A nitrilase mutant and its application in the synthesis of 2-chloronicotinic acid

Abstract

The present invention discloses a nitrilase mutant and its application in the synthesis of 2-chloronicotinic acid, wherein the nitrilase mutant is obtained by single mutation or multiple mutation of the 146th or 194th amino acid of the nitrilase amino acid sequence shown in SEQ ID No.2. The nitrilase mutant of the present invention is greatly improved in activity compared with the wild type, and when a crude extract of the enzyme or a whole cell of an engineered bacterium is used for catalysis, the reaction enzyme activity remains at a high state. In addition, the nitrilase mutant of the present invention can adapt to a catalytic temperature of 30 to 45 ° C. The nitrilase mutant of the present invention is more than 80 times more active than the parent and has a yield increased by 23.2 times, laying the foundation for the industrial enzymatic synthesis of 2-chloronicotinic acid.

Description

(1) Technical field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to the transformation of a nitrilase derived from Gibberella and its application in catalytic synthesis of 2-chloronicotinic acid. (2) Background technology

[0002] 2-Chloronicotinic acid is an important building block for pesticides and pharmaceutical chemicals, with broad application prospects. In the pesticide field, it can be used to synthesize a variety of fungicides, insecticides, and herbicides, such as the sulfonylurea herbicide nicosulfuron, the amide fungicide boscalid, and a series of triazolethione fungicidal compounds. In the pharmaceutical field, it can be used to synthesize antibiotics and cardiovascular disease treatment drugs, such as the anti-AIDS drug nevirapine, the antidepressant mirtazapine, the anti-inflammatory drug pranoprofen, and the anti-inflammatory analgesics niflumic acid and nicotinic acid.

[0003] Due to the widespread application of 2-chloronicotinic acid in pharmaceuticals and pesticides, the development of its synthesis methods has become a research hotspot. Lonza, Switzerland, first applied for the first synthesis patent (US4144238) in 1977, synthesizing 2-chloronicotinic acid from nicotinic acid via nitrogen oxidation, chlorination, and hydrolysis. Subsequently, Japanese Organic Synthesis and Koei Chemical, Japan, among others, have pursued synthetic methods (JP59144759 and JP56169672). Currently, methods for synthesizing 2-chloronicotinic acid include using 3-cyanopyridine as a raw material via nitrogen oxidation, chlorination, and hydrolysis; using 2-chloro-3-trifluoromethylpyridine as a raw material via chlorination and hydrolysis; and using ethyl cyanoacetate as a raw material via chlorination, Michael addition, and hydrolysis. However, all of these routes suffer from drawbacks such as difficult raw material preparation, numerous byproducts, and significant waste discharge. Therefore, developing a green and efficient synthesis process for 2-chloronicotinic acid is of great significance.

[0004] Nitrilases (EC 3.5.5.1) are important enzymes for the synthesis of carboxylic acids, hydrolyzing nitriles to form carboxylic acids. Nitrilase-mediated biocatalytic processes offer advantages such as high atom economy and environmental friendliness, making them an important alternative to traditional synthetic methods and a green manufacturing approach. For example, the nitrilase from Acidovorax facilis ZJB09122 catalyzes the synthesis of 1-cyanocyclohexylacetic acid, a key intermediate of gabapentin, from 1-cyanocyclohexylacetonitrile with high regioselectivity (ZL201710396386.6; ZL202010127927.7). The nitrilase from Arabidopsis thaliana catalyzes the synthesis of (S)-3-cyano-5-methylhexanoic acid, a key chiral intermediate of pregabalin, from isobutylsuccinonitrile with high stereoselectivity and regioselectivity (US10100297B2; ZL201810765047.5).

[0005] However, currently discovered nitrilases that catalyze the hydrolysis of 2-chloronicotinonitrile to 2-chloronicotinic acid generally suffer from low activity and poor reaction selectivity. The present invention utilizes protein engineering technology to molecularly modify nitrilases, constructing a nitrilase that can efficiently catalyze the conversion of 2-chloronicotinonitrile to 2-chloronicotinic acid. This is of great significance for the development of a green synthesis process for 2-chloronicotinic acid. (3) Summary of the invention

[0006] The present invention aims to provide a nitrilase mutant and its application in catalyzing the synthesis of 2-chloronicotinonitrile into 2-chloronicotinic acid. The nitrilase (Gi-Nit) gene sequence from Gibberella intermedia is molecularly modified by site-directed saturation mutagenesis to improve its hydrolysis activity towards 2-chloronicotinonitrile, thereby facilitating the application of the nitrilase in the industrial preparation of 2-chloronicotinic acid and solving the problems of low activity and poor reaction selectivity commonly encountered in existing methods for catalyzing the hydrolysis of 2-chloronicotinonitrile into 2-chloronicotinic acid by nitrilase.

