Nitrilase mutant capable of reducing selectivity of 5-chloronicotinonitrile and improving selectivity of 6-chloronicotinonitrile and application of nitrilase mutant

Abstract

The invention discloses a nitrilase mutant capable of reducing the selectivity of 5-chloronicotinonitrile and improving the selectivity of 6-chloronicotinonitrile and application of the nitrilase mutant. The nitrilase mutant is obtained by performing single mutation or multiple mutation on the 128 site, the 144 site and the 146 site of an amino acid sequence shown in SEQ ID No.2. The nitrilase mutant with obviously improved selectivity on 6-chloronicotinonitrile is obtained by screening, the 6-chloronicotinonitrile can be selectively catalyzed by taking recombinant cells containing nitrilase mutant genes or broken crude enzyme liquid of the recombinant cells as a catalyst and 6-chloronicotinonitrile and 5-chloronicotinonitrile as a common substrate, the high-purity 6-chloronicotinic acid can be synthesized in one step, the process is simple, the cost is low, and the method is suitable for industrial production. And the industrial cost is low. Compared with a wild type, the mutant has the advantages that the catalytic activity on 5-chloronicotinonitrile is reduced, and the selectivity on 6-chloronicotinonitrile is improved.

Description

(I) Technical field

[0001] The present invention relates to a nitrilase mutant capable of reducing the selectivity of 5-chloronicotinonitrile and improving the selectivity of 6-chloronicotinonitrile and an application thereof, in particular to a nitrilase mutant derived from Paraburkholderia graminis and an application thereof in selectively catalyzing 6-chloronicotinonitrile to synthesize 6-chloronicotinic acid. (II) Background technology

[0002] Chlorinated nicotinic acid compounds are important intermediates in the synthesis of pesticides and pharmaceutical chemicals. They can be divided into 2-chloronicotinic acid, 4-chloronicotinic acid, 5-chloronicotinic acid, and 6-chloronicotinic acid according to the different positions of chlorine atoms. For example, 2-chloronicotinic acid is a type of nitrogen heterocyclic compound. In medicine, it can be used to synthesize the anti-AIDS drug nevirapine and the anti-inflammatory drug pranoprofen. In pesticides, it can be used to synthesize the herbicides nicosulfuron, difluanid, and fungicide boscalid. 6-Chloronicotinic acid is widely used in the synthesis of the third-generation aromatic retinoid drug tazarotene for the treatment of psoriasis and acne, as well as other pharmaceutical intermediates and emerging pesticide products. Therefore, chloronicotinic acid has a very broad industrial application prospect.

[0003] The traditional chemical synthesis process of chloronicotinic acid uses 3-cyanopyridine as the raw material, and then produces a mixture of chloronicotinonitrile through oxidation and chlorination, followed by high-temperature alkaline hydrolysis. However, due to the lack of selectivity of chemical hydrolysis, it is necessary to undergo multiple recrystallization separations to obtain a single high-purity product. Due to the many steps, low yield, and large amount of waste salt in the chemical synthesis method, the enzymatic synthesis technology of high-purity chloronicotinic acid has attracted widespread attention.

[0004] Nitrilases (EC 3.5.5.1) are enzymes that can catalyze the hydrolysis of nitrile compounds to produce carboxylic acids and ammonia. The biocatalytic process mediated by nitrilase has the characteristics of mild reaction conditions, high catalytic efficiency, and environmental friendliness. It has important applications in the preparation of high-value-added carboxylic acids and other chemicals. Using nitrilase as a catalyst, it can selectively catalyze the synthesis of chloronicotinonitrile from the corresponding chloronicotinic acid, thereby reconstructing the one-step enzymatic synthesis route of chloronicotinic acid, significantly improving the atom economy, greatly reducing the amount of alkali used, and reducing the discharge of three wastes. However, nitrilase with different structures has different selectivity for chloronicotinonitrile. For example, in previous studies, we screened and obtained nitrilase from Paraburkholderia graminis. Through molecular modification, we obtained mutant PgNit-A55S, which has good catalytic performance for 6-chloronicotinonitrile and eliminates the hydration activity for 2-chloronicotinonitrile. No 2-chloronicotinamide is produced in the process (CN202211100878.3), which significantly improves the selectivity of 6-chloronicotinonitrile when 6-chloronicotinonitrile and 2-chloronicotinonitrile are co-substrates. However, in our further research, we found that the enzyme is also active for 5-chloronicotinonitrile, and has a conversion rate of more than 90% when a mixture of 6-chloronicotinonitrile and 5-chloronicotinonitrile is used as a substrate. The traditional method of using 3-cyanopyridine as raw material and preparing it through oxidation and chlorination usually produces a mixture of chloronicotinonitrile, that is, in addition to 6-chloronicotinonitrile, there are also 2-chloronicotinonitrile and 5-chloronicotinonitrile in the substrate. Therefore, the low substrate selectivity of the nitrilase leads to the problem of difficulty in separating and purifying a single product. Therefore, it is of great significance to further regulate the substrate selectivity of the enzyme and eliminate its catalytic activity against 5-chloronicotinonitrile. (III) Summary of the invention

