Nitrilase and application thereof in preparation of R-mandelic acid

Abstract

The invention discloses nitrilase and an application of the nitrilase in preparation of R-mandelic acid. The nitrilase is wild type nitrilase or a nitrilase mutant, the amino acid sequence of the wild type nitrilase is as shown in SEQ ID NO: 1, and compared with the amino acid sequence as shown in SEQ ID NO: 1, the nitrilase mutant contains one or more amino acid residue differences in S192G, A197S or S285I. When the nitrilase is used for preparing the R-mandelic acid, the conversion rate is high, the ee value of the R-mandelic acid is high, and the nitrilase is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biocatalysis, and particularly relates to a nitrilase and its application in the preparation of R-mandelic acid. Background Art

[0002] Mandelic acid is an important pharmaceutical intermediate and a bulk chiral resolving agent, and has wide applications in the pharmaceutical industry. Compared with racemic mandelic acid, drugs synthesized from single-configuration mandelic acid or its derivatives have not only higher efficacy but also fewer side effects than drugs synthesized from racemic mandelic acid or its derivatives. Therefore, the synthesis of many drugs requires single-configuration mandelic acid.

[0003] Among them, among single-configuration mandelic acids, R-mandelic acid and its analogs are an important class of chiral synthetic building blocks, and are widely used in the synthesis of various drugs, such as cephalosporins, penicillins, anti-tumor preparations, anti-obesity drugs, optically pure amino acids, angiotensin-converting enzyme inhibitors, coenzyme A, etc., and can also be used as chiral resolving reagents for resolving other chiral drugs or pharmaceutical intermediates. Therefore, finding a method for preparing R-mandelic acid with low cost and simple operation has become a research and development hotspot.

[0004] The chemical structural formula of R-mandelic acid is:

[0005]

[0006] There are generally the following three methods for preparing R-mandelic acid monomer: asymmetric synthesis method, optical isomer resolution method, and biosynthesis method, among which the biosynthesis method is the most ideal method.

[0007] The biosynthesis method for preparing R-mandelic acid generally includes the following three methods:

[0008] (1) First synthesize the racemic mandelic acid, and then esterify or ammonolyze it to obtain mandelic acid ester or mandelic acid amide, and then under the action of esterification hydrolase or amide hydrolase, obtain single-enantiomer mandelic acid. For example, Ganapati et al. first catalytically converted racemic mandelic acid into methyl mandelate with an ion exchange resin, and then stereochemically hydrolyzed it into R-mandelic acid with a hydrolase in Candida sp., however, the optical purity of the obtained R-mandelic acid was only 78%.

[0009] (2) Directly use microorganisms with oxidoreductase to catalytically synthesize chiral mandelic acid with phenylglyoxylic acid as the substrate, and mostly form R-mandelic acid. For example, Takao M et al. stereochemically reduced phenylglyoxylic acid to R-mandelic acid with reductases in Streptococcus sp., Candida sp., Enterococcus sp., Rhodotorula sp., Saccharomyces sp., etc., but the yield was low.

[0010] (3) Using benzaldehyde and hydrocyanic acid as raw materials, mandelonitrile is first prepared, and then R-mandelic acid is obtained under the action of nitrilase. Microorganisms reported to contain nitrilase that can catalyze the production of R-mandelic acid from mandelonitrile mainly include: Alcaligenes faecalis ATCC 8750, Alcaligenes ECU0401, Pseudomonas putida MTCC 5110, etc. However, this method has difficulties in industrialization due to the low activity of the microorganisms used. SUMMARY OF THE INVENTION

[0011] The technical problem to be solved by the present invention is that the conversion rate and the ee value of R-mandelic acid prepared from the current nitrilase are relatively low. The present invention provides a nitrilase and its application in the preparation of R-mandelic acid. When using the nitrilase of the present invention to prepare R-mandelic acid, the conversion rate of the substrate is relatively high, and the ee value of R-mandelic acid is relatively high, which is suitable for industrial production.

[0012] The present invention solves the above technical problems through the following technical solutions.

[0013] The first aspect of the present invention provides a nitrilase mutant, which contains one or more amino acid residue differences of S192G, A197S or S285I compared with the amino acid sequence shown in SEQ ID NO: 1.

[0014] In the present invention, S192G means that the amino acid at position 192 is mutated from S to G, A197S means that the amino acid at position 197 is mutated from A to S, and S285I means that the amino acid at position 285 is mutated from S to I.

[0015] In some embodiments, the nitrilase mutant contains the following amino acid residue differences compared with the amino acid sequence shown in SEQ ID NO: 1:

[0016] S192G, A197S, S285I, S192G / A197S, S192G / S285I, A197S / S285I or S192G / A197S / S285I.

[0017] In the present invention, S192G / A197S represents a double mutant containing S192G and A197S, and S192G / A197S / S285I represents a triple mutant containing S192G, A197S and S285I.

[0018] In some preferred embodiments, the nitrilase mutant has an amino acid sequence shown in any one of SEQ ID NOs: 9-12.

[0019] The second aspect of the present invention provides an isolated nucleic acid molecule encoding the nitrilase mutant as described in the first aspect.

[0020] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 13-16.

[0021] The third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid molecule as described in the second aspect.

[0022] In some embodiments, the backbone of the recombinant expression vector is the pET28a plasmid or the pET21a plasmid.

[0023] The fourth aspect of the present invention provides a transformant comprising the isolated nucleic acid molecule as described in the second aspect, or the recombinant expression vector as described in the third aspect.

[0024] In some embodiments, the host cell used in the construction of the transformant is Escherichia coli or Bacillus subtilis.

[0025] In some embodiments, the Escherichia coli is Escherichia coli BL21(DE3).

[0026] The fifth aspect of the present invention provides a method for preparing the nitrilase mutant as described in the first aspect, comprising culturing the transformant as described in the fourth aspect to obtain a fermentation product, and obtaining the nitrilase mutant from the fermentation product.

[0027] In some embodiments, the culture medium used for the culturing is selected from LB liquid medium or TB liquid medium.

[0028] In some embodiments, the conditions for the culturing are: shaking culture at a temperature of 37±1°C.

[0029] The sixth aspect of the present invention provides an enzyme composition comprising two or more of the nitrilase mutants as described in the first aspect, or comprising one or more of the nitrilase mutants as described in the first aspect and a nitrilase having the amino acid sequence as shown in SEQ ID NO: 1.

[0030] The seventh aspect of the present invention provides the use of the nitrilase having the amino acid sequence as shown in SEQ ID NO: 1, the nitrilase mutant as described in the first aspect, the isolated nucleic acid molecule as described in the second aspect, the recombinant expression vector as described in the third aspect, the transformant as described in the fourth aspect or the enzyme composition as described in the sixth aspect in the preparation of R-mandelic acid.

[0031] In some embodiments, R-mandelic acid is prepared using mandelonitrile as a substrate or using hydrocyanic acid and benzaldehyde as substrates.