[0007] The technical solution adopted in the present invention is:

[0008] The present invention provides a nitrilase mutant, which is obtained by subjecting the 146th or 194th amino acid of the nitrilase amino acid sequence shown in SEQ ID No. 2 to single or multiple mutations.

[0009] Preferably, the nitrilase mutant is a mutant in which the amino acid shown in SEQ ID No. 2 is mutated to one of the following: (1) valine (V) at position 194 is replaced by aspartic acid (D), the nucleotide sequence is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4; (2) valine (V) at position 194 is replaced by leucine (L), the nucleotide sequence is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 6; (3) leucine (L) at position 146 is replaced by phenylalanine (F), the nucleotide sequence is shown in SEQ ID No. 7, and the amino acid sequence is shown in SEQ ID No. 8; (4) leucine (L) at position 146 is replaced by serine (S), the nucleotide sequence is shown in SEQ ID No. 9, and the amino acid sequence is shown in SEQ ID No. 10; (5) valine (V) at position 194 is mutated to aspartic acid (D) and leucine (L) at position 146 is mutated to phenylalanine (F), the nucleotide sequence is shown in SEQ ID No. 11, and the amino acid sequence is shown in SEQ ID No. 12; (6) the valine (V) at position 194 is mutated to aspartic acid (D), and the leucine (L) at position 146 is mutated to serine (S), the nucleotide sequence is shown in SEQ ID No. 13, and the amino acid sequence is shown in SEQ ID No. 14.

[0010] The present invention also relates to a coding gene of the nitrilase mutant, a recombinant vector containing the coding gene, and a recombinant genetic engineering bacterium constructed by the recombinant vector. The recombinant vector preferably uses pET28a as a basic vector and preferably uses E. coli BL21 (DE3) as a host bacterium.

[0011] The present invention also provides a use of the mutant nitrilase in catalyzing the conversion of 2-chloronicotinonitrile to 2-chloronicotinic acid. The biocatalyst comprises wet cells obtained by centrifuging the culture broth of an engineered bacterium containing the mutant nitrilase encoding gene in a shake flask, or an enzyme extracted from the crushed wet cells. The biocatalyst comprises 2-chloronicotinonitrile as a substrate and a buffer solution having a pH of 7.0 to 7.5 (preferably pH 7.2) as a reaction medium to form a conversion system. The conversion reaction is carried out at 30 to 45° C. and 100 to 1000 rpm (preferably 30° C. and 600 rpm). After the reaction, the reaction solution is separated and purified to obtain 2-chloronicotinic acid. The substrate is added to the reaction system at a concentration of 50 to 1500 mM (preferably 1200 mM), and the catalyst is used in an amount of 1 to 5 g / L (preferably 3 g / L) based on the dry weight of the bacterial cells.

[0012] Preferably, the buffer is KH2PO4-K2HPO4 buffer (100 mM, pH 7.2).

[0013] Preferably, the substrate is added in batches, with an initial concentration of 50-300 mM (preferably 200 mM), and 50-300 mM (preferably 300 mM) added every 0.5-2 h (preferably 1.5 h) to a total addition of 50-1500 mM. The reaction time is 5-20 h (preferably 15 h).

[0014] Preferably, the wet cell is prepared as follows: the engineered bacteria containing the nitrilase mutant encoding gene is inoculated into LB medium containing a final concentration of 50 mg / L kanamycin, cultured at 37°C and 180 r / min for 12 h, then transferred to a fresh LB medium containing a final concentration of 50 mg / L kanamycin at a volume concentration of 2%, and cultured at 37°C and 180 r / min until the cell concentration OD 600 The pH value is 0.4-0.8, and IPTG is added to the culture medium at a final concentration of 0.1-1 mM (preferably 0.1 mM). The culture is induced at 28°C and 180 rpm for 12 hours. The culture is centrifuged and the precipitate is collected to obtain wet cells. The composition of LB liquid culture medium is (g / L): peptone 10, yeast extract 5, NaCl 10, solvent is water, pH 7.0; the composition of LB plate culture medium is (g / L): peptone 10, yeast extract 5, NaCl 10, agar 20, solvent is water, pH 7.0.