[0005] The invention aims to provide a nitrilase mutant capable of reducing the selectivity of 5-chloronicotinonitrile and improving the selectivity of 6-chloronicotinonitrile and application thereof. The nitrilase PgNit derived from Paraburkholderia graminis is modified to improve the selectivity of 6-chloronicotinonitrile and reduce the selectivity of 5-chloronicotinonitrile. In particular, a mutant PgNit-R128A / D144K / S146R (PgNit-M3) is obtained. The conversion rates of catalytic hydrolysis of 100 g / L of 5-chloronicotinonitrile and 6-chloronicotinonitrile are 2.8% and 99.8% respectively, which significantly reduces the catalytic efficiency of 5-chloronicotinonitrile, improves the substrate selectivity of the enzyme, and solves the problem of poor selectivity of the chlorinated substrate reaction catalyzed by the traditional nitrilase.

[0006] The technical solution adopted by the present invention is:

[0007] The invention provides a nitrilase mutant capable of reducing the selectivity of 5-chloronicotinonitrile and improving the selectivity of 6-chloronicotinonitrile. The nitrilase mutant is obtained by performing single mutation or multiple mutation on the 128th, 144th and 146th positions of the amino acid sequence shown in SEQ ID No.2.

[0008] Furthermore, 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) the arginine at position 128 is mutated to alanine (PgNit-R128A), the nucleotide sequence is shown in SEQ ID No.3, and the amino acid sequence is shown in SEQ ID No.4; (2) the arginine at position 128 is mutated to alanine, and the aspartic acid at position 144 is mutated to lysine (PgNit-R128A / D144K), the nucleotide sequence is shown in SEQ ID No.5, and the amino acid sequence is shown in SEQ ID No.6; (3) the arginine at position 128 is mutated to alanine, the aspartic acid at position 144 is mutated to lysine, and the serine at position 146 is changed to arginine (PgNit-R128A / D144K / S146R), the nucleotide sequence is shown in SEQ ID No.7, and the amino acid sequence is shown in SEQ ID No.8.

[0009] The invention also provides a recombinant vector constructed with the coding gene of the nitrilase mutant and a recombinant genetic engineering bacterium.

[0010] The basic vector of the recombinant vector is not limited, as long as it can maintain the replication of the coding gene or autonomous replication in various host cells of prokaryotic and / or eukaryotic cells, and the vector can be various conventional vectors in the art, such as various plasmids, phages or viral vectors, etc., preferably pET-28b (+). The host cell of the recombinant genetic engineering bacteria is not limited, as long as a recombinant expression system has been established for it, and the recombinant expression vector can stably self-replicate and the nitrilase gene of the present invention carried can be effectively expressed. For example, Escherichia coli, Bacillus subtilis, yeast, actinomycetes, Aspergillus, as well as animal cells and higher plant cells. Escherichia coli is preferred in the present invention, and E. coli BL21 (DE3) is more preferred.

[0011] The present invention selects pET28b(+) as an expression vector for the expression of nitrilase and its mutants. Specifically, the target gene is inserted between NcoI and XhoI on the plasmid pET28b(+) and transformed into an E.coli BL21(DE3) host cell.

