Nitrilase mutants and their use in hydrolyzing pyridine nitrile derivatives

Abstract

The application discloses a nitrilase mutant and application thereof in hydrolysis of pyridine nitrile derivatives, wherein the nitrilase mutant is obtained by single mutation or multiple mutations of amino acids at positions 59, 134, 135 or 192 of the amino acid sequence shown in SEQ ID No. 2. The application is semi-rational design, and the protein is molecularly modified. The catalytic activity of the nitrilase four-mutant AcNR-W59A / T134G / H135L / S192G to 4-TCN is increased by about 142 times, and the catalytic activity to ricinine is increased by about 77 times, while the complete hydrolysis activity is maintained. The nitrilase mutant reported in the application has high catalytic activity to various pyridine nitrile substances, and has a wide and good application prospect in pesticide and feed production.

Description

(1) Technical field

[0001] The present invention relates to a nitrilase mutant and application thereof in hydrolyzing pyridine nitrile derivatives. (2) Background technology

[0002] Nitrilases are enzymes that catalyze the hydrolysis of cyano groups to carboxyl groups. They feature mild reaction conditions, high stereoselectivity, and environmental friendliness. Currently, nitrilases are widely used in industry. Lonza, Switzerland, pioneered the use of nitrilases to catalyze the production of nicotinic acid. Through the combined action of nitrilases and nicotinamide dehydrogenase, they degrade the substrates 2-cyanopyridine and 2-cyanopyrazine into the pharmaceutical intermediates 5-hydroxypyridine-2-carboxylic acid and 5-hydroxypyrazine-2-carboxylic acid, respectively. The reaction is highly selective, with a conversion rate of nearly 100%, significantly superior to traditional chemical methods. BASF, Germany, uses nitrilases to perform chiral resolution of racemic mandelonitrile to produce (R)-mandelic acid, achieving a conversion rate of up to 99%. DuPont has developed a chemical-enzymatic process for converting 2-methylglutaronitrile (MGN) to 1,5-dimethyl-2-piperidone (1,5-DMPD). Immobilized nitrilase (Acidovorax facilis 72w) was used as a catalyst to hydrolyze MGN to 4-cyanovaleric acid (4-CAP) ammonium salt. The hydrolysis reaction achieved a selectivity greater than 98% and a conversion rate of 100%. Compared to traditional chemical processes, this chemoenzymatic production process offers higher yields, generates less waste, and produces a single lactam isomer.

[0003] With the advancement of green and sustainable development strategies, new green pesticides characterized by high efficiency, low toxicity, and easy decomposition have become mainstream in the market. Among them, pyridine chloride pesticides, represented by imidacloprid, have become a hot topic in research and development. 3,4,5,6-tetrachloropyridine-2-carboxylic acid (4-TCA) is a key intermediate in the synthesis of this type of pesticide. Similar pesticides such as piclopram (4-amino-3,5,6-trichloropyridine-2-carboxylic acid) and pyridine (3,6-dichloropyridine-2-carboxylic acid) require 4-TCA. 4-TCA can also be used as a pharmaceutical intermediate and chemical intermediate, and its market demand continues to grow. Chemical synthesis is currently the only means of obtaining 4-TCA industrially, but this process produces a large amount of waste acid, consumes a lot of energy, and the resulting 4-TCA is of low purity, making it unfavorable for 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 has the advantages of being safe, environmentally friendly, and having low emissions of three wastes. It can also utilize the high catalytic efficiency of nitrilase to greatly improve production efficiency.

[0004] Castor, one of the world's top ten oil crops, has a wide range of applications in fuel and chemical industries. Castor meal, a byproduct of oil extraction from castor beans, is rich in crude protein and minerals such as calcium and phosphorus, making it highly valuable. However, it contains a series of toxic substances, primarily ricinine, which hinders its utilization. Ricinine, chemically known as 3-cyano-4-methoxy-1-methyl-2-pyridone, is a naturally occurring piperidine alkaloid that is highly toxic. However, it can also be synthesized into a variety of organic compounds, including pesticides and toxic gases. Studies have shown that accidentally ingesting 20 castor beans can be fatal in adults, while ingesting 2 to 7 seeds can cause fatal poisoning in children aged 4 to 7. Therefore, detoxifying castor meal can allow it to be used in feed or to produce products such as plant protein. Finding efficient methods to detoxify castor meal without compromising its nutritional value is highly valuable.