[0015] The nitrilase mutants of the present invention can be used in the form of whole engineered bacterial cells, in the form of unpurified crude enzymes, or in the form of partially purified or completely purified enzyme proteins. If desired, the nitrilase mutants of the present invention can also be used in the form of immobilized enzymes or immobilized cells using immobilization techniques known in the art.

[0016] Compared with the prior art, the present invention exhibits significant advantages: the nitrilase mutant exhibits significantly increased activity compared to the wild-type, maintaining high enzyme activity even when using crude extracts or whole cells of the engineered bacteria for catalysis. Furthermore, the nitrilase mutant can adapt to catalytic temperatures ranging from 30°C to 45°C. Compared to the parent enzyme, the nitrilase mutant exhibits over 80-fold increased activity and 23.2-fold increased yield, laying the foundation for the industrialized enzymatic synthesis of 2-chloronicotinic acid. (IV) Description of the accompanying drawings

[0017] Figure 1 , the fed-batch reaction process of 2-chloronicotinonitrile to 2-chloronicotinic acid catalyzed by mutant L146F / V194D whole cells. (V) Specific implementation methods

[0018] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0019] The composition of LB liquid medium is (g / L): peptone 10, yeast extract 5, NaCl 10, the solvent is water, and the pH value is 7.0.

[0020] The composition of LB plate medium is (g / L): peptone 10, yeast extract 5, NaCl 10, agar 20, the solvent is water, and the pH value is 7.0.

[0021] Example 1. Expression of wild-type nitrilase

[0022] Construction of a wild-type nitrilase-producing strain: A nitrilase gene (Gi-Nit, Genbank: JN236216) from Gibberella intermedia was synthesized; the nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2. The nitrilase gene was ligated between the NcoI and HindIII sites of the pET-28a plasmid and transformed into competent E. coli BL21(DE3) cells to obtain the wild-type nitrilase-producing strain E. coli BL21(DE3) / pET-28a-Gi-Nit.

[0023] SEQ ID No.1

[0024] ATGAGCAAAACGCTGAAAGTTGCGGCGATTCAGGCGGAACCGGTGTGGAACGACCTGCAGGGCGGGGTGAACAAAAGCATTGGGCTGATTCAGGAAGCAGCGAAAAATGGGGCAAATGTGATTGGATTTCCGGAGGTTTTTATTCCGGGTTATCCGTGGAGCATTTGGGCAAACAGCCCTACAGAAAATGCACCGTGGGTTAATGAATATTTTAAAAATAGCCTGGAGCGTGAAAGTCCGGAAATGGATCAGATTCGTGCGGCAGTTCGTGAAGCGGGAGTTTTTGTTGTGCTGGGTTATAGTGAACGTTATCGTGGTACCCTGTATATTGCACAGAGCTTTATTGATGAGACAGGTACGATTGTTCTGCATCGTCGTAAAATTAAACCGACCCATGTTGAGCGTGCAATTTATGGTGATGGACAAGGTGAAAGCCTGACCAACGTTGCAGACACCAAATTTGGCCGCGTTGCTGGTCTGAACTGTTGGGAACATACCCAAACACTGCTGCGTTATTATGAATATAGCCAGGATGTTGATATCCATGTGTCATCCTGGCCGAGTATTTTTCCGCAGAATGTTCCTGAATGGCCGTATCATATTACCCCTGAATGCTGTAAAGCATTTTCTCACGTTGTTTCTATGGAAGGTGCATGTTTTGTTCTGCTGGCAAGCCAAATTATGACGGAAGAAAACCATAAAAAGGCGAATGTTGAAGGTTATGATTATACGAAAAAGAGTGGTGGTGGTTTTAGTATGATTTTTTCTCCGTTTGGTGAGGAACTGGTTAAACCGCTGGCCCCGAATGAAGAAGGTATCCTGTATGCCGATATTAATCTGGAGGAGAAATATAAAGCGAAACAGAATCTGGATATCGTGGGTCATTATAGCCGTCCGGATCAACTGAGCCTGCGTGTTAACAAACATGCAGCAAAACCGGTGTTTTTTGCAAACGATCTG。

