CN117965504A

CN117965504A - Nitrilase mutant and application thereof in preparation of chloropyridine carboxylic acid

Info

Publication number: CN117965504A
Application number: CN202410117369.4A
Authority: CN
Inventors: 薛亚平; 史高婷; 熊能; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2024-01-29
Filing date: 2024-01-29
Publication date: 2024-05-03

Abstract

The invention discloses a nitrilase mutant and application thereof in preparing 3,4,5, 6-tetrachloropyridine-2-carboxylic acid, wherein the nitrilase mutant comprises a mutant sequence obtained by carrying out single mutation or double mutation on the following sites of an amino acid sequence shown in SEQ ID NO. 2: (1) mutating tryptophan at position 166 to glycine; (2) mutation of asparagine 164 to alanine. The catalytic activities of the nitrilase mutants W166G/N164A and T-W171G/N169A are respectively improved by 38 times and 56 times, the hydration activity is reduced to 50% of the wild type enzyme, and the molar ratio of the amide byproducts to the total products is respectively 3.1% and 2.7%. The recombinant escherichia coli containing the nitrilase mutants W166G/N164A and T-W171G/N169A is used for hydrolyzing 50mM of 3,4,5, 6-tetrachloropyridine-2-carbonitrile at 40 ℃ to generate 3,4,5, 6-tetrachloropyridine-2-carboxylic acid, and the 3,4,5, 6-tetrachloropyridine-2-carboxylic acid can be completely converted in 25 hours or 20 hours respectively, and the final product yield reaches 75% or 80%. The nitrilase mutant can improve the enzyme activity and reduce the proportion of byproduct amide.

Description

Nitrilase mutant and application thereof in preparation of chloropyridine carboxylic acid

Technical Field

The invention relates to a mutant derived from rhodococcus rhodochrous (Rhodococcus rhodochrous J1) nitrilase and application thereof in chloropyridine carboxylic acid synthesis.

Background

Chloropyridine carboxylic acid is an important pesticide intermediate, such as 3,4,5,6-tetrachloropyridine-2-carboxylic acid (3, 4,5, 6-tetrachloropyridine-2-carboxilic acid, 4-TCA for short), and is a typical chloropyridine compound. 4-TCA is a fine chemical intermediate for various pesticides and medicines, and can be used for synthesizing pesticides such as picloram (4-amino-3, 5, 6-trichloropyridine-2-carboxylic acid), pickle grass (3, 6-dichloropyridine-2-carboxylic acid) and the like.

The method for synthesizing the chloropyridine compound takes pyridine as a raw material, and the chloropyridine compound is obtained through nitridation and chlorination. At present, the common synthetic method of 4-TCA is to take pyridine and chlorine as raw materials to catalyze and obtain pentachloropyridine at 250-800 ℃, then take fluorine and cyano to replace and obtain 3,4,5, 6-tetrachloropyridine-2-carbonitrile (4-TCN) at 220-245 ℃, and hydrolyze by strong acid to obtain 4-TCA, but the method has poor selectivity, easily generates byproducts which are difficult to separate, and is difficult to meet the requirement of the subsequent production on the purity of a substrate.

Nitrilases (NITRILASE EC.5.5.1) are a class of enzymes which are capable of directly hydrolysing nitriles (containing-CN) to the corresponding carboxylic acids. The catalytic reaction of the nitrilase has the characteristics of high selectivity, high catalytic rate, mild reaction conditions, small environmental pollution and the like, is an environment-friendly green synthesis method, and has important practical significance on energy conservation and emission reduction. For example, (R) -mandelic acid, a product of BASF, germany, is quantitatively converted into (R) -mandelic acid by reacting benzaldehyde with hydrocyanic acid to form racemic mandelonitrile, and then selecting appropriate reaction conditions, and subjecting the racemic mandelonitrile to chiral kinetic resolution catalyzed by stereoselective nitrilase. The methylene glutaronitrile is firstly hydrolyzed into 4-cyano valeric acid (4-CPA) ammonium salt by an immobilized microbial cell catalyst (Acidovorax facilis W) containing nitrilase, the selectivity of the hydrolysis reaction is more than 98 percent, the conversion rate is 100 percent, and one half of cyano carboxylic acid ammonium salt can be obtained, and 1 to 2 percent of the only reaction byproduct of 2-methyl glutarate diammonium salt is generated. As another example, the Zhejiang university of industry Zheng Yuguo, xue Ya et al reported a series of studies on chemical-enzymatic synthesis of gabapentin, including the discovery of nitrilase, the biocatalytic process of nitrilase converting intermediate 1-cyanocyclohexyl acetonitrile into 1-cyanocyclohexyl acetic acid, and the subsequent hydrogenation processes, etc., reported a triple mutant nitrilase AcN-T (T151V/C223 AC 250G); when the concentration of the whole cell catalyst is 50g/L, the reaction can completely convert 1.0M substrate in 2 hours, the product yield reaches 93%, and a complete gabapentin Ding Huaxue-enzymatic process route is formed. Compared with the traditional chemical method, the production process of the nitrilase method has the advantages of improved yield, reduced waste, high selectivity and the like. There are also a number of nitrilases that have been developed and used in the synthesis of various pharmaceutical intermediates and fine chemicals.

