A nitrile hydrolase mutant and its application

Abstract

This invention belongs to the fields of genetic engineering and enzyme engineering technology, and discloses a nitrile hydrolase mutant. The mutant is obtained by substituting one or more amino acid residues at positions 14, 33, 54, 64, 74, 101, 153, 168, 197, 232, 267, and 296 of the amino acid sequence shown in SEQ ID NO. 1; and / or by truncating amino acids at positions 330-335 of the amino acid sequence shown in SEQ ID NO. 1. It can catalyze the synthesis of 4-acetyl-2-methylbenzoic acid in a short time and with high efficiency. Furthermore, the bio-enzymatic catalysis method is green, environmentally friendly, and pollution-free, making it more suitable for green industrial processing and production.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and relates to a nitrile hydrolase mutant and its application, specifically to a nitrile hydrolase mutant and its application in the catalytic synthesis of 4-acetyl-2-methylbenzoic acid. Background Technology

[0002] Fluranar is the most typical and best-selling isoxazoline drug globally. It exhibits broad-spectrum activity, high efficacy, and safety against both feline and canine parasites, along with good biological activity and excellent safety profile. Currently, marketed products are racemic compounds, but the configuration in which fluranar exhibits antiparasitic activity is the S-configuration. 4-Acetyl-2-methylbenzoic acid is an indispensable intermediate fragment in the synthesis of fluranar.

[0003] Currently, the main methods for synthesizing 4-acetyl-2-methylbenzoic acid include the following three: The first method uses 4-bromo-3-methylacetophenone as a raw material, 1,3-diphenylphosphine propane and palladium on carbon as catalysts, and reacts with carbon monoxide pressurized to 0.8 MPa / 120℃ to obtain 2-methyl-4-acetylbenzoic acid. However, the carbon monoxide used in this method is a highly toxic gas, and its use under high temperature and pressure conditions is highly corrosive to equipment, posing a high safety risk. Furthermore, the catalyst used is expensive, increasing the production cost. The second method uses 2-fluorotoluene as a raw material, obtaining 4-acetyl-2-methylbenzoic acid through acetylation, cyanation, and hydrolysis. However, this method involves many reaction steps, and hydrolysis produces a large amount of acidic wastewater, which is highly corrosive to equipment and has a low yield. The raw materials are also expensive. The third method uses 4-bromo-2-methylbenzoic acid as a raw material to obtain 4-acetyl-2-methylbenzoic acid via Suzuki coupling reaction. However, this method is applied to Grignard reaction, which is difficult to control, has a high risk factor, requires harsh process conditions, and has low reaction yield and high cost.

[0004] Therefore, there is an urgent need to develop a method for preparing 4-acetyl-2-methylbenzoic acid with mild process conditions and high purity and yield to meet the requirements of industrial production. Biocatalysis offers advantages over chemical methods, including milder reaction conditions, environmental friendliness, and no heavy metal pollution. 4-Acetyl-2-methylbenzonitrile can be converted to 2-methyl-4-acetylbenzoic acid and ammonia under the catalysis of nitrile hydrolases. Although nitrile hydrolases are widely used and exhibit good catalytic activity for aliphatic substrates such as acetonitrile and acrylonitrile, as well as heterocyclic aromatic substrates such as imidazole and pyridine, their catalytic effect on aromatic substrates such as m-chlorobenzonitrile, p-methoxybenzonitrile, and p-toluenenitrile is poor. 2-Methyl-4-acetylbenzoic acid is an aromatic substrate, and no corresponding nitrile hydrolases were found in the literature. Therefore, the enzymatic synthesis of 2-methyl-4-acetylbenzoic acid presents significant technical challenges. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, this application provides a nitrile hydrolase mutant and its application, which can be heterologously expressed in high-density fermentation host bacteria, has high enzyme activity, good stability, high reaction substrate concentration, and high spatiotemporal yield of the target product.

[0006] To address the above problems, the present invention provides the following technical solution: In a first aspect, a nitrile hydrolase mutant is obtained by substituting one or more amino acid residues at positions 14, 33, 54, 64, 74, 101, 153, 168, 197, 232, 267, and 296 of the amino acid sequence shown in SEQ ID NO. 1; and / or; It was obtained by truncating amino acids 330-335 of the amino acid sequence shown in SEQ ID NO.1.

[0007] In one embodiment of this application, the nitrile hydrolase is named NITA, derived from... Burkholderiales bacterium .

