Nitrilase and its use in the preparation of r-mandelic acid

Abstract

The application discloses a nitrilase and application of the nitrilase in preparation of R-mandelic acid. The nitrilase is a wild-type nitrilase or a nitrilase mutant. The amino acid sequence of the wild-type nitrilase is shown as SEQ ID NO:1. The nitrilase mutant comprises one or more amino acid residue differences in S192G, A197S or S285I compared with the amino acid sequence shown as SEQ ID NO:1. When the nitrilase is used to prepare 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] This invention relates to the field of biocatalysis, specifically to a nitrile hydrolase and its application in the preparation of R-mandelic acid. Background Technology

[0002] Mandelic acid is an important pharmaceutical intermediate and a major chiral resolving agent with wide applications in the pharmaceutical industry. Compared to racemic mandelic acid, drugs synthesized from monoform mandelic acid or its derivatives are not only more potent but also have fewer side effects. Therefore, the synthesis of many drugs requires monoform mandelic acid.

[0003] Among mono-configured mandelic acids, R-mandelic acid and its analogues are important chiral synthetic building blocks, widely used in the synthesis of various drugs, such as cephalosporins, penicillins, antitumor agents, anti-obesity drugs, optically pure amino acids, angiotensin-converting enzyme inhibitors, and coenzyme A. They can also be used as chiral resolving agents to separate other chiral drugs or pharmaceutical intermediates. Therefore, finding low-cost and simple methods for preparing R-mandelic acid has become a research hotspot.

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

[0005]

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

[0007] The biosynthetic preparation of R-mandelic acid can be broadly categorized into the following three methods:

[0008] (1) First, the racemic form of mandelic acid is synthesized, followed by esterification or ammonolysis to obtain mandelic acid esters or mandelic acid amides. Then, under the action of esterification hydrolases or amide hydrolases, a single enantiomeric mandelic acid is obtained. For example, Ganapati et al. first converted the racemic mandelic acid to methyl mandelic acid ester using an exchange resin catalyst, and then stereohydrolyzed it to R-mandelic acid using hydrolases from Candida albicans. However, the optical purity of the obtained R-mandelic acid was only 78%.

[0009] (2) Using phenylacetic acid as a substrate, chiral mandelic acid is synthesized directly by microorganisms with oxidoreductases, mostly forming R-mandelic acid. For example, Takao M et al. used reductases from Streptococcus, Candida, Enterococcus, Rhodotorula, and yeast to stereoreduc phenylacetic acid to R-mandelic acid, but the yield was low.

[0010] (3) Using benzaldehyde and hydrocyanic acid as raw materials, mandelic acid nitrile is first prepared, and then R-mandelic acid is obtained under the action of nitrile hydrolase. Currently reported microorganisms containing nitrile hydrolase that can catalyze the production of R-mandelic acid from mandelic acid mainly include: Alcaligenes faecalis ATCC 8750, Alcaligenes ECU0401, Pseudomonas putida MTCC 5110, etc. However, this method is difficult to industrialize due to the low activity of the microorganisms used. Summary of the Invention

[0011] The technical problem this invention aims to solve is the low conversion rate and ee value of R-mandelic acid prepared by current nitrile hydrolases. This invention provides a nitrile hydrolase and its application in the preparation of R-mandelic acid. When using the nitrile hydrolase of this invention to prepare R-mandelic acid, the substrate conversion rate is high, and the ee value of R-mandelic acid is also high, making it suitable for industrial production.

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

[0013] A first aspect of the present invention provides a nitrile hydrolase mutant, said nitrile hydrolase mutant containing one or more amino acid residues differing from the amino acid sequence shown in SEQ ID NO:1, namely S192G, A197S, or S285I.

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

[0015] In some embodiments, the nitrile hydrolase mutant differs from the amino acid sequence shown in SEQ ID NO:1 in the following amino acid residues:

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

[0017] In this 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 nitrile hydrolase mutant has an amino acid sequence as shown in any of SEQ ID NO:9-12.

[0019] A second aspect of the invention provides an isolated nucleic acid molecule that encodes a nitrile hydrolase mutant as described in the first aspect.

[0020] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence as shown in any of SEQ ID NO:13-16.

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

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

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

[0024] In some implementations, the host cells used in the construction of the transformant are Escherichia coli or Bacillus subtilis.