Through review, there is currently no report about the conversion of 4-TCN to 4-TCA by nitrilase, other than the present laboratory techniques. Technical development and industrial application of nitrilase hydrolysis (such as 2-chloronicotinic acid and the like) of chloropyridine nitrile compounds have been realized in the laboratory. The laboratory technology takes 4-TCN as a starting substrate, synthesizes 4-TCA by using a biocatalysis method, and can synthesize the 4-TCA by using a nitrilase direct hydrolysis method or a nitrile hydratase coupling amidase method. The high catalytic efficiency and selectivity of the nitrilase lead the biocatalysis method to have the advantages of mild reaction conditions, low energy consumption, economy and the like in the synthesis of 4-TCA. In theory, compared with the nitrile hydratase-amidase method, the nitrilase method has the advantages of no need of considering cascade reaction, optimizing reaction flow, reducing limiting factors and the like. In the reaction principle, the synthesis of 4-TCA by using a nitrilase method has higher feasibility.

Disclosure of Invention

The invention aims to solve the problems of low activity of wild nitrilase on substrate 3,4,5, 6-tetrachloropyridine-2-carbonitrile enzyme, high byproduct amide ratio and the like by enzyme modification, and provides a high-activity nitrilase mutant protein and application thereof in synthesis of 3,4,5, 6-tetrachloropyridine-2-carboxylic acid.

The technical scheme adopted by the invention is as follows:

In a first aspect, the present invention provides a nitrilase mutant (with increased enzymatic activity and low by-product amide content) comprising a mutant sequence obtained by single or double mutation of the amino acid sequence of SEQ ID NO. 2 at the following positions: (1) mutating tryptophan at position 166 to glycine; (2) mutation of asparagine 164 to alanine.

Preferably, the nitrilase mutant is a mutant sequence obtained by double mutation of the following sites of the amino acid sequence shown in SEQ ID NO. 2: (1) mutating tryptophan at position 166 to glycine; (2) mutation of asparagine 164 to alanine.

Further, the nitrilase mutant further comprises a polypeptide tag GKGKG connected to the N end of the mutant sequence.

Further preferably, the nitrilase mutant consists of the mutant sequence and a polypeptide tag GKGKGKG connected with the N end of the mutant sequence, wherein the mutant sequence is obtained by double mutation of the amino acid sequence shown in SEQ ID NO. 2 at the following sites: (1) mutating tryptophan at position 166 to glycine; (2) mutation of asparagine 164 to alanine.

Namely, the nitrilase mutant is a mutant sequence obtained by double mutation of the following sites of the amino acid sequence shown in SEQ ID NO. 10: (1) mutating tryptophan at position 171 to glycine; (2) mutation of asparagine to alanine at position 169.

It is known to those skilled in the art that attachment of a polypeptide tag to the N-or C-terminus of a protease may enhance the activity of the enzyme or impart novel properties (e.g., water solubility, thermostability, etc.), and therefore, a nitrilase mutant comprising the mutant sequence and having a function of the polypeptide sequence attached to one or both ends of the sequence is also within the scope of the present invention.

The nitrilase mutant specifically disclosed by the invention adopts a semi-rational design method and a full-plasmid PCR technology, and comprises the following amino acid sequences of SEQ ID NO:1, and the strain E.coli BL21 (DE 3)/Pet 28 (+) -Nit-RR-J1 derived from the gene encoding the nitrilase of rhodococcus XP_75546.1 is subjected to site-directed mutagenesis, positive mutants are screened and detected after induced expression, and then the mutant protein with improved enzyme activity and low by-product amide ratio is obtained.

