Nitrilase mutant and application thereof in synthesis of nicotinic acid

Abstract

The invention discloses a nitrilase mutant and application thereof in synthesis of nicotinic acid, and belongs to the technical field of bioengineering. Comprising a nitrilase mutant of a new amino acid sequence formed by replacing one or two amino acid residues in 95 alanine and 124 asparagine of an original nitrilase BbNit amino acid sequence with other amino acid residues, a coding gene of the nitrilase mutant, and a recombinant expression vector and a recombinant expression transformant containing the coding gene sequence, the invention relates to a catalyst containing a recombinant nitrilase mutant and application of the catalyst to catalytic hydrolysis of 3-cyanopyridine to synthesize nicotinic acid. Compared with the prior art, the nitrilase mutant prepared by the invention has the advantages that the enzyme catalytic activity is improved, the tolerance to a substrate 3-cyanopyridine is also improved, and the problems of long reaction time, low space time yield and the like in synthesis of nicotinic acid by biologically catalyzing hydrolysis of 3-cyanopyridine are solved, so that the nitrilase mutant is beneficial to practical application in industrial preparation of nicotinic acid.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a nitrilase mutant with high-concentration nitrile substrate tolerance, a coding gene thereof, a recombinant expression vector containing the gene sequence and a recombinant expression transformant, and application of the nitrilase mutant or the recombinant expression transformant in improving the efficiency of synthesizing nicotinic acid from 3-cyanopyridine. BACKGROUND

[0002] Nicotinic acid (NA), also known as nicotinic acid, vitamin B3 and pyridine-3-carboxylic acid, is a vitamin necessary for maintaining human physiological functions, and has a broad market prospect in the fields of medicine, chemical industry and food industry. The synthesis of nicotinic acid mainly includes chemical synthesis and biological catalytic synthesis: the chemical synthesis method is the main production means in the market at present, but it has the problems of serious environmental pollution, high energy consumption, low production capacity, serious lack of advanced technology in the industry, lack of competitiveness in the international market, and inability to meet the market demand; the biological catalytic synthesis method is a rapidly developing alternative technology in recent years, and has the advantages of environmental friendliness and low production cost. Nitrilase (EC 3.5.5.1) can catalyze nitrile substrates to generate the corresponding carboxylic acid and ammonia, and has the advantages of mild reaction conditions, high catalytic efficiency, green and environmentally friendly process, and high application potential. The natural nitrilase has low activity, and most nitrilase-producing bacteria have the problem of poor tolerance to nitrile substrates, which seriously hinders the industrial application of biological catalytic method.

[0003] Directed evolution based on mutant library construction and screening has been proven to be an effective strategy for enzyme molecular modification. Through directed evolution, protein engineering, etc., nitrilase mutants with higher activity, higher stability, or better adaptation to specific reaction conditions can be designed and prepared. There are reports on nitrilases with high substrate tolerance in the prior art. For example, a Chinese patent application with publication number CN118773178A discloses a nitrilase mutant with high substrate tolerance and its application. The nitrilase mutant is obtained by mutating the wild-type nitrilase with NCBI accession number WP_154591789.1. The nitrilase mutant has a yield of up to 99% under the condition of 400mM 3-cyanopyridine for 8h. Although the nitrilase mutant can adapt to a higher substrate concentration, it has the problems of long catalytic reaction time and low space-time yield. A Chinese patent application with publication number CN119265168A discloses a novel nitrilase and its application in nicotinic acid biosynthesis. The nitrilase gene GdNit is derived from Gordonia sp. A recombinant Escherichia coli with high expression of nitrilase is successfully constructed through promoter optimization. The resting cells can completely convert 200mM 3-cyanopyridine substrate within 30min. However, its adaptability to 3-cyanopyridine is only 25-200mM, which has the problem of low nitrile substrate tolerance. A Chinese patent application with publication number CN118652873A discloses a nitrilase mutant with high thermal stability and its application. The nitrilase mutant is obtained by mutating the nitrilase with NCBI accession number ESS14479.1. It can tolerate higher temperature (60℃) and wider pH value (4-10) under the condition of 3-cyanopyridine concentration of 50mM. However, there is no report on its good catalytic performance in high-concentration nitrile substrates.