[0032] In some embodiments, the mandelonitrile is racemic mandelonitrile.

[0033] The eighth aspect of the present invention provides a method for preparing R-mandelic acid, the preparation method comprising: reacting at least any one of a wild-type nitrilase having the amino acid sequence shown in SEQ ID NO: 1, a nitrilase mutant as described in the first aspect, or an enzyme composition as described in the sixth aspect with a substrate to form a reaction system and reacting to obtain R-mandelic acid; the substrate is mandelonitrile, or hydrocyanic acid and benzaldehyde.

[0034] In some embodiments, the mandelonitrile is racemic mandelonitrile.

[0035] In some embodiments, the wild-type nitrilase, nitrilase mutant or enzyme composition is used in the form of wet bacterial cells, bacterial powder, liquid enzyme, solid enzyme powder or immobilized enzyme.

[0036] When the substrate is mandelonitrile, the added concentration of mandelonitrile in the reaction system is 0.05 - 50 mg / mL.

[0037] In some preferred embodiments, the added concentration of mandelonitrile is 0.1 - 10 mg / mL.

[0038] When the substrate is hydrocyanic acid and benzaldehyde, the ratio of the total added amount of benzaldehyde in the reaction system to the volume of the reaction system is (100 - 150) mg:1 mL.

[0039] In some preferred embodiments, the ratio of the total added amount of benzaldehyde in the reaction system to the volume of the reaction system is 106 mg:1 mL or 112 mg:1 mL.

[0040] In some embodiments, when the substrate is hydrocyanic acid and benzaldehyde, the molar ratio of the added amounts of hydrocyanic acid and benzaldehyde is (1 - 1.5) mol:1 mol.

[0041] In some preferred embodiments, when the substrate is hydrocyanic acid and benzaldehyde, the molar ratio of the added amounts of hydrocyanic acid and benzaldehyde is 1.3 mol:1 mol.

[0042] In some embodiments, the pH value of the reaction is 6.5 - 8.5.

[0043] In some embodiments, the pH value of the reaction is 7.0 - 8.0.

[0044] In some embodiments, the reaction temperature is 20 - 40 °C.

[0045] In some embodiments, the temperature of the reaction is 28 - 35 °C.

[0046] In some embodiments, the form of use of the nitrilase mutant or enzyme composition is wet cells, and the mass ratio of the wet cells to the substrate benzaldehyde added in the reaction system can be (0.05 - 0.5) g:1 g.

[0047] In some embodiments, the form of use of the nitrilase mutant or enzyme composition is liquid enzyme.

[0048] In some preferred embodiments, the liquid enzyme is crude enzyme solution or purified enzyme solution.

[0049] In some embodiments, the crude enzyme solution is an enzyme solution obtained by resuspending and homogenizing the wet cells with a solvent, and the mass ratio of the added wet cells to the added volume of the solvent is 1 g:(5 - 10) mL.

[0050] In some embodiments, when calculating the added mass of the liquid enzyme based on the mass of the wet cells that produce the liquid enzyme, the mass ratio of the liquid enzyme to the substrate is (5 - 10) mg:1 mg; the mass of the substrate benzaldehyde and the mass of the substrate mandelonitrile can be converted, for example, 1.33 mg of mandelonitrile is equivalent to 1.06 mg of benzaldehyde.

[0051] The ninth aspect of the present invention provides a reaction end - product system for catalytically preparing mandelic acid using benzaldehyde and hydrocyanic acid as substrates, and the reaction end - product system includes:

[0052] Mandelic acid and mandelonitrile; the percentage of mandelic acid in the sum of the masses of mandelic acid and mandelonitrile in the reaction end - product system is 99% - 100%; the ee value of R - mandelic acid contained in mandelic acid is 96% - 100%; for example, it is 96% - 97%, 96% - 98% or 96% - 99%; the percentage of mandelonitrile in the sum of the masses of mandelic acid and mandelonitrile in the reaction end - product system is 0 - 1%, for example, it is 0 - 0.3%, 0 - 0.5%, 0 - 0.7% or 0 - 0.9%; optionally, it further includes a wild - type nitrilase having an amino acid sequence as shown in SEQ ID NO:1, the nitrilase mutant as described in the first aspect, or the enzyme composition as described in the sixth aspect.

[0053] In some embodiments, it further includes a nitrilase having an amino acid sequence as shown in SEQ ID NO:1.

[0054] On the basis of conforming to common general knowledge in the art, the above - mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention. The reagents and raw materials used in the present invention are all commercially available.

[0055] The positive and progressive effects of the present invention are as follows:

[0056] When the wild-type nitrilase and the nitrilase mutant of the present invention are used in the preparation of R-mandelic acid, the conversion rate and the ee value of R-mandelic acid are relatively high, which is suitable for industrial production.

[0057] In addition to being able to use mandelonitrile as a substrate to prepare R-mandelic acid, the wild-type nitrilase and the nitrilase mutant of the present invention can also use hydrocyanic acid and benzaldehyde as substrates to prepare R-mandelic acid. The substrates have a wide range of sources and low prices, and the enzymatic catalysis conditions are mild, with basically no by-products or fewer by-products. Therefore, it is suitable for large-scale industrial production. Detailed implementation manners

[0058] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0059] The experimental methods in the present invention are all conventional methods unless otherwise specified. For gene cloning operations, reference can be made to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0060] The reagents used in the present invention are as follows:

[0061] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0062] TB liquid medium: 2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, 0.4% glycerol.

[0063] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar, 50 μg / mL kanamycin.

[0064] Escherichia coli BL21 was purchased from Beijing Dingguo ChangSheng Biotechnology Co., Ltd.

[0065] The pET28a plasmid was purchased from Novagen.

[0066] IPTG was purchased from Bangtai Bioengineering Co., Ltd.

[0067] The measurement methods of the present invention are as follows:

[0068] 1. Calculation of conversion rate:

[0069] The concentrations of each component in the reaction solution after the reaction were detected by high performance liquid chromatography (HPLC). The detection instrument was a high performance liquid chromatograph equipped with a UV detector. The specific chromatographic conditions were as follows: the chromatographic column was Agilent Eclipse plus C18 (3.5 μm, 150×4.6 mm); the mobile phase composition was: 0.1% TFA aqueous solution as mobile phase A, 0.1% TFA acetonitrile solution as mobile phase B, and gradient elution was carried out according to Table 1 below. All components in the mobile phase were of chromatographic grade. The column temperature was 30 °C; the detection wavelength was 210 nm; the flow rate of the mobile phase was 1 mL / min; the injection volume was 5 μL.

