A nitrilase and its application in the synthesis of 3-hydroxy-3-methylbutyric acid

Abstract

The present invention discloses a nitrilase and its application in the synthesis of 3-hydroxy-3-methylbutyric acid. The amino acid sequence of the nitrilase disclosed in the present invention is shown in SEQ ID NO:2, and the amino acid sequence of its mutant is shown in SEQ ID NO:6. The nitrilase of the present invention can efficiently catalyze 3-hydroxy-3-methylbutyronitrile to generate 3-hydroxy-3-methylbutyric acid, especially the mutant EbNLE-R126A substrate conversion rate reaches 99%, and the production efficiency of 3-hydroxy-3-methylbutyric acid reaches 29.5g / L / d, which has important industrial application value for the green and efficient preparation of 3-hydroxy-3-methylbutyric acid.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and relates to a nitrilase derived from Erwinia billingiae and a mutant thereof, nucleic acid molecules encoding the nitrilase, vectors and cells containing the nucleic acid molecules, and a preparation method thereof and an application thereof in preparing 3-hydroxy-3-methylbutyric acid. Background Art

[0002] 3-Hydroxy-3-methylbutyrate (HMB) is used as a nutritional supplement in humans, especially in strength and endurance sports, and as a drug to treat muscle wasting. In addition, HMB can also be used as a feed additive or feed supplement for poultry and pigs.

[0003] EP2744489A1 and others disclose a technical solution of preparing HMB sodium salt by oxidizing diacetone alcohol of Abbott Pharmaceuticals with sodium hypochlorite, and then neutralizing with hydrochloric acid to obtain HMB, as shown in formula (I). This solution not only produces a stoichiometric amount of salt, but also produces a large amount of toxic and harmful substances such as chloroform, and the yield of the oxidation reaction is low.

[0004]

[0005] The advantages of using biocatalysis to transform nitriles are not only mild reaction conditions, less environmental pollution, and fewer by-products, but also compliance with the requirements of atom economy, green environmental protection, and sustainable development.

[0006] CN113646293A discloses a technical route for preparing HMB by chemical-enzymatic cascade method of Evonik, which uses commercial nitrilase NIT59 and NIT60 to catalyze the hydrolysis of 3-hydroxy-3-methylbutyronitrile to prepare HMB. The reaction takes 24 hours at 37°C, with a conversion rate of 26% and a production efficiency of only 3.28 g / L / d.

[0007] In summary, the existing biocatalytic conversion of nitrile substances to prepare HMB still has problems such as poor enzyme tolerance to substrates and low conversion intensity, which limits its application on an industrial scale. Therefore, it is of great industrial application value to develop efficient nitrilase with strong substrate tolerance through methods such as genomic data mining, and to prepare HMB in a green and efficient manner. Summary of the invention

[0008] The object of the present invention is to provide a nitrilase and a mutant thereof, nucleic acid molecules encoding the same, vectors and cells containing the nucleic acid molecules, and methods for preparing the same, and use of the same as a catalyst for green and efficient hydrolysis preparation of HMB, thereby solving the problems of poor enzyme tolerance to substrates and low conversion intensity in the existing technology for catalyzing the synthesis of HMB by nitrilase.

[0009] The inventors of the present invention obtained a nitrilase EbNLE that can catalyze the hydrolysis of 3-hydroxy-3-methylbutyronitrile to prepare 3-hydroxy-3-methylbutyric acid from Erwinia billingiae with a strain deposit number of DSM 17872 in the German Collection of Microorganisms (DSMZ), and 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 EbNLE by molecular means, a mutant EbNLE-R126A in which the arginine at position 126 is replaced with alanine is obtained, and its amino acid sequence is shown in SEQ ID NO:6, and its nucleotide sequence is shown in SEQ ID NO:5; compared with EbNLE, the production efficiency of 3-hydroxy-3-methylbutyric acid of EbNLE-R126A is significantly improved, reaching 29.5 g / L / d.

[0010] In a first aspect, the present invention provides a nitrilase EbNLE, whose amino acid sequence is shown in SEQ ID NO: 2, which can catalyze the hydrolysis of 3-hydroxy-3-methylbutyronitrile to prepare 3-hydroxy-3-methylbutyric acid.

[0011] The nitrilase EbNLE provided by the present invention can be a natural, recombinant or synthetic active polypeptide. Specifically, the active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants) using recombinant technology.

