Nitrilase mutants and their use in the synthesis of a chiral intermediate for brivaracetam

Abstract

The application discloses a nitrilase mutant and application thereof in synthesis of a chiral intermediate of brivaracetam, and establishes a high-efficiency (R)-3-cyanohexanoic acid nitrilase synthesis process by constructing a nitrilase mutant with improved catalytic activity and stereoselectivity, so that the demand for industrial synthesis of a key chiral intermediate of brivaracetam is met, and a foundation is laid for industrial production of brivaracetam synthesized by a chemical-enzyme method.

Description

(1) Technical field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a nitrilase mutant, an encoding gene, an engineered bacterium, and applications thereof in hydrolyzing racemic 3-cyanohexanenitrile to synthesize (R)-3-cyanohexanoic acid, a key chiral intermediate of brivaracetam. (2) Background technology

[0002] Brivaracetam is a new anti-epileptic drug developed by UCB and approved for marketing in 2016. Its pharmacological action primarily involves binding to synaptic vesicle protein 2A (SV2A), thereby regulating neurotransmitter release and reducing neuronal excitability, resulting in an anti-epileptic effect. Compared with traditional anti-epileptic drugs such as levetiracetam and gabapentin, brivaracetam offers significant advantages, including higher affinity, lower dosage, and fewer side effects. It is widely used to treat partial-onset epileptic seizures in adults and children. Since its launch, sales of brivaracetam have continued to climb, demonstrating its enormous market potential.

[0003] At present, the main method for synthesizing Bu Waxitan is still based on chemical methods. Among them, patent CN107216276A uses (R)-4-propyldihydro-2(3H)-one as a raw material, performs a ring-opening reaction in an anhydrous organic solvent, and uses a chlorination reagent to form an intermediate for a halogenation reaction, and further synthesizes Bu Waxitan through amidation and ring-closure reactions. Patent CN106432030B uses a β-lactone compound as a raw material, and then uses the Grignard reagent EtMgBr for directional ring opening and then removes the ester group to obtain the key intermediate (R)-4-propyldihydrofuran-2(3H)-one. The intermediate product obtained by ring opening and decyclization is condensed with (S)-2-aminobutanamide and then amidated to synthesize the product Bu Waxitan. However, the above-mentioned synthetic routes all face the defects of complex operation, low product optical purity, and difficulty in separation and purification, making it difficult to achieve industrial application.

[0004] Nitrilases are important industrial enzyme catalysts for the synthesis of chiral carboxylic acids, catalyzing the hydrolysis of various nitrile compounds to produce the corresponding carboxylic acids. Using nitrilase to stereoselectively catalyze the synthesis of the key chiral intermediate (R)-3-cyanohexanoic acid from 3-cyanohexanenitrile, followed by hydrogenation, cyclization, and chiral resolution, is an ideal route for the industrial synthesis of brivaracetam (CN202210350130.2). Therefore, the development of highly stereoselective and catalytically active nitrilase catalysts to achieve efficient synthesis of (R)-3-cyanohexanoic acid is of great significance for the enzymatic synthesis of brivaracetam. (3) Summary of the invention

[0005] The application aims to provide a nitrilase mutant with high catalytic activity and stereoselectivity and its application in synthesis of a chiral intermediate (R)-3-cyanohexanoic acid of brivaracetam, and establishes a high-efficiency (R)-3-cyanohexanoic acid nitrilase synthesis process by constructing a nitrilase mutant with improved catalytic activity and stereoselectivity, so as to meet the demand of industrial synthesis of a key chiral intermediate of brivaracetam, and lay a foundation for industrial production of chemical-enzyme synthesis of brivaracetam.