[0012] The present invention provides an application of a nitrilase mutant in selectively catalyzing 6-chloronicotinonitrile to synthesize 6-chloronicotinic acid. The application is as follows: using wet cells obtained by fermentation culture of recombinant Escherichia coli containing a nitrilase mutant encoding gene or a crude enzyme solution extracted from the wet cells as a catalyst, using 6-chloronicotinonitrile and 5-chloronicotinonitrile as substrates, using a buffer solution with a pH of 6.0-9.0 as a reaction medium, and reacting at 25-40°C to obtain a reaction solution with a significantly reduced 5-chloronicotinic acid content and rich in 6-chloronicotinic acid. The amount of the crude enzyme solution added is based on the weight of the wet cells before crushing, and the amount of the wet cells added is 2-20 g / L, preferably 10 g / L, based on the volume of the buffer solution; the amounts of 6-chloronicotinonitrile and 5-chloronicotinonitrile added are each independently 100-500 g / L, and each is preferably 100 g / L. The buffer solution is preferably a 50 mM PB buffer solution with a pH of 7.0. The reaction temperature is preferably 30°C.

[0013] The catalyst is prepared as follows:

[0014] 1) Plate culture: The recombinant Escherichia coli containing the gene encoding the nitrilase mutant was inoculated into LB solid medium containing 50 μg / mL kanamycin by streaking on a plate, and activated overnight at 37° C. to obtain a single colony; the final concentration of the LB solid medium was: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, the solvent was water, and the pH was 7.0;

[0015] 2) Seed culture: a single colony was inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 180 rpm for 10-12 hours to obtain a seed solution; the final concentration of the LB liquid medium was: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent was water, pH 7.0;

[0016] 3) Fermentation culture: The seed liquid was inoculated into LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 2%, and cultured at 37°C and 180 rpm until OD600 was 0.6-0.8, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM, and induced culture was carried out at 28°C and 180 rpm for 10-12 h. The bacterial liquid was centrifuged at 8000 rpm and 4°C to collect wet cells;

[0017] 4) Wet cell crushing: The wet cells are suspended in a buffer solution of pH 5.0 to 9.0, placed in an ultrasonic crusher, crushed at a frequency of 10-50kHz (preferably 20kHz) for 5-30min (preferably crushed for 2min, with an interval of 10s and crushed for 10min), and the crushed mixed liquid is collected to obtain a crude enzyme solution; the volume of the buffer solution of pH 5.0 to 9.0 is 10mL / g based on the wet weight of the wet cells.

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0019] The invention screens and obtains a nitrilase mutant with reduced selectivity for 5-chloronicotinonitrile and significantly improved selectivity for 6-chloronicotinonitrile. The invention uses a recombinant cell containing a nitrilase mutant gene or a crushed crude enzyme solution thereof as a catalyst, and uses 6-chloronicotinonitrile and 5-chloronicotinonitrile as common substrates to selectively catalyze 6-chloronicotinonitrile to synthesize high-purity 6-chloronicotinic acid in one step. The process is simple and the industrial cost is low. Compared with the wild type, the mutant has reduced catalytic activity for 5-chloronicotinonitrile, improved selectivity for 6-chloronicotinonitrile, and is conducive to improving substrate specificity. In the wet cell dosage system of 10 g / L, the wild-type nitrilase PgNit catalyzed the synthesis of 6-chloronicotinic acid and 5-chloronicotinic acid from 100 g / L 6-chloronicotinonitrile and 100 g / L 5-chloronicotinonitrile, with conversion rates of >90%; while the nitrilase mutant PgNit-R128A / D144K / S146R catalyzed the synthesis of 100 g / L 6-chloronicotinonitrile and 100 g / L 5-chloronicotinonitrile, with a 6-chloronicotinic acid conversion rate of more than 99.8%, and a 5-chloronicotinic acid conversion rate reduced to 2.8%. (IV) Description of the drawings

[0020] Figure 1 , PgNit and its PgNit mutants catalyze the reaction process of chloronicotinonitrile substrates.

[0021] Figure 2 , catalyzing the reaction of 6-chloronicotinonitrile to produce 6-chloronicotinic acid liquid phase peak spectrum.

[0022] Figure 3 , catalyzing the reaction of 5-chloronicotinonitrile to produce 5-chloronicotinic acid liquid phase peak spectrum. (V) Specific implementation methods

[0023] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0024] The final concentration of the LB solid culture medium of the present invention is as follows: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, the solvent is water, and the pH is 7.0.

[0025] The final concentration of the LB liquid culture medium is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent is water, and the pH is 7.0.

[0026] The PB buffer solution (50 mM, pH 7.0) is prepared by weighing 2.28 g of K2HPO4·H2O, adding 200 mL of ultrapure water to completely dissolve it; then weighing 1.36 g of KH2PO4·H2O and adding it to 200 mL of ultrapure water, mixing the two, and adjusting the pH of the buffer solution to 7.0.