[0012] In some embodiments, the nitrilase is obtained by transforming a recombinant vector containing its encoding gene into an Escherichia coli expression host (eg, E. coli BL21 (DE3)) to construct a recombinant genetic engineering strain, 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, whose nucleotide sequence is shown as SEQ ID NO: 1.

[0014] The nucleic acid molecules provided by the present invention can usually be obtained by amplification using a PCR instrument or artificial synthesis.

[0015] In a third aspect, the present invention provides a mutant of the above-mentioned nitrilase, whose amino acid sequence is shown in SEQ ID NO:6. Compared with the amino acid sequence shown in SEQ ID NO:2, the arginine at position 126 is mutated to alanine, and the production efficiency of 3-hydroxy-3-methylbutyrate is significantly improved compared with the above-mentioned nitrilase.

[0016] The mutant provided by the present invention can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically, such as expressed in prokaryotes (Escherichia coli) using recombinant technology.

[0017] In some embodiments, the mutant is obtained by transforming a recombinant vector containing its encoding gene into an Escherichia coli expression host (eg, E. coli BL21 (DE3)) to construct a recombinant genetic engineering bacterium, 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 mutant, whose nucleotide sequence is shown in SEQ ID NO:5.

[0019] The nucleic acid molecules provided by the present invention can usually be obtained by amplification using a PCR instrument or artificial synthesis.

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

[0021] In some embodiments, the recombinant vector is pET26b(+)-EbNLE or pET-26b(+)-EbNLE-R126A, which is obtained by replacing the sequence between the BamHI and EcoRI restriction sites of pET-26b(+) with the nucleic acid molecule encoding the nitrilase or the nucleic acid molecule encoding the mutant, while the other sequences remain unchanged.

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

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

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

[0025] The recombinant vector is transformed into an expression host cell, cultured and induced to express by adding an inducer to obtain the above 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 the recombinant vector can stably replicate itself and the genes it carries can be effectively expressed. It can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., preferably the Escherichia coli expression host E. coli BL21 (DE3).

[0027] In some embodiments, the recombinant cell is a recombinant bacterium E. coli BL21 (DE3) / pET-26b (+) -EbNLE and a recombinant bacterium E. coli BL21 (DE3) / pET-26b (+) -EbNLE-R126A, and the recombinant cell can be a recombinant genetically engineered bacterium. The culture medium used when the recombinant genetically engineered bacterium expresses the nitrilase or its mutant can be a culture medium in the art that can grow the recombinant genetically engineered bacterium and express the nitrilase or its mutant of the present invention, such as LB culture medium.

[0028] There are no special requirements for the culture method and culture conditions, as long as the normal growth of the recombinant genetic engineering strain is ensured, and the expression of nitrilase and its mutants is induced under appropriate temperature conditions. The preferred culture method is: inoculate the recombinant bacteria E. coli BL21 (DE3) / pET-26b (+) -EbNLE or the recombinant bacteria E. coli BL21 (DE3) / pET-26b (+) -EbNLE-R126A into a test tube containing LB liquid culture medium containing kanamycin, culture at 37 ° C, 220 rpm for 12 hours, transfer to a 500 mL triangular shake flask containing 100 mL LB liquid culture medium containing kanamycin at an inoculum volume of 1-2% (v / v), place it at 37 ° C, 220 rpm, and culture it at OD of the culture solution. 600 When the pH reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 100-500 μM as an inducer. After induction at 16°C for 16-24 hours, the culture solution is centrifuged, the bacterial precipitate is collected, and washed with physiological saline to obtain recombinant cells.

[0029] More specifically, the method for constructing the above-mentioned recombinant cell comprises the following steps:

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

[0031] (2) Construction of recombinant expression plasmid pET26b(+)-EbNLE;

[0032] (3) Obtaining the recombinant expression plasmid pET26b(+)-EbNLE-R126A;

[0033] (4) The recombinant expression plasmids pET26b(+)-EbNLE and pET26b(+)-EbNLE-R126A were transformed into host cells;

[0034] (5) Screening of positive clones using plate resistance culture medium.

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

[0036] Cultivating any of the above-mentioned recombinant cells and adding an inducer to induce them to obtain a culture;

[0037] isolating the nitrilase or a mutant thereof from the culture;

[0038] Among them, the method of culturing and inducing the recombinant cells and the method of separating the nitrilase and its mutants from the culture are 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, any of the above-mentioned recombinant vectors, any of the above-mentioned recombinant cells and / or the nitrilase or its mutant prepared by the above-mentioned method in the preparation of 3-hydroxy-3-methylbutyric acid.

