Nitrilase mutant and application thereof in synthesis of brivaracetam chiral intermediate

Abstract

The invention discloses a nitrilase mutant and application of the nitrilase mutant in synthesis of a brivaracetam chiral intermediate, and provides an efficient (R)-3-cyanohexanoic acid nitrilase synthesis process by constructing the nitrilase mutant with improved catalytic activity and stereoselectivity. The requirement of industrially synthesizing the brivaracetam key chiral intermediate is met, and a foundation is laid for industrial production of brivaracetam synthesized by a chemical-enzyme method.

Description

(I) Technical field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a nitrilase mutant, a coding gene, an engineering bacterium and an application thereof in hydrolyzing racemic 3-cyanohexanonitrile to synthesize (R)-3-cyanohexanoic acid, a key chiral intermediate of brivaracetam. (II) Background technology

[0002] Brivaracetam is a new type of anti-epileptic drug developed by UCB and approved for marketing in 2016. Its pharmacological action is mainly through binding to synaptic vesicle protein 2A (SV2A), thereby regulating the release of neurotransmitters, reducing the excitability of neurons, and producing an anti-epileptic effect. Compared with traditional anti-epileptic drugs such as levetiracetam and gabapentin, Brivaracetam has significant advantages such as higher affinity, less dosage, and fewer side effects. It is widely used to treat partial epileptic seizures in adults and children. Since its launch, the sales of Brivaracetam have continued to rise, and it has huge market prospects.

[0003] At present, the main method for synthesizing Bu Wa Xi Tan is still mainly chemical method. Among them, patent CN107216276A uses (R)-4-propyldihydro-2 (3H)-ketone as raw material, performs ring opening reaction in anhydrous organic solvent, uses chlorination reagent to perform halogenation reaction after forming intermediate, and further synthesizes Bu Wa Xi Tan through amidation and ring closing reaction. Patent CN106432030B uses β-lactone compound as raw material, and then removes the ester group to obtain the key intermediate (R)-4-propyldihydrofuran-2 (3H)-ketone through directional ring opening of Grignard reagent EtMgBr, and then removes the ester group to obtain the key intermediate (R)-4-propyldihydrofuran-2 (3H)-ketone, and the intermediate product obtained by ring opening and ring removal is condensed with (S)-2-aminobutyramide and amidated to synthesize the product Bu Wa Xi Tan. However, the above-mentioned synthetic routes all face the defects of complex operation, low optical purity of products, and difficulty in separation and purification, which makes it difficult to achieve industrial application.

[0004] Nitrilases are important industrial enzyme catalysts that catalyze the synthesis of chiral carboxylic acids, and can catalyze the hydrolysis of various nitrile compounds to generate corresponding carboxylic acids. Using nitrilase to stereoselectively catalyze 3-cyanohexanenitrile to synthesize the key chiral intermediate (R)-3-cyanohexanoic acid, and then synthesizing buvacethamine through hydrogenation, cyclization and chiral resolution is an ideal route for its industrial synthesis (CN202210350130.2). Therefore, the development of nitrilase catalysts with high stereoselectivity and high catalytic activity to achieve efficient synthesis of (R)-3-cyanohexanoic acid is of great significance for the enzymatic synthesis of buvacethamine. (III) Summary of the invention

[0005] The present invention aims to provide a nitrilase mutant with high catalytic activity and stereoselectivity and application thereof in the synthesis of (R)-3-cyanohexanoic acid, a chiral intermediate of brivaracetam. The present invention establishes an efficient (R)-3-cyanohexanoic acid nitrilase synthesis process by constructing a nitrilase mutant with improved catalytic activity and stereoselectivity, thereby meeting the demand for industrial synthesis of key chiral intermediates of brivaracetam and laying a foundation for the industrial production of brivaracetam synthesized by chemical-enzymatic method.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a nitrilase mutant with high catalytic activity and stereoselectivity, wherein the nitrilase mutant is obtained by mutating the 52nd, 62nd and 107th positions of the amino acid sequence shown in SEQ ID NO.2. The amino acid sequence shown in SEQ ID NO.2 of the present invention is a nitrilase mutant Pg NITmut / F135L / R199W / T59D (CN202310202126.6) (hereinafter referred to as Pg NIT M0 ), the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. M0 Site-directed mutagenesis was performed to obtain a nitrilase mutant with improved catalytic performance such as catalytic activity and stereoselectivity.