[0025] SEQ ID No.2

[0026] MSKTLKVAAIQAEPVWNDLQGGVNKSIGLIQEAAKNGANVIGFPEVFIPGYPWSIWANSPTENAPWVNEYFKNSLERESPEMDQIRAAVREAGVFVVLGYSERYRGTLYIAQSFIDETGTIVLHRRKIKPTHVERAIYGDGQGESLTNVADTKFGRVAGL NCWEHTQTLLRYYEYSQDVDIHVSSWPSIFPQNVPEWPYHITPECCKAFSHVVSMEGACFVLLASQIMTEENHKKANVEGYDYTKKSGGGFSMIFSPFGEELVKPLAPNEEGILYADINLEEKYKAKQNLDIVGHYSRPDQLSLRVNKHAAKPVFFANDL.

[0027] Expression of wild-type nitrilase gene engineered bacteria: Take 10 μL of wild-type nitrilase gene engineered bacteria from a glycerol tube stored in a -80°C refrigerator and inoculate into 10 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37°C, 200 rpm overnight. Transfer the inoculum to 100 mL of fresh LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 2%. Continue to culture until OD 600 The pH value was 0.4 to 0.8, IPTG was added to a final concentration of 0.1 mM, and the cells were induced at 28°C for 12 hours. After the culture was completed, the cells were centrifuged at 8000 rpm for 10 minutes at 4°C, and the cells were collected and washed twice with 0.9% saline to obtain wild-type nitrilase genetically engineered bacterial cells.

[0028] Example 2: Saturation Mutation and Screening of Nitrilase

[0029] 1. Saturation mutation

[0030] Through homology modeling and molecular docking, we screened and identified the key amino acid site V194 that affects nitrilase activity. Saturation mutagenesis was performed on the amino acid at position 194 in the amino acid sequence of the wild-type nitrilase from Example 1. Primers V194 (see Table 1) were designed and amplified using the pET28a-Gi-Nit plasmid harboring the wild-type nitrilase Gi-Nit gene (nucleotide sequence SEQ ID No. 1) as a template.

[0031] The PCR system was as follows: 25 μL of 2× phanta Max buffer, 1 μL of dNTP mixture (10 mM), 1 μL of each of the 10 μM mutant primers listed in Table 1, 1 μL of plasmid pET28a-Gi-Nit, 1 μL of Phanta Max DNA polymerase, and ddH2O to 50 μL.

[0032] PCR conditions included 5-minute pre-denaturation at 95°C, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 6.5 minutes, followed by a final extension at 72°C for 10 minutes. PCR products were analyzed by 0.9% agarose gel electrophoresis. 1 μL of Dpn I was added to 20 μL of the PCR product and digested at 37°C for 3 hours to remove the template plasmid DNA. The DNA was then inactivated at 65°C for 10 minutes.

[0033] 2. Transformation of nitrilase mutants

[0034] Take competent cells E. coli BL21 (DE3), add 10 μL of the PCR product in step 1, let it stand on ice for 30 minutes, heat shock at 42°C for 90 seconds, add 600 μL of LB liquid medium without kanamycin, incubate at 37°C at 180 rpm for 1 hour, spread on an LB plate containing 50 mg / L kanamycin, and incubate at 37°C overnight.

[0035] 3. Bacteria culture and high-throughput screening

[0036] Single colonies from step 2 were transferred to a 96-well plate. 1 mL of LB medium containing 50 μg / mL kanamycin was added to each well and incubated at 37°C for 12 h. 200 μL of the bacterial culture was then transferred to 800 μL of fresh LB medium containing 50 μg / mL kanamycin and 0.1 mM IPTG at a final concentration of 50 μg / mL and incubated at 28°C for 18 h. The cells in the 96-well plate were centrifuged for 30 min (4000 rpm, 4°C). The supernatant was discarded and the cells were washed and resuspended in 200 μL of KH2PO4-K2HPO4 buffer (100 mM, pH 7.0). 200 μL of the substrate 2-chloronicotinonitrile dissolved in KH2PO4-K2HPO4 buffer was added to each well and the reaction was continued at 30°C for 30 min. After the reaction is completed, 10 μL of 6M HCl is added to terminate the reaction. The reaction solution in the 96-deep-well plate is centrifuged for 15 minutes (4000 rpm, 4°C). 30 μL of the supernatant is transferred to a 96-well microplate containing 150 μL of a mixture of o-phthalaldehyde and mercaptoethanol (0.3 g o-phthalaldehyde, 150 μL of mercaptoethanol, 20 mL of anhydrous ethanol) per well. The plate is then incubated at 37°C for 30 minutes. Fluorescence intensity is then measured using a microplate reader (excitation wavelength 412 nm, emission wavelength 467 nm). Mutants with increased fluorescence intensity are selected as positive bacteria for subsequent liquid chromatography screening and sequencing analysis.