[0006] The technical scheme adopted by the application is as follows:

[0007] The application provides a nitrilase mutant with high catalytic activity and stereoselectivity, which is obtained by mutating the 52th, 62th and 107th positions of the amino acid sequence shown in SEQ ID NO. 2. M0 The nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2. M0 The nitrilase mutant with improved catalytic activity and stereoselectivity is obtained by site-directed mutagenesis on Pg NIT

[0008] Preferably, the nitrilase mutant is mutated from the amino acid sequence shown in SEQ ID NO. 2 to one of the following:

[0009] (1) the 52th proline is mutated to tryptophan; (Pg NIT M0 / P52W, the amino acid sequence is shown in SEQ ID NO. 4, and the nucleotide sequence is shown in SEQ ID NO. 3); (2) the 52th proline is mutated to tryptophan, and the 62th glutamic acid is mutated to leucine; (Pg NIT M0 / P52W / E62L, the amino acid sequence is shown in SEQ ID NO. 6, and the nucleotide sequence is shown in SEQ ID NO. 5); (3) the 52th proline is mutated to tryptophan, the 62th glutamic acid is mutated to leucine, and the 107th glycine is mutated to methionine; (Pg NIT M0 / P52W / E62L / G107M, the amino acid sequence is shown in SEQ ID NO. 8, and the nucleotide sequence is shown in SEQ ID NO. 7). Conservative substitutions at other amino acid positions, additions or deletions of one or more amino acids, amino-terminal truncation, and carboxyl-terminal truncation of the above-mentioned nitrilase mutants are also included in the scope of the present invention.

[0010] The present invention also provides a gene encoding the nitrilase mutant. The nucleotide sequence of the gene encoding the mutant 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 comprising a gene encoding the nitrilase mutant and a recombinant genetically engineered bacterium comprising the recombinant vector; the original vector of the recombinant vector is pET-28b(+). The recombinant vector is used to transform a host cell to obtain a recombinant genetically engineered bacterium; the host cell can be any conventional host cell in the art, preferably Escherichia coli BL21.

[0012] The present invention also provides an application of the nitrilase mutant in catalyzing the preparation of (R)-3-cyanohexanoic acid, a chiral intermediate of brivaracetam, from racemic 3-cyanohexanenitrile. The application method comprises the following steps: using wet bacteria obtained by fermentation and culturing an engineered bacterium containing a gene encoding the nitrilase mutant as a catalyst, racemic 3-cyanohexanenitrile as a substrate, and a buffer solution with a pH of 7 to 8 as a reaction medium to form a reaction system; after completion of the water bath reaction at 20 to 40° C. and 300 to 600 rpm, separating and purifying the reaction solution to obtain (R)-3-cyanohexanoic acid.

[0013] The nitrilase mutant of the present invention can be used in the form of whole cells of an engineered bacterium, in the form of an unpurified crude enzyme, or in the form of a partially purified or completely purified enzyme. The nitrilase mutant of the present invention can also be prepared into a biocatalyst in the form of an immobilized enzyme or immobilized cells using immobilization techniques known in the art.

[0014] Preferably, the substrate concentration in the reaction system is 100-200 g / L (preferably 100 g / L), and the amount of the catalyst used is 5-25 g / L (preferably 10 g / L) based on the weight of the wet cells.

[0015] Preferably, the reaction medium is a 100 mM phosphate buffer solution with a pH value of 8, and the catalytic reaction conditions are: a temperature of 30° C. and a rotation speed of 350 rpm.

[0016] The culture medium used for the engineered bacteria of the present invention can be any culture medium known in the art that can grow the engineered bacteria and produce the nitrilase of the present invention, preferably LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as the solvent, pH 7.5. There are no particular requirements for the culture method or conditions, as long as the engineered bacteria can grow and produce the nitrilase.