[0004] In summary, in the research of biocatalytic synthesis of nicotinic acid, the known nitrilases and nitrilase mutants still have the problems of low catalytic efficiency and low substrate tolerance for 3-cyanopyridine. Therefore, it is of great significance to construct a nitrilase with high nitrile substrate tolerance and better catalytic performance for improving its application in the biocatalytic synthesis of nicotinic acid. SUMMARY

[0005] The purpose of the present application is to provide a nitrilase mutant and its application in the synthesis of nicotinic acid. The original nitrilase BbNit is modified by protein engineering to improve the catalytic activity of the enzyme and the tolerance to the substrate 3-cyanopyridine, thereby facilitating the application of the nitrilase in the industrial preparation of nicotinic acid and solving the problems of long reaction time and low space-time yield in the synthesis of nicotinic acid by hydrolysis of 3-cyanopyridine catalyzed by nitrilase in the prior art.

[0006] The purpose of the present application can be achieved by the following technical solutions: One of the technical solutions of the present application provides a nitrilase mutant with significantly improved tolerance in high-concentration 3-cyanopyridine substrates, taking the original nitrilase (referred to as BbNit) from a Betaproteobacteria bacterium MOLA814 as the parent, the NCBI accession number of the original nitrilase is ESS14479.1, the amino acid sequence is shown in the sequence table SEQ ID No. 2, and the coding gene sequence is shown in SEQ ID No. 1; SEQ ID No. 1:

[0007] SEQ ID No. 2: MVTYTNKFRAATVQAEPVWFDAAATVEKSIGLIKEAASNDAQIIAFPEVFIPGYPYHIWLDSPFAAMGKFAVRYHEQSLPIDSPLIVRLRDAARANKISVVMGFSERDGGTLYMSQIIINEHGNIVAHRRKLKPTHVERTVFGEGDGSDIAVYDMAVGRVGALNCWEHFQTLTKYAMYAMHEQIHIAAWPGMSLYQPEVYAFSSEAQSVATQMYAMEGQTFVLCATQVVGKAAHQFFCESPMHEKLIGYGGGFAQIFGPDGRALADRLPGDGEGILYAEINLAEIAMAKQAADPVGHYSRRDVFTVTFNDQPRDPIKRDKDTTEASFLGRALPQTTVVAPSLQHEASDLEIPKLPLEHENIENEVQK.

[0008] The nitrilase mutant of the present application is a derivative protein of a new amino acid sequence formed by replacing one or both of the amino acid residues of alanine at position 95 and asparagine at position 124 of the amino acid sequence shown in SEQ ID No. 2 with other amino acid residues, and has higher catalytic performance and substrate tolerance than the original nitrilase.

[0009] Further, the amino acid sequence of the nitrilase mutant is selected from one of the following: (1) replacing the alanine at position 95 of the amino acid sequence shown in SEQ ID No. 2 with glutamic acid; (2) replacing the asparagine at position 124 of the amino acid sequence shown in SEQ ID No. 2 with histidine; (3) replacing the alanine at position 95 of the amino acid sequence shown in SEQ ID No. 2 with glutamic acid, and replacing the asparagine at position 124 of the amino acid sequence shown in SEQ ID No. 2 with histidine.

[0010] The second technical solution of the present application provides a coding gene of a nitrilase mutant, which codes the nitrilase mutant as described in the first technical solution. The coding gene sequence of the nitrilase mutant is obtained by converting the amino acid sequence of the nitrilase mutant into the corresponding nucleic acid sequence, or the coding gene sequence of the nitrilase mutant is obtained by adjusting the coding gene sequence of SEQ ID No. 1 according to the difference between the amino acid sequence of the nitrilase mutant and the original nitrilase.

[0011] The third aspect of the present application provides a recombinant expression vector containing the gene encoding the nitrilase mutant. The present application obtains the recombinant expression vector containing the gene encoding the nitrilase mutant by site-directed mutagenesis and combinatorial mutagenesis of BbNit.

