Nitrilase mutant, engineered bacteria and its application in synthesis of (r)-3-cyanohexanoic acid

Abstract

This invention discloses a nitrile hydrolase mutant, an engineered bacterium, and its application in the synthesis of (R)-3-cyanohexanoic acid. The nitrile hydrolase mutant is obtained by single or multiple mutations at positions 135, 199, and 59 of the amino acid sequence shown in SEQ ID NO.2. This invention utilizes directed evolution technology to enhance the activity and stereoselectivity of the nitrile hydrolase by constructing a nitrile hydrolase mutant. Compared to the parental nitrile hydrolase PgNITmut, the enantiomeric selectivity (E value) of the mutant PgNITmut / F135L for the substrate 3-cyanohexanonilonitrile is increased from 332 to 419; the activity of the mutant PgNITmut / F135L / R199W is increased by 1.36 times, and the E value is increased to 461; the activity of the mutant PgNITmut / F135L / R199W / T59D is increased by 2.05 times, and the E value is increased to 569. The nitrile hydrolase mutant of this invention has high activity and stereoselectivity. It can be used to synthesize the chiral intermediate (R)-3-cyanohexanoic acid of buvasidan by a highly efficient nitrile hydrolase method, which greatly reduces the cost of industrial production and has good application prospects in the industrial production of buvasidan.

Description

(I) Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to nitrile hydrolase mutants, encoding genes, engineered bacteria, and their application in the synthesis of buvasidan key chiral intermediate (R)-3-cyanohexanoic acid from racemic 3-cyanohexonitrile. (II) Background Technology

[0002] Brivaracetam is a next-generation antiepileptic drug developed by UCB (Universal Pharma) of Belgium, and was first approved for marketing by the U.S. Food and Drug Administration (FDA) in 2016. Brivaracetam binds to central synaptic vesicle protein 2A (SV2A) and simultaneously reduces the release of excitatory neurotransmitters by inhibiting voltage-dependent sodium ion channels, thereby achieving an antiepileptic effect. As a structural analogue of levetiracetam, brivaracetam has a 15-30 times greater affinity for SV2A, thus offering advantages such as lower dosage and fewer side effects. Furthermore, it is the only intravenous formulation approved by the FDA in the past seven years for the treatment of partial-onset epilepsy in pediatric patients aged one month and older, indicating a very promising market prospect.

[0003] In recent years, efficient and green synthetic methods for buvascarbamazepine have become a research hotspot in pharmaceutical engineering. (R)-3-aminomethylhexanoic acid is a key chiral intermediate in the synthesis of buvascarbamazepine, and currently there are two main synthetic methods: Patent CN113045468A reports a method that uses chiral phenylethylamine to resolve 3-acetylhexanoic acid, followed by Hoffmann degradation to synthesize (R)-3-aminomethylhexanoic acid; Patent WO2016075082 reports a route that uses n-pentanal as a raw material, reacts with diisobutylamine and bromoacetate to synthesize 3-aldehyde hexanoate ester, and then synthesizes (R)-3-aminomethylhexanoic acid through reductive amination. However, the above methods suffer from drawbacks such as complex process steps, low yield, and low optical purity of the product.

[0004] Nitrile hydrolases are important industrial catalysts. Their mediated biocatalytic processes offer advantages such as mild reaction conditions, high catalytic efficiency, strict selectivity, and environmental friendliness, making them a crucial method for the bio-organic synthesis of chiral carboxylic acids. For example, nitrile hydrolases can stereoselectively hydrolyze and synthesize chiral drug intermediates such as (S)-3-cyano-5-methylhexanoic acid and (R)-o-chloromandelic acid (US10100297B2; ZL201810765047.5; ZL201610035695.6). The regio- and stereoselective hydrolysis of racemic 3-cyanohexanilide by nitrile hydrolases to synthesize (R)-3-cyanohexanoic acid, followed by hydrogenation to prepare the key chiral intermediate (R)-3-aminomethylhexanoic acid for buvascarbamazine, demonstrates significant advantages such as high process efficiency and atom economy, showing promising industrial application prospects (CN202210350130.2). (III) Summary of the Invention