[0027] Example 1. Construction of PgNit recombinant bacteria

[0028] The nitrilase (PgNit) gene from Paraburkholderia graminis (GenBank: EDT09464.1, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) was fully synthesized at Beijing Qingke Biotechnology Co., Ltd., connected to the NcoI and XhoI restriction sites of plasmid pET28b(+), and transformed into E. coli DH5 competent cells, coated with LB plates containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37°C overnight, and then the positive transformants were picked and sequenced for identification. The verified positive monoclonal clone was inoculated into 5 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37°C overnight, and after extracting the plasmid for verification, the recombinant expression vector was transferred into the E. coli BL21 (DE3) strain to obtain the recombinant bacteria E. coli BL21 (DE3) / pET28b-PgNit, which was cultured overnight at 37°C on an LB plate containing a final concentration of 50 μg / mL kanamycin. A monoclonal clone was picked and inoculated into 5 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37°C overnight, and the bacterial solution was mixed with 30% glycerol (v / v = 1:1) in a glycerol tube and stored in a -80°C refrigerator.

[0029] SEQ ID NO.2:

[0030] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYF

[0031] AAVQTGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGGT

[0032] LYNTQLLFDADGTLIQRR R KITPTHFERMIWGQGD G S GLRAVDSAVGRIGQLACFE

[0033] HNNPLARYAMIADGEQIHSAMYPGSAFGEGFAQRMEINIRQHALESGAFVVNATA

[0034] WLDADQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQ

[0035] IDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI.

[0036] Example 2. Obtaining PgNit mutants and constructing genetically engineered bacteria containing the mutants

[0037] 1. Construction of mutation library

[0038] Using the recombinant expression vector pET28b-PgNit obtained in Example 1 as a template, the amino acid sites (126, 128, 135, 144, 146, 147, and 192) that affect the enzyme activity were screened according to the docking results, the primers shown in Table 1 were designed, and site-directed saturation mutagenesis was performed on the key amino acid sites to establish a mutation library.

[0039] Table 1 PgNit site-directed saturation mutagenesis primer sequence list

[0040]

[0041] N=A / G / C / T, K=G / T, M=A / C

[0042] The 50mL amplification system is: 25mL 2x Phanta Max Buffer; 1mL Phanta Max Super-Fidelity DNA Polymerase; 1mL dNTP Mix (10mM); 1mL DNA template; 2mL each of the upstream primer and downstream primer at a concentration of 50mM; and make up the system with double distilled water.

[0043] The PCR reaction conditions were: pre-denaturation at 95°C for 5 min, followed by temperature cycling at 95°C for 30 s, 55°C for 30 s, and 72°C for 4 min, for a total of 30 cycles, and finally extension at 72°C for 10 min. The obtained PCR product was purified using a clean-up kit.

[0044] The gene fragment obtained by the above-mentioned site-directed saturation mutation was used as a primer, and the whole plasmid pET-28a(+)-PgNit was used as a template for whole plasmid PCR amplification. A high-fidelity DNA polymerase was used for PCR reaction, and finally a linear whole plasmid PCR product was obtained. After the whole plasmid PCR product was positive by agarose gel electrophoresis, it was demethylated using restriction endonuclease DpnⅠ, purified using a clean-up kit, and transformed into E.coli BL21(DE3) competent cells to obtain recombinant cells, which were the mutation library.

[0045] 2. Screening of mutants

[0046] (1) Use a sterilized toothpick to select a single clone from the above mutation library, inoculate it into a 96-well plate with 600 μL of LB liquid medium containing 50 mg / mL kanamycin, and culture it at 37°C, 180 rpm for 20-22 hours as a seed solution. Then take out 200 μL of the seed solution and transfer it to a new 96-well plate, culture it at 37°C, 180 rpm for 2-3 hours until the OD600 reaches 0.6-0.8, add IPTG with a final concentration of 0.1 mM to each well, and culture it at 28°C, 180 rpm for 12 hours. Then centrifuge the 96-well plate at 4°C, 4000 rpm for 15 minutes, discard the supernatant, and keep the bacteria for later use.

[0047] (2) In step (1), 200 μL of PB buffer (50 mM, pH 7.0) was added to each well of the 96-well plate to resuspend the nitrilase bacteria, and 5-chloronicotinonitrile (5-CA) was added to a final concentration of 50 mM. The reaction was carried out at 30° C. and 180 rpm for 5 min. 15 μL of 6 M HCl was added to terminate the reaction, and then centrifuged at 4° C. and 4000 rpm for 15 min.