[0017] Preferably, the wet cells are prepared as follows:

[0018] The engineered bacteria containing the nitrilase mutant encoding gene were inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 8-12 hours to obtain seed liquid; the seed liquid was then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 2% (v / v), and cultured at 37°C and 180 rpm until the bacterial concentration OD 600 When the pH value reaches 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium at a final concentration of 0.1 mM, induce culture at 28°C and 180 rpm for 12 h, centrifuge at 4°C and 12000 rpm for 10 min, and collect wet bacteria.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] The present invention improves the activity and stereoselectivity of nitrilase by directed evolution technology, and compares it with the parent nitrilase PgNIT M0 In comparison, the mutant Pg NIT M0 / P52W activity increased by 1.1 times, and the enantiomeric selectivity (E value) for the substrate 3-cyanohexanenitrile increased from 569 to 714; the mutant Pg NIT M0 / P52W / E62L activity increased by 1.23 times, and the E value increased to 807; the mutant PgNIT M0 / P52W / E62L / G107M vitality increased by 1.3 times, and E value increased to 1388.

[0021] The nitrilase mutant of the present invention has high activity and stereoselectivity, significantly improving substrate conversion rate and product ee value. M0 After 16 hours of catalytic reaction of 100g / L 3-cyanohexanenitrile, the substrate conversion rate was 44.82% and the product ee value was 99.12%; Pg NIT M0 After 16 h of reaction, the substrate conversion rate of P52W was 40.92% and the product ee value was 99.44%. M0The substrate conversion rate of P52W / E62L catalyzed reaction after 14h is 44.91%, and the product ee value is 99.35%. M0 The substrate conversion rate of P52W / E62L / G107M catalyzed reaction after 12h is 45.5%, and the product ee value is 99.35%.

[0022] The nitrilase mutant has high activity and stereoselectivity, and the chiral intermediate (R)-3-cyanohexanoic acid of brivudine is synthesized by using the high-efficiency nitrilase method, which greatly reduces the industrial production cost, and has good application prospect in the industrial production of brivudine. (IV) FIGURES

[0023] Figure 1 The recombinant E. coli (10g / L) containing the nitrilase mutant Pg NIT M0 / P52W and Pg NIT before modification M0 The reaction progress of 3-cyanohexanenitrile (100g / L) hydrolysis catalyzed by the recombinant E. coli (10g / L) containing the nitrilase mutant Pg NIT

[0024] Figure 2 The reaction progress of 3-cyanohexanenitrile (100g / L) hydrolysis catalyzed by the recombinant E. coli (10g / L) containing the nitrilase mutant Pg NIT M0 / P52W / E62L and Pg NIT before modification M0 The reaction progress of 3-cyanohexanenitrile (100g / L) hydrolysis catalyzed by the recombinant E. coli (10g / L) containing the nitrilase mutant Pg NIT

[0025] Figure 3 The reaction progress of 3-cyanohexanenitrile (100g / L) hydrolysis catalyzed by the recombinant E. coli (10g / L) containing the nitrilase mutant Pg NIT M0 / P52W / E62L / G107M and Pg NIT before modification M0 The reaction progress of 3-cyanohexanenitrile (100g / L) hydrolysis catalyzed by the recombinant E. coli (10g / L) containing the nitrilase mutant Pg NIT

[0026] Figure 4 The reaction progress of 3-cyanohexanenitrile (100g / L) hydrolysis catalyzed by the recombinant E. coli (containing the nitrilase mutant Pg NIT M0 / P52W / E62L / G107M) of different cell concentrations. (V) DETAILED DESCRIPTION

[0027] The application will be further described in conjunction with specific examples, but the protection scope of the application is not limited to this:

[0028] The parent nitrilase used in the present invention is a nitrilase mutant Pg NITmut / F135L / R199W / T59D (hereinafter referred to as Pg NIT) from Paraburkholderia graminis. M0 ), whose amino acid sequence is shown in SEQ ID NO.2, and nucleotide sequence is shown in SEQ ID NO.1, which have been disclosed in patent application CN202310202126.6.

[0029] Example 1: Nitrilase mutant Pg NIT M0 Construction of / P52W and recombinant Escherichia coli

[0030] 1. Screening of mutation sites

[0031] The nitrilase mutant Pg NIT from Paraburkholderia graminis was used to M0 is the parent, its amino acid sequence is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.1.

