Nitrilase mutant and application thereof in hydrolysis of pyridine nitrile derivative

Abstract

The invention discloses a nitrilase mutant and application of the nitrilase mutant in hydrolysis of pyridine nitrile derivatives. The nitrilase mutant is obtained by performing single mutation or multiple mutations on 59th, 134, 135th or 192th amino acids of an amino acid sequence shown in SEQ ID No.2. The invention further discloses a preparation method of the nitrilase mutant. According to the nitrilase four-mutant AcNR-W59A / T134G / H135L / S192G, the protein is subjected to molecular modification through semi-rational design, the catalytic activity of the nitrilase four-mutant AcNR-W59A / T134G / H135L / S192G to 4-TCN is improved by about 142 times, the catalytic activity of the nitrilase four-mutant AcNR-W59A / T134G / H135L / S192G to ricinine is improved by about 77 times, and meanwhile, the complete hydrolytic activity is kept. The nitrilase mutant reported by the invention has very high catalytic activity on various pyridine nitrile substances, and has wide and good application prospects in pesticide and feed production.

Description

(I) Technical field

[0001] The invention relates to a nitrilase mutant and application thereof in hydrolyzing pyridine nitrile derivatives. (II) Background technology

[0002] Nitrilases are a class of enzymes that can catalyze the hydrolysis of cyanide groups to carboxyl groups. They have the characteristics of mild reaction conditions, high stereoselectivity, and environmental friendliness. Currently, nitrilase is widely used in industry. Lonza of Switzerland first used nitrilase to catalyze the production of nicotinic acid. Through the joint action of nitrilase and nicotinamide dehydrogenase, the substrates 2-cyanopyridine and 2-cyanopyrazine are degraded into drug intermediates 5-hydroxypyridine-2-carboxylic acid and 5-hydroxypyrazine-2-carboxylic acid, respectively. The reaction is highly selective and the conversion rate is nearly 100%, which is a great advantage over traditional chemical methods. BASF of Germany uses nitrilase to chirally resolve racemic mandelonitrile to generate (R)-mandelic acid in a directional manner, with a conversion rate of up to 99%. DuPont has developed a chemical-enzymatic process to convert 2-methylglutaronitrile (MGN) into 1,5-dimethyl-2-piperidone (1,5-DMPD). Immobilized nitrilase (Acidovorax facilis 72w) was used as a catalyst for the hydrolysis of MGN to hydrolyze MGN into 4-cyanovaleric acid (4-CAP) ammonium salt, with a selectivity of more than 98% and a conversion rate of 100%. Compared with traditional chemical processes, the chemical-enzymatic production process has a high yield, produces less waste, and can produce a single lactam isomer.

[0003] With the advancement of the green sustainable development strategy, new green pesticides with the characteristics of high efficiency, low toxicity and easy decomposition have become the mainstream of the market. Among them, pyridine chloride pesticides represented by imidacloprid have become a hot spot for research and development, and 3,4,5,6-tetrachloropyridine-2-carboxylic acid (4-TCA) is a key intermediate for the synthesis of such pesticides. Similar pesticides such as chlorpyrifos (4-amino-3,5,6-trichloropyridine-2-carboxylic acid) and pyridine-2-carboxylic acid (3,6-dichloropyridine-2-carboxylic acid) require 4-TCA for synthesis, and 4-TCA can also be used as a pharmaceutical intermediate and chemical intermediate, and its market demand is growing. Chemical synthesis is currently the only means to obtain 4-TCA in industry, but the process has a large amount of waste acid, high energy consumption, and the purity of the obtained 4-TCA is low, which is not conducive to large-scale production. The bio-enzymatic method uses nitrilase to catalyze the hydrolysis of 3,4,5,6-tetrachloropyridine-2-carbonitrile (4-TCN) to synthesize 4-TCA. It is safe, environmentally friendly, and has the advantages of low emissions of three wastes. It can also utilize the high catalytic efficiency of nitrilase to greatly improve production efficiency.

[0004] On the other hand, as one of the world's top ten oil crops, castor is widely used in fuel, chemical industry and other fields. The byproduct of castor oil extraction, castor meal, is not only rich in crude protein and minerals such as calcium and phosphorus, but also has high utilization value. However, it contains a series of toxic substances, mainly ricinine, which hinders its utilization. The chemical name of ricinine is 3-cyano-4-methoxy-1-methyl-2-pyridone, which is a natural piperidine alkaloid with high toxicity. However, it can also synthesize a variety of organic compounds and can be used to make pesticides and poisonous gases. Studies have shown that adults who accidentally eat 20 castor seeds can be fatal, and those aged 4 to 7 years old who accidentally eat 2 to 7 seeds can be poisoned and killed. Therefore, after detoxification, castor meal can be used as feed or to prepare products such as plant protein. Finding a way to efficiently detoxify castor meal without destroying its nutritional components has high application value.

[0005] After reviewing the relevant literature, it was found that, except for the research of this research group, there are currently few reports of nitrilase that can catalyze the hydrolysis of 4-TCN, and there is no report of nitrilase that can catalyze the hydrolysis of ricinine. This may be because the substrate molecules of 4-TCN or ricinine are large, and there are multiple chlorine and other substituent groups on their molecules. Overly large substrates are not easy to enter the active center of the enzyme, so they cannot bind well with the enzyme, resulting in low catalytic efficiency. Studies have shown that when there are three or more chlorine atoms on the substrate, the chlorine atoms form a conjugated effect with the pyridine ring, which hinders the substrate from entering the enzyme catalytic pocket and limits the activity of nitrilase on these substrates. Therefore, the development of nitrilase that can degrade pyridine nitrile derivatives such as 4-TCN or ricinine has great application value. (III) Summary of the invention

[0006] The present invention aims to provide a nitrilase mutant and its application in hydrolyzing pyridine nitrile derivatives, especially the application in catalyzing the synthesis of 3,4,5,6-tetrachloropyridine-2-carboxylic acid (4-TCA) and catalyzing the degradation of ricinine in castor meal. The present invention screens and obtains a variety of nitrilase mutant proteins that can hydrolyze the substrate 4-TCN to generate 4-TCA and have hydrolysis activity on the substrate ricinine, including the encoding gene of the mutant protein, the recombinant vector containing the gene, and the recombinant genetic engineering bacteria transformed by the recombinant vector. The present invention solves the problems of low catalytic activity and poor stability of nitrilase on the substrates 4-TCN and ricinine.