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

[0010] (1) Proline 52 was 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 proline at position 52 is mutated to tryptophan, and the glutamic acid at position 62 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 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; (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 substitution forms of other amino acid positions of the above-mentioned nitrilase mutants, forms of adding or deleting one or more amino acids, forms of amino-terminal truncation, forms of carboxyl-terminal truncation, these mutant forms are also included in the scope of the present invention.

[0011] The present invention also provides a gene encoding the nitrilase mutant, wherein the nucleotide sequence of the gene encoding is shown as SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7.

[0012] The present invention also relates to a recombinant vector comprising a gene encoding the nitrilase mutant and a recombinant genetic engineering bacterium comprising the recombinant vector; the original vector of the recombinant vector is pET-28b(+). The recombinant vector transforms a host cell to obtain a recombinant genetic engineering bacterium, and the host cell can be any conventional host cell in the art, and preferably the host cell is Escherichia coli BL21.

[0013] The present invention also provides the use of the nitrilase mutant in catalyzing racemic 3-cyanohexanenitrile to prepare buvacecanoic acid, a chiral intermediate of brivaracetam. The method of the use is: 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 the water bath reaction is completed at 20 to 40° C. and 300 to 600 rpm, the reaction solution is separated and purified to obtain (R)-3-cyanohexanoic acid.

[0014] The nitrilase mutant 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 mutant of the present invention can also be made into a biocatalyst in the form of immobilized enzymes or immobilized cells using immobilization techniques known in the art.

[0015] 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.

[0016] Preferably, the reaction medium is a 100 mM phosphate buffer with a pH value of 8, and the catalytic reaction conditions are: a temperature of 30° C. and a rotation speed of 350 rpm.

[0017] The culture medium used for the engineered bacteria of the present invention can be a culture medium in the art that can grow the engineered bacteria and produce the nitrilase of the present invention, preferably LB culture medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, solvent is water, pH 7.5. There are no special requirements for the culture method and culture conditions, as long as the engineered bacteria can grow and produce nitrilase.

[0018] Preferably, the wet cells are prepared as follows:

[0019] 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) with a final concentration of 0.1 mM into the culture medium, induce 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.

[0020] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0021] The invention uses directed evolution technology to improve the activity and stereoselectivity of nitrilase, and compares it with the parent nitrilase PgNIT M0 Compared with 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 has 1.3 times more vitality and its E value is increased to 1388.

[0022] The nitrilase mutant of the present invention has high activity and stereoselectivity, and significantly improves the substrate conversion rate and product ee value. M0 After catalyzing 100g / L 3-cyanohexanenitrile for 16h, 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%. M0After 14 h of catalytic reaction, the substrate conversion rate was 44.91% and the product ee value was 99.35%; Pg NIT M0 After 12 hours of catalytic reaction, the substrate conversion rate was 45.5% and the product ee value was 99.35%.

[0023] The nitrilase mutant of the invention has high activity and stereoselectivity, and utilizes the efficient nitrilase method to synthesize the chiral intermediate (R)-3-cyanohexanoic acid of brivaracetam, thereby greatly reducing the industrial production cost, and has good application prospects in the industrial production of brivaracetam. (IV) Description of the drawings

[0024] Figure 1 , containing the nitrilase mutant Pg NIT M0 / P52W and Pg NIT before transformation M0 Comparison of the reaction progress of the hydrolysis of 3-cyanohexanenitrile (100 g / L) catalyzed by recombinant Escherichia coli resting cells (10 g / L).

[0025] Figure 2 , containing the nitrilase mutant Pg NIT M0 / P52W / E62L and Pg NIT before transformation M0 Comparison of the reaction progress of the hydrolysis of 3-cyanohexanenitrile (100 g / L) catalyzed by recombinant Escherichia coli resting cells (10 g / L).

[0026] Figure 3 , containing the nitrilase mutant Pg NIT M0 / P52W / E62L / G107M and Pg NIT before transformation M0 Comparison of the reaction progress of the hydrolysis of 3-cyanohexanenitrile (100 g / L) catalyzed by recombinant Escherichia coli resting cells (10 g / L).