[0048] (3) Take 20 μL of the supernatant after centrifugation of the 96-well plate in step (2) and add it to a black fluorescent ELISA plate. Add 180 μL of fluorescent colorimetric solution to each well, react at 37°C for 30 min, and measure the fluorescence intensity by an ELISA instrument (412 nm excitation wavelength, 467 nm emission wavelength). By comparing the fluorescence values ​​of the template strain and the mutant strain, select the clones with decreased fluorescence values ​​in response to 5-chloronicotinonitrile, corresponding to wells B6, D5, C7, A8, D9, E10, C11, G1, B2, C6, A5, E8, D2, and G5. The fluorescent colorimetric solution is prepared by dissolving 5 mL of solution A and 5 mL of solution B in 90 mL of PB buffer (100 mM, pH 7.2), wherein solution A (75 mM): 100 mg of o-phthalaldehyde dissolved in 10 mL of anhydrous ethanol; solution B (72 mM): 50 μL of mercaptoethanol dissolved in 10 mL of anhydrous ethanol.

[0049] (4) 200 μL of the seed solution from step (1) corresponding to wells B6, D5, C7, A8, E10, C11, G1, B2, E8, D2, and G5 of the clones selected in step (3) was transferred to a new 96-well plate, and the induction step was the same as step (1). The 96-well plate was centrifuged at 4°C, 4000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were retained.

[0050] (5) In step (4), 200 μL of PB buffer (50 mM, pH 7.0) was added to each well of the 96-well plate to resuspend the nitrilase cells, 6-chloronicotinonitrile (6-CA) was added to a final concentration of 50 mM, the reaction was carried out at 30° C., 180 rpm for 5 min, 15 μL of 6 M HCl was added to terminate the reaction, and then centrifuged at 4° C., 4000 rpm for 15 min.

[0051] (6) Take 20 μL of the supernatant after centrifugation of the 96-well plate in step (5) and transfer it to a black fluorescent ELISA plate. The color development step is the same as step (3). By comparing the fluorescence values ​​of the template strain and the mutant strain, clones D5, C7, A8, E10, E8, D2, and G5 with increased fluorescence values ​​for 6-chloronicotinonitrile reaction are selected for rescreening.

[0052] Table 2 Preliminary screening data in PgNit mutant library

[0053]

[0054] 3. Rescreening and verification of mutants

[0055] The positive mutants of D5, C7, A8, E10, E8, D2, and G5 obtained by the above high-throughput screening were further rescreened and verified. Take 100 μL of the seed solution of the corresponding positive strain in the 96-well plate and add it to 100 mL of LB liquid medium containing 50 μg / mL kanamycin, culture it at 37°C and 180 rpm until OD600 is 0.6-0.8, add IPTG with a final concentration of 0.1 mM, and induce culture at 28°C and 180 rpm for 10-12 hours. The harvested bacterial solution was centrifuged at 8000 rpm and 4°C, and the wet bacteria were collected for later use.

[0056] Weigh 0.1 g of bacteria (wet weight) and resuspend in 10 mL of PB buffer (50 mM, pH 7.0), incubate at 30°C for 5 min, add 50 mM 6-chloronicotinonitrile and 50 mM 5-chloronicotinonitrile, and react at 180 rpm and 30°C for 5-10 min. Sample 1 mL of the reaction solution, add 10 μL 6M HCl to terminate the reaction, then let stand for 1 min and centrifuge at 12000 rpm for 2 min. The supernatant was collected and diluted appropriately. The peak areas of 6-chloronicotinic acid and 5-chloronicotinic acid were analyzed by high performance liquid chromatography (HPLC). Mutants with increased 6-chloronicotinic acid area and decreased 5-chloronicotinic acid peak area were selected, and highly selective mutants PgNit-R128A, PgNit-D144K, and PgNit-S146R were screened and obtained. Among them, the single mutant PgNit-R128A (denoted as PgNit-M1) had significantly improved substrate specificity for 6-chloronicotinonitrile. The nucleotide sequence is shown in SEQ ID No.3, and the amino acid sequence is shown in SEQ ID No.4.