[0070] Table 1 Gradient Elution Parameter Table

[0071] Time (min) Mobile Phase A % Mobile Phase B % 0.00 90 10 10.00 0 100 11.00 0 100 11.50 90 10 16.00 90 10

[0072] The reagents used for detection were as follows:

[0073] 1.1 Blank solution / diluent: methanol;

[0074] 1.2 Reference solution: Take a certain amount of each reference substance, weigh accurately, place it in a volumetric flask, dissolve and dilute to the scale with the diluent, and shake well to obtain the reference solution;

[0075] 1.3 Test solution: Take the dry matter with the same mass as the reference substance after removing the enzyme, extracting, and drying the final reaction solution after the reaction, weigh accurately, place it in a volumetric flask, dissolve and dilute to the scale with the diluent, and shake well to obtain the test solution.

[0076] The calculation formula for the conversion rate is: conversion rate = molar amount of mandelic acid / (molar amount of mandelic acid + molar amount of mandelonitrile)

[0077] ×100%. The mass contents of mandelic acid and mandelonitrile were calculated by the area normalization method, and the molar amounts were converted according to the mass contents.

[0078] 2. Calculation of the ee value of R-mandelic acid:

[0079] The detection instrument was a high performance liquid chromatograph equipped with a UV detector. The specific chromatographic conditions were as follows: the chromatographic column was Daicel Chiralpak AD-H (4.6 mm×250 mm, 5 μm); the mobile phase composition was: n-heptane (chromatographic grade): isopropanol (chromatographic grade): trifluoroacetic acid (chromatographic grade) = 95:5:0.1 (v / v); the column temperature was 25 °C; the detection wavelength was 210 nm; the flow rate of the mobile phase was 1 mL / min; the injection volume was 10 μL;

[0080] The reagents used for detection were as follows:

[0081] 2.1 Blank solution / diluent: Isopropanol solution of 0.1% TFA; TFA is trifluoroacetic acid.

[0082] 2.2 Reference solution: Weigh a certain amount of racemic mandelic acid accurately, place it in a volumetric flask, dissolve and dilute it to the mark with the diluent, and shake well to obtain the reference solution.

[0083] 2.3 Test solution: After the reaction is completed, remove the enzyme from the final reaction solution, extract it, dry it, weigh accurately the same mass of the dried product as that of the reference substance, place it in a volumetric flask, dissolve and dilute it to the mark with the diluent, and shake well to obtain the test solution.

[0084] The optical purity of R-mandelic acid is evaluated by calculating the enantiomeric excess value (ee value).

[0085] The calculation formula for the ee value of R-mandelic acid is: where, A R : Peak area of the R-configuration product (R-mandelic acid), A S : Peak area of the S-configuration product (S-mandelic acid).

[0086] Example 1: Preparation of crude nitrilase solution

[0087] 1.1 Screening and acquisition of enzyme genes

[0088] Multiple nitrilases that can be used to prepare R-mandelic acid were screened from the NCBI database, and their sources and sequences are shown in Table 2.

[0089] 1.2 Transformation of enzyme genes

[0090] The above-mentioned nitrilase gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the nitrilase gene was cloned into the expression vector pET28a using the restriction enzyme sites NdeI&HindIII to obtain a recombinant plasmid. The recombinant plasmid was transformed into the competent cells of the host Escherichia coli BL21(DE3) to obtain a genetically engineered bacterium.

[0091] 1.3 Cultivation of strains

[0092] After the genetically engineered bacterium was activated by streaking on a petri dish, a single colony was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37 °C for 12 h. Then, it was transferred to 150 mL of fresh TB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 2% (v / v), and cultured with shaking at 37 °C until OD 600When it reaches about 0.8, cool down to 30 °C, add IPTG to a final concentration of 0.5 mM, induce culture at 30 °C for 16 h. After the culture is completed, centrifuge the culture solution at 10,000 rpm for 10 min, discard the supernatant, collect the precipitate part, and obtain wet cells of nitrilase (containing the nitrilase gene). Store the wet cells of nitrilase in a -20 °C refrigerator for later use.

[0093] 1.4 Preparation of crude nitrilase solution

[0094] Take 10 g of wet cells of nitrilase, resuspend them in 100 mL of 0.05 M sodium phosphate buffer at pH 7.0 (homogenization ratio is 1:10), and break them by high-pressure homogenization to obtain a crude nitrilase solution.

[0095] Example 2: Screening of crude nitrilase solution

[0096] Prepare an enzyme-catalyzed reaction system with a total volume of 1 mL. The enzyme-catalyzed reaction system contains the following components: 1.33 mg of racemic mandelonitrile (molecular weight is 133.15, 0.01 mmol), 50 μL of methanol, 0.1 mL of any one of the crude nitrilase solutions in Example 1 (equivalent to 0.01 g of wet cells of nitrilase), and 0.85 mL of 100 mM Tris-HCl (pH 7.5). Place the 1 mL of enzyme-catalyzed reaction system in a shaker at 37 °C and 220 rpm for reaction. After reacting for 16 h, terminate the reaction to obtain the final reaction solution. The reaction route of this example is shown in the following formula:

[0097]

[0098] Take 100 μL of the final reaction solution, add 900 μL of methanol, shake and mix well, centrifuge and remove the precipitate (containing nitrilase), take the supernatant, and use HPLC to determine the mass content of mandelonitrile and mandelic acid (including S-mandelic acid and R-mandelic acid ) in the final reaction solution, and calculate the conversion rate.

[0099] Take 900 μL of the reaction solution, adjust the pH to 2 with phosphoric acid, add 850 μL of ethyl acetate, shake, centrifuge and remove the precipitate, take the organic phase, evaporate the solvent in the organic phase, and use HPLC to determine the ee value of R-mandelic acid. The results of each conversion rate and ee value are shown in Table 2.

[0100] Table 2 Sequence list of nitrilase

[0101] Enzyme Name Amino Acid Sequence Conversion Rate ee Value of R-Mandelic Acid Enz.1 SEQ ID NO:17 99.90％ 81.16％ Enz.2 SEQ ID NO:18 88.21％ 81.168％ Enz.3 SEQ ID NO:1 90.67％ 86.582％ Enz.4 SEQ ID NO:19 90.95％ 79.52％ Enz.5 SEQ ID NO:20 91.42％ 82.14％ Enz.6 SEQ ID NO:21 18.31％ 72.916％ Enz.7 SEQ ID NO:22 93.16％ 72.782％ Enz.8 SEQ ID NO:23 4.12％ /

[0102] According to the results in Table 2, nitrilase with relatively high conversion rate and ee value was finally screened out. The NCBI accession number of this nitrilase Enz.3 is WP_012492804.1, which is derived from Burkholderia, and its amino acid sequence is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:2.

[0103] Example 3: Preparation of R-mandelic acid by catalyzing racemic mandelonitrile with crude enzyme solution of nitrilase Enz.3

[0104] In this example, R-mandelic acid was prepared using the crude enzyme solution of nitrilase Enz.3 in an enzyme-catalyzed reaction system with a total volume of 50 mL.