[0008] In one embodiment of this application, a combination of any one or more of C14A, A33G, A54V, K64C, P74E, E101K, W153F, A168N, Q197N, G232A, L267P, and D296E is used as a substitute.

[0009] In one embodiment of this application, the cysteine ​​at position 14 of C14A, i.e., SEQ ID NO.1, is mutated to alanine and named NITA_C14A.

[0010] In one embodiment of this application, the alanine at position 33 of SEQ ID NO.1 in A33G is mutated to glycine, and named NITA_A33G.

[0011] In one embodiment of this application, the alanine at position 54 of SEQ ID NO.1 in A54V is mutated to valine, and named NITA_A54V.

[0012] In one embodiment of this application, the lysine at position 64 of K64C (SEQ ID NO.1) is mutated to cysteine ​​and named NITA_K64C.

[0013] In one embodiment of this application, the 74th proline in P74E (SEQ ID NO.1) is mutated to glutamic acid, and named NITA_P74E.

[0014] In one embodiment of this application, the glutamic acid at position 101 of E101K (SEQ ID NO.1) is mutated to lysine, and named NITA_E101K.

[0015] In one embodiment of this application, W153F, i.e., tryptophan at position 153 of SEQ ID NO.1, is mutated to phenylalanine and named NITA_W153F.

[0016] In one embodiment of this application, the alanine at position 168 of A168N, i.e., SEQ ID NO.1, is mutated to asparagine and named NITA_A168N.

[0017] In one embodiment of this application, Q197N, i.e., the glutamine at position 197 of SEQ ID NO.1, is mutated to asparagine and named NITA_Q197N.

[0018] In one embodiment of this application, the glycine at position 232 of G232A (SEQ ID NO.1) is mutated to alanine and named NITA_G232A.

[0019] In one embodiment of this application, the leucine at position 267 of L267P (SEQ ID NO.1) is mutated to proline, and named NITA_L267P.

[0020] In one embodiment of this application, the aspartic acid at position 296 of D296E (SEQ ID NO.1) is mutated to glutamic acid and named NITA_D296E.

[0021] In one embodiment of this application, CUT(330-335) is the truncation of amino acids 330 to 335 of SEQ ID NO.1, named NITA_CUT(330-335).

[0022] In one embodiment of this application, the nitrile hydrolase mutant is obtained by combining mutations of A33G, E101K, W153F, A168N, Q197N, G232A and CUT (330-335) based on the amino acid sequence shown in SEQ ID NO.1, and is named NITA_M, with its amino acid sequence shown in SEQ ID NO.2.

[0023] In one embodiment of this application, the alanine at position 33 of SEQ ID NO.1 is mutated to glycine, the glutamic acid at position 101 is mutated to lysine, the tryptophan at position 153 is mutated to phenylalanine, the alanine at position 168 is mutated to asparagine, the glutamine at position 197 is mutated to asparagine, the glycine at position 232 is mutated to alanine, and the amino acids at positions 330 to 335 are truncated.

[0024] In one embodiment of this application, the fusion protein obtained by attaching a tag to the protein end defined by the nitrile hydrolase mutant is also within the scope of protection of this application.

[0025] Secondly, this application provides a gene encoding a nitrile hydrolase mutant.

[0026] In one embodiment of this application, the nucleotide sequence of the gene encoding NITA is shown in SEQ ID NO.3.

[0027] In one embodiment of this application, the nucleotide sequence of the gene encoding NITA_M is shown in SEQ ID NO.4.

[0028] Thirdly, this application provides a recombinant vector containing a gene encoding a nitrile hydrolase mutant.

[0029] In one embodiment of this application, the recombinant vector is constructed as follows: the nitrile hydrolase mutant gene is inserted into the pRSFDuet-1 vector. Nde I / Xho A recombinant vector containing a nitrile hydrolase mutant gene was constructed between the I sites.

[0030] Fourthly, this application provides a recombinant genetically engineered bacterium containing a gene encoding a nitrile hydrolase mutant.

[0031] In one embodiment of this application, recombinant genetically engineered bacteria are prepared by transferring a constructed recombinant vector into a host bacterium to obtain recombinant genetically engineered bacteria.

[0032] In one embodiment of this application, the host bacteria include, but are not limited to, various conventional engineered bacteria in the art.

[0033] In one embodiment of this application, the host bacterium may be E. coli BL21.

[0034] Fifthly, this application provides the use of a nitrile hydrolase mutant in the catalytic synthesis of 4-acetyl-2-methylbenzoic acid from 4-acetyl-2-methylbenzonitrile.