[0032] About PgNIT M0 Homology modeling, molecular docking and MM / PBSA were performed to analyze the binding mode of the enzyme and substrate. The key amino acids in the catalytic pocket that affect the stereoselective recognition of the substrate were screened based on steric hindrance, interaction force, etc., and finally sites 52, 62 and 107 were selected for modification.

[0033] SEQ ID NO.2

[0034] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYFAAVQDGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGG TLYNTQLLFDADGTLIQRRRKITPTHLERMIWGQGDGSGLRAVDSAVGRIGQLACFEHNNPLARYAMIADGEQIHSAMYPGSAFGEGFAQWMEINIRQHALESGAFVVNATAWLDA DQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQIDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI.

[0035] 2. Design of site-directed mutagenesis primers

[0036] According to the mutation sites screened in step 1, the corresponding primers were designed, as shown in Table 1.

[0037] Table 1 Primers for site-directed saturation mutagenesis

[0038]

[0039] Note: N=A / G / C / T, K=G / T, M=A / C

[0040] 3. Construction of mutants

[0041] The recombinant plasmid pET-28b(+)-Pg NIT containing the target gene fragment M0 As a template, the primers in Table 1 were used to amplify the template according to the overlap extension PCR method, and the 52nd, 62nd, and 107th positions in the parent amino acid sequence were subjected to site-directed mutagenesis.

[0042] PCR amplification system (50 μL): dNTP (1 μL, 2 mM), buffer (25 μL), ultrapure water (make up to 50 μL), PgNIT M0 The template plasmid DNA (1 μL, 10 ng / μL), primers (2 μL, 10 pmol / μL) and Phanta Max Super-Fdelity DNA polymerase (1 μL, 5 U / μL) were used for PCR amplification using the PCR amplification program in Table 2.

[0043] Table 2 PCR amplification program

[0044]

[0045] After the PCR product was positive after 0.9% agarose gel electrophoresis analysis, 20 μL of the PCR reaction solution was taken and 1 μL of endonuclease Dpn I was added to remove the template plasmid DNA by enzyme digestion at 37°C for 2 h and inactivated at 65°C for 10 min.

[0046] Take competent cells E. coli BL21 (DE3), add 10 μL of PCR product after removing the template plasmid, let it stand on ice for 30 minutes, heat shock at 42°C for 90 seconds, add 600 μL of LB liquid medium without kanamycin, culture at 37°C at 180 r / min for 1 hour, spread on LB plates containing 50 mg / L kanamycin, and culture at 37°C overnight.

[0047] Bacterial culture and high-throughput screening: Use a sterilized toothpick to pick up the monoclonal colonies growing on the plate and transfer them to a 96-well plate pre-filled with 600 μL sterile LB liquid medium (containing a final concentration of 50 μg / mL kanamycin). The first three wells and the last three wells of the 96-well plate were filled with the control group monoclonal colonies (transferred into the parent Pg NIT M0 ), then tightly seal the 96-deep-well plate. Cultivate in a constant temperature shaker at 37°C and 180 rpm for 20-24 hours to obtain a seed solution. Remove 200 μL of seed solution from the cultured 96-deep-well plate and transfer it to a new 96-deep-well plate pre-filled with 600 μL of sterile LB liquid medium (containing a final concentration of 50 μg / mL kanamycin and 0.1 mM IPTG). Incubate at 28°C and 180 rpm for approximately 12 hours to induce expression of the nitrilase protein. Subsequently, centrifuge at 4000 rpm at room temperature for 10 minutes, discard the supernatant, and collect the expressed bacterial cells for subsequent high-throughput screening.