The nitrilase mutant is obtained by connecting a polypeptide tag GKGKGKG at the N end of an amino acid sequence shown in SEQ ID NO. 2 to obtain an amino acid sequence shown in SEQ ID NO. 10, and then mutating one or more amino acids at position 171 and position 169 of the amino acid sequence shown in SEQ ID NO. 10.

The PCR amplification reaction is carried out by taking a recombinant plasmid pET28b (+)/Nit-RR-J1 containing a coding gene of a nitrilase (Nit-RR-J1) shown in SEQ ID NO.1 as a template, and the nucleotide sequences of the upstream primer and the downstream primer of a polypeptide tag are shown in the following table 1, and the polypeptide tag GKGKG is directly added to the N end of the nitrilase gene through PCR amplification, thus obtaining a PCR product containing the recombinant plasmid pET28b (+)/Nit-RR-J1-T.

TABLE 1 primer design table for polypeptide tags

In a second aspect, the present invention provides a gene encoding the above nitrilase mutant.

Further, it is preferable that the nitrilase mutant is one of the following: (1) The 166 th tryptophan of the amino acid sequence shown in SEQ ID NO. 2 is mutated into glycine (W166G), the amino acid sequence is shown in SEQ ID NO. 4, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 3; (2) The 164 th asparagine of the amino acid sequence shown in SEQ ID NO. 2 is mutated into alanine (N164A), the amino acid sequence is shown in SEQ ID NO. 6, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 5; (3) The 166 th tryptophan of the amino acid sequence shown in SEQ ID NO. 2 is mutated into glycine, the 164 th asparagine is mutated into alanine, the amino acid sequence is shown in SEQ ID NO. 8, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 7. (4) The 171 th tryptophan of the amino acid sequence shown in SEQ ID NO. 10 is mutated into glycine (W171G), the amino acid sequence is shown in SEQ ID NO. 12, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 11; (5) Mutation of 169 th asparagine of an amino acid sequence shown in SEQ ID NO. 10 into alanine (N169A), wherein the amino acid sequence is shown in SEQ ID NO. 14, and the nucleotide sequence of a coding gene is shown in SEQ ID NO. 13; (6) The 171 th tryptophan of the amino acid sequence shown in SEQ ID NO. 10 is mutated into glycine, the 169 th asparagine is mutated into alanine, the amino acid sequence is shown in SEQ ID NO. 16, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 15.

In a third aspect, the present invention provides a biological material comprising the above-described coding gene, which biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid, a viral vector or a cell.

The plasmid may employ any suitable vector. For example, suitable vectors include, but are not limited to, prokaryotic expression vectors pET28, pET20, pGEX4T1, pTrC A, and pBV220; including but not limited to eukaryotic expression vectors pPIC9K, pPICZ α, pYD1 and pYES2/GS; including but not limited to cloning vectors pUC18/19 and pBluscript-SK. In the present example, pET28b.

In an embodiment of the invention, the plasmid is obtained as follows:

Method one (mutant unlabeled recombinant plasmid): -comparing said SEQ ID NO:1 is inserted between the cleavage sites NcoI and XhoI of the pET-28b (+) plasmid to obtain a wild type plasmid; taking the wild type plasmid as a template, and carrying out full plasmid PCR amplification by one or two of the following primer pairs to obtain the plasmid;

Method two (mutant tagged recombinant plasmid): (1) Taking the wild plasmid as a template, and carrying out full plasmid PCR amplification by using the following primer pair to obtain a plasmid carrying a tag;

(2) Taking the plasmid carrying the tag as a template, and carrying out full plasmid PCR amplification by one or two of the following primer pairs to obtain the plasmid;

the cells may employ any suitable host cell, including animal cells, eukaryotes, and prokaryotes. In the example of the present invention is E.coli BL21 (DE 3).

In a fourth aspect, the invention also provides the use of the nitrilase mutant in the catalysis of 3,4,5, 6-tetrachloropyridine-2-carbonitrile to 3,4,5, 6-tetrachloropyridine-2-carboxylic acid.

Specifically, the application is as follows: the preparation method comprises the steps of taking wet thalli, wet thalli immobilized cells or pure enzymes extracted after ultrasonic crushing of the wet thalli obtained by fermenting and culturing engineering bacteria containing nitrilase mutant encoding genes as a catalyst, taking 3,4,5, 6-tetrachloropyridine-2-carbonitrile as a substrate, adding a reaction medium to form a reaction system, reacting at 25-50 ℃ (preferably 40 ℃) in a constant temperature water bath, and separating and purifying obtained reaction liquid after the reaction is completed, thereby obtaining 3,4,5, 6-tetrachloropyridine-2-carboxylic acid.