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

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

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

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

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

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

[0031] In some implementations, R-mandelic acid is prepared using mandelic nitrile as a substrate or using hydrogen cyanide and benzaldehyde as substrates.

[0032] In some implementations, mandenitrile is racemic mandenitrile.

[0033] The eighth aspect of the present invention provides a method for preparing R-mandelic acid, the method comprising: reacting at least one of a wild-type nitrile hydrolase having the amino acid sequence shown in SEQ ID NO:1, a nitrile hydrolase 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 react to obtain R-mandelic acid; wherein the substrate is mandelic nitrile, or hydrocyanic acid and benzaldehyde.

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

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

[0036] When the substrate is mandelic acid nitrile, the concentration of mandelic acid nitrile added to the reaction system is 0.05-50 mg / mL.

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

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

[0039] In some preferred embodiments, the ratio of the total amount of benzaldehyde added 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 hydrogen cyanide and benzaldehyde, the molar ratio of added hydrogen cyanide to benzaldehyde is (1-1.5) mol: 1 mol.

[0041] In some preferred embodiments, when the substrate is hydrogen cyanide and benzaldehyde, the molar ratio of added hydrogen cyanide to benzaldehyde is 1.3 mol: 1 mol.

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

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

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

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

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

[0047] In some embodiments, the nitrile hydrolase mutant or enzyme composition is used in the form of a liquid enzyme.

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

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

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

[0051] A ninth aspect of the present invention provides a reaction end product system for the catalytic synthesis of mandelic acid from benzaldehyde and hydrogen cyanide as substrates, the reaction end product system comprising:

[0052] Mandelic acid and mandelinnitriles; the mandelic acid constitutes 99%-100% of the total mass of mandelic acid and mandelinnitriles in the final product system; the ee value of R-mandelic acid contained in the mandelic acid is 96%-100%, for example, 96%-97%, 96%-98%, or 96%-99%; the mandelinnitriles constitute 0-1% of the total mass of mandelic acid and mandelinnitriles in the final product system, for example, 0-0.3%, 0-0.5%, 0-0.7%, or 0-0.9%; optionally, it also includes a wild-type nitrile hydrolase having the amino acid sequence shown in SEQ ID NO: 1, a nitrile hydrolase mutant as described in the first aspect, or an enzyme composition as described in the sixth aspect.

[0053] In some embodiments, a nitrile hydrolase having an amino acid sequence as shown in SEQ ID NO:1 is also included.

[0054] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention. All reagents and raw materials used in the present invention are commercially available.

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

[0056] The wild-type nitrile hydrolase and nitrile hydrolase mutant of the present invention exhibit high conversion rates and high ee values ​​for R-mandelic acid during preparation, making them suitable for industrial production.

[0057] In addition to being able to prepare R-mandelic acid using mandelic acid as a substrate, the wild-type nitrile hydrolase and nitrile hydrolase mutant of the present invention can also prepare R-mandelic acid using hydrocyanic acid and benzaldehyde as substrates. The substrates are widely available and inexpensive, and the enzyme catalysis conditions are mild, with virtually no or few byproducts. Therefore, they are suitable for large-scale industrial production. Detailed Implementation

[0058] The present invention is further illustrated below by way of examples, but the invention is not limited to the scope of the examples described. Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions, or as selected in the product instructions.

[0059] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

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

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

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

[0063] LB solid medium: 10 g / L trypsin, 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] pET28a plasmid was purchased from Novagen.

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

[0067] The measurement method of the present invention is as follows:

[0068] 1. Conversion rate calculation:

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

[0070] Table 1 Gradient elution parameters

[0071] Time (minutes) 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 in the test are shown below:

[0073] 1.1 Blank solution / diluent: methanol;

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

[0075] 1.3 Test solution: After the reaction is completed, take the final reaction solution, remove the enzyme, extract, and dry it, and accurately weigh the same mass of the dried substance as the control substance. Place it in a volumetric flask, dissolve and dilute it to the mark with diluent, and shake well to obtain the test solution.

[0076] The conversion rate is calculated using the formula: Conversion rate = Molar amount of mandelic acid / (Molar amount of mandelic acid + Molar amount of mandelinnitrile)

[0077] ×100%. The mass content of mandelic acid and mandelic nitrile was calculated using the area normalization method, and the molar amount was converted according to the mass content.