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

[0008] The present invention provides a nitrilase mutant with improved enzyme activity. The nitrilase mutant is obtained by subjecting the 59th, 134th, 135th or 192nd amino acid of the amino acid sequence shown in SEQ ID No. 2 to single mutation or multiple mutation.

[0009] Furthermore, it is preferred that the nitrilase mutant is a mutant in which the amino acid sequence shown in SEQ ID No. 2 is mutated to one of the following:

[0010] (1) the tryptophan at position 59 is mutated to alanine (W59A), denoted as AcNR-W59A, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4;

[0011] (2) Threonine 134 was mutated to glycine (T134G), denoted as AcNR-T134G, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 6;

[0012] (3) the histidine at position 135 was mutated to leucine (H135L), denoted as AcNR-H135L, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 7, and the amino acid sequence is shown in SEQ ID No. 8;

[0013] (4) Serine at position 192 was mutated to glycine (S192G), denoted as AcNR-S192G, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 9, and the amino acid sequence is shown in SEQ ID No. 10;

[0014] (5) mutants obtained by combining the single point mutations described in (1) to (4) in pairs;

[0015] (6) A mutant obtained by combining the single point mutations described in (1) to (4) with three mutations;

[0016] (7) The mutant (W59A / T134G / H135L / S192G) obtained by combining the single point mutations described in (1) to (4) with four mutations was recorded as AcNR-W59A / T134G / H135L / S192G, the nucleotide sequence of the encoding gene is shown in SEQ ID No.11, and the amino acid sequence is shown in SEQ ID No.12.

[0017] The present invention also relates to a coding gene of the nitrilase mutant, a recombinant vector constructed by the coding gene, and a recombinant genetic engineering bacterium obtained by transforming a host cell with the recombinant vector. The basic vector of the recombinant vector is mainly the pET series of Escherichia coli, including pET22b, pET24a, and pET28a, and the recombinant genetic engineering bacterium mainly uses Escherichia coli BL21 (DE3) as a host bacterium.

[0018] The present invention also provides an application of the nitrilase mutant in catalyzing 3,4,5,6-tetrachloropyridine-2-carbonitrile (4-TCN) to prepare 3,4,5,6-tetrachloropyridine-2-carboxylic acid (4-TCA). Specifically, the application comprises: using wet bacterial bodies obtained by fermentation culture of genetically engineered bacteria expressing the nitrilase mutant, or crude enzyme liquid extracted after ultrasonic crushing of wet bacterial bodies, or pure enzyme liquid obtained by purifying the crude enzyme liquid, or immobilized cells obtained by immobilizing wet bacterial bodies on diatomaceous earth as catalysts, using 4-TCN as substrate, and using pH 4.0-10.5 and 200mM buffer as reaction medium to form a reaction system 1 (preferably 200mM NaH2PO4-Na2HPO4, pH=8.0), reacting under the conditions of 800rpm, 35-50°C (preferably 45°C) constant temperature water bath, and separating and purifying the reaction liquid after the reaction is complete to obtain 4-TCA.

[0019] Furthermore, in the reaction system 1, the final concentration of the substrate added is 20-200 mM, preferably 50 mM, based on the volume of the reaction system; when the catalyst is a crude enzyme solution or a pure enzyme solution, the added amount is 0.1-3.5 g / L, preferably 1.0 g / L, based on the protein content; when the catalyst is wet bacteria, the added amount is 10-100 g / L, preferably 10 g / L, based on the wet weight of the bacteria; when the catalyst is immobilized cells, the added amount is 10-200 g / L, preferably 50 g / L, based on the weight of the wet bacteria.

[0020] The present invention also provides an application of the nitrilase mutant in degrading ricin in castor meal. Specifically, the application comprises: using wet bacteria obtained by fermentation culture of genetically engineered bacteria expressing the nitrilase mutant, or crude enzyme liquid extracted after ultrasonic crushing of the wet bacteria as a catalyst, using castor meal containing ricin as a substrate, and using water or a pH 4.0-10.5 buffer solution (preferably 200 mM, pH=7.0 sodium phosphate buffer solution) as a reaction medium to form a reaction system 2, reacting at 300 rpm and 30-50° C. (preferably 40° C.) until the ricinine content in the castor meal is less than 10 ppm, thereby obtaining castor meal with greatly reduced toxicity.

[0021] Furthermore, in the reaction system 2, the amount of reaction medium added is 10-400 mL / kg, preferably 400 mL / kg, based on the mass of castor meal; the amount of catalyst added is based on the wet weight of the bacteria, and the wet bacteria is 1-10 g / kg, preferably 5 g / kg, based on the mass of castor meal.

[0022] The wet bacterial cells were prepared as follows: the engineered bacteria containing the nitrilase mutant encoding gene were inoculated into LB medium, cultured at 37° C. for 10-12 hours, the culture solution was inoculated into LB medium containing kanamycin at a final concentration of 50 mg / L at an inoculum concentration of 1% by volume, and cultured at 37° C. until the culture solution OD reached 0.1%. 600is between 0.6 and 0.8, for about 2 to 3 hours; add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, induce culture at 28°C for 10 hours, centrifuge, collect the bacteria, wash twice with physiological saline, and obtain wet bacteria.