Further, the reaction medium is a buffer solution or a mixed solution of the buffer solution and an organic solvent. Preferably, the pH of the reaction medium is=7.

Further, the organic solvent is one or a mixture of two of alkane solvents, n-octane, isooctane, cyclohexane and n-dodecane.

In an embodiment of the present invention, the volume ratio of the buffer solution to the organic solvent is 9:1.

Further, the buffer was 0.2M, pH 7.0Na ₂HPO₄-NaH₂PO₄ buffer.

Further, the final concentration of the substrate in the reaction system is 10 to 50mM, and when the catalyst is wet bacteria, the concentration of the catalyst is 10 to 50g/L (preferably 20 g/L) of the reaction system based on the mass of the wet bacteria; when the catalyst is immobilized cells, the concentration of the catalyst is 50g/L of the reaction system based on the mass of the immobilized cells; when the catalyst is crude enzyme liquid, the catalyst dosage is 3g/L of reaction system based on the protein quantity of the crude enzyme liquid; when the catalyst is pure enzyme, the catalyst dosage is 2g/L of the reaction system based on the mass of the pure enzyme.

Further, the wet cell is prepared as follows: inoculating the nitrilase-containing mutant coding genetically engineered bacteria into LB culture medium, culturing at 37 ℃ for 10-12h, inoculating the strain into LB culture medium containing 50mg/L kanamycin at a final concentration of 2% by volume, culturing at 37 ℃ until the OD ₆₀₀ of the culture solution is 0.6-0.8, adding isopropyl-beta-D-thiopyran galactoside at a final concentration of 0.1mM, culturing at 28 ℃ for 10h in an induction way, centrifuging, collecting thalli, and washing with physiological saline for 2 times to obtain wet thalli.

Further, the crude enzyme solution and the pure enzyme are prepared according to the following method: (1) Resuspension of wet thalli with pH 8.0 containing final concentration 300mM NaCl and 50mM NaH ₂PO₄ buffer solution, ultrasonic crushing (power 400W,25min, crushing 1s pause 1 s), centrifuging (12000 rpm,10 min), taking supernatant as crude enzyme solution; (2) Passing the crude enzyme solution through a Ni-NTA column washed by an equilibrium buffer solution at a flow rate of 1mL/min, and eluting the weakly adsorbed impurity protein by using an elution buffer solution at a flow rate of 1.5mL/min; eluting with protein eluting buffer solution, and collecting target protein with flow rate of 1.5mL/min; finally, dialyzing the collected target protein by using 50mM NaH ₂PO₄ buffer solution as a dialyzate, and taking the trapped fluid as pure enzyme; the balance buffer is pH 8.0 buffer solution containing 300mM NaCl with the final concentration and 50mM NaH ₂PO₄ buffer solution; the eluting buffer is pH 8.0 buffer solution containing 300mM NaCl and 50mM imidazole with final concentration and 50mM NaH ₂PO₄ buffer solution; the protein elution buffer was pH 8.0, 50mM NaH ₂PO₄ buffer containing final concentrations of 300mM NaCl and 500mM imidazole.

Further, the wet cell-immobilized cells were prepared as follows: suspending wet thalli in a buffer solution system with pH=7.0 and 200mM NaH ₂PO₄-Na₂HPO₄, adding diatomite with a final concentration of 6mg/mL, stirring for 1h at room temperature, then adding an aqueous solution of polyethylenimine with a mass concentration of 5%, and stirring for 1h at room temperature; finally, adding glutaraldehyde water solution with the mass concentration of 25%, stirring for 0.5h, and vacuum filtering to obtain immobilized cells; the volume addition of the polyethyleneimine water solution is 3 percent based on the volume of the buffer solution, and the volume addition of the glutaraldehyde water solution is 1 percent based on the volume of the buffer solution.

Further, the aqueous phase of the two-phase system is 0.2M, the pH value is 7.0NaH ₂PO₄-Na₂HPO₄ buffer solution, the organic phase is organic matters such as xylene, cyclohexane, n-octane, isooctane, ethyl acetate, isobutyl acetate, n-dodecane, dichloromethane and the like, and the aqueous phase and the organic phase are mixed according to the volume ratio of 9:1 (v/v) to obtain the two-phase system.