[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: column: Daicel Chiralpak AD-H (4.6 mm × 250 mm, 5 μm); mobile phase composition: n-heptane (chromatographic grade): isopropanol (chromatographic grade): trifluoroacetic acid (chromatographic grade) = 95:5:0.1 (v / v); column temperature: 25℃; detection wavelength: 210 nm; mobile phase flow rate: 1 mL / min; injection volume: 10 μL.

[0080] The reagents used in the test are shown below:

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

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

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

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

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

[0086] Example 1: Preparation of crude nitrile hydrolase solution

[0087] 1.1 Screening and Acquisition of Enzyme Genes

[0088] Several nitrile hydrolases that can be used to prepare R-mandelic acid were screened from the NCBI database. Their sources and sequences are shown in Table 2.

[0089] 1.2 Transformation of enzyme genes

[0090] The aforementioned nitrile hydrolase gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and cloned into the expression vector pET28a using the NdeI & HindIII restriction sites to obtain a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells to obtain genetically engineered bacteria.

[0091] 1.3 Culture of the strain

[0092] After activating the genetically engineered bacteria by streak plating, a single colony was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C with shaking for 12 h. Then, at a 2% (v / v) inoculation rate, the culture was transferred to 150 mL of fresh TB liquid medium also containing 50 μg / mL kanamycin and incubated at 37°C with shaking until OD (out of control) was reached. 600When the concentration reaches approximately 0.8, the temperature is lowered to 30°C, and IPTG is added to a final concentration of 0.5 mM. The mixture is then induced and cultured at 30°C for 16 hours. After culturing, the culture medium is centrifuged at 10,000 rpm for 10 minutes, the supernatant is discarded, and the precipitate is collected to obtain wet cells containing the nitrile hydrolase gene. These wet cells are stored at -20°C for later use.

[0093] 1.4 Preparation of crude nitrile hydrolase solution

[0094] Take 10g of wet nitrile hydrolase cells, resuspend them in 100mL of 0.05M pH7.0 sodium phosphate buffer (homogenization ratio 1:10), and homogenize under high pressure to obtain crude nitrile hydrolase solution.

[0095] Example 2: Screening of crude nitrile hydrolase solution

[0096] A 1 mL enzyme-catalyzed reaction system was prepared. This system contained the following components: 1.33 mg racemic mandelic acid nitrile (molecular weight 133.15, 0.01 mmol), 50 μL methanol, 0.1 mL of any one of the crude enzyme solutions of various nitrile hydrolases from Example 1 (equivalent to 0.01 g of wet nitrile hydrolases), and 0.85 mL of 100 mM Tris-HCl (pH 7.5). The 1 mL enzyme-catalyzed reaction system was placed at 37 °C and 220 rpm with shaking for 16 h, after which the reaction was terminated 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, vortex to mix, centrifuge and remove the precipitate (containing nitrile hydrolase), take the supernatant, and use HPLC to determine the mandelic acid (including S-mandelic acid) in the final reaction solution. and R-mandelic acid The conversion rate is calculated based on the mass content of the product.

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

[0100] Table 2 Sequence listing of nitrile hydrolases

[0101] enzyme name amino acid sequence Conversion rate R-mandelic acid ee value 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] Based on the results in Table 2, nitrile hydrolases with high conversion rates and ee values ​​were finally selected. The NCBI accession number for this nitrile hydrolase, Enz.3, is WP_012492804.1. It 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 catalysis of crude enzyme solution of nitrile hydrolase Enz.3 using racemic mandelic nitrile as substrate.

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

[0105] A 50 mL enzyme-catalyzed reaction system was prepared. This system contained the following components: 66.5 mg racemic mandelic acid nitrile (0.5 mmol), 2.5 mL methanol, 5 mL crude Enz.3 nitrile hydrolase solution (equivalent to 0.5 g Enz.3 nitrile hydrolase wet cells), and 42.5 mL 100 mM Tris-HCl (pH 7.5). The 50 mL enzyme-catalyzed reaction system was placed at 37 °C and shaken at 220 rpm for 16 h. The reaction was then terminated to obtain the final reaction solution.

[0106] Take 100 μL of the final reaction solution, add 900 μL of methanol, shake to mix, centrifuge and remove the precipitate (containing nitrile hydrolase), take the supernatant, and use HPLC to determine the mass content of mandelic acid and mandelic acid in the final reaction solution. The conversion rate was calculated 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, shake and centrifuge, take the organic phase, evaporate the solvent in the organic phase to dryness, and determine the ee value of R-mandelic acid by HPLC, which is 87.54%.