[0023] The crude enzyme solution was prepared as follows: the wet bacteria were resuspended in pH 8.0, 200 mM NaH2PO4-Na2HPO4 buffer, ultrasonically disrupted (400 W, 20 min, 1 s disruption and 1 s pause), the disrupted product was centrifuged (4000 rpm, 10 min) and the supernatant was taken as the crude enzyme solution.

[0024] The pure enzyme solution is prepared as follows: the crude enzyme solution is passed through a Ni-NTA column washed with a binding buffer at a flow rate of 1 mL / min, and weakly adsorbed impurities are eluted with an equilibrium buffer at a flow rate of 1.5 mL / min for 1-5 column volumes (preferably 3); the target protein is then eluted and collected with an elution buffer at a flow rate of 1.5 mL / min for 1-5 column volumes (preferably 3); finally, the collected target protein is dialyzed with a pH=8.0, 50 mM sodium phosphate buffer as a dialysate (the molecular weight cut-off of the dialysis bag is 30 kDa), and the retained solution is the pure enzyme solution. The binding buffer is a pH 8.0, 200 mM NaH2PO4 buffer containing a final concentration of 300 mM NaCl, the equilibration buffer is a pH 8.0, 200 mM NaH2PO4 buffer containing a final concentration of 300 mM NaCl and 50 mM imidazole, and the elution buffer is a pH 8.0, 200 mM NaH2PO4 buffer containing a final concentration of 300 mM NaCl and 500 mM imidazole.

[0025] The immobilized cells are prepared as follows: the wet cells of the recombinant genetic engineering bacteria containing the nitrilase mutant encoding gene are suspended in a NaH2PO4-Na2HPO4 buffer system (200mM, pH=8.0) at a concentration of 100g / L, diatomaceous earth with a final concentration of 6g / L is added, and the mixture is stirred at room temperature for 1h. Subsequently, a polyethyleneimine aqueous solution with a mass concentration of 5% is added, and the mixture is stirred at room temperature for 1h. Finally, a glutaraldehyde aqueous solution with a mass concentration of 25% is added, stirred for 0.5h, and finally vacuum filtered, washed with deionized water for 3 to 5 times, and dried at room temperature to obtain immobilized cells. The volume amount of the polyethyleneimine aqueous solution added is 3% based on the volume of the buffer, and the volume amount of the glutaraldehyde aqueous solution added is 1% based on the volume of the buffer. The immobilized cells are then stored in a 4°C refrigerator for later use.

[0026] The catalyst of the present invention can be wet cells of recombinant genetically engineered bacteria containing the nitrilase mutant gene (preferably Escherichia coli BL211 (DE3)), can be crude enzyme liquid extracted after ultrasonic disruption of wet cells of recombinant genetically engineered bacteria, can also be pure enzyme liquid obtained after purification of crude enzyme liquid, and can also be immobilized cells obtained by immobilizing wet cells.

[0027] The LB liquid culture medium of the present invention comprises: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, the solvent is water, and the pH value is natural. The LB solid culture medium is prepared by adding 15 g / L agar to the LB liquid culture medium.

[0028] Compared with the current existing technology, the beneficial effects of the present invention are mainly reflected in: the present invention has obtained a series of nitrilase mutants with improved enzyme activity by semi-rational design and molecular modification of nitrilase AcNR, among which the catalytic activity of the nitrilase quadruple mutant AcNR-W59A / T134G / H135L / S192G is improved by about 142 times (for substrate 4-TCN) and about 77 times (for substrate ricinine) compared with the wild-type AcNR, while maintaining complete hydrolysis activity. Recombinant Escherichia coli containing the nitrilase mutant AcNR-W59A / T134G / H135L / S192G hydrolyzes 50mM (4-TCN) to synthesize 4-TCA at 45°C, and complete conversion can be achieved in 48.5h. The recombinant Escherichia coli containing the nitrilase mutant AcNR-W59A / T134G / H135L / S192G was used to hydrolyze ricin in castor cake at 35°C, and basic conversion was achieved in 48 hours. The ricinine concentration in the castor cake was reduced from 0.36% to 0.0007-0.0005%, and the ricinine content was greatly reduced (<10ppm). The nitrilase mutant of the present invention can improve the enzyme catalytic activity, and can catalyze the hydrolysis of various pyridine nitrile derivatives, and has good application prospects. (IV) Description of the drawings

[0029] Figure 1 , SDS-PAGE images of nitrilase and its mutant transformants, lane 1 is the crude enzyme solution of nitrilase AcNR, lane 2 is the crude enzyme solution of AcNR-W59A / T134G / H135L / S192G, lane 3 is the pure enzyme solution of nitrilase AcNR, and lane 4 is the pure enzyme solution of AcNR-W59A / T134G / H135L / S192G.

[0030] Figure 2 , and a bar graph comparing the enzyme activities of nitrilase AcNR and its mutants towards substrate 4-TCN.

[0031] Figure 3, and a bar graph comparing the enzyme activities of nitrilase AcNR and its mutants towards the substrate ricinine.

[0032] Figure 4 , the optimal temperature curve of the nitrilase quadruple mutant AcNR-W59A / T134G / H135L / S192G for the substrate 4-TCN.

[0033] Figure 5 , the optimal temperature curve of the nitrilase quadruple mutant AcNR-W59A / T134G / H135L / S192G for the substrate ricinine.

[0034] Figure 6 , the optimal pH curve of the nitrilase quadruple mutant AcNR-W59A / T134G / H135L / S192G for the substrate 4-TCN.

[0035] Figure 7 , the optimal pH curve of the nitrilase quadruple mutant AcNR-W59A / T134G / H135L / S192G for the substrate ricinine.