[0035] The bioactive nitrile hydrolase mutant of this application can be used in the form of engineered bacterial wet cells, crude enzyme solution, or purified enzyme solution. Furthermore, the nitrile hydrolase mutant of this invention can also be prepared into an immobilized enzyme using immobilization methods known in the art.

[0036] In one embodiment of this application, wet bacterial cells obtained by inducing expression of nitrile hydrolase mutant recombinant genetically engineered bacteria are used as catalysts, and 4-acetyl-2-methylbenzonitrile is used as a substrate to carry out a transformation reaction at 30-60°C to obtain 4-acetyl-2-methylbenzoic acid.

[0037] In one embodiment of this application, the synthetic route is as follows: .

[0038] In one embodiment of this application, obtaining wet bacterial cells includes the following steps: activating recombinant engineered bacteria, transferring them to an induction medium, adding an inducer for induction culture, centrifuging and collecting the bacterial cells, and resuspending the bacterial cells in a buffer solution.

[0039] In one embodiment of this application, the final concentration of the added inducer is 0.001-10 g / L, and the induction time is 4-60 h.

[0040] In one embodiment of this application, the final concentration of the added inducer is 0.02-1 g / L, and the induction time is 4-50 h.

[0041] In one embodiment of this application, the reaction temperature is 40-45°C.

[0042] In one embodiment of this application, the pH value of the reaction is 6.0-9.5.

[0043] In one embodiment of this application, the pH value of the reaction is 8.0.

[0044] In one embodiment of this application, the concentration of 4-acetyl-2-methylbenzonitrile is 1-10000 g / L.

[0045] In one embodiment of this application, the amount of catalyst used is 1-10000 g / L based on the weight of wet bacterial cells.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The nitrile hydrolase mutant provided in this application can be heterologously expressed in high-density fermentation host bacteria, which can increase the substrate catalytic concentration to 1000 g / L, effectively solving the problem of substrate inhibition of enzyme activity, improving production efficiency, reducing substrate loss, and promoting the application of biotechnology industry.

[0047] (2) The nitrile hydrolase mutant provided in this application can catalyze the synthesis of 4-acetyl-2-methylbenzoic acid in a short time and with high efficiency. Moreover, the bio-enzyme catalysis method is green, environmentally friendly and pollution-free, and is more suitable for green industrial processing and production.

[0048] (3) The nitrile hydrolase mutant provided in this application has high catalytic efficiency, high product conversion rate and longer half-life. Attached Figure Description

[0049] Figure 1 The graph shows the effect of temperature on the activity of nitrile hydrolase NITA. Figure 2 The graph shows the effect of pH on the activity of nitrile hydrolase NITA. Detailed Implementation

[0050] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0051] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of the particular range. The range defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range.

[0052] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; and the remainder is weight / weight percentage.

[0053] The present invention will be further described below with reference to specific embodiments. Molecular biology experimental methods not specifically described in the following embodiments can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0056] Example 1: Preparation of recombinant bacteria expressing nitrile hydrolase NITA and its mutants The dual-gene expression vector pRSFDuet-1 (disclosed in Han Guangwei, Co-expression and Immunogenicity of Clostridium perfringens α,β_1,β_2,ε toxin proteins [D]. Chinese Academy of Agricultural Sciences, 2014) was selected. Nde I / Xho I site insertion Burkholderiales bacterium The nucleotide sequence of the nitrile hydrolase NITA encoding gene or the nucleotide sequence of the mutant encoding gene is used. When the inserted sequence is the nucleotide sequence of the nitrile hydrolase NITA encoding gene, the recombinant expression plasmid pRSFDuet-1-NITA is obtained. The recombinant plasmid is inoculated into E. coli BL21(DE3) for induction and expression (when the fermentation broth OD... 600 When the concentration reaches 0.3-0.8, an inducer is added to induce expression (the final concentration of the inducer is 0.02-1 g / L, and the induction time is 4-50 h), resulting in BL21-pRSFDuet-1-NITA. The amino acid sequence of the nitrile hydrolase NITA is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.3.

[0057] The PCR amplification technique is as follows: PCR amplification of genes Using plasmids or the E. coli BL21(DE3) genome as templates, PCR amplification was performed using the 2×PhantaR Max Master Mix kit from Nanjing Novizan Biotechnology Co., Ltd. to obtain the coding gene fragments of cysQ, eda, nusA, and NITA and its mutants. The reaction procedure was: 95℃, 1 min; 68℃, 2 min; 72℃, 2 min, for a total of 30 cycles. The PCR reaction system is shown in Table 1.