[0040] In a ninth aspect, the present invention provides a method for preparing 3-hydroxy-3-methylbutyric acid, comprising the following steps: using the above-mentioned nitrilase, the above-mentioned mutant, the nitrilase or its mutant prepared by any of the above-mentioned recombinant cells and / or the above-mentioned method as a catalyst to catalyze the hydrolysis reaction of 3-hydroxy-3-methylbutyronitrile to prepare 3-hydroxy-3-methylbutyric acid.

[0041] In some embodiments, in the above method, the temperature of the hydrolysis reaction is 30-42°C, such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, or a value or range between any two of these values, wherein 37°C is preferred;

[0042] The pH of the hydrolysis reaction is 6.5-7.5, such as pH 6.5, pH 7, pH 7.5, or a value or range between any two of these values, preferably pH 7;

[0043] The final concentration of the substrate 3-hydroxy-3-methylbutyronitrile is 0.01-1M, for example 0.01M, 0.02M, 0.04M, 0.06M, 0.08M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, or a value or range between any two of these values, preferably 0.1M.

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

[0045] In some embodiments, any of the above methods comprises using any of the above recombinant cells to catalyze the hydrolysis of 3-hydroxy-3-methylbutyronitrile to prepare 3-hydroxy-3-methylbutyric acid;

[0046] Specifically, the lyophilized cells or lyophilized powder of the above recombinant cells can be used as catalysts for whole-cell catalytic production of 3-hydroxy-3-methylbutyric acid, and the amount of lyophilized cells or lyophilized powder is 30-90 g / g 3-hydroxy-3-methylbutyronitrile, and can be 60 g / g 3-hydroxy-3-methylbutyronitrile. Wherein, the lyophilized powder is a lyophilized enzyme powder obtained by crushing any of the above-mentioned recombinant cells to obtain a cell crushing liquid, and then freeze-drying the cell crushing liquid, and the lyophilized cells are obtained by directly freeze-drying the recombinant cells without crushing.

[0047] It should be understood that the nitrilase EbNLE or its mutant EbNLE-R126A of the present invention can be used in the form of whole cells of engineered bacteria, in the form of crude enzymes without purification, or in the form of partially purified or completely purified enzymes. The nitrilase EbNLE or its mutant EbNLE-R126A of the present invention can also be prepared into catalysts in the form of immobilized enzymes or immobilized cells using immobilization techniques known in the art.

[0048] In some embodiments, in any of the above methods, the hydrolysis reaction is carried out under stirring or shaking, for example, 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 3-hydroxy-3-methylbutyronitrile to generate 3-hydroxy-3-methylbutyric acid, especially the mutant EbNLE-R126A has a substrate conversion rate of 99%, and a production efficiency of 3-hydroxy-3-methylbutyric acid of 29.5 g / L / d, which has important industrial application value for green and efficient preparation of HMB. DETAILED DESCRIPTION

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

[0052] Erwinia billingiae is from the German Collection of Microorganisms (DSMZ), and the strain collection number is DSM 17872.

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

[0054] Example 1: Obtaining the gene sequence of the nitrilase EbNLE from Erwinia billingiae DSM 17872

[0055] 1. Extract genomic DNA of Erwinia billingiae DSM 17872.

[0056] 2. Using the genomic DNA of Erwinia billinii DSM 17872 as a template and EbNLE-FP and EbNLE-RP as primers, PCR amplification was performed to obtain a fragment containing the nitrilase gene, wherein the nucleotide sequence of the nitrilase gene is shown in SEQ ID NO: 1, and the amino acid sequence of the nitrilase EbNLE encoded by it is shown in SEQ ID NO: 2.

[0057] EbNLE-FP: 5'-CGCGGATCCATGAACAAACCGGTTACCGTT-3' (SEQ ID NO: 3);

[0058] EbNLE-RP: 5'-CCGGAATTCCTACTCCTCCTCTTCTTGTCC-3' (SEQ ID NO: 4).