[0036] Figure 8 Efficiency curves of crude enzyme solutions of nitrilase AcNR and quadruple mutant AcNR-W59A / T134G / H135L / S192G catalyzing the hydrolysis of 50 mM 4-TCN.

[0037] Fig. 9 Efficiency curves of pure enzyme solutions of nitrilase AcNR and quadruple mutant AcNR-W59A / T134G / H135L / S192G catalyzing the hydrolysis of 50mM 4-TCN. (V) Specific implementation methods

[0038] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0039] Example 1: Site-directed mutagenesis and screening of nitrilase

[0040] 1. Construction of wild-type nitrilase engineered bacteria

[0041] Based on nitrilase (GenBank: QFR55171.1), pET-28b(+)-AcNR plasmid was constructed, and the host bacteria E. coli BL21(DE3) was transformed to construct the engineering bacteria E. coli BL21(DE3) / pET-28b(+)-AcNR. The nitrilase was recorded as nitrilase AcNR, the nucleotide sequence was SEQ ID No.1, and the amino acid sequence was SEQ ID No.2.

[0042] 2. Mutation site selection

[0043] First, homology modeling of the nitrilase AcNR protein structure was performed using Alphafold3.0, and then the model was molecularly docked with 4-TCN and ricinine using Discovery Studio to predict the active site. Alanine scanning was then performed to obtain the 59th, 134th, 135th, and 192nd amino acids near the catalytic triad of nitrilase. Modifying the corresponding amino acid species can change the active pocket of the enzyme and thus affect the enzyme activity.

[0044] After successful site-directed mutagenesis using whole-plasmid PCR, the expression vector containing the target gene was transferred into the Escherichia coli host. After inducing expression, positive mutants were screened out by detecting enzyme activity. The enzyme activity was then repeatedly detected to determine mutants with improved enzyme activity, thereby obtaining nitrilase mutants that can efficiently catalyze the production of 4-TCA from 4-TCN and can efficiently catalyze the hydrolysis of ricinine.

[0045] 3. Single point mutation

[0046] Using the pET-28b(+)-AcNR plasmid of the engineered bacteria E. coli BL21(DE3) / pET-28b(+)-AcNR as a template and the primers in Table 1, site-directed mutagenesis was performed on positions 59, 134, 135, and 192 by whole-plasmid PCR amplification.

[0047] PCR system (50 μL): template 0.5-20 ng, forward and reverse primers 10-15 pmol each, 5× Prime STAR Buffer (Mg 2+ plus), 0.2 mM dNTP, 1.25 U Prime STAR HSDNA Polymerase, and water to make up to 50 μL.

[0048] PCR conditions: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 30 s; (3) annealing at 64°C for 30 s; (4) extension at 72°C for 3.5 min; steps (2) to (4) for a total of 35 cycles; (5) final extension at 72°C for 10 min and storage at 4°C.

[0049] The PCR product was verified by agarose gel electrophoresis, digested with DpnI, introduced into E. coli BL21 (DE3), and spread on LB plates containing 50 μg / mL kanamycin to obtain a single clone.

[0050] Table 1 Primer design of mutation sites

[0051]

[0052] 4. Combination mutation

[0053] Using the pET-28b(+)-AcNR-W59A plasmid (nucleotide sequence SEQ ID No. 3) of the mutant transformant E. coli BL21(DE3) / pET-28b(+)-AcNR-W59A as a template, site-directed mutation of the T134G position was performed by whole plasmid amplification, and the PCR system was the same as that of the single mutation. The PCR product was verified by agarose gel electrophoresis, digested with DpnI, introduced into E. coli BL21(DE3), and coated on an LB plate containing 50 μg / mL kanamycin to obtain a double mutant transformant E. coli BL21(DE3) / pET-28b(+))-AcNR-W59A / T134G. Similarly, the pET-28b(+)-AcNR-W59A / T134G plasmid of the obtained double mutant transformant E. coli BL21(DE3) / pET-28b(+))-AcNR-W59A / T134G was used as a template to perform site-directed mutagenesis at the H135L position by whole plasmid amplification, and then transformed with the same PCR system as mentioned above to obtain the triple mutant transformant E. coli BL21(DE3) / pET-28b(+))-AcNR-W59A / T134G / H135L. The pET-28b(+)-AcNR-W59A / T134G / H135L plasmid of the triple mutant transformant E. coli BL21(DE3) / pET-28b(+)-AcNR-W59A / T134G / H135L was used as a template again, and the site-directed mutation of S192G was performed by full plasmid amplification, and the mutation points were superimposed by the PCR system again to obtain the quadruple mutant E. coli BL21(DE3) / pET-28b(+)-AcNR-W59A / T134G / H135L / S192G (nucleotide sequence SEQ ID No.11, amino acid sequence SEQ ID No.12). The construction methods of other combinations of two mutations and three mutations are the same as above.

[0054] The pure enzyme solution was prepared by the method of Example 3, and the SDS-PAGE results are shown in Figure 1 .

[0055] The method of Example 4 was used to detect the 4-TCN hydrolysis activity of the mutants. Figure 2 As shown in Table 2, the catalytic activity of the nitrilase quadruple mutant AcNR-W59A / T134G / H135L / S192G was increased by about 142 times compared with the wild-type AcNR.

[0056] Table 2. Hydrolysis activity of pure enzyme solution of mutants on 4-TCN

[0057] Mutants Relative enzyme activity U / g AcN 3.7 AcNR-W59A 56 AcNR-T134G 106 AcNR-H135L 83 AcNR-S192G 79 AcNR-W59A / T134G 137 AcNR-W59A / T134G / H135L 341 AcNR-W59A / T134G / H135L / S192G 524

[0058] The method of Example 5 was used to detect the hydrolysis activity of the mutant on ricinine. The results are shown in Figure 3 As shown in Table 3, the catalytic activity of the nitrilase quadruple mutant AcNR-W59A / T134G / H135L / S192G was increased by about 77 times compared with the wild-type AcNR.