[0037] Table 1. Primer design for site-directed saturation mutagenesis at 194 sites

[0038]

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

[0040] Example 3: Rescreening of Nitrilase-Positive Mutants

[0041] The positive bacteria obtained in Example 2 were cultured according to the culture conditions of Example 1 to obtain mutant whole cells. Wet cells were weighed and added to 100 mM PBS (pH 7.2) buffer and mixed to prepare a bacterial suspension with a wet cell concentration of 20 g / L.

[0042] The reaction system for mutant viability assays consisted of a 10 mL total volume, 0.1 g / L wet cell volume, 100 mM PBS (pH 7.2), and 20 mM 2-chloronicotinonitrile. The reaction was incubated at 30°C, shaken at 180 rpm, and allowed to react for 10 minutes. The reaction was terminated by adding 10 μL of 6 M hydrochloric acid to 1 mL of the aliquot. After centrifugation, the contents of the substrate 2-chloronicotinonitrile and the product 2-chloronicotinic acid were determined by liquid chromatography.

[0043] Liquid chromatography detection conditions: the chromatographic column was a C18 column, the mobile phase was acetonitrile:water:phosphoric acid = 250:750:1 (v / v / v), the flow rate was 1 mL / min, and the detection wavelength was 270 nm; the elution time of the substrate 2-chloronicotinonitrile was 8.75 min, and the elution time of the product 2-chloronicotinic acid was 3.75 min.

[0044] Enzyme activity unit (U) is defined as the amount of cells required to catalyze the production of 1 μmol of 2-chloronicotinonitrile per minute at 30°C and pH 7.2. Mutants with higher whole-cell enzyme activity than the wild-type were used to extract plasmids for sequencing.

[0045] DNA sequencing of the positive clones with improved activity showed that the valine at position 194 was replaced by aspartic acid or leucine, and the nitrilase mutant engineered bacteria E. coli BL21(DE3) / pET28-V194D and E. coli BL21(DE3) / pET28-V194L were obtained. The activities of the mutants are shown in Table 2.

[0046] Table 2. Whole-cell enzyme activity of positive mutants

[0047]

[0048] Example 4. Construction of iterative mutation library and activity determination

[0049] The optimal single mutant V194D from Example 3 was subjected to iterative mutagenesis. Primers were designed (see Table 3). Using the nitrilase mutant plasmid pET28-V194D as a template, full plasmid amplification and subsequent transformation were performed according to Step 1 in Example 2. Single colonies were selected and cultured in a test tube containing 10 mL of LB liquid medium containing 50 μg / mL kanamycin, sequenced, and preserved. The iteratively mutagenized recombinant bacteria were cultured according to the method described in Step 1 of Example 1, and whole-cell enzyme activity was determined as described in Example 3. The whole-cell enzyme activity and sequences of each mutant are shown in Table 4. The double mutant L146F / V194D had the highest cell viability, reaching 236 U / g (wet cells), 81 times that of the starting strain.

[0050] Table 3. Primers for iterative mutagenesis

[0051]

[0052] Note: N=A / G / C / T, K=G / T, M=A / C

[0053] Table 4. Whole-cell enzyme activities of iterative mutants

[0054]

[0055] Example 5. Reaction Process of 2-Chloronicotinonitrile Catalyzed by Wild-Type Nitrilase Gi-Nit Whole Cell (I)

[0056] The transformation system and operation were as follows: 4.6 g / L (dry weight) of the wild-type recombinant Gi-Nit nitrilase obtained in Example 1 were added to 20 mL of KH2PO4-K2HPO4 buffer (100 mM, pH 7.2). The substrate 2-chloronicotinonitrile was added at a concentration of 150 mM. The reaction was carried out at 30°C and 600 rpm for 15 hours. The reaction progress was monitored by HPLC using the same conditions as described in Example 3. The results showed that the 2-chloronicotinic acid concentration reached 5 g / L after 15 hours.