[0108] Example 4: Preparation of R-mandelic acid catalyzed by whole-cell nitrile hydrolase using hydrogen cyanide and benzaldehyde as substrates.

[0109] Example 4-1

[0110] At room temperature, 240g of water was added to a 500mL four-necked flask, along with 4.5g of wetted nitrile hydrolase cells (Enz.3 bacterial sludge) obtained in Example 1.3, and the mixture was stirred for 20 minutes. 9.84g of hydrogen cyanide (molecular weight 27.03; 0.36mol) was added, and the pH of the reaction system was adjusted to 7.85 with liquid alkali. The temperature was controlled at 28–35℃, and then 30.0g of benzaldehyde (molecular weight 106.12; 0.28mol) was slowly added dropwise, controlling the dropping rate, and the addition was completed in approximately 2 hours. The mixture was then kept at 28–35℃ for another 2 hours. The ratio of the total added benzaldehyde mass to the final total volume of the reaction system was approximately 112mg:1ml. The final reaction solution was then collected, and the mass content of each component in the final reaction solution was determined by HPLC.

[0111] HPLC analysis showed that the retention time of mandelic acid was 4.557 min, and no benzaldehyde or mandelic nitrile impurities were found in the final reaction solution. Calculations showed that the conversion rate (based on the amount of mandelic nitrile) was 100%.

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

[0113] The reaction route in this embodiment is shown in the following equation:

[0114]

[0115] Example 4-2

[0116] At room temperature, 240g of water was added to a 500mL four-necked flask, along with 5.625g of wetted nitrile hydrolase cells (Enz.3 bacterial sludge) obtained in Example 1, Section 1.3. The mixture was stirred for 20 minutes, and then 9.84g (0.36mol) of hydrogen cyanide was added. The pH of the reaction system was adjusted to 7.85 using liquid alkali. The temperature was controlled at 28–35°C, and 30.0g (0.28mol) of benzaldehyde was slowly added dropwise over approximately 2 hours. The mixture was then kept at 28–35°C for another 2 hours. The ratio of the total added benzaldehyde mass to the final total volume of the reaction system was approximately 112mg:1ml. The final reaction solution was then collected, and the mandelic acid content in the final reaction solution was determined by HPLC.

[0117] Tests showed that the final reaction solution contained no benzaldehyde or mandelic acid impurities, the conversion rate (calculated based on the amount of mandelic acid) was 100%, and the ee value of R-mandelic acid was 96.78%.

[0118] As can be seen from the above examples, when the amount of benzaldehyde added is 0.28 mol, the amount of hydrogen cyanide added is 0.36 mol, and the amount of wet nitrile hydrolase added is 4.5 g, it is sufficient to completely convert benzaldehyde.

[0119] Example 5: Preparation of whole-cell nitrile hydrolase mutant

[0120] This embodiment provides a method for preparing a nitrile hydrolase mutant, which includes the following steps:

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

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

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

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

[0125] Table 3 Primer list for gene mutation

[0126] Primer name Primer sequence Serial Number S192G-F CCGAGCTTCGGACTGTACGCGGGCGCGGCGTAC SEQ ID NO:3 S192G-R CGCGTACAGTCCGAAGCTCGGCCACGCCCGAT 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 PCR reagent was sourced from Takara, catalog number R045A.

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

[0129] Table 4. PCR amplification system of whole plasmid

[0130] reagents 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 procedure is shown in Table 5:

[0132] Table 5 PCR amplification program

[0133]

[0134] (4) The PCR products obtained from single-point or double-point mutations were digested with DpnI enzyme at 37°C for 2 hours. After digestion, the products were transformed into E. coli BL21(DE3) competent cells and then plated on LB agar containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies were picked and sequenced using the Sanger method. The sequences of the successfully mutated nitrile hydrolase mutants are shown in Table 6.

[0135] Table 6 Sequence listing of nitrile hydrolase 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 refers to the mutation of serine (S) at position 192 of wild-type nitrile hydrolase E0 to glycine (G). The same applies to other sites.

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

[0139] Example 6: Preparation of R-mandelic acid catalyzed by whole-cell nitrile hydrolase mutant using hydrogen cyanide and benzaldehyde as substrates.