[0012] The fourth aspect of the present application provides a recombinant expression transformant containing the gene of the nitrilase mutant of the present application or the recombinant expression vector thereof. The recombinant expression transformant is prepared by transforming the recombinant expression vector into a host cell. The host cell is any conventional host cell in the art, provided that the recombinant expression vector can be stably replicated by itself and effectively expressed after induction by an inducer. The preferred host cell is E. coli, and the more preferred host cell is E. coli BL21 (DE3) for efficient expression of the target protein by the recombinant expression vector.

[0013] Further, the recombinant expression transformant is prepared by the following method: The recombinant expression vector is introduced into the competent cells of E. coli BL21 (DE3) by heat shock transformation to obtain the recombinant expression transformant.

[0014] Further, the subsequent culture step of the recombinant expression transformant comprises: The recombinant expression transformant is inoculated into a test tube containing 5 mL of LB liquid medium (containing 50 μg / mL Kan) at 37°C and 200 rpm for 10 h. The seed liquid is transferred into a 250 mL conical flask containing 50 mL of LB liquid medium at a 2% (v / v) inoculation amount, and cultured at 37°C and 180 rpm until the OD 600 is 0.6-0.8. Isopropyl-β-thiogalactoside (IPTG) is added at a final concentration of 0.1 mM, and the culture is induced at 28°C and 180 rpm for 10 h. After the culture, the bacterial cells are collected by centrifugation at 8000 rpm and 4°C for 10 min.

[0015] The fifth aspect of the present application provides a nitrilase mutant catalyst, which is in any of the following forms: (1) culturing the recombinant expression transformant of the present application to separate wet cells containing the nitrilase mutant; (2) culturing the recombinant expression transformant of the present application to separate a crude enzyme solution containing the nitrilase mutant; (3) drying the crude enzyme solution of the nitrilase mutant to obtain a crude enzyme powder.

[0016] The sixth technical solution of the present application provides an application of a nitrilase mutant catalyst in the synthesis of nicotinic acid by a biological catalysis method, that is, a method for efficiently synthesizing nicotinic acid by using a nitrilase mutant catalyst in a high-concentration 3-cyanopyridine substrate.

[0017] Further, the concentration of the 3-cyanopyridine is 100-500 mM.

[0018] Further, the amount of the nitrilase mutant catalyst used in the reaction system is 0.05-0.3 g of wet cells per 10 mL of the reaction system; and the reaction system is a 200 mM, pH=7.0 phosphate buffer.

[0019] Further, the synthesis condition is 30°C, 200 rpm, and 10-60 min of reaction.

[0020] Compared with the prior art, the present application has the following beneficial effects: 1. By comparing the catalytic activities of original nitrilases from three different strains in a high-concentration substrate 3-cyanopyridine, and by comparing and analyzing the amino acid sequences of the three original nitrilases, nine key amino acid sites are selected, BbNit is used as the parent to perform back-mutation, and the back-mutant with lower activity is screened from the nitrilases with successful back-mutation, the mutation sites of the back-mutant with lower activity are used as the key points for subsequent point saturation mutation, and the nitrilase mutant of the present application is finally obtained through point saturation mutation and combination mutation. The present application accurately finds the key sites and improvement direction affecting the catalytic activity of the original nitrilase through the research of the inventor, has stronger purpose and higher mutation efficiency compared with the prior art which directly uses the random mutation method. Moreover, the nitrilase mutant prepared by the present application not only has significantly improved catalytic activity and catalytic efficiency compared with the original nitrilase BbNit, but also has significantly improved tolerance to high-concentration substrates, especially the nitrilase mutant N124H / A95E obtained through combination mutation, which can reach a yield of 97% under the condition of 450 mM substrate concentration and only 1 h of reaction, greatly shortening the catalytic reaction time of nicotinic acid synthesis and having extremely high industrial application value. 2. The nitrilase mutant of the present application not only has high tolerance to the substrate 3-cyanopyridine, but also has the advantage of low catalytic dosage. Compared with the original nitrilase BbNit, the product concentration and yield obtained under the same catalyst dosage and reaction time condition are higher, showing great low-cost production advantage, so as to significantly reduce the production cost of nicotinic acid. BRIEF DESCRIPTION OF DRAWINGS

[0021] The present application will be further described below in combination with the drawings.