[0058] Table 1. PCR system ;

[0059] Digestion reaction of restriction endonucleases The plasmid vectors were digested overnight at 30°C using restriction endonucleases from TaKaRa. The composition of the three dual-enzyme digestion systems is shown in Table 2.

[0060] Table 2. Double enzyme digestion reaction system ;

[0061] Connection reaction The linearized plasmid and the target gene fragment were ligated using the CloneEZR recombinant cloning kit from Nanjing Genscript Biotech Co., Ltd. The reaction system is shown in Table 3. The ligation conditions were 22℃ for 30 min and 4℃ for 5 min.

[0062] Table 3 Connection System ;

[0063] The ligation solution was transferred into E. coli BL21(DE3) competent cells. Single colonies were picked from plates containing kanamycin (50 mg / L) and inoculated into LB medium containing the same concentration of kanamycin. The cells were incubated overnight at 37°C and 200 rpm.

[0064] PCR amplification of the coding gene of the site-directed mutant of NITA: Rapid mutation was performed using PCR amplification technology with unmutated strain pRSFDuet-1-NITA as template DNA. NITA mutants include the following single-point or multi-point mutations, which include the substitution, deletion or addition of amino acid residues: NITA_C14A, NITA_A33G, NITA_A54V, NITA_K64C, NITA_P74E, NITA_E101K, NITA_W153F, NITA_A168N, NITA_Q197N, NITA_G232A, NITA_L267P, NITA_D296E, and NITA_CUT(330-335).

[0065] The primers for site-directed mutagenesis of C14A are: Forward primer: 5'-GAGCgccCCGTTTGATACCGCGGCGGCGACCC-3'; Reverse primer: 5'-TATCAAACGGggcGCTCGCCAGCTGCGCCACG-3'.

[0066] The primers for site-directed mutagenesis of A33G are: Forward primer: 5'-ATGCGCGAAGCGggcGCGGCGGGCGCGCGCCTG-3'; Reverse primer: 5'-GCgccCGCTTCGCGCATCGCGGTCACCGCGCG-3'.

[0067] The primers for site-directed mutagenesis of A54V are: Forward primer: 5'-GAAAGGCgttACCTTTGGCGCGCCGGTGGGCA-3'; Reverse primer: 5'-CAAAGGTaacGCCTTTCGGATAGCCGCCCAGA-3'.

[0068] The primers for site-directed mutagenesis of K64C are: Forward primer: 5'-CATGCGCtgtCCGGAAGGCCGCGATGCGTGCT-3'; Reverse primer: 5'-CTTCCGGacaGCGCATGCCCACCGGCGCGCCA-3'.

[0069] The primers for site-directed mutagenesis of P74E are: Forward primer: 5'-CTTTAGCgaaGCGGAACGCTATTATGAAGCGG-3'; Reverse primer: 5'-GTTCCGCttcGCTAAAGCACGCATCGCGGCCT-3'.

[0070] The primers for site-directed mutagenesis of E101K are: Forward primer: 5'-GGAAACCaagCTGTTTGTGGTGATTGGCGTGA-3'; Reverse primer: 5'-CAAACAGcttGGTTTCCGCCGCCGCTTCCGCA-3'.

[0071] The primers for site-directed mutagenesis of W153F are: Forward primer: 5'-CGATCGCttcGAACAGTTTGGCGGCAGCACCA-3'; Reverse primer: 5'-ACTGTTCgaaGCGATCGCCATAGCCGCGCCAA-3'.

[0072] The primers for site-directed mutagenesis of A168N are: Forward primer: 5'-ATTAGCCCGaacAGCGTGTTTGATACCAGCCTG-3'; Reverse primer: 5'-ACGCTgttCGGGCTAATATAATGGCACATGGT-3'.

[0073] The primers for site-directed mutagenesis of Q197N are: Forward primer: 5'-GAACAGCaacATGAGCTGGGCGCCGTATAGCC-3'; Reverse primer: 5'-AGCTCATgttGCTGTTCACCATGCGCAGCATC-3'.

[0074] The primers for site-directed mutagenesis of G232A are: Forward primer: 5'-TGGAAgctCGCTGCTTTGTGCTGACCGCGTGC-3'; Reverse primer: 5'-AAAGCAGCGagcTTCCAGCGCAATATGGCGCA-3'.