[0005] After reviewing relevant literature, we found that, aside from our group's research, there are relatively few nitrilases reported to catalyze the hydrolysis of 4-TCN, and none to catalyze the hydrolysis of ricinine. This may be due to the large size of the substrate molecules, such as 4-TCN or ricinine, and the presence of multiple chlorine- and other substituent groups. Excessively large substrates are less accessible to the enzyme's active site, preventing them from binding well, resulting in lower catalytic efficiency. Studies have shown that when three or more chlorine atoms are present on the substrate, they form a conjugated effect with the pyridine ring, hindering access to the enzyme's catalytic pocket and limiting the nitrilases' activity against these substrates. Therefore, developing nitrilases capable of degrading pyridinenitrile derivatives, such as 4-TCN or ricinine, has significant application potential. (3) Summary of the invention

[0006] The present invention aims to provide a nitrilase mutant and its use in the hydrolysis of pyridinenitrile derivatives, particularly in the catalytic synthesis of 3,4,5,6-tetrachloropyridine-2-carboxylic acid (4-TCA) and the catalytic degradation of ricinine in castor meal. The present invention screens and obtains multiple nitrilase mutant proteins capable of hydrolyzing the substrate 4-TCN to produce 4-TCA and having hydrolytic activity against the substrate ricinine. These proteins include genes encoding these mutant proteins, recombinant vectors containing these genes, and recombinant genetically engineered bacteria transformed with these recombinant vectors. These mutant proteins address the problems of low catalytic activity and poor stability of nitrilase towards the substrates 4-TCN and ricinine.

[0007] The technical solution adopted in 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 or multiple mutations.

[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 was 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) 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 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) mutants obtained by combining the single point mutations described in (1) to (4) with triple 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 designated 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 gene encoding the nitrilase mutant, a recombinant vector constructed from the gene, and a recombinant genetically engineered bacterium obtained by transforming a host cell with the recombinant vector. The base vector of the recombinant vector is primarily the pET series of Escherichia coli, including pET22b, pET24a, and pET28a. The recombinant genetically engineered bacterium primarily uses Escherichia coli BL21 (DE3) as a host bacterium.

[0018] The present invention also provides an application of the nitrilase mutant in catalyzing the preparation of 3,4,5,6-tetrachloropyridine-2-carbonitrile (4-TCN) to 3,4,5,6-tetrachloropyridine-2-carboxylic acid (4-TCA). Specifically, the application comprises: using wet cells obtained by fermentation and culture of genetically engineered bacteria expressing the nitrilase mutant, or a crude enzyme solution extracted after ultrasonic disruption of the wet cells, or a pure enzyme solution obtained by purifying the crude enzyme solution, or immobilized cells obtained by immobilizing the wet cells on diatomaceous earth as a catalyst, using 4-TCN as a substrate, and using a 200 mM buffer solution with a pH of 4.0-10.5 as a reaction medium to form a reaction system (preferably 200 mM NaH2PO4-Na2HPO4, pH=8.0), reacting at 800 rpm and a constant temperature water bath of 35-50°C (preferably 45°C). After the reaction is complete, the reaction solution is separated and purified 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 ricinine in castor meal. Specifically, the application comprises: using wet cells obtained by fermentation and culture of genetically engineered bacteria expressing the nitrilase mutant, or a crude enzyme solution extracted after ultrasonic crushing of the wet cells, as a catalyst; using castor meal containing ricinine 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 second reaction system. The reaction is carried out 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 cell was prepared as follows: the engineered bacteria containing the nitrilase mutant encoding gene was 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 a volume concentration of 1%, and cultured at 37° C. until the culture solution OD reached 0. 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, and wash twice with physiological saline to obtain wet bacteria.