[0027] Figure 4 The recombinant Escherichia coli (containing the nitrilase mutant Pg NIT) with different cell concentrations M0 / P52W / E62L / G107M) catalyzed the hydrolysis of 3-cyanohexanenitrile (100 g / L). (V) Specific implementation methods

[0028] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0029] The parent nitrilase used in the present embodiment is a nitrilase mutant Pg NITmut / F135L / R199W / T59D (hereinafter referred to as Pg NIT M0 ), whose amino acid sequence is shown in SEQ ID NO.2, and whose nucleotide sequence is shown in SEQ ID NO.1, has been disclosed in patent application CN202310202126.6.

[0030] Example 1: Nitrilase mutant Pg NIT M0 Construction of / P52W and recombinant Escherichia coli

[0031] 1. Screening of mutation sites

[0032] The nitrilase mutant Pg NIT from Paraburkholderia graminis M0 is the parent, whose amino acid sequence is shown in SEQ ID NO.2, and whose nucleotide sequence is shown in SEQ ID NO.1.

[0033] About PgNIT M0 Homology modeling, molecular docking and MM / PBSA were performed to analyze the binding mode of the enzyme and the 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.

[0034] SEQ ID NO.2

[0035] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYFAAVQDGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGG TLYNTQLLFDADGTLIQRRRKITPTHLERMIWGQGDGSGLRAVDSAVGRIGQLACFEHNNPLARYAMIADGEQIHSAMYPGSAFGEGFAQWMEINIRQHALESGAFVVNATAWLDA DQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQIDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI.

[0036] 2. Design of site-directed mutagenesis primers

[0037] According to the mutation sites screened in step 1, the corresponding primers were designed, as shown in Table 1.

[0038] Table 1 Primers for site-directed saturation mutagenesis

[0039]

[0040] Note: N=A / G / C / T, K=G / T, M=A / C

[0041] 3. Construction of mutants

[0042] 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 mutation.

[0043] The PCR amplification system is (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 amplification using the PCR amplification program in Table 2.

[0044] Table 2 PCR amplification program

[0045]

[0046] After the PCR product was positive after 0.9% agarose gel electrophoresis analysis, 20 μL of the PCR reaction solution was taken, 1 μL of endonuclease Dpn I was added to remove the template plasmid DNA at 37°C for 2 h, and inactivated at 65°C for 10 min.

[0047] 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 min, heat shock at 42°C for 90 s, add 600 μL LB liquid culture medium without kanamycin, culture at 37°C at 180 r / min for 1 h, spread on LB plates containing 50 mg / L kanamycin, and culture at 37°C overnight.

[0048] Bacterial culture and high-throughput screening: Use a sterilized toothpick to pick the monoclonal colonies growing on the plate and transfer them to a 96-well plate pre-filled with 600 μL sterile LB liquid culture medium (containing kanamycin at a final concentration of 50 μg / mL). 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 ), and then strictly seal the 96 deep-well plate. Cultivate in a constant temperature shaker at 37°C and 180r / min for 20 to 24 hours to obtain seed solution. Take out 200μL of seed solution from the cultured 96 deep-well plate and transfer it to a new 96 deep-well plate pre-filled with 600μL of sterile LB liquid culture medium (containing final concentration of 50μg / mL kanamycin and 0.1mM IPTG), and culture at 28°C and 180r / min for about 12 hours to induce expression of nitrilase protein. Then centrifuge at 4000r / min at room temperature for 10 minutes, discard the supernatant, and collect the expressed bacterial cells for subsequent high-throughput screening.