The nitrilase mutant of the invention may be in the form of a recombinant expression transformant (i.e., wet cell, preferably E.coli BL21 (DE 3)) containing the nitrilase mutant gene as a catalyst, or may be an unpurified crude enzyme, or may be a partially purified or completely purified enzyme, or may be an immobilized enzyme or immobilized cell produced by immobilizing the nitrilase mutant of the invention using immobilization techniques known in the art.

The final concentration composition of the LB liquid medium comprises: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, water as solvent and natural pH value. Final concentration composition of LB solid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 20g/L agar, water as solvent and natural pH value.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, through semi-rational design, the protein is subjected to molecular transformation, the enzyme activity of the nitrilase mutant Nit-RR-J1-W166G/N164A is improved by 38 times compared with that of the wild nitrilase, the proportion of amide byproducts is greatly reduced, the ratio of the amide byproducts is reduced from 83.6% of the wild nitrilase to 3.1%, and the enzyme activity of the mutant Nit-RR-J1-T-W171G/N169A is improved by 56 times compared with that of the wild nitrilase. Therefore, the mutant and the application thereof lay a foundation for catalyzing the synthesis of 3,4,5, 6-tetrachloropyridine-2-carbonitrile by the chemical enzyme method of 3,4,5, 6-tetrachloropyridine-2-carboxylic acid.

Drawings

FIG. 1 shows an electrophoretogram of a nitrilase mutant protein after purification. Lane 1 is Nit-RR-J1, lane 2 is Nit-RR-J1-T-W171G, lane 3 is Nit-RR-J1-T-N169A, and lane 4 is Nit-RR-J1-T-W171G/N169A.

FIG. 2 optimum pH of nitrilase mutant Nit-RR-J1-T. (line graph)

FIG. 3 optimum temperature of nitrilase mutant Nit-RR-J1-T. (line graph)

FIG. 4 is a high performance liquid chromatogram of 3,4,5, 6-tetrachloropyridine-2-carboxylic acid.

FIG. 5 shows the effect of co-solvent on 4-TCN hydrolysis using E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A wet cells.

FIG. 6 shows an enzyme-catalyzed reaction in a two-phase system using E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A wet cells.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

The final concentration composition of the LB liquid medium is as follows: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, water as solvent and natural pH value.

Final concentration composition of LB solid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 20g/L agar, water as solvent and natural pH value.

Example 1: semi-rational design and site-directed mutagenesis

The plasmid pET-28b (+) -Nit-RR-J1 cloned in R.rhodochrous J1 nitrilase Nit-RR-J1 (nucleotide sequence SEQ ID NO:1, amino acid sequence of encoded protein SEQ ID NO: 2) was used as a template, a protein with highest homology to the R.rhodochrous J1 nucleic acid sequence was searched on-line website by SWISS MODEL as a template, and nitrilase from P.fluoroscins EBC191 (PDB ID:6 ZBY.1) was obtained by sequence alignment screening, the homology of the nitrilase with the nitrilase used herein was 50.5% at the highest, modeling was performed by using Discovery site, the modeling was scored by Procheck Program to determine the feasibility, and protein receptor and substrate ligand were pre-treated prior to docking by docking with nitrilase and 4-TCN. The docking results were visually analyzed using Pymol. Proximity of the selection substrateThe amino acid residues in the amino acid residues are subjected to the next screening step. And (3) carrying out catalytic channel prediction on the result of molecular docking, wherein the process is completed by CAVER ANALYST, and according to the predicted result, comparing with amino acid residues obtained by docking, selecting repeated amino acid residues for further experiments.

Predicting by using the Hot Spot software on-line website, submitting our protein sequence, and further screening according to the generated result. PCR amplification was performed with full plasmid site-directed mutagenesis (Table 2). PCR reaction System (50. Mu.L): template 0.5-20 ng (pET-28 b (+) -Nit-RR-J1 plasmid synthesized between two cleavage sites NcoI and XhoI of pET-28b (+) plasmid is used as template) from R.rhodochrous J1 nitrilase coding gene (GenBank accession No. XP_ 75546.1), nucleotide sequence SEQ ID NO:1, the sequence of coding protein amino acid sequence SEQ ID NO:2, 2X Phanta max Buffer mu L,0.2mM dNTP, primers each 0.2 mu M, phanta Max Super-FIDELITY DNA Polymerase 1 mu L, and water is added to make up to 50 mu L. PCR conditions: (1) pre-denaturation at 95℃for 3min; (2) denaturation at 95℃for 15s; (3) annealing at 60 ℃ for 15s; (4) Extending at 72 ℃ for 5.5min, wherein the total time of the steps (2) to (4) is 30 cycles; (5) finally, extending at 72 ℃ for 10min and preserving at 16 ℃. The PCR products were verified by agarose gel electrophoresis, digested with DpnI, then introduced into E.coli BL21 (DE 3), plated onto LB plates containing 50. Mu.g/mL kanamycin, and monoclonal obtained, followed by sequencing. And further performing reaction verification according to the sequencing result.