[0022] Figure 1 is the amino acid sequence alignment chart of three nitrilases BbNit, PpNit and PsNit derived from different bacterial species of the present application. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0024] Unless otherwise specified, all technical and scientific terms used in the present application have the same meanings as commonly understood by a person of ordinary skill in the art. The experimental materials used in the embodiments of the present application are all conventional experimental materials in the art, and can be purchased through commercial channels. The experimental methods without detailed conditions are performed according to conventional experimental methods or according to the operation instructions recommended by the suppliers.

[0025] Example 1: Construction and primary screening of original nitrilases The Nit sequences were synthesized by codon optimization of the target gene fragments of original nitrilases BbNit from Betaproteobacteria bacterium MOLA814, PpNit from Pseudomonas putida and PsNit from Pusillimonas sp. T2, respectively. The target gene fragments of BbNit, PpNit and PsNit were digested with NdeI and XhoI, respectively, and then the digested products were recovered. The recovered products were then ligated by T7 ligase to obtain original plasmids pET-28b-BbNit, pET-28b-PpNit and pET-28b-PsNit, respectively. Finally, the original nitrilase strains E. coli BL21(DE3) / pET-28b-BbNit, E. coli BL21(DE3) / pET-28b-PpNit and E. coli BL21(DE3) / pET-28b-PsNit were obtained by introducing the original plasmids into E. coli BL21(DE3) competent cells by E. coli heat shock transformation method. Take 0.05 g of each of the three strains of wet bacterial bodies, resuspend in 10 mL of 200 mM, pH = 7.0 phosphate buffer, 30°C for 10 min, add the substrate 3-cyanopyridine with a final concentration of 300-500 mM, 200 rpm, 30°C constant temperature oscillation reaction for 5-40 min, after the reaction is completed, add 200 μL of 6.0 M HCl to terminate the reaction, centrifuge and take the supernatant, and use HPLC to detect the product concentration of the three original nitrilases under different substrate concentrations, and the results are shown in Table 1. Table 1 As can be seen from the data in Table 1, BbNit shows the highest catalytic activity, followed by PsNit, and PpNit has almost no activity; with the increase of substrate concentration, PsNit shows a significant downward trend in product concentration, while BbNit shows a gradual downward trend in product concentration, and when the substrate concentration is 500 mM, the product concentration of the BbNit group is only 111.82 mM, indicating that high-concentration substrate has a significant inhibitory effect on BbNit, which needs to be further improved.

[0026] Example 2: Construction of nitrilase point mutant The inventors analyzed the amino acid sequences of the three original nitrilases obtained in Example 1, and the results are shown in Table 2: Figure 1 The inventors found that the sequence similarity of PpNit and PsNit was as high as 94.15%, while the sequence similarity of PpNit and BbNit was only 76.65%. Although PpNit and PsNit are highly similar in amino acid sequence, there is a significant difference in their catalytic activity. The activity difference between PpNit and BbNit is small, but the sequence similarity is low. Based on this, the inventors speculate that the sequence difference between PpNit and PsNit and BbNit has an important influence on their catalytic activity, so they choose Figure 1 the nine key amino acid sites indicated by the black arrows to perform back mutation test with BbNit as the parent, and the specific sites and mutation methods are as follows: P80S, A95S, A95N, N124K, N124E, A127S, V157L, V157I, I184V, E244K, L268M, T322I, T322M.

[0027] The amplification full-plasmid primers used are shown in Table 2; Table 2 The PCR reaction program is: pre-denaturation 94°C, 5 min; enter cycle 98°C, 10 s, 68°C, 8 min, for a total of 30 cycles; finally 68°C for 10 min. The PCR amplification system is shown in Table 3. Table 3 After successful identification by 1% agarose gel electrophoresis, 1 μL Dpn I restriction endonuclease was added to 40 μL reaction solution and incubated at 37°C for 3 h to remove the plasmid DNA template in the reaction solution. Then, the solution was transferred into E. coli BL21(DE3) competent cells, which were cultured on LB plates containing kanamycin overnight, and the clones were picked for sequencing verification.