[0057] Example 6. Reaction Progress of 2-Chloronicotinonitrile Catalyzed by Nitrilase Mutant L146F / V194D Whole Cells (I)

[0058] The transformation system composition and transformation operation are as follows: 20 mL of KH2PO4-K2HPO4 buffer (100 mM, pH 7.2) was added to the recombinant nitrilase mutant L146F / V194D obtained in Example 4, with the bacterial cell addition amount being 2.3 g / L (dry weight). The initial substrate concentration was 300 mM, and 300 mM was added every 0.5-1 hour until the total feed amount reached 1200 mM. The reaction was carried out at 30°C and 600 rpm for 15 hours. The reaction progress was monitored by HPLC, and the HPLC detection conditions were as described in Example 3. The results showed that after 15 hours of reaction, the 2-chloronicotinic acid concentration could reach 71 g / L ( Figure 1 ).

[0059] Example 7. Reaction Progress of 2-Chloronicotinonitrile Catalyzed by Nitrilase Mutant L146F / V194D Whole Cells (II)

[0060] The transformation system composition and transformation operation are as follows: 20 mL of KH2PO4-K2HPO4 buffer (100 mM, pH 7.2) was added to the recombinant nitrilase mutant L146F / V194D obtained in Example 4, with the bacterial cell addition amount being 3 g / L (dry weight). The initial substrate concentration was 300 mM, and 300 mM was added every 0.5-1 hour until the total feed amount reached 1200 mM. The reaction was carried out at 30°C and 600 rpm for 15 hours. The reaction progress was monitored by HPLC under the same conditions as in Example 3. The results showed that after 15 hours of reaction, the 2-chloronicotinic acid concentration could reach 116 g / L ( Figure 1 ).

Claims

1. A nitrilase mutant, characterized in that The nitrilase mutant is a mutant in which the amino acid shown in SEQ ID No. 2 is mutated into one of the following: (1) the valine at position 194 is replaced by aspartic acid; (2) the valine at position 194 is mutated into aspartic acid and the leucine at position 146 is mutated into phenylalanine; (3) the valine at position 194 is mutated into aspartic acid and the leucine at position 146 is mutated into serine.

2. A gene encoding the nitrilase mutant according to claim 1.

3. A recombinant vector containing the coding gene according to claim 2.

4. A recombinant genetically engineered bacterium constructed with the recombinant vector according to claim 3.

5. Use of the nitrilase mutant according to claim 1 in catalyzing the preparation of 2-chloronicotinonitrile into 2-chloronicotinic acid.

6. The use according to claim 5, characterized in that The application uses wet cells obtained by centrifuging the culture fluid of an engineered bacterium containing the nitrilase mutant encoding gene after shake flask culture, or enzymes extracted after the wet cells are crushed, as a biocatalyst; 2-chloronicotinonitrile is used as a substrate; a buffer solution with a pH of 7.0-7.5 is used as a reaction medium to form a conversion system; the conversion reaction is carried out under conditions of 30-45° C. and 100-1000 r / min; after the reaction is completed, the reaction liquid is separated and purified to obtain 2-chloronicotinic acid.

7. The use according to claim 6, characterized in that In the transformation system, the substrate is added at a concentration of 50-1500 mM, and the catalyst is used in an amount of 1-5 g / L based on the dry weight of bacterial cells.

8. The use according to claim 6, characterized in that The substrate is added in batches, with an initial concentration of 50-300 mM, and 50-300 mM is added every 0.5-2 h until the total addition amount is 50-1500 mM.

9. The use according to claim 6, characterized in that The wet bacteria were prepared as follows: the engineered bacteria containing the nitrilase mutant encoding gene were inoculated into LB medium containing a final concentration of 50 mg / L kanamycin, cultured at 37°C and 180 rpm for 12 h, then transferred to fresh LB medium containing a final concentration of 50 mg / L kanamycin at a volume concentration of 2%, and cultured at 37°C and 180 rpm until the bacteria concentration OD 600 The final concentration of IPTG was 0.1-1 mM, and the culture was induced at 28 °C and 180 r / min for 12 h. The culture was centrifuged and the precipitate was collected to obtain wet bacteria.

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