[0140] Example 6-1 Preparation of R-mandelic acid catalyzed by whole-cell nitrile hydrolase mutant in 240g water system

[0141] At room temperature (25℃, the same below), 240g of water was added to a 500mL four-necked flask, along with 4.5g of any of the bacterial sludge obtained in Example 5. The mixture was stirred for 20 minutes, and then 9.84g of hydrogen cyanide (molecular weight 27.03; 0.36mol) was added. The pH of the reaction system was adjusted to 7.85 with liquid alkali, and the temperature was controlled at 28–35℃. 30.0g of benzaldehyde (molecular weight 106.12; 0.28mol) was slowly added dropwise, controlling the dropping rate, and the addition was completed in approximately 2 hours. The mixture was then kept at 28–35℃ for another 2 hours. The ratio of the total added benzaldehyde mass to the final total volume of the reaction system was approximately 112mg:1ml. The final reaction solution was then collected, and the mass content of each component in the final reaction solution was determined by HPLC.

[0142] The percentages of mandelic acid and mandelic nitrile in the final reaction solution of different whole-cell nitrile hydrolase mutant bacterial sludge were determined (as shown in Table 7), and the conversion rate was calculated (based on the amount of mandelic nitrile). The retention time of mandelic acid was 4.569 min, and the retention time of mandelic nitrile was 6.044 min.

[0143] The calculated ee values ​​of R-mandelic acid are 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 catalyzed by whole-cell nitrile hydrolase mutant in 80g water system

[0145] At room temperature, 80g of water and 4g of any of the bacterial sludge obtained in Example 5 were added to a 250mL four-necked flask. The mixture was stirred for 20 minutes, and then 3.3g (0.12mol) of hydrogen cyanide was added. The pH of the reaction system was adjusted to 7.85 with liquid alkali. The temperature was controlled at 28–35℃, and 10.0g (0.094mol) of benzaldehyde was slowly added dropwise over approximately 2 hours. The mixture was then kept at 28–35℃ for another 2 hours. The ratio of the total added benzaldehyde mass to the final total volume of the reaction system was approximately 106mg:1ml. The final reaction solution was then collected, and the content of each component in the final reaction solution was determined by HPLC.

[0146] The percentage of mandelic acid and mandelic nitrile in the final reaction solution of different whole-cell nitrile hydrolase mutant bacterial sludge was obtained by detection (as shown in Table 7), and the conversion rate was calculated (based on the amount of mandelic nitrile).

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

[0148] Table 7 Catalytic Results of Mutant Whole-Cell Nitrile Hydrolases

[0149]

[0150] The results above show that the ee value of R-mandelic acid is increased in the whole-cell nitrile hydrolase mutant compared to the whole-cell nitrile hydrolase.

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

[0152] SEQ ID NO:1 Nitrile hydrolase Enz.3 amino acid sequence

[0153] MTINHPRYVVAAVQAAPVFLDLEATVTKTIELIEEAARNGATLIAFPETWIPGYPLFSWLGSPAWSLQFFQRYHDNSLVINSEQYRLIEQAAARNKIMVVLGFSERDAGSLYISQSIINSEGITISTRRKLKPTHVERTVFGEGDGSDLSVHETELGRVGALCCWEHLQPLTRYAMFAQNEQVHIGAWPSFSLYAGAAYTLGPEVNTAVSQIYAVEGQCFVVAPSAVVSEQMIELLCSTPEHHALLQAGGGHARIFGPDGRSLAEPIPENVEGILYAEIDLSVISLAKAAADPAGHYSRPDVTRLLLDPTPKSRVVHVRAEPAAPEMQPATAVVQVDQPTEPLERVTPA

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

[0155]

[0156] SEQ ID NO:9S192G

[0157] MTINHPRYVVAAVQAAPVFLDLEATVTKTIELIEEAARNGATLIAFPETWIPGYPLFSWLGSPAWSLQFFQRYHDNSLVINSEQYRLIEQAAARNKIMVVLGFSERDAGSLYISQSI INSEGITISTRRKLKPTHVERTVFGEGDGSDLSVHETELGRVGALCCWEHLQPLTRYAMFAQNEQVHIGAWPSFGLYAGAAYTLGPEVNTAVSQIYAVEGQCFVVAPSAVVSEQMI ELLCSTPEHHALLQAGGGHARIFGPDGRSLAEPIPENVEGILYAEIDLSVISLAKAAADPAGHYSRPDVTRLLLDPTPKSRVVHVRAEPAAPEMQPATAVVQVDQPTEPLERVTPA