TABLE 2 primer design Table

And (3) utilizing liquid chromatography screening to verify that the enzyme activity improvement mutant is determined to be W166G, and the nucleotide sequences of N164A are respectively SEQ ID NO:3, SEQ ID NO: shown at 5. And, by using the same method, a combined mutant W166G/N164A is constructed, and the nucleotide sequence of the combined mutant W166G/N164A is shown as SEQ ID NO: shown at 7.

The mutants were introduced into E.coli BL21 (DE 3) to construct mutants E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G, E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-N164A and a combination mutant E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G/N164A, respectively.

Adding a polypeptide tag GKGKG at the N end of the sequence, and mutating the nucleotide at the W171G, N A position, wherein the sequences are SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: shown at 13. By the same method, a combined mutant W171G/N169A is constructed, and the nucleotide sequence of the combined mutant W171G/N169A is shown as SEQ ID NO: 15.

The mutants were introduced into E.coli BL21 (DE 3) to construct mutants E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G, E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-N169A and a combination mutant E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A, respectively.

Example 2: expression of nitrilase mutants

The mutants E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G, E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-N164A and the combined mutants E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G/N164A obtained in example 1, the mutants E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G, E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-N169A and the combined mutants E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A were inoculated with the original strain E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T and the final strain E.coli BL21 (. Beta.3)/pET-37B-10 mg to a final medium of 37 mg (37 mg, 25 mg, 37 mg, 50 mg) of the medium was inoculated to the final medium and the final medium was inoculated with the medium of 50mg medium. And centrifuging the culture solution to collect thalli, and washing the thalli for 2 times by using normal saline to obtain corresponding wet thalli.

Example 3: purification of nitrilase mutant proteins

(1) To 0.1g of the wet cells collected in example 2, 15mL of an equilibration buffer (50 mM NaH ₂PO₄, 300mM NaCl buffer, pH 8.0) was added, and the cells were resuspended and then sonicated (400W, 25min, 1s of disruption were suspended for 1 s). The product obtained after crushing was centrifuged (12000 Xg, 10 min), and the obtained supernatant was crude enzyme solution, which was prepared for separation and purification.

(2) After pre-loading the 15mL Ni-NTA affinity column, equilibration was performed using equilibration buffer (50 mM NaH ₂PO₄, 300mM NaCl,pH 8.0) at a flow rate of 1.5mL/min.

(3) After washing 8-10 column volumes, the crude enzyme solution obtained was passed through a Ni-NTA column at a flow rate of 1.5mL/min, and the target protein was mounted on the column. After loading, a large amount of non-adsorbed foreign proteins are not bound to the resin and are directly removed.

(4) Weakly adsorbed heteroproteins were eluted using elution buffer (50 mM NaH ₂PO₄, 300mM NaCl,50mM imidazole, pH 8.0) at a flow rate of 1.5mL/min.

(5) The target protein was eluted and collected using a protein elution buffer (50 mM NaH ₂PO₄, 300mM NaCl,500mM imidazole, pH 8.0) at a flow rate of 1.5mL/min.

(6) The collected enzyme solution was used in a dialysis bag (Economical Biotech Membrane,14kDa,34mm Width, available from Shanghai Biotechnology Co., ltd.) and was sodium dihydrogen phosphate-disodium hydrogen phosphate (50 mM, pH 7.0) buffer solution, and the retentate was taken as purified protein to obtain the corresponding pure enzyme solution.

(7) The purified proteins were analyzed by SDS-PAGE and the results of protein electrophoresis are shown in FIG. 1.

Example 4: determination of crude enzyme Activity of nitrilase

The crude enzyme obtained in example 3 was subjected to enzyme activity measurement. Nitrilase activity assay reaction system (1 mL): naH ₂PO₄-Na₂HPO₄ (200 mM, pH 7.0) buffer, 50mM 3,4,5, 6-tetrachloropyridine-2-carbonitrile, 100. Mu.L crude enzyme solution. After the reaction solution is preheated at 40 ℃ for 5min, a constant-temperature water bath is performed for 30min at 400rpm, 100 mu L of acetonitrile is added to terminate the reaction, 200 mu L of supernatant is sampled, the conversion rate of the conversion solution 3,4,5, 6-tetrachloropyridine-2-carbonitrile is measured by using a liquid chromatography (Shimadzu LC-16) external standard method, and a high performance liquid chromatogram of the 3,4,5, 6-tetrachloropyridine-2-carboxylic acid is shown in FIG. 4.