[0057] The HPLC equipment and analysis conditions were as follows: the chromatographic column type was a Welchrom C18 column (250 mm × 4.6 mm, 5 μm); the chromatographic column operating temperature was set at 30°C, the UV detection wavelength was 210 nm, the mobile phase was 25% acetonitrile and 75% water (containing 0.1% H3PO4), and the flow rate was 1.0 mL / min.

[0058] 4. Construction of combined iterative mutants

[0059] The single mutant PgNit-R128A wet bacteria were cultured according to the method in step 3, and the plasmid was extracted as a template for whole-plasmid PCR amplification. The aspartic acid at position 144 was mutated to lysine to obtain the double mutant PgNit-R128A / D144K (denoted as PgNit-M2), the nucleotide sequence of which is shown in SEQ ID No.5, and the amino acid sequence of which is shown in SEQ ID No.6;

[0060] Subsequently, based on PgNIT-M2, a saturation mutation was performed on position 146 to obtain a mutant PgNit-R128A / D144K / S146R (denoted as PgNIT-M3) with significantly improved substrate specificity for 6-chloronicotinonitrile and significantly reduced yield of 5-chloronicotinic acid. The nucleotide sequence is shown in SEQ ID No.7, and the amino acid sequence is shown in SEQ ID No.8.

[0061] Example 3: Preparation of wet cells of PgNit and its mutants

[0062] 1) Plate culture: The recombinant genetically engineered bacteria containing nitrilase PgNit and the recombinant genetically engineered bacteria of mutants (PgNit-M1, PgNit-M2, PgNit-M3) were streaked onto solid LB medium containing 50 μg / mL kanamycin, and activated at 37° C. overnight to obtain single colonies.

[0063] 2) Seed culture: A single colony was inoculated into 5 μL of LB liquid culture medium containing 50 μg / mL kanamycin, and cultured at 37° C., 180 rpm for 10-12 h to obtain seed solution.

[0064] 3) Fermentation culture: Add kanamycin with a final concentration of 50 μg / mL to a shake flask containing 100 mL of liquid LB medium, inoculate with 2% (v / v) seed solution, culture at 37°C, 180 rpm until OD600 is 0.6-0.8, add IPTG with a final concentration of 0.1 mM, and induce culture at 28°C, 180 rpm for 10-12 h. The harvested bacterial solution was centrifuged at 8000 rpm, 4°C, and wet cells were collected to obtain wet cells of PgNit and its mutants for later use.

[0065] Example 4: Reaction progress of chloronicotinonitrile substrate catalyzed by PgNit and its PgNit mutants

[0066] 1. In 20 mL PB buffer (pH 7.0, 50 mM), 10 g / L of Pg Nit wet cells and PgNit-M1 wet cells prepared by the method of Example 3 were added respectively, and a mixture of 100 g / L 5-chloronicotinonitrile and 100 g / L 6-chloronicotinonitrile was used as a substrate. The reaction was carried out under the conditions of 30 ° C and 600 rpm magnetic stirring water bath, and the high performance liquid chromatography described in Example 2 was used for detection. The results showed that under the optimal system, the nitrilase PgNit catalyzed the synthesis of 6-chloronicotinonitrile from 100 g / L 6-chloronicotinonitrile with a conversion rate of >99%, and catalyzed the synthesis of 5-chloronicotinic acid from 100 g / L 5-chloronicotinonitrile with a conversion rate of 92%; under the optimal system, the nitrilase mutant PgNit-M1 catalyzed the synthesis of 6-chloronicotinic acid from 100 g / L 6-chloronicotinonitrile with a conversion rate of >99%, while the conversion rate of PgNit-M1 catalyzed the synthesis of 5-chloronicotinic acid from 100 g / L 5-chloronicotinonitrile was reduced to 21.4%.

[0067] 2. In 20 mL PB buffer (pH 7.0, 50 mM), 10 g / L of PgNit-M2 wet cells prepared by the method of Example 3 were added respectively, and a mixture of 100 g / L 5-chloronicotinonitrile and 100 g / L 6-chloronicotinonitrile was used as a substrate, and the reaction was carried out under the conditions of 30 ° C and 600 rpm magnetic stirring water bath. The high performance liquid chromatography described in Example 2 was used for detection. Under the optimal system, the nitrilase mutant PgNit-M2 catalyzed 100 g / L 6-chloronicotinonitrile to synthesize 6-chloronicotinic acid, and the conversion rate was>99%. PgNit-M2 catalyzed 100 g / L 5-chloronicotinonitrile to synthesize 5-chloronicotinic acid, and the conversion rate was reduced to 15.3%.