[0049] Resuspend the bacterial cells in the wells with 200 μL of phosphate buffer (10 mM pH 8), take 50 μL of the bacterial solution and react with racemic 3-cyanohexanenitrile (final concentration 20 mM), and react for 30 min at 200 r / min in a constant temperature reaction oscillator at 30 ° C. After the reaction, add 30 μL of 2M HCl to each well to terminate the reaction, and then centrifuge at 4000 r / min at room temperature for 10 min to obtain the reaction solution. Take 30 μL of the supernatant and transfer it to a 96-well microplate, and each well has been pre-added with 150 μL of fluorescent colorimetric solution (the configuration method is: weigh 0.1 g of o-phthalaldehyde and dissolve it in 20 mL of anhydrous ethanol, add 50 μL of mercaptoethanol, and dilute it 4 times with phosphate buffer (10 mM pH 8) to prepare the fluorescent reaction solution), and incubate it in a constant temperature incubator at 37 ° C for 30 min to fully develop the color. After the incubation, the fluorescence intensity in the 96-well plate was measured using an ELISA reader (excitation wavelength 412 nm, emission wavelength 467 nm). According to the fluorescence intensity value compared with the control group, the strain with significantly improved fluorescence intensity was preliminarily determined to be a strain with significantly improved catalytic activity.

[0050] Screening resulted in the superior mutant Pg NIT with improved activity and stereoselectivity M0 / P52W、PgNIT M0 / E62L、Pg NIT M0 / G107M, for further iterative mutation.

[0051] Example 2: Nitrilase mutant Pg NIT M0 / P52W / E62L and the construction of recombinant Escherichia coli

[0052] The recombinant plasmid pET-28b(+)-Pg NIT containing the target gene fragment obtained by the method of Example 1 M0 / P52W as a template (amino acid sequence as shown in SEQ ID NO.4, nucleotide sequence as shown in SEQ ID NO.3), using primers E62-F and E62-R in Table 1, and using the method of Example 1, the recombinant engineering bacteria E. coli BL21 (DE3) / pET28b (+) -PgNIT M0 / P52W / E62L, the amino acid sequence of which is shown in SEQ ID NO.6, and the nucleotide sequence of which is shown in SEQ ID NO.5.

[0053] Example 3: Nitrilase mutant Pg NIT M0 / P52W / E62L / G107M and the construction of recombinant Escherichia coli

[0054] The recombinant plasmid pET-28b(+)-Pg NIT containing the target gene fragment obtained by the method of Example 2 M0 / P52W / E62L was used as a template, and primers G107-F and G107-R in Table 1 were used according to the method of Example 1 to obtain the recombinant engineering bacteria E. coli BL21(DE3) / pET28b(+)-Pg NIT M0 / P52W / E62L / G107M, the amino acid sequence of which is shown in SEQ ID NO.8, and the nucleotide sequence of which is shown in SEQ ID NO.7.

[0055] Example 4: Inducible expression of recombinant Escherichia coli containing nitrilase mutants

[0056] Pick the parent nitrilase E.coli BL21(DE3) / pET28b(+)-Pg NIT from the plate M0 and the mutant 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 the recombinant Escherichia coli with / 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 r / min for 8 h; then the bacterial solution in the test tube was inoculated into a shake flask containing sterile LB liquid medium (containing kanamycin at a final concentration of 50 μg / mL) at 2% (v / v), and then cultured at 37°C and 180 r / min. OD 600 When the pH reaches 0.6-0.8, take it out, add IPTG with a final concentration of 0.1 mM in a sterile environment to induce expression, culture at 28°C, 180 rpm for about 12 hours, collect the wet cells by centrifugation at 4°C, 12000 rpm for 10 minutes, wash the wet cells with 0.85% saline, and store at -20°C for later use (i.e., resting cells, used for hydrolysis reaction).

[0057] Example 5: Determination of Viability and Stereoselectivity of Recombinant Escherichia coli Containing Nitrilase Mutants

[0058] 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 of / P52W / E62L / G107M were used as catalyst to carry out the hydrolysis reaction of 3-cyanohexanenitrile at 30°C.

[0059] The reaction system is as follows: 10 mL phosphate buffer (100 mM, pH 8), 1 g 3-cyanohexanenitrile, 0.1 g wet bacteria. React at 30°C, 350 rpm for 30 min, take a sample of 200 μL, add 30 μL 6M HCl to terminate the reaction and extract with ethyl acetate. Take 200 μL of the upper organic phase and dry it with anhydrous sodium sulfate, add 7.5 μL methanol and 3.75 μL diazomethane, and use gas chromatography to determine the conversion rate of the substrate and the enantiomeric excess (ee) of the product (R)-3-cyanohexanoic acid.