[0075] The primers for site-directed mutagenesis of L267P are: Forward primer: 5'-ATACCGTGcctATGCGCGGCGGCAGCGCGATT-3'; Reverse primer: 5'-GCGCATaggCACGGTATCCGGCGCATCGCCCA-3'.

[0076] The primers for site-directed mutagenesis of D296E are: Forward primer: 5'-GCTGTATGCGgaaATTGATCTGGGCCAGGTGG-3'; Reverse primer: 5'-CAATttcCGCATACAGCAGCTGTTCGCCTTCA-3'.

[0077] The primers for the NITA_CUT (330-335) mutation are: Forward primer: 5'-TTTGATGTGGTGGGCCATTATGCGCGCCCGGA-3'; Reverse primer: 5'-TGGCCCACCACATCAAAATCATATTTGCCGCG-3'.

[0078] The NITA_A33G-E101K-W153F-A168N-Q197N-G232A-CUT (330-335) protein mutant includes the above-mentioned partial mutations and is named NITA_M. Its amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule encoding the mutant protein is shown in SEQ ID NO.4.

[0079] Example 2: Obtaining the whole-cell fermentation products of nitrile hydrolase NITA and its mutants The recombinant expression strains containing the nitrile hydrolase NITA and its mutants prepared in Example 1 were plated onto LB agar plates containing 50 µg / L kanamycin (NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L, agar 20 g / L) and incubated at 37°C for 12 h. The next day, single colonies were selected from the plates and transferred to shake tubes containing 5 mL of LB liquid medium (containing 50 µg / L kanamycin) and cultured at 37°C and 200 rpm for 12 h as seed culture. The seed culture was then transferred at a 1% (v:v) inoculation rate to 100 mL of TB medium (yeast extract 25 g / L, peptone 15 g / L, NaCl 10 g / L, glucose 2 g / L, lactose 0.5 g / L, containing 50 µg / L kanamycin). The medium was then incubated at 37°C and 200 rpm with shaking. After 2 hours, adjust the temperature to 25°C and continue culturing for 20-22 hours.

[0080] Collect the fermentation broth and freeze-centrifuge (4℃, 7000 rpm, 6 min). Resuspend the broth in a buffer (1g of wet bacterial sludge re-dissolved in 5mL buffer) as the whole cell product and store it at 4℃ for later use.

[0081] Example 3 Enzyme activity assay of nitrile hydrolase NITA and its mutants The method for determining the enzyme activity of nitrile hydrolase NITA and its mutants was as follows: 0.1 g of 4-acetyl-2-methylbenzonitrile and 0.2 mg of wet bacterial sludge were added to a 1.5 mL enzyme-catalyzed reaction system, and the pH was then adjusted to 7.5 with 100 mM phosphate buffer. The reaction conditions were 30℃ and 200 rpm; sampling times were 0 min, 20 min, and 30 min. Sample preparation: the sample was diluted with 50% acetonitrile aqueous solution, followed by a 100-fold dilution of the extract, and then analyzed by GC. Enzyme activity (U) was defined as the amount of enzyme required to convert 1 µmol of 4-acetyl-2-methylbenzoic acid within 1 minute. The relative enzyme activities of other mutant enzymes were calculated using the wild-type enzyme activity as 100%.

[0082] Example 4 Comparison of catalytic activities of nitrile hydrolase NITA and its mutants The enzyme activity changes of nitrile hydrolase NITA and its mutants were compared (Table 4). The results showed that the catalytic activity of the nitrile hydrolase NITA_A33G-E101K-W153F-A168N-Q197N-G232A-CUT (330-335) (NITA_M) mutant was 417.85 times that of NITA. The enzyme modification effect was significant, greatly improving the catalytic efficiency of nitrile hydrolase NITA.

[0083] Table 4. Relative enzyme activities of nitrile hydrolase NITA and its mutants ; ; Example 5 Half-life of nitrile hydrolase NITA and its mutants at 40°C The half-life detection method at 40℃ was as follows: wild-type enzyme and mutant enzyme were incubated at 40℃ for different times, and enzyme activity was measured. Half-life curves were plotted and the half-life time was calculated. The enzyme activity detection scheme was the same as above. Enzyme activity detection showed that the multi-point mutant NITA_A33G-E101K-W153F-A168N-Q197N-G232A-CUT (330-335) (NITA_M) also had a half-life extended by 46.11 days at 40℃. The enzyme structure was more stable and could catalyze the substrate for a longer period of time. Specific data are shown in Table 5.