[0005] The purpose of this invention is to provide a nitrile hydrolase mutant, gene, engineered bacteria, and its application in the synthesis of (R)-3-cyanohexanoic acid. This invention establishes an efficient synthetic process for (R)-3-cyanohexanoic acid nitrile hydrolase by constructing a nitrile hydrolase mutant with further improved catalytic activity and stereoselectivity, laying the foundation for the industrial production of buvasidan by nitrile hydrolase and meeting the demand for key chiral intermediates in the industrial synthesis of buvasidan.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a nitrile hydrolase mutant with high catalytic activity and stereoselectivity. The nitrile hydrolase mutant is obtained by single or multiple mutations at positions 135, 199, and 59 of the amino acid sequence shown in SEQ ID NO.2. The amino acid sequence shown in SEQ ID NO.2 is a nitrile hydrolase mutant PgNit / W140G / M175T (hereinafter referred to as PgNITmut) derived from *Paraburkholderia graminis*, with the nucleotide sequence shown in SEQ ID NO.1. By performing site-directed mutagenesis on PgNITmut, a nitrile hydrolase mutant with simultaneously improved catalytic activity and stereoselectivity is obtained.

[0008] Preferably, the nitrile hydrolase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.2 to one of the following: (1) phenylalanine at position 135 is mutated to leucine (PgNITmut / F135L, amino acid sequence as shown in SEQ ID NO.4, nucleotide sequence as shown in SEQ ID NO.3); (2) phenylalanine at position 135 is mutated to leucine, and arginine at position 199 is mutated to tryptophan (PgNITmut / F135L / R199W, amino acid sequence as shown in SEQ ID NO.6, nucleotide sequence as shown in SEQ ID NO.5); (3) phenylalanine at position 135 is mutated to leucine, arginine at position 199 is mutated to tryptophan, and threonine at position 59 is mutated to aspartic acid (PgNITmut / F135L / R199W / T59D, amino acid sequence as shown in SEQ ID NO.8, nucleotide sequence as shown in SEQ ID NO.7).

[0009] Conservative substitutions, additions or deletions of one or more amino acids, amino-terminal truncation, and carboxyl-terminal truncation of the above-mentioned nitrile hydrolase mutants are also included within the scope of this invention.

[0010] The present invention also provides the encoding gene of the above-mentioned nitrile hydrolase mutant, the nucleotide sequence of which is shown in SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7.

[0011] The present invention also relates to a recombinant vector containing the coding gene of the nitrile hydrolase mutant and a recombinant genetically engineered bacterium containing the recombinant vector; the original vector of the recombinant vector is pET28b. The recombinant vector is transformed into host cells to obtain recombinant genetically engineered bacteria. The host cells can be various conventional host cells in the art, preferably Escherichia coli BL21.

[0012] The present invention also provides a method for preparing the nitrile hydrolase mutant, the method comprising the following steps:

[0013] (1) Design site-directed mutagenesis primers, use the recombinant plasmid carrying the gene fragment with the nucleotide sequence shown in SEQ ID NO.1 as a template, perform overlap extension PCR to obtain the mutant product with F mutated to L at position 135 in the amino acid sequence of the parent nitrile hydrolase.

[0014] (2) Using the mutant product carrying the PgNITmut / F135L gene obtained in step (1) as a template, overlap extension PCR was performed to obtain the mutant product with the 199th R mutated to W.

[0015] (3) Using the mutant product carrying the PgNITmut / F135L / R199W gene obtained in step (2) as a template, overlap extension PCR was performed to obtain the mutant product with the 59th T mutation changed to D.

[0016] (4) The mutant products obtained in steps (1), (2) and (3) are transformed into host bacteria respectively, and strains expressing nitrile hydrolase mutants are screened. The strains with correct sequencing results are the target strains. The nitrile hydrolase mutants PgNITmut / F135L, PgNITmut / F135L / R199W and PgNITmut / F135L / R199W / T59D are obtained by induction expression.

[0017] This invention also provides the application of the nitrile hydrolase mutant in the catalytic preparation of (R)-3-cyanohexonitrile from racemic 3-cyanohexonitrile. The method of application is as follows: using wet bacterial cells obtained by fermentation and centrifugation of engineered bacteria containing the nitrile hydrolase mutant encoding gene as a catalyst, using racemic 3-cyanohexonitrile as a substrate, and using a buffer solution with a pH of 5-10 as a reaction medium to form a reaction system, after the water bath reaction is completed at 20-55℃ and 200-400rpm, the reaction solution is separated and purified to obtain (R)-3-cyanohexonitrile.