[0059] Example 2: Construction of nitrilase EbNLE expression strain

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

[0061] 2. The recombinant expression plasmid pET26b(+)-EbNLE obtained in step 1 was transferred into the expression host E. coli BL21(DE3) by chemical transformation, and LB solid medium containing 50 μg / mL kanamycin was applied for screening to obtain the recombinant bacteria E. coli BL21(DE3) / pET-26b(+)-EbNLE expressing nitrilase EbNLE.

[0062] Example 3: Obtaining the gene sequence of mutant EbNLE-R126A

[0063] The recombinant expression plasmid pET-26b(+)-EbNLE obtained in Example 2 was used as a template, and whole plasmid PCR was performed using EbNLE-R126A-FP and EbNLE-R126A-RP as primers to introduce site-directed mutations to obtain a mutant recombinant expression plasmid pET26b(+)-EbNLE-R126A, wherein the nucleotide sequence of the nitrilase mutant is shown in SEQ ID NO: 5, and the amino acid sequence of the nitrilase mutant EbNLE-R126A encoded thereby is shown in SEQ ID NO: 6.

[0064] EbNLE-R126A-FP: 5'-ACCATTGGAATCCGGGCG AAGCTTAAGGCTACG-3' (SEQ IDNO: 7);

[0065] EbNLE-R126A-RP: 5'-CGTAGCCTTAAGCTTCGCCCGGATTCCAATGGT-3' (SEQ ID NO: 8).

[0066] Example 4: Construction of mutant EbNLE-R126A expression strain

[0067] The mutant recombinant expression plasmid pET26b(+)-EbNLE-R126A obtained in Example 3 was transferred into the expression host E. coli BL21(DE3) by chemical transformation, and LB solid culture medium containing 50 μg / mL kanamycin was applied for screening to obtain the recombinant bacteria E. coli BL21(DE3) / pET-26b(+)-EbNLE-R126A expressing the mutant EbNLE-R126A.

[0068] Example 5: Preparation of enzyme

[0069] The recombinant bacteria E. coli BL21 (DE3) / pET-26b (+) -EbNLE obtained in Example 2 and the recombinant bacteria E. coli BL21 (DE3) / pET-26b (+) -EbNLE-R126A obtained in Example 4 were inoculated into LB test tube medium containing 50 μg / mL kanamycin, cultured at 37 ° C, 220 rpm for 12 hours, and then transferred to 100 mL LB shake flask medium containing 50 μg / mL kanamycin at an inoculum volume of 1% (v / v), and cultured at 37 ° C, 220 rpm until OD 600=0.6-0.8, add the inducer IPTG with a final concentration of 300μM, induce and culture at 16℃ for 20 hours, centrifuge the induced culture solution at 9000rpm for 10 minutes, collect the bacterial precipitate, wash with physiological saline to obtain resting cells. The resting cells are directly freeze-dried to obtain freeze-dried cells, or freeze-dried after ultrasonic disruption to obtain freeze-dried powder, and stored at 4℃.

[0070] Example 6: Enzymatic Preparation of 3-Hydroxy-3-Methylbutyric Acid

[0071] The original enzyme EbNLE and the mutant enzyme EbNLE-R126A prepared in Example 5 were used as biocatalysts in the preparation reaction of 3-hydroxy-3-methylbutyric acid. The reaction formula is as follows:

[0072]

[0073] 5 mL reaction system: 0.1 M substrate 3-hydroxy-3-methylbutyronitrile, 3.0 g lyophilized cells (equivalent to 60 g / g 3-hydroxy-3-methylbutyronitrile), 100 mM Tris-HCl buffer, stirred at 200 rpm at 37°C, pH 7.0 for 9.5 hours.

[0074] After the reaction was completed, 1 mL of the reaction solution was centrifuged to remove the biocatalyst, and the supernatant was analyzed by high performance liquid chromatography to detect the concentration of 3-hydroxy-3-methylbutyric acid. According to calculations, when the original enzyme EbNLE was used as the biocatalyst, the conversion rate of the substrate 3-hydroxy-3-methylbutyric acid (conversion rate = (initial amount of reactants - remaining amount of reactants) / initial amount of reactants × 100%) was 31%, and the production efficiency of the product 3-hydroxy-3-methylbutyric acid (production efficiency = the amount of product generated by the reaction / (reaction time * amount of catalyst)) was 9.3 g / L / d. In contrast, when the mutant enzyme EbNLE-R126A was used as the biocatalyst, the conversion rate of the substrate 3-hydroxy-3-methylbutyric acid reached 99%, and the production efficiency of the product 3-hydroxy-3-methylbutyric acid reached 29.5 g / L / d.