[0059] Table 3. Hydrolysis activity of pure enzyme solution of mutants on ricinine

[0060] Mutants Relative enzyme activity U / g AcN 1.8 AcNR-W59A 26 AcNR-T134G 41 AcNR-H135L 37 AcNR-S192G 39 AcNR-W59A / T134G 56 AcNR-W59A / T134G / H135L 89 AcNR-W59A / T134G / H135L / S192G 139

[0061] After site-directed mutagenesis, a combined mutant transformant E. coli BL21 (DE3) / pET-28b (+) -AcNR-W59A / T134G / H135L / S192G with significantly improved enzyme activity was finally screened and obtained, which was recorded as the nitrilase mutant AcNR-W59A / T134G / H135L / S192G. The nucleotide sequence of the encoding gene is shown in SEQ ID No. 11, and the amino acid sequence is shown in SEQ ID No. 12.

[0062] Example 2: Expression of nitrilase mutants

[0063] The original strain E. coli BL21 (DE3) / pET-28b (+) -AcNR, the single point or combination mutant transformants selected in Example 1 were inoculated into LB medium and cultured at 37 ° C for 10-12 hours. The culture solution was inoculated into LB medium containing kanamycin (final concentration 50 mg / L) at a volume inoculum of 1%, and the culture was expanded at 37 ° C and 150 rpm until the OD of the culture solution reached 600 is between 0.6 and 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mM, and the culture is induced at 28°C for 10 hours. After the culture solution is centrifuged, the precipitate is washed twice with physiological saline to obtain the corresponding wet bacteria.

[0064] Example 3: Preparation of crude enzyme solution and pure enzyme solution of nitrilase and its mutants

[0065] 1. Preparation of crude enzyme solution

[0066] 1 g of the wet cells collected in Example 2 was added to 20 mL of sodium phosphate buffer solution (200 mM NaH2PO4-Na2HPO4, pH 8.0) and the cells were resuspended and then ultrasonically disrupted (400 W, 20 min, 1 s disruption and 1 s pause). The disrupted product was centrifuged (8000 rpm, 10 min) and the supernatant was taken as the crude enzyme solution.

[0067] 2. Preparation of pure enzyme solution

[0068] (1) After pre-packing a 15 mL Ni-NTA affinity chromatography column, flush it with binding buffer (200 mM NaH2PO4, 300 mM NaCl, pH 8.0) at a flow rate of 1 mL / min.

[0069] (2) After flushing for 8-10 column volumes, the crude enzyme solution is passed through the Ni-NTA column at a flow rate of 1 mL / min, and the target protein is mounted on the column. After loading, a large amount of unadsorbed impurities will not bind to the resin and will be directly removed.

[0070] (3) Use equilibrium buffer (200 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 8.0) to elute weakly adsorbed impurities at a flow rate of 1 mL / min for 3 column volumes.

[0071] (4) Use elution buffer (200 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0) to elute and collect the target protein at a flow rate of 1 mL / min, elute for 3 column volumes, and collect the eluate.

[0072] (5) The collected eluate was dialyzed with 200 mM sodium phosphate buffer at a pH of 8.0 as the dialysate (the molecular weight of the retained protein was 30 kDa). The retained fluid after dialysis was the pure enzyme solution.

[0073] Example 4: Determination of the hydrolysis activity of nitrilase on 4-TCN

[0074] The pure enzyme solution obtained in Example 3 was subjected to enzyme activity assay. 10 mL of sodium phosphate buffer (200 mM, pH 8.0), 50 mM substrate 4-TCN, and 100 mg of pure enzyme solution (based on protein content) were added to a 50 mL stoppered conical flask, and the reaction solution was shaken at 800 rpm in a constant temperature water bath at 45°C for 30 min. After 100 μL of acetonitrile was added to terminate the reaction, 200 μL of the supernatant was sampled, and the 4-TCN conversion rate in the conversion solution was determined by liquid chromatography (Thermo Fisher) external standard method.

[0075] The HPLC column used was an Ascentis C18 column (5μM, 15cm×4.6mm), the mobile phase was ultrapure water: acetonitrile: orthophosphoric acid = 150:100:1 (v / v), the detection temperature was 40°C, the detection wavelength was 224nm, and the flow rate was 0.8mL / min. 4-TCA showed an absorption peak at 15.8min, with a single sharp peak shape.

[0076] Definition of enzyme activity: At 45°C and pH 8.0, the amount of enzyme required to catalyze the production of 1 μM of product 4-TCA per minute is 1 enzyme activity unit (U). Specific enzyme activity (U / g) is defined as the ratio of unit enzyme activity to unit mass.

[0077] Example 5: Determination of the hydrolysis activity of nitrilase on ricinine

[0078] The wet cells obtained in Example 2 were subjected to enzyme activity determination. 0.25 g of the wet cells were put into a 250 mL triangular flask, and then 50 g of castor meal and 20 mL of sodium phosphate buffer (200 mM, pH 7.0) were added and mixed thoroughly, and cultured in a water bath at 35 ° C and 300 rpm for 48 h. During the reaction, samples were taken at intervals of 6 h, filtered, and the filter cake was dried and crushed at 50 ° C. 0.5 g of the crushed sample was weighed, 20 mL of methanol was added, and the mixture was shaken at 75 ° C and 600 rpm for 4 h. The supernatant was taken by centrifugation at 8000 rpm and 4 ° C for 10 min, and the residual ricinine was analyzed by liquid chromatography.