[0018] The nitrile hydrolase mutant described in this invention can be used in whole-cell engineered bacteria, as unpurified crude enzyme, or as partially or fully purified enzyme. Furthermore, the nitrile hydrolase mutant of this invention can be prepared into immobilized enzymes or immobilized cell-based biocatalysts using immobilization techniques known in the art.

[0019] Preferably, the substrate concentration in the reaction system is 100-200 g / L (preferably 100 g / L), and the amount of catalyst used is 5-20 g / L (preferably 10 g / L) based on the weight of wet bacterial cells.

[0020] Preferably, the reaction medium is a phosphate buffer solution with a pH of 7.4, and the catalytic reaction temperature is 30°C.

[0021] The culture medium used for the engineered bacteria described in this invention can be any culture medium in the art that enables the engineered bacteria to grow and produce the nitrile hydrolase of this invention, preferably LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as solvent, pH 7.0. There are no special requirements for the culture method and conditions, as long as the engineered bacteria can grow and produce the nitrile hydrolase.

[0022] Preferably, the wet bacterial cells are prepared according to the following method:

[0023] Engineered bacteria containing the gene encoding the nitrile hydrolase mutant were inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 h to obtain a seed culture. The seed culture was then inoculated at a volume concentration of 2% (v / v) into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C until the bacterial cell concentration reached OD500. 600 Once the concentration reaches 0.5–0.7, add isopropyl-β-D-thiopyranogalactopyranoside (IPTG) to the culture medium to a final concentration of 0.1 mM, induce culture at 28°C for 12 h, centrifuge at 4°C and 12,000 rpm for 10 min, and collect the wet cells.

[0024] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0025] This invention utilizes directed evolution technology to enhance the activity and stereoselectivity of nitrile hydrolases by constructing nitrile hydrolases mutants. Compared to the parental nitrile hydrolase PgNITmut, the enantioselectivity (E value) of the mutant PgNITmut / F135L for the substrate 3-cyanohexonitrile increased from 332 to 419; the activity of the mutant PgNITmut / F135L / R199W increased by 1.36 times, and the E value increased to 461; the activity of the mutant PgNITmut / F135L / R199W / T59D increased by 2.05 times, and the E value increased to 569.

[0026] The nitrile hydrolase mutants of this invention exhibit high activity and stereoselectivity, significantly improving substrate conversion and product ee value. After 24 h of reaction with 100 g / L 3-cyanohexonitrile catalyzed by PgNITmut, the substrate conversion was 44.98% and the product ee value was 98.51%. After 24 h of reaction with PgNITmut / F135L, the substrate conversion was 45.38% and the product ee value was 98.78%. After 20 h of reaction with PgNITmut / F135L / R199W, the substrate conversion was 45.37% and the product ee value was 98.89%. After 16 h of reaction with PgNit / F135L / R199W / T59D, the substrate conversion was 44.82% and the product ee value was 99.12%.

[0027] The nitrile hydrolase mutant of this invention has high activity and stereoselectivity. It can be used to synthesize the chiral intermediate (R)-3-cyanohexanoic acid of buvasidan by a highly efficient nitrile hydrolase method, which greatly reduces the cost of industrial production and has good application prospects in the industrial production of buvasidan. (iv) Description of the attached drawings

[0028] Figure 1 The reaction process of catalyzing the hydrolysis of 3-cyanohexonitrile (100 g / L) by recombinant Escherichia coli resting cells (10 g / L) containing nitrile hydrolase mutants PgNITmut, PgNITmut / F135L, PgNITmut / F135L / R199W, and PgNITmut / F135L / R199W / T59D.

[0029] Figure 2 The reaction process of 3-cyanohexonitrile (100 g / L) hydrolysis catalyzed by recombinant Escherichia coli (containing nitrile hydrolase mutant PgNITmut / F135L / R199W / T59D) at different cell concentrations was described. (V) Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0031] The parental nitrile hydrolase used in the specific embodiment is a nitrile hydrolase mutant PgNit / W140G / M175T (hereinafter referred to as PgNITmut) derived from Paraburkholderia graminis. Its amino acid sequence is shown in SEQ ID NO.2 and its nucleotide sequence is shown in SEQ ID NO.1. It has been disclosed in CN202210350130.2.