The chromatographic column isC-18column (250 mm. Times.4.6 mm,5 μm), mobile phase deionized water): acetonitrile=60: 40 (v/v), flow rate was 0.8mL/min, UV detection wavelength 224nm, column temperature 40 ℃.

Definition of enzyme activity (U): the amount of enzyme required to catalyze the production of 1. Mu. Mol of 3,4,5, 6-tetrachloropyridine-2-carboxylic acid per minute at 35℃and pH 7.0 in 200mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer was defined as 1U, and the results are shown in Table 3.

TABLE 3 crude enzyme Activity of nitrilase mutants

Example 5: determination of pure enzyme Activity of nitrilase

The enzyme activity of the purified protein of example 3 was measured. Nitrilase activity assay reaction system (1 mL): naH ₂PO₄-Na₂HPO₄ (200 mM, pH 7.0) buffer, 10mM 3,4,5, 6-tetrachloropyridine-2-carbonitrile, 100. Mu.L of pure enzyme solution. After the reaction solution is preheated at 40 ℃ for 5min, a constant-temperature water bath is performed for 30min at 400rpm, 100 mu L of acetonitrile is added to terminate the reaction, 200 mu L of supernatant is sampled, the conversion rate of the conversion solution 3,4,5, 6-tetrachloropyridine-2-carbonitrile is measured by using a liquid chromatography (Shimadzu LC-16) external standard method, and a high performance liquid chromatogram of the 3,4,5, 6-tetrachloropyridine-2-carboxylic acid is shown in FIG. 5.

Conditions for HPLC analysis were as described in example 4, enzyme activity definition (U): the amount of enzyme required to catalyze the production of 1. Mu. Mol of 3,4,5, 6-tetrachloropyridine-2-carboxylic acid per minute at 35℃and pH 7.0 in 200mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer was defined as 1U. The relative activities of the mutant Nit-RR-J1-W166G and the mutant Nit-RR-J1-N164A were 9.8 and 2.5 times that of the original nitrilase Nit-RR-J1, respectively, and the initial activities of the combined mutant Nit-RR-J1-W166G/N164A were improved by 38 times as compared with the original nitrilase Nit-RR-J1, and the results are shown in Table 4.

TABLE 4 pure enzymatic Activity of nitrilase mutants

Example 6: determination of optimum pH containing nitrilase mutants

100. Mu.L of nitrilase from the purified protein of example 3 was added to a buffer system of different pH values (pH 3.0-6.0 was a citrate-sodium citrate buffer, pH 6.0-8.0 was a Na ₂HPO₄-NaH₂PO₄ buffer, pH 8-9 was a Tris-HCl buffer, pH 9.0-10.0 was a Glycine-NaOH buffer) in a total volume of 1mL, and after preheating for 5min in a 40℃constant temperature water bath shaker, 4-TCN was added at a final concentration of 10mM, 400rpm, and reacted at 45℃for 10min. After the reaction was terminated, the reaction mixture was sampled and centrifuged at 12,000rpm for 3 minutes to obtain a supernatant, and the reaction mixture was analyzed for 4-TCA concentration by HPLC. The results are shown in FIG. 2, with the enzyme activity of the original strain as a control, and the optimum pH is 7.0.

Example 7: determination of optimum temperature for nitrilase mutants

100. Mu.L of nitrilase was obtained from the purified protein of example 3, and after mixing with 100. Mu.L of nitrilase using 200mM Na ₂HPO₄-NaH₂PO₄ buffer solution having pH of 7.0 (1 mL reaction system), the mixture was placed on a water bath shaking table at a predetermined temperature and incubated for 5 minutes at 30℃at 35℃at 40℃at 45℃at 50℃at 55℃at 60℃with a final concentration of 10mM 4-TCN, and after reacting for 30 minutes in a constant temperature water bath shaking table at 400rpm, 100. Mu.L of acetonitrile was added to terminate the reaction, and the reaction was centrifuged by sampling to obtain the supernatant, and the reaction solution was analyzed for the concentration of 4 chloropicolinic acid by HPLC. The results are shown in FIG. 3, with the viability of the original strain as a control, and the optimum temperature is 40 ℃.