[0068] 3. In 20mL PB buffer (pH 7.0, 50mM), 10g / L of PgNit-M3 wet cells prepared by the method of Example 3 were added respectively, and a mixture of 100g / L 5-chloronicotinonitrile and 100g / L 6-chloronicotinonitrile was used as a substrate, and the reaction was carried out under the conditions of 30°C and 600rpm magnetic stirring water bath. The high performance liquid chromatography described in Example 2 was used for detection. Under the optimal system, the nitrilase mutant PgNit-M3 catalyzed 100g / L 6-chloronicotinonitrile to synthesize 6-chloronicotinic acid, and the conversion rate was 99.8%, while PgNit-M3 catalyzed 100g / L 5-chloronicotinonitrile to synthesize 5-chloronicotinic acid, and the conversion rate was reduced to 2.8%. Compared with the wild nitrilase, the substrate specificity of the mutant PgNit-M3 was significantly improved.

[0069] The present invention is not limited by the above specific textual description. The present invention can be modified in various ways within the scope of the claims, and these modifications are within the scope of the present invention.

Claims

1. A nitrilase mutant that reduces the selectivity of 5-chloronicotinonitrile and improves the selectivity of 6-chloronicotinonitrile, characterized in that: The nitrilase mutant is obtained by performing single mutation or multiple mutation on the 128th, 144th and 146th positions of the amino acid sequence shown in SEQ ID No.

2.

2. The nitrilase mutant according to claim 1, wherein The nitrilase mutant is a mutant in which the amino acid shown in SE QID No. 2 is mutated into one of the following: (1) the arginine at position 128 is mutated into alanine; (2) the arginine at position 128 is mutated into alanine, and the aspartic acid at position 144 is mutated into lysine; (3) the arginine at position 128 is mutated into alanine, the aspartic acid at position 144 is mutated into lysine, and the serine at position 146 is changed into arginine.

3. A recombinant genetically engineered bacterium constructed with the gene encoding the nitrilase mutant according to claim 1.

4. Use of the nitrilase mutant of claim 1 in selectively catalyzing the synthesis of 6-chloronicotinonitrile from 6-chloronicotinic acid.

5. The use according to claim 4, characterized in that The application is as follows: using wet bacteria obtained by fermentation culture of recombinant Escherichia coli containing a gene encoding a nitrilase mutant or a crude enzyme solution extracted from the wet bacteria as a catalyst, using 6-chloronicotinonitrile and 5-chloronicotinonitrile as substrates, using a buffer solution with a pH of 6.0-9.0 as a reaction medium, and reacting at 25-40° C. to obtain a reaction solution with a significantly reduced 5-chloronicotinic acid content and rich in 6-chloronicotinic acid.

6. The use according to claim 5, characterized in that The amount of the crude enzyme solution added is based on the weight of the wet cells before crushing, and the amount of the wet cells added is 2-20 g / L based on the volume of the buffer solution; the amounts of 6-chloronicotinonitrile and 5-chloronicotinonitrile added are each independently 100-500 g / L based on the volume of the buffer solution.

7. The use according to claim 5, characterized in that The buffer is 50 mM PB buffer with a pH of 7.0; and the reaction temperature is 30°C.

8. The use according to claim 5, characterized in that The catalyst is prepared as follows: 1) Plate culture: The recombinant E. coli containing the gene encoding the nitrilase mutant was inoculated onto a solid LB medium containing 50 μg / mL kanamycin by streaking on a plate, and activated at 37°C overnight to obtain a single colony; 2) Seed culture: Inoculate a single colony into LB liquid medium containing 50 μg / mL kanamycin, and culture at 37°C, 180 rpm for 10-12 h to obtain seed solution; 3) Fermentation culture: The seed liquid was inoculated into LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 2%, and cultured at 37°C and 180 rpm until OD600 was 0.6-0.8, and isopropyl-β-D-thiogalactopyranoside was added at a final concentration of 0.1 mM, and induced culture was carried out at 28°C and 180 rpm for 10-12 h. The bacterial liquid was centrifuged at 8000 rpm and 4°C to collect the wet cells; 4) Wet cell crushing: The wet cells are suspended in a buffer solution of pH 5.0 to 9.0, placed in an ultrasonic crusher, crushed at a frequency of 10 to 50 kHz for 5 to 30 minutes, and the crushed mixed liquid is collected to obtain a crude enzyme solution.

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