[0079] The liquid chromatography conditions were Diamonsil TM C18 column, column temperature: 40°C, mobile phase: ultrapure water: ethanol = 9:1 (v / v), flow rate: 1 mL / min, detection wavelength: 308 nm, injection volume: 20 μL.

[0080] Definition of enzyme activity: At 40°C and pH 7.0, the amount of enzyme required to catalyze the production of 1 μM product per minute is 1 unit of enzyme activity (U). Specific enzyme activity (U / g) is defined as the ratio of unit enzyme activity to unit mass.

[0081] Example 6: Determination of Kinetic Parameters of Nitrilase and Its Mutants

[0082] 1. The kinetic parameters of the pure enzyme solution in Example 3 were determined, with 4-TCN as substrate and pure enzyme solutions of AcNR and AcNR-W59A / T134G / H135L / S192G as catalysts.

[0083] The reaction system is 10 mL: the pure enzyme solution is diluted 10 times with pH 8.0, 200 mM sodium phosphate buffer and added to the reaction container so that the final concentration is 0.2 mg / mL in terms of protein content, and substrates are added at a final concentration of 10-300 mM, respectively, and the mixture is made up to 10 mL with 200 mM pH = 8 sodium phosphate buffer as the reaction medium. The reaction is carried out at 45 ° C and 800 rpm for 30 min, and 200 μL of the sample is taken. The concentration of 4-TCA in the reaction solution is detected by HPLC described in Example 4.

[0084] The K values ​​of nitrilase AcNR and nitrilase combination mutant AcNR-W59A / T134G / H135L / S192G were obtained by nonlinear fitting of the obtained data in Origin. m and K cat As shown in Table 4, it can be found that the K cat Compared with the original nitrilase, it is significantly improved, which shows that the activity of the modified nitrilase is indeed improved, and their K m This reflects that the affinity of the modified quadruple mutant enzyme for the substrate has slightly decreased.

[0085] Table 4 Kinetic parameters of nitrilase mutants for 4-TCN

[0086]

[0087] 2. The kinetic parameters of the wet cells obtained in Example 2 were determined using castor meal as substrate and cells of AcNR and AcNR-W59A / T134G / H135L / S192G as catalysts.

[0088] Take 0.25g of wet cells and put them into a 250mL triangular flask, then add 5-50g of castor meal and 20mL of sodium phosphate buffer (200mM, pH 7.0) and mix thoroughly, and culture in a water bath at 35°C and 300rpm for 48h. During the reaction, take samples every 6h, filter, and dry and crush the filter cake at 50°C. Weigh 0.5g of the crushed sample, add 20mL of methanol, and shake at 75°C and 600rpm for 4h. Centrifuge at 8000rpm and 4°C for 10min to take the supernatant, and perform liquid chromatography analysis on the residual ricinine.

[0089] The K values ​​of nitrilase AcNR and nitrilase combination mutant AcNR-W59A / T134G / H135L / S192G were obtained by nonlinear fitting of the obtained data in Origin. m and K cat As shown in Table 5, it can be found that the K cat Compared with the original nitrilase, the activity of the modified nitrilase is significantly improved. m This reflects that the affinity of the modified quadruple mutant enzyme for the substrate is slightly reduced.

[0090] Table 5 Kinetic parameters of nitrilase mutants to ricinine

[0091]

[0092] Example 7: Determination of the optimal temperature of nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G in substrate 4-TCN

[0093] The optimum temperature of the wet cells in Example 2 was determined, using 4-TCN as a substrate and wet cells of nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G as catalysts.

[0094] The reaction system was 10 mL: 0.1 g of wet cells was dissolved in 10 mL of 200 mM sodium phosphate buffer at pH 8.0 in a conical flask, and a final concentration of 50 mM substrate was added. The temperature was set to 20-60 ° C (20, 25, 30, 35, 40, 45, 50, 55, 60 ° C), and the reaction was carried out at 800 rpm for 30 min. 200 μL of the sample was taken, and the concentration of 4-TCA in the reaction solution was detected by HPLC described in Example 4 and the enzyme activity was calculated. The results are shown in Figure 4 As shown, the optimal temperature of the four mutants is 45°C. Under the same conditions, the optimal temperature of the original nitrilase AcNR was 35°C.

[0095] Example 8: Determination of the optimal temperature of nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G in the substrate ricinine

[0096] The optimum temperature of the wet cells in Example 2 was determined using castor meal as a substrate and wet cells of nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G as a catalyst.

[0097] Take 0.25g of wet bacteria and put them into a 250mL triangular flask, add 50g of castor cake and 20mL of sodium phosphate buffer (200mM, pH 7.0) and mix well, and culture in a water bath at 20-60℃ (20, 25, 30, 35, 40, 45, 50, 55, 60℃) and 300rpm for 48h. During the reaction, samples were taken at intervals of 6h, filtered, and the filter cake was dried and crushed at 50℃. Weigh 0.5g of the crushed sample, add 20mL of methanol, and shake at 75℃ and 600rpm for 4h. Centrifuge at 8000rpm and 4℃ for 10min to take the supernatant, and perform liquid chromatography analysis on the residual ricinine. The HPLC described in Example 4 was used to detect the residual concentration of ricinine in the reaction solution and calculate the enzyme activity. The results are shown in Table 2. Figure 5 As shown, the optimal temperature of the four mutants is 40°C. Under the same conditions, the optimal temperature of the original nitrilase AcNR was 35°C.

[0098] Example 9: Determination of the optimal pH of nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G in substrate 4-TCN

[0099] The optimal pH of the wet cells in Example 2 was determined, with 4-TCN as the substrate and the wet cells of nitrilase AcNR and its four mutants AcNR-W59A / T134G / H135L / S192G as the catalyst. Sodium citrate buffer was used for pH 4.0-6.5; sodium phosphate buffer was used for pH 6.5-8.0; Tris-hydrochloric acid buffer was used for pH 8-9; and glycine-sodium hydroxide buffer was used for pH 9-10.5.