[0075] The HPLC detection method is:

[0076] Agilent high performance liquid chromatograph was used, with an RP18 Grace Alltima column (size 4.5 mm×250 mm); the mobile phase was 0.1 M phosphoric acid: acetonitrile = 9:1 (volume ratio); the flow rate was 1.0 mL / min; the detection wavelength was 229 nm; the temperature was 20°C; all samples were centrifuged at 10000 rpm and filtered through a 0.22 μm organic filter membrane, and the sample volume was 10 μL.

[0077] The elution time of the 3-hydroxy-3-methylbutyronitrile standard was 8.9 min, and the elution time of the 3-hydroxy-3-methylbutyric acid standard was 4.5 min.

[0078] The elution time of 3-hydroxy-3-methylbutyronitrile in the reaction solution is 8.9 min, and the elution time of 3-hydroxy-3-methylbutyric acid is 4.5 min.

[0079] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0080] sequence

[0081] SEQ ID NO:1

[0082] ATGAACAAACCGGTTACCGTTGCCTGCGTTCAGGCCGCCCCTGTTTTTATGAATCTTG

[0083] AAGGAACCATAGACAAAACGATCGCCCTCATATCAGAAGCCGCACAGAAAGGCGCGG

[0084] AGCTCATCGCTTTTCCTGAGACCTGGATACCCGGTTATCCGTGGTTCTTATGGCTTAAC

[0085] TCGCCCGCCATTAATATGCCCCTGGTTTATCAGTATCATCAGAACTCCCTCGTGCTGGA

[0086] CAGTGCTCAGGCGAAGCGAATTGCGGCTGCGGCGCAGCAGAATAATATCGTTGTGGTT

[0087] CTGGGCTTCAGCGAGCGCGATCATGGCAGCCTCTATATCTCACAATGGCTGATTGGAA

[0088] GCGACGGGGAGACCATTGGAATCCGGCGCAAGCTTAAGGCTACGCACGTGGAGCGTA

[0089] CGCTGTTCGGCGAAGGCGACGGTTCCTCCCTGACTACCTGGGAGACACCGTTGGGGA

[0090] ACGTTGGAGCCCTCTGCTGCTGGGAGCATCTGCAGCCACTGTCCCGTTATGCGATGTA

[0091] TTCCCAGCATGAGGAAATACATATCGCCGCCTGGCCCAGTTTCAGTCTTTACACCAGC

[0092] GCAACGGCTGCGCTGGGGCCCGAGGTCAACACGGCTGCTTCTCGCCTGTATGCCGCA

[0093] GAGGGACAATGCTTCGTGATAGCCCCGTGTGCCGTGGTTTCTGATGAGATGATTGATTT

[0094] CCTTTGTCCAGATGATGACCGTAGGGCGTTACTCAGTGCCGGGGGGGGACATGCCCGT

[0095] ATTTACGGCCCGGACGGAAGAGAACTCGTCACACCTCTCGGGGAGAATGAGGAAGG

[0096] ACTGCTTATCGCTGAGCTCGACTCTGCTGCGATTACTTTTGCCAAACTTGCGGCTGAC

[0097] CCGATAGGCCACTATTCACGTCCTGACGTGACCCGCCTCCTTTTTAATCCTTCATCCAA

[0098] CAAGACTGTGATTAAACGGTATTCGCCTCCAGAGCTAATTGCCGAACAGGCTGGACAA

[0099] GAAGAGGAGGAGTAG

[0100] SEQ ID NO:2

[0101] MNKPVTVACVQAAPVFMNLEGTIDKTIALISEAAQKGAELIAFPETWIPGYPWFLWLNSP

[0102] AINMPLVYQYHQNSLVLDSAQAKRIAAAAQQNNIVVVLGFSERDHGSLYISQWLIGSDGE

[0103] TIGIRRKLKATHVERTLFGEGDGGSSLTTWETPLGNVGALCCWEHLQPLSRYAMYSQHEEI

[0104] HIAAWPSFSLYTSATAALGPEVNTAASRLYAAEGQCFVIAPCAVVSDEMIDFLCPDDDRRA

[0105] LLSAGGHARIYGPDGRELVTPLGENEEGLLIAELDSAAITFAKLAADPIGHYSRPDVTRL

[0106] LFNPSSNKTVIKRYSPPELIAEQAGQEEEE

[0107] SEQ ID NO:5

[0108] ATGAACAAACCGGTTACCGTTGCCTGCGTTCAGGCCGCCCCTGTTTTATGAATCTTG

[0109] AAGGAACCATAGACAAAACGATCGCCCTATATCAGAAGCCGCACAGAAAGGCGCG