[0023] The crude enzyme solution was prepared as follows: wet cells were resuspended in 200 mM NaH2PO4-Na2HPO4 buffer at pH 8.0, and ultrasonically disrupted (400 W, 20 min, 1 s disruption, 1 s pause), and 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 flushed with a binding buffer at a flow rate of 1 mL / min, weakly adsorbed impurities are eluted with an equilibration 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 dialysis bag has a molecular weight cut-off of 30 kDa), and the retentate 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 genetically engineered 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 is added at a final concentration of 6g / L, and the cells are stirred at room temperature for 1h. Subsequently, a polyethyleneimine aqueous solution with a mass concentration of 5% is added and the cells are stirred at room temperature for 1h. Finally, a glutaraldehyde aqueous solution with a mass concentration of 25% is added and stirred for 0.5h. Finally, the cells are vacuum filtered, washed with deionized water 3 to 5 times, and dried at room temperature to obtain immobilized cells. The volume of the polyethyleneimine aqueous solution added is 3% based on the volume of the buffer solution, and the volume of the glutaraldehyde aqueous solution added is 1% based on the volume of the buffer solution. The immobilized cells are then stored in a 4°C refrigerator for later use.

[0026] The catalyst of the present application can be a wet bacterium body of a recombinant genetically engineered bacterium containing the nitrile hydratase mutant gene (preferably E. coli BL211 (DE3)), can be a crude enzyme solution extracted after ultrasonic crushing of the wet bacterium body, can also be a pure enzyme solution obtained after purification of the crude enzyme solution, and can also be a fixed cell obtained by immobilization of the wet bacterium body.

[0027] The LB liquid medium of the present application is composed of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with water as the solvent and the pH value being natural. The LB solid culture medium is obtained by adding agar 15 g / L to the LB liquid medium.

[0028] Compared with the prior art, the present application has the following beneficial effects: the present application is semi-rational design, the nitrile hydratase AcNR is molecularly modified, a series of nitrile hydratase mutants with improved enzyme activity are obtained, the catalytic activity of the nitrile hydratase four mutant AcNR-W59A / T134G / H135L / S192G is about 142 times (for the substrate 4-TCN) and about 77 times (for the substrate ricinine) higher than that of the wild type AcNR, and the complete hydrolysis activity is maintained. The recombinant E. coli containing the nitrile hydratase mutant AcNR-W59A / T134G / H135L / S192G can completely convert 4-TCA synthesized from 50 mM (4-TCN) at 45℃ in 48.5 h. The recombinant E. coli containing the nitrile hydratase mutant AcNR-W59A / T134G / H135L / S192G can completely convert ricinine in castor meal at 35℃ in 48 h, and the concentration of ricinine in the castor meal is reduced from 0.36% to 0.0007-0.0005%, and the content of ricinine is greatly reduced (<10 ppm). The nitrile hydratase mutant of the present application can improve the enzyme catalytic activity and can catalyze the hydrolysis of various pyridine nitrile derivatives, and has good application prospect. (V) DESCRIPTION OF DRAWINGS

[0029] Figure 1 , SDS-PAGE diagram of nitrile hydratase and its mutant transformant, lane 1 is nitrile hydratase AcNR crude enzyme solution, lane 2 is AcNR-W59A / T134G / H135L / S192G crude enzyme solution, lane 3 is nitrile hydratase AcNR pure enzyme solution, and lane 4 is AcNR-W59A / T134G / H135L / S192G pure enzyme solution.

[0030] Figure 2 , columnar diagram of comparison of enzyme activity of nitrile hydratase AcNR and its mutant on substrate 4-TCN.

[0031] Figure 3, 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 the crude enzyme solutions of AcNR, AcNR-W59A / T134G / H135L / S192G, and AcNR nitrilase catalyzing the hydrolysis of 50 mM 4-TCN.

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

[0038] The present invention is further described below with reference to 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-producing bacteria

[0041] A pET-28b(+)-AcNR plasmid was constructed based on the nitrilase, and the host bacteria E. coli BL21(DE3) was transformed to construct the engineered bacteria E. coli BL21(DE3) / pET-28b(+)-AcNR. The nitrilase was designated as nitrilase AcNR, with a nucleotide sequence of SEQ ID No. 1 and an amino acid sequence of SEQ ID No. 2.