[0048] Resuspend the cells in each well in 200 μL of phosphate buffer (10 mM, pH 8). 50 μL of each cell suspension was reacted with racemic 3-cyanohexanenitrile (final concentration 20 mM) for 30 min at 30°C in a shaker at 200 rpm. After completion of the reaction, 30 μL of 2 M HCl was added to each well, followed by centrifugation at 4000 rpm for 10 min at room temperature to obtain the resulting solution. 30 μL of the supernatant was transferred to a 96-well microplate. 150 μL of fluorescent colorimetric solution (prepared by weighing 0.1 g of o-phthalaldehyde and dissolving it in 20 mL of anhydrous ethanol, adding 50 μL of mercaptoethanol, and then diluting the solution 4-fold with phosphate buffer (10 mM, pH 8)) was added to each well. The plates were incubated in a 37°C incubator for 30 min to allow for full color development. After incubation, the fluorescence intensity in the 96-well plate was measured using a microplate reader (excitation wavelength 412 nm, emission wavelength 467 nm). Based on the fluorescence intensity values ​​compared with the control group, strains with significantly increased fluorescence intensity were preliminarily determined to have significantly increased catalytic activity.

[0049] Screening yielded a superior mutant Pg NIT with improved activity and stereoselectivity M0 / P52W、PgNIT M0 / E62L、Pg NIT M0 / G107M, for further iterative mutation.

[0050] Example 2: Nitrilase mutant Pg NIT M0 / P52W / E62L and construction of recombinant Escherichia coli

[0051] The recombinant plasmid pET-28b(+)-Pg NIT containing the target gene fragment was obtained by the method of Example 1 M0 The recombinant engineering bacteria E. coli BL21(DE3) / pET28b(+)-Pg NIT was obtained by using the primer E62-F, E62-R in Table 1 and the method of Example 1, with / P52W as the template (the amino acid sequence is shown as SEQ ID NO. 4, and the nucleotide sequence is shown as SEQ ID NO. 3). M0 / P52W / E62L, the amino acid sequence of which is shown as SEQ ID NO. 6, and the nucleotide sequence of which is shown as SEQ ID NO. 5.

[0052] Example 3: Mutant nitrile hydratase Pg NIT M0 / P52W / E62L / G107M and the construction of recombinant E. coli

[0053] The recombinant plasmid pET-28b(+)-Pg NIT containing the target gene fragment was obtained by the method of Example 2 M0 The recombinant engineering bacteria E. coli BL21(DE3) / pET28b(+)-Pg NIT was obtained by using the primer G107-F, G107-R in Table 1 and referring to the method of Example 1, with / P52W / E62L as the template. M0 / P52W / E62L / G107M, the amino acid sequence of which is shown as SEQ ID NO. 8, and the nucleotide sequence of which is shown as SEQ ID NO. 7.

[0054] Example 4: Induced expression of recombinant E. coli containing mutant nitrile hydratase

[0055] The parent nitrile hydratase E. coli BL21(DE3) / pET28b(+)-Pg NIT was picked from the plate M0 and the mutants E. coli BL21(DE3) / pET28b(+)-Pg NIT obtained in Examples 1, 2 and 3 M0 / P52W, E. coli BL21(DE3) / pET28b-Pg NIT M0 / P52W / E62L, E. coli BL21(DE3) / pET28b-Pg NIT M0A single colony of recombinant E. coli with the strain of / P52W / E62L / G107M was inoculated into 10 mL of LB liquid medium (containing kanamycin at a final concentration of 50 μg / mL) and cultured at 37°C and 180 rpm for 8 h. The bacterial suspension in the test tube was then inoculated into a shake flask containing sterile LB liquid medium (containing kanamycin at a final concentration of 50 μg / mL) at a concentration of 2% (v / v), and the flask was cultured at 37°C and 180 rpm. OD 600 When the pH value reaches 0.6-0.8, the cells are taken out and IPTG is added at a final concentration of 0.1 mM in a sterile environment to induce expression. The cells are cultured at 28°C and 180 rpm for about 12 h. The wet cells are collected by centrifugation at 4°C and 12,000 rpm for 10 min. The wet cells are washed with 0.85% physiological saline and stored at -20°C for later use (i.e., resting cells for hydrolysis reaction).