[0110] AGCTCATCGCTTTTCCTGAGACCTGGATACCCGGTTATCCGTGGTTCTTATGGCTTAAC

[0111] TCGCCCGCCATTAATATGCCCCTGGTTTATCAGTATCATCAGAACTCCCTCGTGCTGGA

[0112] CAGTGCTCAGGCGAAGCGAATTGCGGCTGCGGCGCAGCAGAATAATCGTTGTGGTT

[0113] CTGGGCTTCAGCGAGCGCGATCATGGCAGCCTCTATATCTCACAATGGCTGATTGGAA

[0114] GCGACGGGGAGACCATTGGAATCCGGGCGAAGCTTAAGGCTACGCACGTGGAGCGTA

[0115] CGCTGTTCGGCGAAGGCGACGGTTCCTCCCTGACTACCTGGGAGACACCGTTGGGGA

[0116] ACGTTGGAGCCCTCTGCTGCTGGGAGCATCTGCAGCCACTGTCCCGTTATGCGATGTA

[0117] TTCCCAGCATGAGGAAATACATATCGCCGCCTGGCCCAGTTTCAGTCTTTACACCAGC

[0118] GCAACGGCTGCGCTGGGGCCCGAGGTCAACACGGCTGCTTCTCGCCTGTATGCCGCA

[0119] GAGGGACAATGCTTCGTGATAGCCCCGTGTGCCGTGGTTTCTGATGAGATGATTGATTT

[0120] CCTTTGTCCAGATGATGACCGTAGGGCGTTACTCAGTGCCGGGGGGGGACATGCCCGT

[0121] ATTTACGGCCCGGACGGAAGAGAACTCGTCACACCTCTCGGGGAGAATGAGGAAGG

[0122] ACTGCTTATCGCTGAGCTCGACTCTGCTGCGATTACTTTTGCCAAACTTGCGGCTGAC

[0123] CCGATAGGCCACTATTCACGTCCTGACGTGACCCGCCTCCTTTTTAATCCTTCATCCAA

[0124] CAAGACTGTGATTAAACGGTATTCGCCTCCAGAGCTAATTGCCGAACAGGCTGGACAA

[0125] GAAGAGGAGGAGTAG

[0126] SEQ ID NO:6

[0127] MNKPVTVACVQAAPVFMNLEGTIDKTIALISEAAQKGAELIAFPETWIPGYPWFLWLNSP

[0128] AINMPLVYQYHQNSLVLDSAQCRIAAAAQQNNIVVVLGFSERDHGSLYISQWLIGSDGE

[0129] TIGIRAKLKATHVERTLFGEGDGSSLTTWETPLGNVGALCCWEHLQPLSRYAMYSQHEEI

[0130] HIAAWPSFSLYTSATAALGPEVNTAASRLYAAEGQCFVIAPCAVVSDEMIDFLCPDDDRRA

[0131] LLSAGGGHARIYGPDGRELVTPLGENEEGLLIAELDSAAITFAKLAADPIGHYSRPDVTRL

[0132] LFNPSSNKTVCROSSPELIAEQAGQEEEE

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, wherein the nucleotide sequence is shown in SEQ ID NO:

5. 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 of animal or plant variety.

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

6. Use of 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 mutant of nitrilase prepared by the preparation method according to claim 5 in the preparation of 3-hydroxy-3-methylbutyric acid.

7. A method for preparing 3-hydroxy-3-methylbutyric acid, comprising the following steps: The mutant according to claim 1, the recombinant cell according to claim 4 and / or the mutant of the nitrilase prepared by the preparation method according to claim 5 is used as a catalyst to catalyze the hydrolysis reaction of 3-hydroxy-3-methylbutyronitrile to prepare 3-hydroxy-3-methylbutyric acid.

8. The preparation method according to claim 7, characterized in that: The temperature of the hydrolysis reaction is 30-42° C., the pH is 6.5-7.5, and the final concentration of the substrate 3-hydroxy-3-methylbutyronitrile is 0.01-1M.

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