[0105] Prepare an enzyme-catalyzed reaction system with a total volume of 50 mL. This enzyme-catalyzed reaction system contains the following components: 66.5 mg of racemic mandelonitrile (0.5 mmol), 2.5 mL of methanol, 5 mL of crude enzyme solution of Enz.3 nitrilase (equivalent to 0.5 g of wet cells of Enz.3 nitrilase), and 42.5 mL of 100 mM Tris-HCl (pH 7.5). Place the 50 mL enzyme-catalyzed reaction system at 37 °C and oscillate the reaction at 220 rpm. After reacting for 16 h, terminate the reaction to obtain the final reaction solution.

[0106] Take 100 μL of the final reaction solution, add 900 μL of methanol, mix well by oscillation, centrifuge and remove the precipitate (containing nitrilase), take the supernatant, and use HPLC to determine the mass content of mandelonitrile and mandelic acid in the final reaction solution, and calculate the conversion rate to be 91.12%.

[0107] Take 900 μL of the reaction solution, adjust the pH to 2 with phosphoric acid, add 850 μL of ethyl acetate, then oscillate and centrifuge, take the organic phase, evaporate the solvent in the organic phase, and use HPLC to determine the ee value of R-mandelic acid to be 87.54%.

[0108] Example 4: Preparation of R-mandelic acid by catalyzing hydrocyanic acid and benzaldehyde with whole-cell nitrilase

[0109] Example 4-1

[0110] Under room temperature conditions, add 240 g of water to a 500 mL four-necked flask, add 4.5 g of the wet cells of nitrile hydratase (Enz.3 cell sludge) obtained in Example 1.3, and stir for 20 minutes. Add 9.84 g of hydrocyanic acid (molecular weight 27.03; 0.36 mol), adjust the pH value of the reaction system to 7.85 with liquid alkali, control the temperature at 28 - 35 °C, then slowly dropwise add 30.0 g of benzaldehyde (molecular weight 106.12; 0.28 mol), control the dropping rate, and finish dropping in about 2 hours. Then keep it warm at 28 - 35 °C for 2 hours. The ratio of the total added mass of benzaldehyde to the final total volume of the reaction system is about 112 mg:1 mL. Then take the final reaction solution and detect the mass content of each component in the final reaction solution by HPLC method.

[0111] It can be known from HPLC detection that the retention time of mandelic acid is 4.557 min, and there are no benzaldehyde and mandelonitrile impurities in the final reaction solution. It can be calculated that the conversion rate (calculated based on the amount of mandelonitrile) is 100%.

[0112] The retention time of S-mandelic acid is 29.555 min, and the retention time of R-mandelic acid is 36.446 min. It can be calculated that the ee value of R-mandelic acid is 97.23%.

[0113] The reaction route of this example is shown in the following formula:

[0114]

[0115] Example 4 - 2

[0116] Under room temperature conditions, add 240 g of water to a 500 mL four-necked flask, add 5.625 g of the wet cells of nitrile hydratase (Enz.3 cell sludge) obtained in 1.3 of Example 1, stir for 20 minutes, add 9.84 g of hydrocyanic acid (0.36 mol), adjust the pH of the reaction system to 7.85 with liquid alkali, control the temperature at 28 - 35 °C, slowly dropwise add 30.0 g of benzaldehyde (0.28 mol), control the dropping rate, and finish dropping in about 2 hours. Then keep it warm at 28 - 35 °C for 2 hours. The ratio of the total added mass of benzaldehyde to the final total volume of the reaction system is about 112 mg:1 mL. Then take the final reaction solution and detect the mass content of mandelic acid in the final reaction solution by HPLC method.

[0117] It can be detected that there are no benzaldehyde and mandelonitrile impurities in the final reaction solution, the conversion rate (calculated based on the amount of mandelonitrile) is 100%, and the ee value of R-mandelic acid is 96.78%.

[0118] As can be seen from the above embodiments, when the addition amount of benzaldehyde is 0.28 mol and the addition amount of hydrocyanic acid is 0.36 mol, an addition amount of 4.5 g of wet cells of nitrilase is sufficient to completely convert benzaldehyde.

[0119] Example 5: Preparation of Whole-Cell Nitrilase Mutants

[0120] This example provides a method for preparing nitrilase mutants, which includes the following steps:

[0121] (1) According to the nucleotide sequence of the wild-type nitrilase Enz.3 recorded in Section 1.1 of Example 1 (the enzyme that has not been mutated in this example is defined as the wild-type nitrilase), the gene fragment of this nitrilase was synthesized by Shanghai Sangon Biological Engineering Co., Ltd., and the gene fragment was cloned into the expression vector pET28a using the restriction enzyme sites NdeI&HindIII to obtain a recombinant plasmid.

[0122] (2) Using this recombinant plasmid as a template, with the forward primer and reverse primer of the site to be mutated, a single-point mutant gene was obtained by the method of whole-plasmid PCR.

[0123] (3) Using the constructed single-point mutant plasmid as a template, with the forward and reverse primers of another site, a two-point mutant gene was obtained by the method of whole-plasmid PCR.

[0124] The primers used for the above gene mutations are shown in Table 3.

[0125] Table 3 Gene Mutation Primer Table

[0126] Primer Name Primer Sequence Sequence Number S192G-F CCGAGCTTCGGACTGTACGCGGGCGCGGCGTAC SEQ ID NO:3 S192G-R CGCGTACAGTCCGAAGCTCGGCCACGCCCCGAT SEQ ID NO:4 A197S-F TACGCGGGCTCAGCGTACACCCTCGGTCCGGAA SEQ ID NO:5 A197S-R GGTGTACGCTGAGCCCGCGTACAGGCTGAAGCT SEQ ID NO:6 S285I-F TCTGTGATCATACTGGCGAAAGCTGCTGCGGAC SEQ ID NO:7 S285I-R TTTCGCCAGTATGATCACAGACAGATCGATTTCC SEQ ID NO:8

[0127] The source of the PCR reagent is Takara, and the product number is R045A.

[0128] The PCR amplification system is shown in Table 4:

[0129] Table 4 Whole-Plasmid PCR Amplification System

[0130] Reagent Dosage (μL) 2×Primer Star Mix 12.5 Forward Primer (F) 1 Reverse Primer (R) 1 Template 0.5 Deionized Water 10

[0131] The PCR amplification program is shown in Table 5:

[0132] Table 5 PCR Amplification Program

[0133]

[0134] (4) Take the PCR products obtained by single-point mutation or two-point mutation, and digest them with DpnI enzyme at 37 °C for 2 hours. After digestion, transform them into E. coli BL21(DE3) competent cells, and then spread them on LB solid medium containing 50 μg / mL kanamycin and culture overnight at 37 °C. Pick single colonies and perform sequencing using the Sanger method. Obtain the successfully mutated nitrilase mutants, and their sequences are shown in Table 6.