[0060] The gas chromatograph model was 6890N (Agilent), and the capillary column model was BGB-175 (30m×0.25mm, 0.25μm) (BGB Analytik Switzerland). The chromatographic conditions were: injection volume 1.0μL, injection port and detector temperature were both 250℃, column temperature was maintained at 120℃ for 12min, and then increased to 200℃ at 10℃ / min and maintained for 5min. The carrier gas was high-purity helium, with a flow rate of 1.5mL / min and a split ratio of 50:1.

[0061] 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).

[0062] Enzyme activity definition: the amount of enzyme required to catalyze the production of 1 μmol of product (R)-3-cyanohexanoic acid at 30°C.

[0063] The results are shown in Table 3, and compared with the parent nitrilase Pg NIT M0 Compared with 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 E value increased to 1388.

[0064] Table 3. Comparison of nitrilase activity and stereoselectivity

[0065]

[0066] Example 6: Synthesis of (R)-3-cyanohexanoic acid from 3-cyanohexanenitrile catalyzed by recombinant Escherichia coli containing a nitrilase mutant (I)

[0067] 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 with / P52W / E62L / G107M were used as catalyst, 1 g of 3-cyanohexanenitrile was used as substrate, and 10 mL of phosphate buffer (100 mM, pH 8) was used as 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 in Example 5.

[0068] The results showed that Pg NIT M0 After catalyzing 100g / L 3-cyanohexanenitrile for 16h, the substrate conversion rate was 44.82% and the product ee value was 99.12% (blank control); Pg NIT M0 / P52W After 16 hours of reaction, the substrate conversion rate was 40.92% and the product ee value was 99.44% ( Figure 1 ); Pg NIT M0After 14h 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 ).

[0069] 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 and subjected to reduced pressure rotary evaporation to obtain the unreacted residual substrate; the upper aqueous phase was filtered to obtain a clear liquid, which was the product (R)-3-cyanohexanoic acid aqueous solution.

[0070] Example 7: Synthesis of (R)-3-cyanohexanoic acid (II) from 3-cyanohexanenitrile catalyzed by recombinant Escherichia coli containing a nitrilase mutant

[0071] 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 bacteria were used as catalyst (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 substrate, and 10 mL of phosphate buffer (100 mM, pH 8) was used as the reaction medium. The reaction was hydrolyzed at 30°C and 350 rpm for 8-20 h, and samples were taken. The substrate conversion rate and product ee value were determined by the method of Example 5.

[0072] 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%; 10g / L nitrilase mutant catalyzed the reaction for 14 hours, the conversion rate was 45.5%, and the product ee value was 99.35%; 15g / L nitrilase mutant catalyzed the reaction for 12 hours, the conversion rate was 45.74%, and the product ee value was 99.31%; 20g / L nitrilase mutant catalyzed the reaction for 10 hours, the conversion rate was 46.88%, and the product ee value was 99.27%; 25g / L nitrilase mutant catalyzed the reaction for 8 hours, the conversion rate was 47.89%, and the product ee value was 99.20%.

[0073] The present invention is not limited by the above specific textual description. The present invention can make various changes within the scope summarized 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 52nd, 62nd and 107th positions of the amino acid sequence shown in SEQ ID NO.

2.

2. The nitrilase mutant according to claim 1, wherein The nitrilase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into one of the following: (1) Proline at position 52 mutated to tryptophan; (2) Proline at position 52 mutated to tryptophan, and Glutamic acid at position 62 mutated to Leucine; (3) Proline at position 52 mutated to tryptophan, Glutamic acid at position 62 mutated to Leucine, and Glycine at position 107 mutated to Methionine.

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

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

5. The use according to claim 4, characterized in that The application method comprises the following steps: using wet bacteria obtained by fermentation culture of engineered bacteria containing a gene encoding a nitrilase mutant 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 a water bath reaction is completed at 20 to 40° C. and 300 to 600 rpm, separating and purifying the reaction liquid to obtain (R)-3-cyanohexanoic acid.

6. The use according to claim 5, 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.

7. The use according to claim 5, 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.

8. The use according to claim 5, 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 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%, and cultured at 37°C and 180 rpm until the bacterial concentration OD 600 When it reaches 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside with a final concentration of 0.1 mM to the culture medium, 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.

Images