[0084] Table 5. Half-life of nitrile hydrolase NITA and its mutants at 40℃ ; ; Example 6: Optimization of reaction temperature in whole-cell reaction system For NITA, while increasing the reaction temperature can yield higher enzyme activity, it often comes at the cost of enzyme protein inactivation. Therefore, selecting a suitable reaction temperature to balance the effects of enzyme activity and protein inactivation is a crucial step in optimizing the catalytic reaction system. The reaction system was incubated at 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃, with other conditions consistent with enzyme activity requirements. The highest activity was defined as 100%. The results showed that the relative activity was highest at 45℃. Figure 1 However, in order to coordinate enzyme activity and protein inactivation, the preferred catalytic reaction temperature is 40°C.

[0085] Example 7 Optimization of the reaction pH of the whole-cell reaction system For reaction pH, a suitable buffer environment helps NITA achieve higher enzyme activity; therefore, selecting a suitable reaction pH is also an important part of optimizing the catalytic reaction system. The reaction system was incubated at pH 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively, with other conditions consistent with enzyme activity requirements. The results showed that, among the pH conditions of 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, pH 8.0 exhibited the highest relative activity. Figure 2 Therefore, the preferred pH for the catalytic reaction is 8.0. Example 8 Biocatalytic synthesis of 4-acetyl-2-methylbenzoic acid from 4-acetyl-2-methylbenzonitrile The biocatalytic synthesis of 4-acetyl-2-methylbenzonitrile from 4-acetyl-2-methylbenzonitrile was carried out using a reaction system consisting of 1000 g of 4-acetyl-2-methylbenzonitrile, a pH 8.0 buffer environment, and 10 g of whole-cell bacterial sludge. After 12 h of reaction at 40 °C, the yields of NITA and its mutants are shown in Table 6. NITA_A33G-E101K-W153F-A168N-Q197N-G232A-CUT (330-335) (NITA_M) catalyzed the conversion of 1000 g of 4-acetyl-2-methylbenzonitrile into 99.8% 4-acetyl-2-methylbenzoic acid using 10 g of whole-cell bacterial sludge after 12 h. Clearly, the mutant NITA_M significantly improved the conversion rate of 4-acetyl-2-methylbenzonitrile, increasing it by 73.92 times compared to the wild type.

[0086] Table 6. Conversion rates of 1000 g of 4-acetyl-2-methylbenzonitrile catalyzed by nitrile hydrolase NITA and its mutants ; ; The present application has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present application. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and implementation methods of the present application without departing from the spirit and scope of the present application, and all such modifications and improvements fall within the scope of the present application.

Claims

1. A nitrile hydrolase mutant, characterized in that, The nitrile hydrolase mutant is obtained by substituting one or more amino acid residues at positions 14, 33, 54, 64, 74, 101, 153, 168, 197, 232, 267, and 296 of the amino acid sequence shown in SEQ ID NO. 1; and / or; It was obtained by truncating amino acids 330-335 of the amino acid sequence shown in SEQ ID NO.

1.

2. The nitrile hydrolase mutant according to claim 1, characterized in that, The replacements include any combination of one or more of C14A, A33G, A54V, K64C, P74E, E101K, W153F, A168N, Q197N, G232A, L267P, and D296E.

3. The nitrile hydrolase mutant according to claim 2, characterized in that, The nitrile hydrolase mutant was obtained by combining mutations of A33G, E101K, W153F, A168N, Q197N, G232A and CUT (330-335) based on the amino acid sequence shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.

2.

4. A gene encoding a nitrile hydrolase mutant as described in any one of claims 1-3.

5. A recombinant vector containing the gene of claim 4.

6. A recombinant genetically engineered bacterium containing the gene of claim 4.

7. The use of the nitrile hydrolase mutant according to any one of claims 1-3 in the catalytic synthesis of 4-acetyl-2-methylbenzoic acid from 4-acetyl-2-methylbenzonitrile.

8. The application according to claim 7, characterized in that, Using wet bacterial cells obtained by inducing expression of recombinant genetically engineered bacteria with nitrile hydrolase mutant as a catalyst, and 4-acetyl-2-methylbenzonitrile as a substrate, a transformation reaction was carried out at 30-60℃ to obtain 4-acetyl-2-methylbenzoic acid.

9. The application according to claim 8, characterized in that, The pH value of the reaction is 6.0-9.

5.

10. The application according to claim 8, characterized in that, The concentration of 4-acetyl-2-methylbenzonitrile is 1-10000 g / L, and the amount of catalyst used is 1-10000 g / L based on the weight of wet bacterial cells.

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