[0056] Example 5: Determination of activity and stereoselectivity of recombinant Escherichia coli containing nitrilase mutants

[0057] The nitrilase Pg NIT prepared in Example 4 M0 、Pg NIT M0 / P52W、Pg NIT M0 / P52W / E62L and Pg NIT M0 The wet cells obtained by culturing the recombinant Escherichia coli expressing / P52W / E62L / G107M were used as catalyst to carry out the hydrolysis reaction of 3-cyanohexanenitrile at 30°C.

[0058] The reaction system was as follows: 10 mL of 100 mM phosphate buffer (pH 8), 1 g of 3-cyanohexanenitrile, and 0.1 g of wet bacterial cells. The reaction was incubated at 30°C and 350 rpm for 30 min. A 200 μL sample was removed, quenched with 30 μL of 6 M HCl, and extracted with ethyl acetate. A 200 μL sample of the upper organic phase was dried over anhydrous sodium sulfate, followed by the addition of 7.5 μL of methanol and 3.75 μL of diazomethane. The substrate conversion and enantiomeric excess (ee) of the product (R)-3-cyanohexanoic acid were determined by gas chromatography.

[0059] The gas chromatograph was an Agilent 6890N model, with a BGB-175 capillary column (30 m × 0.25 mm, 0.25 μm) (BGB Analytik, Switzerland). Chromatographic conditions were as follows: injection volume 1.0 μL, injection and detector temperatures at 250°C, column temperature at 120°C for 12 min, then ramped to 200°C at 10°C / min and held for 5 min. The carrier gas was high-purity helium at a flow rate of 1.5 mL / min, with a split ratio of 50:1.

[0060] The enantiomeric excess (ee) and conversion rate (c) were calculated according to the method of Rakels et al. (Enzyme Microb. Technol., 1993, 15: 1051-1056).

[0061] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of (R)-3-cyanohexanoic acid at 30°C.

[0062] The results are shown in Table 3, and compared with the parent nitrilase Pg NIT M0 In comparison, the mutant Pg NIT M0 / P52W activity increased by 1.1 times, and the enantiomeric selectivity (E value) for the substrate 3-cyanohexanenitrile increased from 569 to 714; the mutant Pg NIT M0 / P52W / E62L activity increased by 1.23 times, and the E value increased to 807; the mutant Pg NIT M0 / G107M activity increased by 1.3 times, and the E value increased to 1388.

[0063] Table 3. Comparison of nitrilase activity and stereoselectivity

[0064]

[0065] Example 6: Synthesis of (R)-3-cyanohexanoic acid from 3-cyanohexanenitrile by recombinant Escherichia coli containing a nitrilase mutant (I)

[0066] 0.1 g of the nitrilase Pg NIT prepared in Example 4 was used to M0 、Pg NIT M0 / P52W、Pg NIT M0 / P52W / E62L and Pg NIT M0 The wet cells of recombinant Escherichia coli expressing / P52W / E62L / G107M were used as a catalyst, 1 g of 3-cyanohexanenitrile was used as a substrate, and 10 mL of phosphate buffer (100 mM, pH 8) was used as a reaction medium to form a reaction system. The hydrolysis reaction was carried out at 30°C and 350 rpm for 12-16 h. Samples were taken, and the substrate conversion rate and product ee value were detected using the method of Example 5.

[0067] The results showed that Pg NIT M0 After 16 hours of catalytic reaction of 100 g / L 3-cyanohexanenitrile, the substrate conversion rate was 44.82% and the product ee value was 99.12% (blank control); Pg NIT M0 After 16 h of reaction, the substrate conversion rate was 40.92% and the product ee value was 99.44%. Figure 1 ); Pg NIT M0After 14 h of catalytic reaction, the substrate conversion rate was 44.91% and the product ee value was 99.35% ( Figure 2 ); Pg NIT M0 After 12 h of catalytic reaction, the substrate conversion rate was 45.5% and the product ee value was 99.35% ( Figure 3 ).

[0068] The 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 and subjected to reduced pressure rotary evaporation to obtain the remaining unreacted substrate. The upper aqueous phase was filtered to obtain a clear liquid, which was the product (R)-3-cyanohexanoic acid aqueous solution.