[0028] The enzyme activity of the successfully mutated nitrilase point mutants was detected, and the relative activity of each group of nitrilase point mutants relative to BbNit was calculated based on the enzyme activity of BbNit, and the results are shown in Table 4. Table 4 The relative activity of all nitrilase point mutants was lower than that of BbNit, wherein the activity of P80S, A95N, N124K, A127S and V157I was lower than 54% of BbNit, and the activity of the remaining point mutants was higher than 54%. The inventors believe that the lower activity of the back-mutants indicates that the mutation sites have a more critical structural or functional contribution to the catalytic function of BbNit, which is the innovation point of the present application. These sites will be screened for higher catalytic efficiency or substrate tolerance through subsequent point saturation mutation. Since these sites were found to have a greater impact on the activity of BbNit through site-directed back-mutation, point saturation mutation was performed on the P80, A95, N124, A127, V157, I184 and E244 sites.

[0029] Example 3 Construction of nitrilase mutants by point saturation mutation and combination mutation First, point saturation mutation was performed on the P80, A95, N124, A127, V157, I184 and E244 sites, and the point saturation mutation primers are shown in Table 5. Table 5 The PCR reaction procedure, PCR amplification system and transformation culture method are the same as those in Example 2, which are not repeated here. The mutant library colonies obtained by the above procedure were inoculated into 300 μL LB medium (96-well deep well plate) containing 50 μg / mL kanamycin, and incubated at 37°C, 200 rpm for 18-20 h for seed activation; then half of the seed liquid (150 μL) was transferred into a new well containing 850 μL LB medium (containing 50 μg / mL kanamycin and 0.1 mM IPTG), and induced to culture at 30°C for 18-20 h; the bacterial cells were collected by centrifugation at 3500 rpm for 30 min, and high-throughput screening was performed on the bacterial cells: 200 mM phosphate buffer (pH = 7.0) and 400 mM 3-cyanopyridine were added to each well to resuspend the bacterial cells, and 1 mL bacterial suspension was prepared, which was placed in a constant temperature water bath shaker at 30°C, 200 rpm for 10 min, and then centrifuged at 3500 rpm for 20 min; the supernatant was collected to screen the bacterial strains with higher activity, and the screened point saturation mutants were sequenced; According to the high-throughput screening results, the bacterial strains with higher activity screened from the other half of the seed liquid were inoculated into a test tube containing 5 mL LB liquid medium (containing 50 μg / mL kanamycin), and incubated at 37°C, 200 rpm for 10 h. The seed liquid was transferred into a 250 mL conical flask containing 50 mL LB liquid medium at a 2% (v / v) inoculation amount, and incubated at 37°C, 180 rpm until the OD 600 was 0.6-0.8; then IPTG was added at a final concentration of 0.1 mM, and the culture was induced at 28°C, 180 rpm for 10 h. After the culture, the bacterial cells were collected by centrifugation at 8000 rpm, 4°C for 10 min.

[0030] In a 10 mL system containing 200 mM phosphate buffer (pH = 7) and 400 mM 3-cyanopyridine substrate, 0.05 g of the wet bacterial cell bodies screened above were added, and the reaction was carried out at 30°C, 200 rpm for 1 h. After the reaction, 200 μL of HCl (6.0 M) was added to terminate the reaction. The reaction mixture was centrifuged at 10000 rpm for 2 min, and the supernatant was collected for HPLC detection. The detection results are shown in Table 6.

[0031] Table 6 As can be seen from the results in Table 6, the mutant P80P obtained by silencing mutation of proline at position 80 is 118% of BbNit; the mutant A95E obtained by mutating alanine at position 95 to glutamic acid is 133% of BbNit; and the mutant N124H obtained by mutating asparagine at position 124 to histidine is 180% of BbNit; thus, three nitrile hydratase mutants with higher catalytic yield at a substrate concentration of 400 mM are obtained.