[0100] The reaction system is 10mL: 0.1g wet bacteria are dissolved in 10mL buffer of different pH values ​​in a conical flask, and then a substrate with a final concentration of 50mM is added. The buffer is 200mM of pH = (4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5) as the reaction medium, and the reaction is carried out at 45°C and 800rpm for 30min. 200μL of the sample is taken, and the concentration of 4-TCA in the reaction solution is detected by HPLC described in Example 4 and the enzyme activity is calculated. The results are shown in Figure 6 As shown, the optimal pH of the four mutants is 8.0. Under the same conditions, the optimal pH of the original nitrilase AcNR was 7.5.

[0101] Example 10: Determination of the optimal pH of nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G in the substrate ricinine

[0102] The optimal pH of the wet cells in Example 2 was determined, with castor meal as substrate and wet cells of nitrilase AcNR and its four mutants AcNR-W59A / T134G / H135L / S192G as catalyst. Sodium citrate buffer was used for pH 4.0-6.5; sodium phosphate buffer was used for pH 6.5-8.0; Tris-hydrochloric acid buffer was used for pH 8-9; and glycine-sodium hydroxide buffer was used for pH 9-10.5.

[0103] Take 0.25g of wet bacteria and put it into a 250mL conical flask, add 50g of castor cake, 20mL, 200mM different pH buffer solutions ((4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5)) and mix thoroughly, and culture in a water bath at 35°C, 300rpm for 48h. During the reaction, samples were taken at intervals of 6h, filtered, and the filter cake was dried and crushed at 50°C. Weigh 0.5g of the crushed sample, add 20mL of methanol, and shake at 75°C and 600rpm for 4h. Centrifuge at 8000rpm and 4°C for 10min to obtain the supernatant, and perform liquid chromatography analysis on the residual ricinine. The HPLC described in Example 5 was used to detect the concentration of residual ricinine in the reaction solution and calculate the enzyme activity. The results are shown in Table 5. Figure 7 As shown, the optimal pH of the four mutants is 7.0. Under the same conditions, the optimal pH of the original nitrilase AcNR was 7.0.

[0104] Example 11: Nitrilase and its mutant AcNR-W59A / T134G / H135L / S192G convert 50 mM 4-TCN

[0105] 1. Crude enzyme solution as catalyst

[0106] 4-TCN was used as a substrate, and the crude enzyme solution of the nitrilase and its quadruple mutant AcNR-W59A / T134G / H135L / S192G prepared in Example 3 was used as a catalyst.

[0107] The reaction system was 10 mL: a crude enzyme solution with a protein content of 1 mg / mL was added to a stoppered flask, a substrate with a final concentration of 50 mM was added, and then the solution was made up to 10 mL with sodium phosphate buffer (200 mM, pH 8.0). The reaction was carried out on a water bath shaker at 45°C and 800 rpm. 200 μL of the sample was taken every 30 minutes and centrifuged. The supernatant was tested for 4-TCA by HPLC as described in Example 4, and the conversion rate was calculated. The results are shown in Figure 8 As shown in the figure, the substrate reaction rate of the crude enzyme solution of the mutant AcNR-W59A / T134G / H135L / S192G was increased by about 2.2 times compared with that of the nitrilase AcNR.

[0108] 2. Pure enzyme solution as catalyst

[0109] 4-TCN was used as the substrate, and the pure enzyme solution of the nitrilase and its quadruple mutant AcNR-W59A / T134G / H135L / S192G prepared in Example 3 was used as the catalyst.

[0110] The reaction system is 10 mL: the pure enzyme solution is diluted 10 times with pH 8.0, 200mM sodium phosphate buffer and then added to the reaction container to make the final concentration of 1 mg / mL in terms of protein content, and then a final concentration of 50mM substrate is added, and the reaction medium is supplemented to 10mL with (200mM, pH 8.0) sodium phosphate buffer as the reaction medium, and the reaction is carried out in a constant temperature water bath at 800rpm and 45°C. 200μL of the sample is taken at intervals of 30min, and the centrifugation is carried out at 12000rpm, and the precipitate is discarded. The HPLC described in Example 4 is used to detect 4-TCA in the reaction solution, and the conversion rate is calculated. The reaction progress results of the nitrilase and its four mutants are shown in Fig. 9 It was determined that the mutant AcNR-W59A / T134G / H135L / S192G could completely react the substrate within 48.5 hours, which was shorter than the original nitrilase, and the reaction speed was 2.1 times higher than that of the nitrilase AcNR.

[0111] Example 12: Immobilization and application of nitrilase mutant AcNR-W59A / T134G / H135L / S192G

[0112] Weigh 2 g of the combined mutant AcNR-W59A / T134G / H135L / S192G wet cells obtained by the method of Example 2 and suspend them in 20 mL of NaH2PO4-Na2HPO4 buffer system (200 mM, pH = 8.0), add diatomaceous earth at a final concentration of 0.006 g / mL, and stir at room temperature for 1 h. Then add 0.6 mL of a 5% polyethyleneimine aqueous solution and stir at room temperature for 1 h. Add 0.2 mL of a 25% glutaraldehyde aqueous solution and stir for 0.5 h, and finally vacuum filter to obtain immobilized cells.

[0113] Take 0.5g of the immobilized cells corresponding to the wet cells and suspend them in 10mL NaH2PO4-Na2HPO4 buffer system (200mM, pH=8.0), add 0.13g 4-TCN (final concentration 50mM), and react in a constant temperature water bath at 45°C and 800rpm. The immobilized cells prepared by the original nitrilase AcNR react for 7-8 hours per batch, and the immobilized cells prepared by the mutant AcNR-W59A / T134G / H135L / S192G react for 4-6 hours per batch. After each batch of reactions, vacuum filtration is performed to separate the solid and liquid, and the reaction solution is analyzed for product concentration by high performance liquid chromatography (see Example 4), and the immobilized cells are then put into the next batch of reactions, and the results are shown in Table 6.