[0135] Table 6 Sequence list of nitrilase mutants

[0136] Enzyme Name Mutation Site Amino Acid Sequence Nucleotide Sequence E0 (Enz.3) Wild Type SEQ ID NO:1 SEQ ID NO:2 E1 S192G SEQ ID NO:9 SEQ ID NO:13 E2 A197S SEQ ID NO:10 SEQ ID NO:14 E3 S285I SEQ ID NO:11 SEQ ID NO:15 E4 A197S / S285I SEQ ID NO:12 SEQ ID NO:16

[0137] In Table 6, the mutation site S192G means that the serine (S) at the 192nd position of the wild-type nitrilase E0 is mutated to glycine (G). The same applies to others.

[0138] (5) Prepare the wet cells of nitrilase mutants according to the method shown in Example 1 for the sequences shown in Table 6, and obtain the whole-cell nitrilase mutants E1 (also known as E1 bacterial sludge), whole-cell nitrilase mutants E2 (also known as E2 bacterial sludge), whole-cell nitrilase mutants E3 (also known as E3 bacterial sludge), and whole-cell nitrilase mutants E4 (also known as E4 bacterial sludge) respectively.

[0139] Example 6: Preparation of R-mandelic acid by catalysis of whole-cell nitrilase mutants using hydrocyanic acid and benzaldehyde as substrates

[0140] Example 6-1 Preparation of R-mandelic acid by catalysis of whole-cell nitrilase mutants in a 240 g water system

[0141] At room temperature (25 °C, the same below), add 240 g of water to a 500 mL four-necked flask, add 4.5 g of any bacterial sludge obtained in Example 5, stir for 20 minutes, add 9.84 g of hydrocyanic acid (molecular weight 27.03; 0.36 mol), adjust the pH of the reaction system to 7.85 with liquid alkali, control the temperature at 28 - 35 °C, slowly dropwise add 30.0 g of benzaldehyde (molecular weight 106.12; 0.28 mol), control the dropping rate, and finish dropping in about 2 hours. Then keep it warm at 28 - 35 °C for 2 hours. The ratio of the total added mass of benzaldehyde to the final total volume of the reaction system is about 112 mg:1 ml. Then take the final reaction solution and detect the mass content of each component in the final reaction solution by HPLC method.

[0142] After detection, obtain the percentages of the mass contents of mandelic acid and mandelonitrile in the final reaction solutions of different whole-cell nitrilase mutant bacterial sludges (shown in Table 7), and calculate the conversion rate (calculated based on the amount of mandelonitrile). The retention time of mandelic acid is 4.569 min, and the retention time of mandelonitrile is 6.044 min.

[0143] It can be calculated that the ee value of R-mandelic acid is shown in Table 7, where the retention time of S-mandelic acid is 32.189 min and the retention time of R-mandelic acid is 39.087 min.

[0144] Example 6-2 Preparation of R-mandelic acid by catalysis of whole-cell nitrilase mutants in an 80 g water system

[0145] At room temperature, 80 g of water was added to a 250 mL four-necked flask, 4 g of any bacterial sludge obtained in Example 5 was added, and the mixture was stirred for 20 minutes. Then 3.3 g (0.12 mol) of hydrocyanic acid was added, and the pH of the reaction system was adjusted to 7.85 with liquid alkali. The temperature was controlled at 28-35 °C, and 10.0 g (0.094 mol) of benzaldehyde was slowly added dropwise. The dropping rate was controlled, and the addition was completed in about 2 hours. Then it was kept warm at 28-35 °C for 2 hours. The ratio of the total added mass of benzaldehyde to the final total volume of the reaction system was about 106 mg:1 ml. Then the final reaction solution was taken, and the contents of each component in the final reaction solution were detected by HPLC.

[0146] After detection, the percentage of the mass content of mandelic acid and mandelonitrile in the final reaction solution of different whole-cell nitrilase mutant bacterial sludges was obtained (as shown in Table 7), and the conversion rate was calculated (calculated based on the amount of mandelonitrile).

[0147] The catalytic results of the above various examples are shown in Table 7.

[0148] Table 7 Catalytic results of whole-cell nitrilase mutant enzymes

[0149]

[0150] From the above results, it can be seen that the ee value of R-mandelic acid has increased for the whole-cell nitrilase mutants compared with the whole-cell nitrilase.

[0151] The sequences used in the present invention are as follows:

[0152] SEQ ID NO:1 Amino acid sequence of nitrilase Enz.3

[0153] MTINHPRYVVAAVQAAPVFLDLEATVTKTIELIEEAARNGATLIAFPETWIPGYPLFSWLGSPAWSLQFFQRYHDNSLVINSEQYRLIEQAAARNKIMVVLGFSERDAGSLYISQSIINSEGITISTRRKLKPTHVERTVFGEGDGSDLSVHETELGRVGALCCWEHLQPLTRYAMFAQNEQVHIGAWPSFSLYAGAAYTLGPEVNTAVSQIYAVEGQCFVVAPSAVVSEQMIELLCSTPEHHALLQAGGGHARIFGPDGRSLAEPIPENVEGILYAEIDLSVISLAKAAADPAGHYSRPDVTRLLLDPTPKSRVVHVRAEPAAPEMQPATAVVQVDQPTEPLERVTPA

[0154] SEQ ID NO:2 Nitrilase Enz.3 nucleotide sequence

[0155]

[0156] SEQ ID NO:9 S192G

[0157] MTINHPRYVVAAVQAAPVFLDLEATVTKTIELIEEAARNGATLIAFPETWIPGYPLFSWLGSPAWSLQFFQRYHDNSLVINSEQYRLIEQAAARNKIMVVLGFSERDAGSLYISQSI INSEGITISTRRKLKPTHVERTVFGEGDGSDLSVHETELGRVGALCCWEHLQPLTRYAMFAQNEQVHIGAWPSFGLYAGAAYTLGPEVNTAVSQIYAVEGQCFVVAPSAVVSEQMIELLCSTPEHHALLQAGGGHARIFGPDGRSLAEPIPENVEGILYAEIDLSVISLAKAAADPAGHYSRPDVTRLLLDPTPKSRVVHVRAEPAAPEMQPATAVVQVDQPTEPLERVTPA

[0158] SEQ ID NO:10 A197S

[0159] MTINHPRYVVAAVQAAPVFLDLEATVTKTIELIEEAARNGATLIAFPETWIPGYPLFSWLGSPAWSLQFFQRYHDNSLVINSEQYRLIEQAAARNKIMVVLGFSERDAGSLYISQSIINSEGITISTRRKLKPTHVERTVFGEGDGSDLSVHETELGRVGALCCWEHLQPLTRYAMFAQNEQVHIGAWPSFSLYAGSAYTLGPEVNTAVSQIYAVEGQCFVVAPSAVVSEQMIELLCSTPEHHALLQAGGGHARIFGPDGRSLAEPIPENVEGILYAEIDLSVISLAKAAADPAGHYSRPDVTRLLLDPTPKSRVVHVRAEPAAPEMQPATAVVQVDQPTEPLERVTPA