[0032] The N124H with the highest catalytic yield was selected as the parent, and the P80 and A95 sites were subjected to superimposed combination mutation, so that the proline at position 80 was subjected to silent mutation, and the alanine at position 95 was mutated into glutamic acid. The primers are shown in Table 7. Table 7 After PCR amplification and bacterial culture, 0.05 g of the above superimposed combination mutant high-activity wet cell bacteria were added into 10 mL of a system containing 200 mM pH = 7 phosphate buffer and 400 mM 3-cyanopyridine substrate, and reacted at 30°C, 200 rpm for 1 h. After the reaction, 200 μL of HCl (6.0 M) was added to terminate the reaction. Centrifugation was performed at 10000 rpm for 2 min, and the supernatant was taken for HPLC detection. The results are shown in Table 8. Table 8 As can be seen from the data in Table 8, the superimposed mutant N124H / A95E with higher activity is 201% of BbNit, which shows significantly improved catalytic performance and high-concentration substrate tolerance.

[0033] Example 4: Effect of crude enzyme liquid dosage on yield of nicotinic acid synthesis 0.075-0.3 g of N124H / A95E mutant bacteria and BbNit bacteria prepared in Example 3 were respectively resuspended in 10 mL of 200 mM, pH = 7.0 phosphate buffer, and then subjected to ultrasonic crushing under ice bath conditions. The power of the crushing instrument was set to 40 W, the ultrasonic working time was 1 s, the interval was 1 s, and the total crushing time was 20 min. Centrifugation was performed at 10000 rpm, 4°C for 20 min, and the supernatant was collected to obtain a crude enzyme liquid reaction system with different wet cell dosages. Then, 400 mM and 450 mM reaction substrates 3-cyanopyridine were respectively added into the above reaction system, and reacted at 30°C, 200 rpm for 1 h. After that, 200 μL of 6.0 M HCl was added to terminate the reaction, and the supernatant was taken for HPLC detection. The results are shown in Table 9. Table 9 As shown in Table 9, when the substrate concentration is 400 mM, the conversion rate of BbNit increases from 84.66% to 90.82% with the increase of the amount of wet cells in the crude enzyme solution, while the yield of N124H / A95E can reach 90% when the amount of wet cells is 0.075 g, and the substrate can be almost completely converted when the amount of wet cells is 0.3 g. When the substrate concentration is increased to 450 mM, the yield of BbNit is only maintained at about 60% due to substrate inhibition, while the yield of N124H / A95E increases from 87.36% to 97.36% with the increase of the amount of wet cells in the crude enzyme solution. The above data fully prove that N124H / A95E has significant anti-inhibition ability and high catalytic properties under high substrate concentration, which provides key technical support for the industrial production of nicotinic acid.

[0034] It should be noted that, in this document, the terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed or inherent to such a process, method, article or device.

[0035] Although the embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made thereto without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A nitrilase mutant, characterized in that, The amino acid sequence of the nitrilase mutant is one of the following sequences: (1) the alanine at position 95 of the amino acid sequence shown as SEQ ID No. 2 is replaced by glutamic acid; (2) the asparagine at position 124 of the amino acid sequence shown as SEQ ID No. 2 is replaced by histidine; (3) the alanine at position 95 of the amino acid sequence shown as SEQ ID No. 2 is replaced by glutamic acid, and the asparagine at position 124 of the amino acid sequence shown as SEQ ID No. 2 is replaced by histidine.

2. A gene encoding a nitrilase mutant, characterized in that, The coding gene encodes the nitrilase mutant as claimed in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the coding gene as claimed in claim 2.

4. A recombinant expression transformant, characterized by, The recombinant expression transformant comprises the recombinant expression vector as claimed in claim 3.

5. A nitrilase mutant catalyst characterized in that, The nitrilase mutant catalyst is in any of the following forms: (1) culturing the recombinant expression transformant as claimed in claim 4, and separating the wet cells containing the nitrilase mutant; (2) culturing the recombinant expression transformant as claimed in claim 4, and separating the crude enzyme solution containing the nitrilase mutant; (3) drying the crude enzyme solution of the nitrilase mutant in (2) to obtain the crude enzyme powder.

6. The use of the nitrilase mutant catalyst as claimed in claim 5 in catalyzing the hydrolysis of 3-cyanopyridine to synthesize nicotinic acid.

7. Use according to claim 6, characterized in that, The concentration of the 3-cyanopyridine is 100-500 mM.

8. Use according to claim 6, characterized in that, The amount of the nitrilase mutant catalyst used in the reaction system is 0.05-0.3 g wet cells per 10 mL reaction system.

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