[0042] 2. Mutation site selection

[0043] First, homology modeling of the nitrilase AcNR protein structure was performed using Alphafold3.0. Then, the model was molecularly docked with 4-TCN and ricinine using Discovery Studio to predict the active site. Then, through alanine scanning, the 59th, 134th, 135th, and 192nd amino acids were found 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 induction of expression, positive mutants were screened out by detecting enzyme activity. The enzyme activity was then repeatedly detected to determine the mutants with improved enzyme activity, thereby obtaining nitrilase mutants that can efficiently catalyze 4-TCN to produce 4-TCA 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 listed in Table 1, site-directed mutagenesis was performed at positions 59, 134, 135, and 192 by whole-plasmid PCR amplification.

[0047] PCR system (50 μL) is as follows: 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 onto 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] Site-directed mutagenesis at position T134G was performed by whole-plasmid amplification using the pET-28b(+)-AcNR-W59A plasmid (nucleotide sequence SEQ ID No. 3) from the mutant transformant E. coli BL21(DE3) / pET-28b(+)-AcNR-W59A as a template. The PCR system was the same as for the single mutation. The PCR product was verified by agarose gel electrophoresis, digested with DpnI, and introduced into E. coli BL21(DE3). The product was then plated onto LB plates containing 50 μg / mL kanamycin to generate the double mutant transformant E. coli BL21(DE3) / pET-28b(+)-AcNR-W59A / T134G. Similarly, using the obtained double mutant transformant E. coli BL21(DE3) / pET-28b(+)-AcNR-W59A / T134G plasmid pET-28b(+)-AcNR-W59A / T134G as a template, site-directed mutagenesis at the H135L position was performed by whole-plasmid amplification. Then, using the same PCR system as above, triple mutant transformants E. coli BL21(DE3) / pET-28b(+)-AcNR-W59A / T134G / H135L were obtained after transformation. The pET-28b(+)-AcNR-W59A / T134G / H135L plasmid of the triple-mutant transformant E. coli BL21(DE3) / pET-28b(+)-AcNR-W59A / T134G / H135L was again used as a template to perform site-directed mutagenesis of S192G by whole-plasmid amplification. The mutation points were then superimposed using a PCR system to obtain a 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 for other combinations of double and triple mutations were 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 FIG. 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 mutant enzymes on 4-TCN

[0057] mutant Relative enzyme activity U / g AcNR 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. 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] mutant Relative enzyme activity U / g AcNR 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 obtained, designated 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, and 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 cultured at 37 ° C and 150 rpm until the OD of the culture solution reached 0. 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 and pure enzyme solutions 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 (200 mM NaH2PO4-Na2HPO4, pH 8.0) and resuspended in the suspension. The suspension was then ultrasonically disrupted (400 W, 20 min, 1 s disruption followed by 1 s pause). The disrupted product was centrifuged (8000 rpm, 10 min) and the supernatant was obtained 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 8-10 column volumes of washing, the crude enzyme solution is passed through the Ni-NTA column at a flow rate of 1 mL / min. The target protein is loaded onto 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 against 200 mM sodium phosphate buffer at pH 8.0 (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 4-TCN Hydrolysis Activity of Nitrilase

[0074] The pure enzyme solution obtained in Example 3 was assayed for enzyme activity. To a 50 mL stoppered Erlenmeyer flask were added 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). The reaction solution was incubated at 45°C in a shaker at 800 rpm in a constant temperature water bath for 30 min. After terminating the reaction by adding 100 μL of acetonitrile, 200 μL of the supernatant was sampled and the 4-TCN conversion rate in the conversion solution was determined using liquid chromatography (Thermo Fisher) with an external standard method.

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

[0076] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μM 4-TCA per minute at 45°C and pH 8.0. Specific 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 assayed for enzyme activity. 0.25 g of the wet cells were placed in a 250 mL triangular flask, followed by addition of 50 g of castor meal and 20 mL of sodium phosphate buffer (200 mM, pH 7.0), thoroughly mixed, and incubated in a water bath at 35°C and 300 rpm for 48 h. During the reaction, samples were taken at 6-h intervals, filtered, and the filter cake was dried and pulverized at 50°C. 0.5 g of the pulverized 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 centrifuged at 8000 rpm and 4°C for 10 min, and the residual ricinine was analyzed by liquid chromatography.