[0032] Example 1: Construction of the nitrile hydrolase mutant PgNITmut / F135L and recombinant Escherichia coli

[0033] Using the nitrile hydrolase mutant PgNit / W140G / M175T (hereinafter referred to as PgNITmut) from Paraburkholderia graminis as the parent, with its amino acid sequence as shown in SEQ ID NO.2 and its nucleotide sequence as shown in SEQ ID NO.1, corresponding primers were designed to perform site-directed mutagenesis on it.

[0034] SEQ ID NO.1

[0035]

[0036] SEQ ID NO.2

[0037] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYFAAVQTGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGGTLYNTQLLFDADGTLIQRRRKITPTHFERMIGGQGDGSGLRAVDSAVGRIGQLACFEHNNPL ARYATIADGEQIHSAMYPGSAFGEGFAQRMEINIRQHALESGAFVVNATAWLDADQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQIDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI.

[0038] Using the recombinant plasmid pET28b-PgNITmut containing the target gene fragment as a template, and using F135L-Forward and F135L-Reverse primers in Table 1 as primers, the template was amplified by overlapping extension PCR, and the Phe at position 135 in the parental amino acid sequence was mutated at a specific site.

[0039] Table 1 Primers

[0040]

[0041] The PCR amplification system was as follows (50 μL): template DNA 0.5 ng, 2×Phanta Max Buffer 25 μL, dNTPs (10 mM each) 1 μL, upstream and downstream of the mutant primers 1 μL each, Phanta Max Super-Fidelity DNA Polymerase 1 U, and the remainder ddH2O to be added to the total volume.

[0042] PCR reaction parameters: (1) 95℃ pre-denaturation for 30s; (2) 95℃ denaturation for 30s; (3) 65℃ annealing for 30s; (4) 72℃ extension for 6min, repeat steps (2)-(4) 32 times; (5) 72℃ extension for 7min, store at 16℃.

[0043] After the PCR product showed a positive result by 0.9% agarose gel electrophoresis, 20 μL of the PCR reaction solution was taken, and 1 μL of the restriction enzyme Dpn I was added for digestion at 37℃ for 3 h to remove the template plasmid DNA. The DNA was then inactivated at 65℃ for 10 min. The cells were heat-shocked into E. coli BL21(DE3) competent cells, and after recovery, they were plated on LB agar plates containing 50 μg / mL kanamycin and cultured overnight. Each plate yielded a mutant library of approximately 300 clones.

[0044] Subsequently, 4-5 clones were selected and cultured in LB medium at 37°C for 8 hours. The bacterial culture was then sequenced to obtain the nitrile hydrolysate recombinant engineered bacteria E. coli BL21(DE3) / pET28b-PgNITmut / F135L, whose amino acid sequence is shown in SEQ ID NO.4 and nucleotide sequence is shown in SEQ ID NO.3.

[0045] Example 2: Construction of the nitrile hydrolase mutant PgNITmut / F135L / R199W and recombinant Escherichia coli

[0046] To construct the nitrile hydrolase double mutant PgNITmut / F135L / R199W, corresponding primers were designed, and the primer sequences are shown in Table 1.

[0047] Using the recombinant plasmid pET28b-PgNITmut / F135L constructed in Example 1 as a template, and employing primers R199W-Forward and R199W-Reverse from Table 1, the recombinant engineered bacterium E. coli BL21(DE3) / pET28b-PgNITmut / F135L / R199W was obtained according to Example 1. Its amino acid sequence is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.5.

[0048] Example 3: Construction of the nitrile hydrolase mutant PgNITmut / F135L / R199W / T59D and recombinant Escherichia coli

[0049] To construct the nitrile hydrolase double mutant PgNITmut / F135L / R199W / T59D, corresponding primers were designed, and the primer sequences are shown in Table 1.

[0050] Using the recombinant plasmid pET28b-PgNITmut / F135L / R199W constructed in Example 2 as a template, and employing primers T59D-Forward and T59D-Reverse from Table 1, the recombinant engineered bacteria E. coli BL21(DE3) / pET28b-PgNITmut / F135L / R199W / T59D was obtained according to Example 1. Its amino acid sequence is shown in SEQ ID NO.8, and its nucleotide sequence is shown in SEQ ID NO.7.

