIGIRRKLKATHVERTLFGESDGSSLTTWETPLGNVGALCCWEHLQMLSRYAMYSQH

[0129] EEIHIAAWPSFSLYTSATAALGPEVNTAASRLYAAEGQCFVIAPCAVVSDEMIDFLCPDDDR

[0130] RALLSAGGGHARIYGPDGRELVTPLGENEEGLLIAELDSSAITFAKLAADPVGHYSRPDV

[0131] TRLLFNPSVNKTVIKRHSPPELIAEQAAAEEEEE

Claims

1. A mutant of nitrilase, the amino acid sequence of which is shown in SEQ ID NO:

6.

2. A nucleic acid molecule encoding the mutant according to claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

5.

3. A recombinant vector comprising the nucleic acid molecule according to claim 2.

4. A recombinant cell comprising the recombinant vector according to claim 3; The cell is not an animal or plant variety.

5. A method for preparing a mutant of nitrilase, which comprises the following steps: 1) Culturing the recombinant cell according to claim 4 and inducing the expression of the mutant of nitrilase; 2) Isolating the mutant according to claim 1 from the culture obtained in 1).

6. Use of the mutant of nitrilase prepared by the mutant according to claim 1, the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3, the recombinant cell according to claim 4 and / or the preparation method according to claim 5 in the preparation of 2-hydroxy-4-methylthiobutyric acid.

7. A method for preparing 2-hydroxy-4-methylthiobutyric acid, comprising the following steps: Using the mutant of nitrilase prepared by the mutant according to claim 1, the recombinant cell according to claim 4 and / or the preparation method according to claim 5 as a catalyst to catalyze the hydrolysis reaction of 2-hydroxy-4-methylthiobutyronitrile to prepare 2-hydroxy-4-methylthiobutyric acid.

8. According to the preparation method according to claim 7, characterized in that: The temperature of the hydrolysis reaction is 20-40 °C, the pH is 5-9, and the final concentration of the substrate 2-hydroxy-4-methylthiobutyronitrile is 30-250 mM.

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