[0079] Liquid chromatography conditions were: a Diamonsil™ 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] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μM product per minute at 40°C and pH 7.0. 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 using 4-TCN as substrate and pure enzyme solutions of AcNR and AcNR-W59A / T134G / H135L / S192G as catalysts.

[0083] The reaction system was 10 mL: pure enzyme solution was diluted 10-fold with 200 mM sodium phosphate buffer, pH 8.0, and added to the reaction vessel to a final concentration of 0.2 mg / mL based on protein content. Substrate was added at a final concentration of 10-300 mM, and the volume was made up to 10 mL with 200 mM sodium phosphate buffer, pH 8, as the reaction medium. The reaction was carried out at 45°C and 800 rpm for 30 min. A 200 μL sample was taken, and the concentration of 4-TCA in the reaction solution was detected by HPLC as described in Example 4.

[0084] The K values ​​of AcNR and 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 catCompared with the original nitrilase, the activity of the modified nitrilase was significantly 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] 0.25g of wet cells were placed in a 250mL Erlenmeyer flask. 5-50g of castor meal and 20mL of 200mM sodium phosphate buffer (pH 7.0) were added and mixed thoroughly. The mixture was incubated in a water bath at 35°C and 300rpm for 48 hours. During the reaction, samples were taken every 6 hours, filtered, and the filter cake was dried and pulverized at 50°C. 0.5g of the pulverized sample was weighed, 20mL of methanol was added, and the mixture was shaken at 75°C and 600rpm for 4 hours. The mixture was centrifuged at 8000rpm and 4°C for 10 minutes. The supernatant was analyzed by liquid chromatography for residual ricinine.

[0089] The K values ​​of AcNR and 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 was 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] Reaction system was 10 mL: 0.1 g wet cell was dissolved in a conical flask with 10 mL, pH 8.0, 200 mM sodium phosphate buffer, and then 50 mM substrate was added to the final concentration, the temperature was set to 20-60 °C (20, 25, 30, 35, 40, 45, 50, 55, 60 °C), 800 rpm for 30 min, 200 μL was taken, and the concentration of 4-TCA in the reaction solution was detected by HPLC as described in Example 4, and the enzyme activity was calculated. The results are shown in Table 2. Figure 4 As shown in Table 2, the optimum temperature of the four mutants was 45 °C. Under the same conditions, the optimum temperature of the original nitrilase AcNR was 35 °C.

[0095] Example 8: Determination of the optimum temperature of nitrilase AcNR and its four mutants AcNR-W59A / T134G / H135L / S192G in the substrate ricinine

[0096] The optimum temperature of the wet cell in Example 2 was determined, with castor meal as the substrate, and the wet cell of nitrilase AcNR and its four mutants AcNR-W59A / T134G / H135L / S192G as the catalyst.

[0097] Each 0.25 g of wet cell was put into a 250 mL triangular flask, 50 g of castor meal, 20 mL of sodium phosphate buffer (200 mM, pH 7.0) was added and mixed thoroughly, and then incubated in a water bath at 20-60 °C (20, 25, 30, 35, 40, 45, 50, 55, 60 °C), 300 rpm for 48 h. During the reaction, samples were taken every 6 h, filtered, and the filter cake was dried at 50 °C and pulverized. 0.5 g of the pulverized sample was weighed, 20 mL of methanol was added, and the mixture was shaken at 75 °C, 600 rpm for 4 h. The supernatant was obtained by centrifugation at 8000 rpm, 4 °C for 10 min, and the residual ricinine was analyzed by liquid chromatography. The residual concentration of ricinine in the reaction solution was detected by HPLC as described in Example 4, and the enzyme activity was calculated. The results are shown in Table 3. Figure 5 As shown in Table 3, the optimum temperature of the four mutants was 40 °C. Under the same conditions, the optimum temperature of the original nitrilase AcNR was 35 °C.