[0051] Example 4: Induced expression of recombinant Escherichia coli containing nitrile hydrolase mutant

[0052] Recombinant *E. coli* containing the parental nitrile hydrolase *E. coli* BL21(DE3) / pET28b-PgNITmut, as well as the mutants *E. coli* BL21(DE3) / pET28b-PgNITmut / F135L, *E. coli* BL21(DE3) / pET28b-PgNITmut / F135L / R199W, and *E. coli* BL21(DE3) / pET28b-PgNITmut / F135L / R199W / T59D obtained in Examples 1, 2, and 3, were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. Then, at a 2% (v / v) inoculation rate, the bacteria were inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 150 rpm until the bacterial concentration reached OD500. 600 =0.6, add IPTG to a final concentration of 0.1mM, induce culture at 28℃ for 12h, centrifuge at 4℃ and 12000rpm for 10min to collect wet cells, wash the wet cells with 0.85% physiological saline, and store at -20℃ for later use (i.e., resting cells, used for hydrolysis reaction).

[0053] Example 5: Determination of the activity and stereoselectivity of recombinant Escherichia coli containing nitrile hydrolase mutants

[0054] Using wet cells obtained from the culture of recombinant *E. coli* containing nitrile hydrolases PgNITmut, PgNITmut / F135L, PgNITmut / F135L / R199W, and PgNITmut / F135L / R199W / T59D prepared in Example 4 as a catalyst, the hydrolysis of 3-cyanohexanoic acid was carried out at 30°C. The reaction system was as follows: 10 mL phosphate buffer (100 mM, pH 7.4), 1 g 3-cyanohexanoic acid, and 0.1 g wet cells. The reaction was carried out at 30°C for 15 min, and 200 μL of the sample was taken. The reaction was terminated by adding 30 μL of 6M HCl and extracted with ethyl acetate. 200 μL of the upper organic phase was dried with anhydrous sodium sulfate, and 7.5 μL of methanol and 3.75 μL of diazomethane were added. The conversion rate of the substrate and the enantiomeric excess (ee) of the product (R)-3-cyanohexanoic acid were determined by gas chromatography.

[0055] The gas chromatograph was a TRACE 1300 (Thermo Fisher Scientific), and the capillary column was a BGB 175 (BGB Analytik Switzerland). Chromatographic conditions were as follows: injection volume 2.0 μL, injector and detector temperatures both 250 °C, column temperature 120 °C held for 20 min, then ramped to 200 °C at a rate of 10 °C / min and held for 5 min. The carrier gas was high-purity helium, with a flow rate of 1.0 mL / min and a split ratio of 30:1.

[0056] The calculation of enantiomeric excess (ee) and conversion rate (c) refers to the calculation method of Rakels et al. (Enzyme Microb. Technol., 1993, 15: 1051-1056).

[0057] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of product (R)-3-cyanohexanoic acid under reaction conditions at 30°C.

[0058] The results are shown in Table 2. The catalytic activity of the nitrile hydrolase mutant PgNit / F135L was 1.03 times that of the parent, with E = 419; while the catalytic activity of the double mutant PgNit / F135L / R199W was 1.36 times that of the parent, with E = 461; and the catalytic activity of the triple mutant PgNit / F135L / R199W / T59D was 2.06 times that of the parent, with E = 569.

[0059] Table 2. Comparison of nitrile hydrolases activity and stereoselectivity

[0060]

[0061] Example 6: Recombinant Escherichia coli containing a nitrile hydrolase mutant catalyzes the synthesis of (R)-3-cyanohexanoic acid from 3-cyanohexonitrile (I)

[0062] Using 0.1g of wet bacterial cells containing nitrile hydrolases PgNITmut, PgNITmut / F135L, PgNITmut / F135L / R199W and PgNit / F135L / R199W / T59D prepared in Example 4 as catalysts, 1g of 3-cyanohexonitrile as substrate, and 10mL of phosphate buffer (100mM, pH 7.4) as reaction medium, the reaction was carried out at 30°C for 16-24h. Samples were taken, and the substrate conversion rate and product ee value were determined using the method in Example 5.

[0063] The results are as follows Figure 1 As shown, after 24 h of reaction with 100 g / L 3-cyanohexonitrile catalyzed by PgNITmut, the substrate conversion rate was 44.98%, and the product ee value was 98.51%; after 24 h of reaction with PgNITmut / F135L, the substrate conversion rate was 45.38%, and the product ee value was 98.78%; after 20 h of reaction with PgNITmut / F135L / R199W catalyzed by PgNITmut, the substrate conversion rate was 45.37%, and the product ee value was 98.89%; after 16 h of reaction with PgNit / F135L / R199W / T59D catalyzed by PgNit, the substrate conversion rate was 44.82%, and the product ee value was 99.12%.