[0158] SEQ ID NO:10 A197S

[0159] MTINHPRYVVAAVQAAPVFLDLEATVTKTIELIEEAARNGATLIAFPETWIPGYPLFSWLGSPAWSLQFFQRYHDNSLVINSEQYRLIEQAAARNKIMVVLGFSERDAGSLYISQSIINSEGITISTRRKLKPTHVERTVFGEGDGSDLSVHETELGRVGALCCWEHLQPLTRY AMFAQNEQVHIGAWPSFSLYAGSAYTLGPEVNTAVSQIYAVEGQCFVVAPSAVVSEQMIELLCSTPEHHALLQAGGGHARIFGPDGRSLAEPIPENVEGILYAEIDLSVISLAKAAADPAGHYSRPDVTRLLLDPTPKSRVVHVRAEPAAPEMQPATAVVQVDQPTEPLERVTPA

[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 nitrile hydrolase mutant, characterized in that, The nitrile hydrolase mutant differs from the amino acid sequence shown in SEQ ID NO: 1 by A197S or A197S / S285I.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nitrile hydrolase mutant as described in claim 1.

3. The nucleic acid molecule as described in claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 14 or 16.

4. A recombinant expression vector comprising the isolated nucleic acid molecule as described in claim 2 or 3.

5. The recombinant expression vector as described in claim 4, characterized in that, The backbone of the recombinant expression vector is either the pET28a plasmid or the pET21a plasmid.

6. A transformant comprising the isolated nucleic acid molecule as described in claim 2 or 3, or the recombinant expression vector as described in claim 4 or 5.

7. The transformant as described in claim 6, characterized in that, The host cell used in the construction of the transformant was *Escherichia coli* (E. coli). Escherichia coli ) or Bacillus subtilis ( Bacillus subtilis ).

8. The transformant as described in claim 7, characterized in that, The Escherichia coli mentioned is Escherichia coli BL21(DE3).

9. A method for preparing the nitrile hydrolase mutant as described in claim 1, comprising culturing the transformant as described in any one of claims 6-8 to obtain a fermentation product, and obtaining the nitrile hydrolase mutant from the fermentation product.

10. The method as described in claim 9, characterized in that, The culture medium used for the culture is selected from LB liquid medium or TB liquid medium; and / or, The culture conditions were: shaking culture at a temperature of 37±1℃.

11. An enzyme composition, characterized in that, The enzyme composition comprises two nitrile hydrolase mutants, the amino acid sequences of which differ from the amino acid sequence shown in SEQ ID NO: 1 as A197S and A197S / S285I, respectively; or The enzyme composition comprises a nitrile hydrolase mutant with an amino acid sequence differing from that shown in SEQ ID NO: 1 by A197S and a wild-type nitrile hydrolase with an amino acid sequence shown in SEQ ID NO: 1; or The enzyme composition comprises a nitrile hydrolase mutant with an amino acid sequence differing from that shown in SEQ ID NO: 1 by A197S and a nitrile hydrolase mutant with an amino acid sequence differing from that shown in SEQ ID NO: 1 by S192G or S285I; or The enzyme composition comprises a nitrile hydrolase mutant with an amino acid sequence differing from that shown in SEQ ID NO: 1 by A197S / S285I and a wild-type nitrile hydrolase with an amino acid sequence shown in SEQ ID NO: 1; or The enzyme composition comprises a nitrile hydrolase mutant with an amino acid sequence difference of A197S / S285I compared to that shown in SEQ ID NO: 1 and a nitrile hydrolase mutant with an amino acid sequence difference of S192G or S285I compared to that shown in SEQ ID NO:

1.

12. The use of the nitrile hydrolase mutant of claim 1, the isolated nucleic acid molecule of claim 2 or 3, the recombinant expression vector of claim 4 or 5, the transformant of any one of claims 6-8, or the enzyme composition of claim 11 in the preparation of R-mandelic acid.

13. The application as described in claim 12, characterized in that, The preparation of R-mandelic acid uses mandelic nitrile as a substrate or hydrocyanic acid and benzaldehyde as substrates.