[0069] Example 7: Synthesis of (R)-3-cyanohexanoic acid (II) from 3-cyanohexanenitrile by recombinant Escherichia coli containing a nitrilase mutant

[0070] 0.05 g, 0.1 g, 0.15 g, 0.20 g, and 0.25 g of the nitrilase PgNIT prepared by the method of Example 4 were used, respectively. M0 / P52W / E62L / G107M wet cells were used as catalysts (the catalyst addition concentration was 5 g / L, 10 g / L, 15 g / L, 20 g / L, and 25 g / L based on the volume of the reaction medium), 1 g of 3-cyanohexanenitrile was used as a substrate, and 10 mL of phosphate buffer (100 mM, pH 8) was used as a reaction medium. The reaction was hydrolyzed at 30°C and 350 rpm for 8-20 h. Samples were taken, and the substrate conversion rate and product ee value were determined using the method of Example 5.

[0071] The results are as follows Figure 4 5g / L nitrilase mutant Pg NIT M0 / P52W / E62L / G107M catalyzed the reaction for 20 hours, the conversion rate was 42.98%, and the product ee value was 99.56%; 10 g / L nitrilase mutant catalyzed the reaction for 14 hours, the conversion rate was 45.5%, and the product ee value was 99.35%; 15 g / L nitrilase mutant catalyzed the reaction for 12 hours, the conversion rate was 45.74%, and the product ee value was 99.31%; 20 g / L nitrilase mutant catalyzed the reaction for 10 hours, the conversion rate was 46.88%, and the product ee value was 99.27%; 25 g / L nitrilase mutant catalyzed the reaction for 8 hours, the conversion rate was 47.89%, and the product ee value was 99.20%.

[0072] The present invention is not limited by the above specific description. Various changes can be made to the present invention within the scope outlined by the claims, and these changes are all within the scope of the present invention.

Claims

1. A nitrilase mutant with high catalytic activity and stereoselectivity, characterized in that: The nitrilase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO. 2 to one of the following: (1) the proline at position 52 is mutated to tryptophan; (2) the proline at position 52 is mutated to tryptophan, and the glutamic acid at position 62 is mutated to leucine; (3) the proline at position 52 is mutated to tryptophan, the glutamic acid at position 62 is mutated to leucine, and the glycine at position 107 is mutated to methionine.

2. A recombinant genetically engineered bacterium containing a gene encoding the nitrilase mutant according to claim 1.

3. A mutant of the nitrilase according to claim 1 in catalyzing the preparation of racemic 3-cyanohexanenitrile ( R )-3-cyanohexanoic acid.

4. The use according to claim 3, characterized in that The application method comprises the following steps: using wet bacteria obtained by fermentation culture of an engineered bacterium containing the nitrilase mutant encoding gene as a catalyst, using racemic 3-cyanohexanenitrile as a substrate, and using a buffer solution with a pH of 7 to 8 as a reaction medium to form a reaction system; after completion of the water bath reaction at 20 to 40° C. and 300 to 600 rpm, separating and purifying the reaction solution to obtain ( R )-3-cyanohexanoic acid.

5. The use according to claim 4, characterized in that The substrate concentration in the reaction system is 100-200 g / L, and the amount of the catalyst used is 5-25 g / L based on the weight of the wet bacteria.

6. The use according to claim 4, characterized in that The reaction medium is a 100 mM phosphate buffer solution with a pH value of 8, and the catalytic reaction temperature is 30°C.

7. The use according to claim 4, characterized in that The wet cells are prepared as follows: The engineered bacteria containing the nitrilase mutant encoding gene were inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 8-12 h to obtain seed liquid; the seed liquid was then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 2%, and cultured at 37°C and 180 rpm until the bacterial concentration OD 600 When the pH value reaches 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.1 mM into the culture medium, induce the culture at 28°C and 180 rpm for 12 h, centrifuge at 4°C and 12000 rpm for 10 min, and collect the wet bacteria.

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