[0160] SEQ ID NO:11 S285I

[0161] MTINHPRYVVAAVQAAPVFLDLEATVTKTIELIEEAARNGATLIAFPETWIPGYPLFSWLGSPAWSLQFFQRYHDNSLVINSEQYRLIEQAAARNKIMVVLGFSERDAGSLYISQSIINSEGITISTRRKLKPTHVERTVFGEGDGSDLSVHETELGRVGALCCWEHLQPLTRYAMFAQNEQVHIGAWPSFSLYAGAAYTLGPEVNTAVSQIYAVEGQCFVVAPSAVVSEQMIELLCSTPEHHALLQAGGGHARIFGPDGRSLAEPIPENVEGILYAEIDLSVIILAKAAADPAGHYSRPDVTRLLLDPTPKSRVVHVRAEPAAPEMQPATAVVQVDQPTEPLERVTPA

[0162] SEQ ID NO:12 A197S / S285I

[0163] MTINHPRYVVAAVQAAPVFLDLEATVTKTIELIEEAARNGATLIAFPETWIPGYPLFSWLGSPAWSLQFFQRYHDNSLVINSEQYRLIEQAAARNKIMVVLGFSERDAGSLYISQSIINSEGITISTRRKLKPTHVERTVFGEGDGSDLSVHETELGRVGALCCWEHLQPLTRYAMFAQNEQVHIGAWPSFSLYAGSAYTLGPEVNTAVSQIYAVEGQCFVVAPSAVVSEQMIELLCSTPEHHALLQAGGGHARIFGPDGRSLAEPIPENVEGILYAEIDLSVIILAKAAADPAGHYSRPDVTRLLLDPTPKSRVVHVRAEPAAPEMQPATAVVQVDQPTEPLERVTPA

[0164] SEQ ID NO:13 S192G

[0165] ATGACCATTAACCACCCGCGTTACGTTGTTGCTGCGGTTCAGGCAGCACCGGTTTTCCTTGATCTGGAAGCTACCGTTACTAAAACGATTGAACTGATTGAAGAAGCGGCGCGTAACGGCGCGACCCTGATTGCATTCCCGGAAACCTGGATTCCGGGTTACCCGCTGTTTTCCTGGTTAGGTTCCCCGGCGTGGTCCCTGCAGTTCTTCCAGCGTTACCATGATAATTCACTGGTTATTAACAGCGAACAGTACCGTCTGATCGAACAGGCGGCGGCGCGTAACAAAATCATGGTGGTGCTGGGCTTCAGCGAACGTGATGCGGGTAGCCTGTACATCTCACAGTCTATCATCAACAGCGAAGGCATCACCATCTCTACCCGTCGTAAACTGAAACCGACCCATGTAGAACGTACCGTTTTTGGTGAAGGCGATGGCTCTGATCTGAGCGTTCACGAAACCGAACTGGGTCGTGTTGGCGCTCTGTGCTGCTGGGAACACCTGCAGCCGCTGACGCGTTACGCGATGTTTGCGCAGAACGAACAGGTTCACATCGGGGCGTGGCCGAGCTTCggaCTGTACGCGGGCGCGGCGTACACCCTCGGTCCGGAAGTTAACACCGCTGTTTCTCAGATCTACGCGGTTGAAGGCCAGTGCTTCGTTGTGGCTCCGAGTGCAGTTGTTTCTGAACAGATGATCGAACTGCTGTGCAGCACCCCGGAACACCACGCTCTGTTGCAGGCTGGCGGTGGCCACGCTCGTATCTTCGGCCCGGATGGCCGTAGCCTGGCTGAACCGATCCCGGAAAACGTTGAAGGCATCCTGTACGCGGAAATCGATCTGTCTGTGATCAGCCTGGCGAAAGCTGCTGCGGACCCGGCGGGTCACTACTCACGTCCGGATGTGACCCGTCTGCTGCTGGACCCGACCCCGAAAAGCCGTGTTGTTCACGTTCGGGCGGAACCGGCGGCTCCGGAAATGCAGCCGGCGACCGCGGTTGTTCAGGTTGATCAGCCGACCGAACCGCTGGAACGTGTTACCCCGGCG

[0166] SEQ ID NO:14 A197S

[0167] ATGACCATTAACCACCCGCGTTACGTTGTTGCTGCGGTTCAGGCAGCACCGGTTTTCCTTGATCTGGAAGCTACCGTTACTAAAACGATTGAACTGATTGAAGAAGCGGCGCGTAACGGCGCGACCCTGATTGCATTCCCGGAAACCTGGATTCCGGGTTACCCGCTGTTTTCCTGGTTAGGTTCCCCGGCGTGGTCCCTGCAGTTCTTCCAGCGTTACCATGATAATTCACTGGTTATTAACAGCGAACAGTACCGTCTGATCGAACAGGCGGCGGCGCGTAACAAAATCATGGTGGTGCTGGGCTTCAGCGAACGTGATGCGGGTAGCCTGTACATCTCACAGTCTATCATCAACAGCGAAGGCATCACCATCTCTACCCGTCGTAAACTGAAACCGACCCATGTAGAACGTACCGTTTTTGGTGAAGGCGATGGCTCTGATCTGAGCGTTCACGAAACCGAACTGGGTCGTGTTGGCGCTCTGTGCTGCTGGGAACACCTGCAGCCGCTGACGCGTTACGCGATGTTTGCGCAGAACGAACAGGTTCACATCGGGGCGTGGCCGAGCTTCAGCCTGTACGCGGGCtcaGCGTACACCCTCGGTCCGGAAGTTAACACCGCTGTTTCTCAGATCTACGCGGTTGAAGGCCAGTGCTTCGTTGTGGCTCCGAGTGCAGTTGTTTCTGAACAGATGATCGAACTGCTGTGCAGCACCCCGGAACACCACGCTCTGTTGCAGGCTGGCGGTGGCCACGCTCGTATCTTCGGCCCGGATGGCCGTAGCCTGGCTGAACCGATCCCGGAAAACGTTGAAGGCATCCTGTACGCGGAAATCGATCTGTCTGTGATCAGCCTGGCGAAAGCTGCTGCGGACCCGGCGGGTCACTACTCACGTCCGGATGTGACCCGTCTGCTGCTGGACCCGACCCCGAAAAGCCGTGTTGTTCACGTTCGGGCGGAACCGGCGGCTCCGGAAATGCAGCCGGCGACCGCGGTTGTTCAGGTTGATCAGCCGACCGAACCGCTGGAACGTGTTACCCCGGCG

[0168] SEQ ID NO:15 S285I

[0169]