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

[0099] The optimal pH of the wet cells from Example 2 was determined using 4-TCN as the substrate and wet cells of the nitrilase AcNR and its quadruple mutant 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-HCl buffer was used for pH 8-9; and glycine-sodium hydroxide buffer was used for pH 9-10.5.

[0100] The reaction system is 10 mL: 0.1 g of wet bacteria is dissolved in 10 mL of buffer of different pH values ​​in a conical flask, and then a final concentration of 50 mM substrate is added. The buffer is 200 mM with 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 800 rpm for 30 minutes. 200 μL of the sample is sampled and the concentration of 4-TCA in the reaction solution is detected by HPLC as described in Example 4 and the enzyme activity is calculated. The results are shown in FIG. Figure 6 As shown, the optimal pH of the quadruple 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 using castor meal as the substrate and wet cells of the nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G as the catalyst. Sodium citrate buffer was used for pH 4.0-6.5; sodium phosphate buffer for pH 6.5-8.0; Tris-HCl buffer for pH 8-9; and glycine-sodium hydroxide buffer for pH 9-10.5.

[0103] Take 0.25g of wet bacteria and put it into a 250mL triangular 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 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. 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. 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 FIG. Figure 7As 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: Conversion of 50 mM 4-TCN by Nitrilase and its mutant AcNR-W59A / T134G / H135L / S192G

[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 (based on protein content) of 1 mg / mL was added to a stoppered Erlenmeyer flask, substrate was added to a final concentration of 50 mM, and the volume was made up to 10 mL with sodium phosphate buffer (200 mM, pH 8.0). The reaction was incubated on a shaker at 45°C and 800 rpm in a water bath. 200 μL of the sample was taken every 30 minutes and centrifuged. The supernatant was tested for 4-TCA using HPLC as described in Example 4, and the conversion rate was calculated. The results are shown in Table 1. Figure 8 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 was 10 mL: the pure enzyme solution was diluted 10-fold with pH 8.0, 200 mM sodium phosphate buffer and added to the reaction vessel to a final concentration of 1 mg / mL based on protein content. Then, substrate was added to a final concentration of 50 mM and the volume was made up to 10 mL with (200 mM, pH 8.0) sodium phosphate buffer as the reaction medium. The reaction was carried out in a water bath at 800 rpm and 45°C. 200 μL of the sample was sampled every 30 minutes and centrifuged at 12,000 rpm. The precipitate was discarded. 4-TCA in the reaction solution was detected by HPLC as described in Example 4, and the conversion rate was calculated. The reaction progress results of the nitrilase and its four mutants are shown in FIG. Figure 9 The mutant AcNR-W59A / T134G / H135L / S192G was found to be able to completely react the substrate within 48.5 h, which was shorter than that of the original nitrilase and 2.1 times faster than that of the AcNR nitrilase.

[0111] Example 12: Immobilization and Application of Nitrilase Mutant AcNR-W59A / T134G / H135L / S192G

[0112] 2 g of wet cells of the combined mutant AcNR-W59A / T134G / H135L / S192G obtained by the method of Example 2 were weighed and suspended in 20 mL of NaH2PO4-Na2HPO4 buffer (200 mM, pH = 8.0). Celite was added to a final concentration of 0.006 g / mL and stirred at room temperature for 1 hour. Subsequently, 0.6 mL of a 5% aqueous polyethyleneimine solution was added and stirred at room temperature for 1 hour. 0.2 mL of a 25% aqueous glutaraldehyde solution was added and stirred for 0.5 hour. Finally, vacuum filtration was performed to obtain immobilized cells.