[0064] The above reaction was terminated, and the E. coli cells were removed by centrifugation. An equal volume of dichloromethane was added for extraction, and the lower organic phase was collected. The organic phase was then subjected to rotary evaporation under reduced pressure to obtain the unreacted residual substrate. The upper aqueous phase was filtered to obtain a clear liquid, which is the product (R)-3-cyanohexanoic acid aqueous solution.

[0065] Example 7: Recombinant Escherichia coli containing a nitrile hydrolase mutant catalyzes the synthesis of (R)-3-cyanohexanoic acid from 3-cyanohexonitrile (II)

[0066] Wet bacterial cells containing nitrile hydrolase PgNit / F135L / R199W / T59D, prepared by the method of Example 4, were used as catalysts (catalyst concentrations were 5 g / L, 10 g / L, and 20 g / L based on the volume of the reaction medium, respectively). 1 g of 3-cyanohexonitrile was used as the substrate, and 10 mL of phosphate buffer (100 mM, pH 7.4) was used as the reaction medium. Hydrolysis was carried out at 30 °C for 10–24 h. Samples were taken, and the substrate conversion rate and product ee value were determined by the method of Example 5.

[0067] The results are as follows Figure 2As shown. With 5 g / L nitrile hydrolase mutant PgNit / F135L / R199W / T59D catalyzing the reaction for 24 h, the conversion rate was 43.1%, and the product ee value was 99.24%; with 10 g / L nitrile hydrolase mutant PgNit / F135L / R199W / T59D catalyzing the reaction for 16 h, the conversion rate was 44.82%, and the product ee value was 99.12%; with 20 g / L nitrile hydrolase mutant PgNit / F135L / R199W / T59D catalyzing the reaction for 10 h, the conversion rate was 44.50%, and the product ee value was 99.08%.

[0068] This invention is not limited to the specific textual description above. Various changes can be made to this invention within the scope outlined in the claims, and all such changes are within the scope of this invention.

Claims

1. A nitrile hydrolase mutant with high catalytic activity and stereoselectivity, characterized in that, The nitrile hydrolase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.2 to one of the following: (1) phenylalanine at position 135 is mutated to leucine, and arginine at position 199 is mutated to tryptophan; (2) phenylalanine at position 135 is mutated to leucine, arginine at position 199 is mutated to tryptophan, and threonine at position 59 is mutated to aspartic acid.

2. The encoding gene of the nitrile hydrolase mutant of claim 1.

3. A recombinant genetically engineered bacterium containing the encoding gene of claim 2.

4. A method for preparing racemic 3-cyanohexonitrile catalytically via the nitrile hydrolase mutant of claim 1 (… R Applications of 3-cyanohexanoic acid.

5. The application as described in claim 4, characterized in that, The method of application is as follows: Using engineered bacteria containing a nitrile hydrolase mutant encoding gene, wet bacterial cells obtained by fermentation and centrifugation are used as a catalyst; racemic 3-cyanohexonitrile is used as a substrate; and a buffer solution with a pH of 5-10 is used as the reaction medium to construct the reaction system. After the reaction is completed in a water bath at 20-55 ℃ and 200-400 rpm, the reaction solution is separated and purified to obtain (…). R )-3-Cyanohexanoic acid.

6. The application as described in claim 5, characterized in that, The substrate concentration in the reaction system is 100-200 g / L, and the amount of catalyst used is 5-20 g / L based on the weight of wet bacterial cells.

7. The application as described in claim 5, characterized in that, The wet bacterial cells were prepared according to the following method: Engineered bacteria containing the gene encoding the nitrile hydrolase mutant were inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37 ℃ for 12 h to obtain a seed culture. The seed culture was then inoculated at a volume concentration of 2% into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37 ℃ until the bacterial cell concentration reached OD0.

05. 600 Once the concentration reaches 0.5-0.7, add 0.1 mM isopropyl-β-D-thiopyranogalactopyranoside to the culture medium, induce culture at 28 ℃ for 12 h, centrifuge at 4 ℃ and 12000 rpm for 10 min, and collect the wet cells.

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