[0114] Table 6 Transformation of 50 mM 4-TCN using immobilized cells

[0115]

[0116] Example 13: Application of nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G in degrading ricinine in castor cake.

[0117] 1. Wet bacteria as catalyst

[0118] According to the method provided in Example 2, wet cells of nitrilase AcNR and its four mutants AcNR-W59A / T134G / H135L / S192G were obtained. 0.25g of wet cells were put into a 250mL triangular flask, 50g of castor cake and 20mL of sodium phosphate buffer (200mM, pH 7.0) were added and mixed thoroughly, and cultured in a water bath at 35°C and 300rpm for 48h. During the reaction, samples were taken at intervals of 6h, filtered, and the filter cake was dried and crushed at 50°C. 0.5g of the crushed sample was weighed, 20mL of methanol was added, and the mixture was shaken at 75°C and 600rpm for 4h. The supernatant was taken by centrifugation at 8000rpm and 4°C for 10min, and the residual ricinine was analyzed by liquid chromatography. After analysis, the ricinine concentration in the castor cake dropped from 0.36% to 0.0007% after 48h, and the ricinine content was <10ppm.

[0119] 2. Crude enzyme solution as catalyst

[0120] According to the method provided in Example 3, the crude enzyme solution of nitrilase AcNR and its four mutants AcNR-W59A / T134G / H135L / S192G was obtained. 20 mL of the crude enzyme solution (equivalent to 0.25 g of wet bacteria) was put into a 250 mL triangular flask, 50 g of castor cake was added and mixed thoroughly, and cultured in a water bath at 35 ° C and 300 rpm for 48 h. During the reaction process, samples were taken at intervals of 6 h, filtered, and the filter cake was dried and crushed at 50 ° C. Weigh 0.5 g of the crushed sample, add 20 mL of methanol, and shake for 4 h at 75 ° C and 600 rpm. Centrifuge at 8000 rpm and 4 ° C for 10 min to take the supernatant, and perform liquid chromatography analysis on the residual ricin. After analysis, the ricinine concentration in the castor cake dropped from 0.36% to 0.0005% after 48 h, and the ricinine content was <10 ppm.

[0121] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A nitrilase mutant, characterized in that The nitrilase mutant is obtained by subjecting the 59th, 134th, 135th or 192nd amino acid of the amino acid sequence shown in SEQ ID No. 2 to single point mutation or superimposed combined mutation.

2. The nitrilase mutant according to claim 1, wherein The nitrilase mutant is obtained by mutating the amino acid sequence of SEQ ID No. 2 into one of the following: (1) the tryptophan at position 59 is mutated into alanine; (2) the threonine at position 134 is mutated into glycine; (3) the histidine at position 135 is mutated into leucine; (4) the serine at position 192 is mutated into glycine; (5) the single point mutations described in (1) to (4) are combined in pairs; (6) the single point mutations described in (1) to (4) are combined into three mutations; (7) the single point mutations described in (1) to (4) are combined into four mutations.

3. The nitrilase mutant according to claim 1, wherein The amino acid sequence of the nitrilase mutant is shown in SEQ ID No.

12.

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

5. Use of the nitrilase mutant according to claim 1 in catalyzing the synthesis of 3,4,5,6-tetrachloropyridine-2-carboxylic acid from 3,4,5,6-tetrachloropyridine-2-carbonitrile.

6. The use according to claim 5, characterized in that The application is as follows: using wet bacteria obtained by fermentation culture of genetically engineered bacteria expressing a nitrilase mutant, or a crude enzyme solution extracted after ultrasonic crushing of the wet bacteria, or a pure enzyme solution obtained by purifying the crude enzyme solution, or immobilized cells obtained by immobilizing the wet bacteria on diatomaceous earth as catalysts, using 3,4,5,6-tetrachloropyridine-2-carbonitrile as substrate, and using pH=4.0-10.5, 200mM buffer as reaction medium to form a reaction system, reacting completely under constant temperature conditions of 800rpm and 35-50℃, centrifuging and separating and purifying to obtain 3,4,5,6-tetrachloropyridine-2-carboxylic acid.

7. The use according to claim 6, characterized in that In the reaction system, the substrate is added at a concentration of 20-200 mM; when the catalyst is a crude enzyme solution or a pure enzyme solution, the added amount is 0.1-3.5 g / L based on the protein content; when the catalyst is wet bacteria, the added amount is 10-100 g / L based on the wet weight of the bacteria; when the catalyst is immobilized cells, the added amount is 10-200 g / L based on the weight of the wet bacteria.

8. Use of the nitrilase mutant according to claim 1 to degrade ricin in castor meal.

9. The use according to claim 8, characterized in that The application is as follows: using wet bacteria obtained by fermentation culture of genetically engineered bacteria expressing a nitrilase mutant, or crude enzyme liquid or pure enzyme liquid extracted after ultrasonic crushing of the wet bacteria as a catalyst, using castor meal containing ricinine as a substrate, and using water or a pH 4.0-10.5 buffer solution as a reaction medium to form a reaction system, reacting at 300 rpm and 30-50° C. until the ricinine content in the castor meal is less than 10 ppm, thereby obtaining castor meal with greatly reduced toxicity.

10. The use according to claim 9, characterized in that In the reaction system, the amount of reaction medium added is 10-400 mL / kg based on the mass of castor meal; the amount of catalyst added is based on the wet weight of bacteria, and the amount of wet bacteria added is 1-10 g / kg based on the mass of castor meal.

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