14. The application as described in claim 13, characterized in that, The amygdalinone is a racemic amygdalinone.

15. A method for preparing R-mandelic acid, characterized in that, The preparation method includes: reacting at least one of the nitrile hydrolase mutant as described in claim 1 or the enzyme composition as described in claim 11 with a substrate to form a reaction system and obtain R-mandelic acid; the substrate is mandelic nitrile, or hydrocyanic acid and benzaldehyde.

16. The preparation method according to claim 15, characterized in that, The amygdalinone is a racemic amygdalinone.

17. The preparation method according to claim 15 or 16, characterized in that, The nitrile hydrolase mutant or the enzyme composition is used in the form of wet cells, liquid enzyme, or solid enzyme powder; and / or, When the substrate is mandelic acid nitrile, the concentration of mandelic acid nitrile added to the reaction system is 0.05-50 mg / mL; When the substrate is hydrogen cyanide and benzaldehyde, the ratio of the total amount of benzaldehyde added to the volume of the reaction system is (100-150) mg:1 mL, and the ratio of the molar amounts of hydrogen cyanide and benzaldehyde added is (1-1.5) mol:1 mol. And / or, the pH of the reaction is 6.5-8.5; And / or, the temperature of the reaction is 20-40°C.

18. The preparation method according to claim 15 or 16, characterized in that, The nitrile hydrolase mutant or the enzyme composition is used as an immobilized enzyme; and / or, When the substrate is mandelic acid nitrile, the concentration of mandelic acid nitrile added to the reaction system is 0.05-50 mg / mL; When the substrate is hydrogen cyanide and benzaldehyde, the ratio of the total amount of benzaldehyde added to the volume of the reaction system is (100-150) mg:1 mL, and the ratio of the molar amounts of hydrogen cyanide and benzaldehyde added is (1-1.5) mol:1 mol. And / or, the pH of the reaction is 6.5-8.5; And / or, the temperature of the reaction is 20-40°C.

19. The preparation method according to claim 17, characterized in that, When the substrate is mandelic acid nitrile, the concentration of mandelic acid nitrile added to the reaction system is 0.1-10 mg / mL; When the substrate is hydrogen cyanide and benzaldehyde, the ratio of the total amount of benzaldehyde added to the volume of the reaction system is 106 mg:1 mL or 112 mg:1 mL, and the molar ratio of hydrogen cyanide to benzaldehyde added is 1.3 mol:1 mol. And / or, the pH of the reaction is 7-8; And / or, the temperature of the reaction is 28-35°C.

20. The preparation method according to claim 15, characterized in that, The nitrile hydrolase mutant or enzyme composition is used in the form of wet bacterial cells, and the mass ratio of the wet bacterial cells to the benzaldehyde added in the reaction system is (0.05-0.5) g:1 g; and / or, When calculating based on the mass of the wet bacterial cells that produce the nitrile hydrolase, the mass ratio of the wet bacterial cells to the added amylopectin in the reaction system is (5-10) mg:1 mg.

21. A reaction end product system for the catalytic synthesis of mandelic acid from benzaldehyde and hydrogen cyanide as substrates, characterized in that, The reaction end product system includes: Mandelic acid, comprising 99%-100% of the total mass of mandelic acid and mandelic nitrile in the final product system of the reaction, wherein the percentage is not 100%; wherein the ee value of R-mandelic acid is 96%-100%; and Mandelinnitrile, which accounts for 0-1% of the sum of the mass of mandelic acid and mandelinnitrile in the final product system of the reaction, and the percentage is not 0; Furthermore, the final product system of the reaction also includes the nitrile hydrolase mutant as described in claim 1 or the enzyme composition as described in claim 11.

22. The reaction end product system as described in claim 21, characterized in that, The ee value of the R-mandelic acid is 96%-99%; and Mandelinnitrile, comprising 0-0.9% of the total mass of mandelic acid and mandelinnitrile in the final product system of the reaction.

23. The reaction end product system as described in claim 21, characterized in that, The ee value of the R-mandelic acid is 96%-98%; and Mandelinnitrile, comprising 0-0.7% of the total mass of mandelic acid and mandelinnitrile in the final product system of the reaction.

24. The reaction end product system as described in claim 21, characterized in that, The ee value of the R-mandelic acid is 96%-97%; and Mandelinnitrile, comprising 0-0.5% of the total mass of mandelic acid and mandelinnitrile in the final product system of the reaction.

25. The reaction end product system as described in claim 24, characterized in that, The percentage of mandelic acid and mandelic acid in the final product system is 0-0.3%.

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