[0113] 0.5 g of immobilized cells corresponding to the wet cells were suspended in 10 mL of NaH PO -Na HPO buffer (200 mM, pH 8.0), 0.13 g of 4-TCN (final concentration 50 mM) was added, and the mixture was reacted in a constant temperature water bath at 45°C and 800 rpm. The immobilized cells prepared from the original AcNR nitrilase were reacted for 7-8 hours per batch, while those prepared from the mutant AcNR-W59A / T134G / H135L / S192G were reacted for 4-6 hours per batch. After completion of each batch reaction, vacuum filtration was performed to separate the solid and liquid. The reaction solution was analyzed for product concentration by high performance liquid chromatography (see Example 4), and the immobilized cells were then added to the next batch of reactions. 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] Wet cells of the nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G were obtained according to the method provided in Example 2. 0.25 g of each wet cell was placed in a 250 mL Erlenmeyer flask, 50 g of castor meal and 20 mL of sodium phosphate buffer (200 mM, pH 7.0) were added, mixed thoroughly, and incubated in a water bath at 35°C, 300 rpm for 48 h. During the reaction, samples were taken every 6 h, filtered, and the filter cake was dried and pulverized at 50°C. 0.5 g of the pulverized sample was weighed, 20 mL of methanol was added, and the mixture was shaken at 75°C, 600 rpm for 4 h. The supernatant was centrifuged at 8000 rpm, 4°C for 10 min, and liquid chromatography analysis was performed for residual ricinine. Analysis showed that the ricinine concentration in the castor meal decreased from 0.36% to 0.0007% after 48 h, with a ricinine content of less than 10 ppm.

[0119] 2. Crude enzyme solution as catalyst

[0120] According to the method provided in Example 3, crude enzyme solutions of the nitrilase AcNR and its quadruple mutant AcNR-W59A / T134G / H135L / S192G were obtained. 20 mL of each crude enzyme solution (equivalent to 0.25 g of wet bacterial cells) was added to a 250 mL Erlenmeyer flask, 50 g of castor meal was added and mixed thoroughly, and the mixture was incubated in a water bath at 35°C and 300 rpm for 48 h. During the reaction, samples were taken every 6 h, filtered, and the filter cake was dried and pulverized at 50°C. 0.5 g of the pulverized sample was weighed, 20 mL of methanol was added, and the mixture was shaken at 75°C and 600 rpm for 4 h. The mixture was centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant was analyzed by liquid chromatography for residual ricinine. Analysis showed that the ricinine concentration in the castor meal decreased from 0.36% to 0.0005% after 48 h, and the ricinine content was less than 10 ppm.

[0121] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with the art may 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 shall be based on the definition of the claims.

Claims

1. A nitrilase mutant, characterized in that The nitrilase mutant is a mutant in which the amino acid sequence shown in SEQ ID No. 2 is mutated 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) a double mutation combination: the tryptophan at position 59 is mutated into alanine and the threonine at position 134 is mutated into glycine; (6) a triple mutation combination: the tryptophan at position 59 is mutated into alanine, the threonine at position 134 is mutated into glycine, and the histidine at position 135 is mutated into leucine; (7) a quadruple mutation combination of the single point mutations described in (1) to (4).

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

12.

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

4. 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.

5. The use according to claim 4, characterized in that The application comprises the following steps: using wet bacteria obtained by fermentation culture of genetically engineered bacteria expressing a nitrilase mutant, or crude enzyme liquid extracted after ultrasonic disruption of the wet bacteria, or pure enzyme liquid obtained by purifying the crude enzyme liquid, 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 a 200 mM buffer solution at pH = 4.0-10.5 as a reaction medium to form a reaction system, reacting at 800 rpm and a constant temperature of 35-50° C., centrifuging, and separating and purifying to obtain 3,4,5,6-tetrachloropyridine-2-carboxylic acid.

6. The use according to claim 5, characterized in that In the reaction system, the substrate is added at a concentration of 20 to 200 mM; when the catalyst is a crude enzyme solution or a pure enzyme solution, the added amount is 0.1 to 3.5 g / L based on the protein content; when the catalyst is wet bacteria, the added amount is 10 to 100 g / L based on the wet weight of the bacteria; when the catalyst is immobilized cells, the added amount is 10 to 200 g / L based on the weight of the wet bacteria.

7. Use of the nitrilase mutant according to claim 1 for degrading ricinine in castor meal.

8. The use according to claim 7, characterized in that The application comprises the following steps: using wet cells 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 cells 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 reduced toxicity.

9. The use according to claim 8, 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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