[0170] SEQ ID NO:16 A197S / S285I

[0171] ATGACCATTAACCACCCGCGTTACGTTGTTGCTGCGGTTCAGGCAGCACCGGTTTTCCTTGATCTGGAAGCTACCGTTACTAAAACGATTGAACTGATTGAAGAAGCGGCGCGTAACGGCGCGACCCTGATTGCATTCCCGGAAACCTGGATTCCGGGTTACCCGCTGTTTTCCTGGTTAGGTTCCCCGGCGTGGTCCCTGCAGTTCTTCCAGCGTTACCATGATAATTCACTGGTTATTAACAGCGAACAGTACCGTCTGATCGAACAGGCGGCGGCGCGTAACAAAATCATGGTGGTGCTGGGCTTCAGCGAACGTGATGCGGGTAGCCTGTACATCTCACAGTCTATCATCAACAGCGAAGGCATCACCATCTCTACCCGTCGTAAACTGAAACCGACCCATGTAGAACGTACCGTTTTTGGTGAAGGCGATGGCTCTGATCTGAGCGTTCACGAAACCGAACTGGGTCGTGTTGGCGCTCTGTGCTGCTGGGAACACCTGCAGCCGCTGACGCGTTACGCGATGTTTGCGCAGAACGAACAGGTTCACATCGGGGCGTGGCCGAGCTTCAGCCTGTACGCGGGCtcaGCGTACACCCTCGGTCCGGAAGTTAACACCGCTGTTTCTCAGATCTACGCGGTTGAAGGCCAGTGCTTCGTTGTGGCTCCGAGTGCAGTTGTTTCTGAACAGATGATCGAACTGCTGTGCAGCACCCCGGAACACCACGCTCTGTTGCAGGCTGGCGGTGGCCACGCTCGTATCTTCGGCCCGGATGGCCGTAGCCTGGCTGAACCGATCCCGGAAAACGTTGAAGGCATCCTGTACGCGGAAATCGATCTGTCTGTGATCataCTGGCGAAAGCTGCTGCGGACCCGGCGGGTCACTACTCACGTCCGGATGTGACCCGTCTGCTGCTGGACCCGACCCCGAAAAGCCGTGTTGTTCACGTTCGGGCGGAACCGGCGGCTCCGGAAATGCAGCCGGCGACCGCGGTTGTTCAGGTTGATCAGCCGACCGAACCGCTGGAACGTGTTACCCCGGCG

[0172] SEQ ID NO:17 Enz.1

[0173] MGIEHPKYKVAVVQAAPAWLDLDASIDKSIALIEEAAQKGAKLIAFPEAFIPGYPWHIWMDSPAWAIGRGFVQRYFDNSLAYDSPQAEKLRAAVRKAKLTAVLGLSERDGGSLYLAQWLIGPDGETIAKRRKLRPTHAERTVYGEGDGSDLAVHNRPDIGRLGALCCWEHLQPLSKYAMYAQNEQVHVAAWPSFSLYDPFAVALGAEVNNAASRVYAVEGSCFVLAPCATVSQAMIDELCDRPDKHTLLHVGGGFAAIYGPDGSQIGDKLAPDQEGLLIAEIDLGAIGVAKNAADPAGHYSRPDVTRLLLNKKPYKRVEQFSPPAEAVEPTDIAAAAS

[0174] SEQ ID NO:18 Enz.2

[0175] MGIEHPKYRVAVVQAAPAWLDLDASIDKSIALIEEAAQKGAKLIAFPEAFIPGYPWHIWMDSPAWAIGRGFVQRYFDNSLAYDSPQAEKLRAAVRKAKLTAVIGLSERDGGSLYLAQWLIGPDGETIAKRRKLRPTHAERTVYGEGDGSDLAVHNRPDIGRLGALCCWEHLQPLSKYAMYAQNEQVHVAAWPSFSLYDPFAVALGAEVNNAASRVYAVEGSCFVLAPCATVSQAMIDELCDRPDKHALLHVGGGFAAIYGPDGSQIGDKLAPDQEGLLIAEIDLGAIGVAKNAADPAGHYSRPDVTRLLLNKKPYKRVEQFSPPSEAVEPTDIAAAAS

[0176] SEQ ID NO:19 Enz.4

[0177] MKEAIKVACVQAAPIYMDLKATVDKTIELMEEAARNNARLIAFPETWIPGYPWFLWLDSPAWAMQFVRQYHENSLELDGPQAKRISDAAKRLGIMVTLGMSERVGGTLYISQWFIGDNGDTIGARRKLKPTFVERTLFGEGDGSSLAVFETSVGRLGGLCCWEHLQPLTKYALYAQNEEIHCAAWPSFSLYPNAAKALGPDVNVAASRIYAVEGQCFVLASCALVSQS MIDMLCTDDEKHALLLAGGGHSRIIGPDGGDLVAPLAENEEGILYANLDPGVRILAKMAADPAGHYSRPDITRLLIDRSPKLPVVEIEGDLRPYALGKASETGAQLEEI

[0178] SEQ ID NO:20 Enz.5

[0179] MGIEHPKYRVAAVQAAPAWLDLDRSIDKAIALIEEAAANGARLIAFPEVFIPGYPWHIWLDSPAWAIGRGFVQRYFDNSLAYDSPQAERLRAAVRKARLTAVIGLSERSGGSLYIAQWLVGPDGETIAKRRKLRPTHAERTVYGEGDGSDLAVHDRPDIGRLGALCCWEHLQPLSKYAMYAQNEQVHVASWPSFSLYDPFAPALGAEVNNAASRVYAVEGSCFVLAPCATVSQAMIDELCDRPDKHALLHAGGGFAAIYGPDGSSLAEKLAPDQEGLLYADIDLGAIGVAKNAADPAGHYSRPDVTRLLLNNKPYKRVEHFALPGDTVAPADVDAAAS

[0180] SEQ ID NO:21 Enz.6

[0181] MAIEHPRYRVAAVQAAPEFLNLEATVDKTIALIEEAARGGASLIAFPETWIPGYPWFAWLGAPIWGMKFIQAYHDNSMVIDGAQFERIAQAASRCNITVVLGFSEKDAGSLYIAQAILSPEGKTIATRRKLKPTHVERAIFGEGDGSDLAVHDTKLGRVGALCCWEHLQPLSKYAMYAQNEQVHIAAWPSFSLYVDAAYALGPEVNNAASRLYAVEGQCFVVAPCATVSQKMIDMLCETPEQQALLKPGGGHAQIYGPDGRSLADPLPPDAEGLLYADIDLAAITLAKAAADPAGHYSRPDVTQLLLDRNPKPRVVHAKPGQSANNSSPGMRAVEHTELEEGEQA

[0182] SEQ ID NO:22 Enz.7

[0183] MGIEHPKYKVAVVQAAPAWLDLDASIDKTIGLIEEAAQKGAKLIAFPEAFIPGYPWHIWMDSPAWAIGRGFVQRYFDNSLAYDSPQAEKLRAAVRKAKLTAVIGLSERDGGSLYLAQWLIGPDGETIAKRRKLRPTHAERTVYGEGDGSDLAVHNRPDIGRLGALCCWEHLQPLSKYAMYAQNEQVHVAAWPSFSLYDPFAVALGAEVNNAASRVYAVEGSCFVLAPCATVSQAMIDELCDRPDKHALLHVGGGFAAIYGPDGSQIGDKLAPDQEGLLIAEIDLGAIGVAKNAADPAGHYSRPDVTRLLLNKKPYKRVEQFSPPAEALEPTDIAAAAS

[0184] SEQ ID NO:23 Enz.8

[0185] MGIEHTKYKVAVVQAAPAWLDLEASIGKSIGLIKEAADKGAKLIAFPEAFIPGYPWYIWMDSPAWAIGRGFVQRYFDNSLSYDSPQAERLRDAVRQAKLTAVIGLSERDGGSLYLAQWLIGPDGETIAKRRKLRPTHAERTVYGEGDGSDLAVHARPDIGRLGALCCWEHLQPLSKYAMYAQNEQVHVAAWPSFSLYDPFAPALGAEVNNAASRVYAVEGSCFVLAPCATVSQAMIDELCDRPDKHALLHAGGGFAAIYGPDGSQIGEKLAPDQEGLLIAEIDLGAIGVAKNAADPAGHYSRPDVTRLLLNKKRYQRVEQFALPADMVEPADIGAAAS。

Claims

1. A nitrilase mutant, characterized in that, The nitrilase mutant contains one or more amino acid residue differences of S192G, A197S or S285I as compared with the amino acid sequence shown in SEQ ID NO:

1.

2. The nitrilase mutant as shown in claim 1, characterized in that The nitrilase mutant contains the following amino acid residue differences as compared with the amino acid sequence shown in SEQ ID NO:1: S192G, A197S, S285I, S192G / A197S, S192G / S285I, A197S / S285I or S192G / A197S / S285I; Preferably, the nitrilase mutant has an amino acid sequence shown in any one of SEQ ID NOs: 9-12.

3. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nitrilase mutant as claimed in claim 1 or 2; Preferably, the nucleic acid molecule contains a nucleotide sequence shown in any one of SEQ ID NOs: 13-16.

4. A recombinant expression vector, which contains the isolated nucleic acid molecule as claimed in claim 3, and the backbone of the recombinant expression vector is preferably a pET28a plasmid or a pET21a plasmid.

5. A transformant, which contains the isolated nucleic acid molecule as claimed in claim 3, or the recombinant expression vector as claimed in claim 4.

6. The transformant according to claim 5, wherein The host cell used in constructing the transformant is Escherichia coli or Bacillus subtilis, and preferably the Escherichia coli is Escherichia coli BL21(DE3).

7. A method for preparing the nitrilase mutant as claimed in claim 1 or 2, which includes culturing the transformant as claimed in claim 5 or 6 to obtain a fermentation product, and obtaining the nitrilase mutant from the fermentation product; Preferably, the culture medium used for the culturing is selected from an LB liquid medium or a TB liquid medium; and / or, the conditions for the culturing are: culturing with shaking at a temperature of 37±1°C.

8. An enzyme composition, characterized in that, The enzyme composition includes two or more of the nitrilase mutants as claimed in claim 1 or 2, or includes one or more of the nitrilase mutants as claimed in claim 1 or 2 and a wild-type nitrilase having the amino acid sequence shown in SEQ ID NO:

1.

9. Use of a wild-type nitrilase having the amino acid sequence shown in SEQ ID NO:1, the nitrilase mutant as claimed in claim 1 or 2, the isolated nucleic acid molecule as claimed in claim 3, the recombinant expression vector as claimed in claim 4, the transformant as claimed in claim 5 or 6 or the enzyme composition as claimed in claim 8 in the preparation of R-mandelic acid.

10. The application according to claim 9, characterized in that, In the preparation of R-mandelic acid, mandelonitrile is used as a substrate or hydrocyanic acid and benzaldehyde are used as substrates; Preferably, the mandelonitrile is racemic mandelonitrile.

11. A method for preparing R-mandelic acid, characterized in that, The preparation method includes: reacting at least any one of a wild-type nitrilase having the amino acid sequence shown in SEQ ID NO:1, the nitrilase mutant as claimed in claim 1 or 2 or the enzyme composition as claimed in claim 8 with a substrate to form a reaction system and reacting to obtain R-mandelic acid; the substrate is mandelonitrile, or hydrocyanic acid and benzaldehyde; Preferably, the mandelonitrile is racemic mandelonitrile.

12. The preparation method according to claim 11, characterized in that, The use form of the wild-type nitrilase, the nitrilase mutant or the enzyme composition is wet cells, cell powder, liquid enzyme, solid enzyme powder or immobilized enzyme; and / or, When the substrate is mandelonitrile, the added concentration of mandelonitrile in the reaction system is 0.05 - 50 mg / mL, preferably 0.1 - 10 mg / mL; When the substrates are hydrocyanic acid and benzaldehyde, the ratio of the total added amount of benzaldehyde in the reaction system to the volume of the reaction system is (100 - 150) mg:1 mL, such as 106 mg:1 mL or 112 mg:1 mL, and the molar ratio of hydrocyanic acid to benzaldehyde added is (1 - 1.5) mol:1 mol, such as 1.3 mol:1 mol; and / or, the pH value of the reaction is 6.5 - 8.5, preferably 7.0 - 8.0; and / or, the temperature of the reaction is 20 - 40 °C, preferably 28 - 35 °C.

13. The preparation method according to claim 11 or 12, characterized in that, The use form of the wild-type nitrilase, the nitrilase mutant or the enzyme composition is wet cells, and the mass ratio of the wet cells to the added substrate benzaldehyde in the reaction system can be (0.05 - 0.5) g:1 g; and / or, The use form of the nitrilase mutant or the enzyme composition is liquid enzyme; calculated according to the mass of the wet cells producing the nitrilase, the mass ratio of the wet cells to the added substrate mandelonitrile in the reaction system can be (5 - 10) mg:1 mg.

14. A reaction end product system for catalytically preparing mandelic acid from benzaldehyde and hydrocyanic acid as substrates, characterized in that, The reaction end product system includes: Mandelic acid, the percentage of which in the sum of the masses of mandelic acid and mandelonitrile in the reaction end product system is 99% - 100%; the ee value of the R-mandelic acid contained therein is 96% - 100%; such as 96% - 97%, 96% - 98% or 96% - 99%; and Mandelonitrile, the percentage of which in the sum of the masses of mandelic acid and mandelonitrile in the reaction end product system is 0 - 1%, such as 0 - 0.3%, 0 - 0.5%, 0 - 0.7% or 0 - 0.9%; Optionally, the reaction end product system further includes a wild-type nitrilase having the amino acid sequence shown in SEQ ID NO:1, a nitrilase mutant as described in claim 1 or 2, or an enzyme composition as described in claim 8.

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