Example 8: determination of the Activity of recombinant E.coli containing a nitrilase mutant

Recombinant E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T containing nitrilase mutants obtained in example 2, E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-N164A, combination mutants E.coli BL21(DE3)/pET28b(+)-Nit-RR-J1-W166G/N164A,E.coli BL21(DE3)/pET28b(+)-Nit-RR-J1-T-W171G,E.coli BL21(DE3)/pET28b(+)-Nit-RR-J1-T-N169A, combination mutants E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A, E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR J1-T and original strain E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T were subjected to viability assay. Nitrilase activity detection reaction system (10 mL): na ₂HPO₄-NaH₂PO₄ (200 mM, pH 7.0) buffer, final concentration 200mM 3,4,5, 6-tetrachloropyridine-2-carbonitrile, recombinant E.coli wet cell 1g/L. After the reaction solution was preheated at 40℃for 10 minutes, it was reacted at 180rpm for 10 minutes. 200. Mu.L of the supernatant was sampled and the conversion of 3,4,5, 6-tetrachloropyridine-2-carboxylic acid in the conversion solution was measured by a liquid chromatography (Shimadzu LC-16) external standard method. The relative viability of recombinant E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G, E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-N164A and E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G/N164A containing the nitrilase mutants was examined as described in example 4 and was 22.4, 2.1 and 47.6 times that of the original strain E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1, respectively, and the results are shown in Table 5.

TABLE 5 resting cell viability of nitrilase mutants

Example 9: transformation of 3,4,5, 6-tetrachloropyridine-2-carbonitrile using recombinant E.coli containing a nitrilase mutant

10G/L of the combined mutant E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G/N164A, E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A wet cells were dissolved in 10mL of Na ₂HPO₄-NaH₂PO₄ buffer system (200 mM, pH=7.0), 50mM 3,4,5, 6-tetrachloropyridine-2-carbonitrile was added and reacted in a constant temperature water bath at 40 ℃. Samples were taken at different times, centrifuged at 12000rpm, the pellet was discarded, and the supernatant was analyzed for product concentration by high performance liquid chromatography. The conditions for HPLC analysis were as described in example 4. As shown in Table 6, the mutant E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G/N164A can completely convert 50mM substrate in about 25 hours, the product yield can reach 75%, the amide by-product is only 3.1% of the product, and the mutant E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A can completely convert 50mM substrate in about 20 hours, and the product yield can reach 80%.

TABLE 6 transformation of recombinant E.coli resting cells containing nitrilase mutants 50mM 3,4,5, 6-tetrachloropyridine-2-carbonitrile

Example 10: conversion of different concentrations of 3,4,5, 6-tetrachloropyridine-2-carbonitrile using recombinant E.coli containing a nitrilase mutant

10G/L,20G/L,30G/L,40G/L or 50G/L of the combined mutant E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G/N164A, E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A wet cells were dissolved in 10mL of Na ₂HPO₄-NaH₂PO₄ buffer system (200 mM, pH=7.0), 10mM,20mM,30mM,40mM,50mM of 3,4,5, 6-tetrachloropyridine-2-carbonitrile were added, respectively, and reacted in a constant temperature water bath at 35 ℃. Samples were taken at the same time, centrifuged at 12000rpm, the precipitate was discarded, and the supernatant was analyzed for product concentration by high performance liquid chromatography. The conditions for HPLC analysis are as described in example 4, and the results are shown in Table 7 and Table 8.

TABLE 7 resting cell transformation of nitrilase mutant Nit-RR-J1-W166G/N164A 3,4,5, 6-tetrachloropyridine-2-carbonitrile

TABLE 8 resting cell transformation of nitrilase mutant Nit-RR-J1-T-W171G/N169A 3,4,5, 6-tetrachloropyridine-2-carbonitrile

Example 11: transformation of 50mM 3,4,5, 6-tetrachloropyridine-2-carbonitrile Using immobilized cells

2G of the combined mutant E.coli BL21(DE3)/pET28b(+)-Nit-RR-J1-W166G/N164A,E.coli BL21(DE3)/pET28b(+)-Nit-RR-J1-T-W171G/N169A,E.coli BL21(DE3)/pET28b(+)-Nit-RR-J1-T obtained by the method of example 2 and the original strain E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1 wet cell were separately weighed and suspended in 20mL of NaH ₂PO₄-Na₂HPO₄ buffer system (200 mM, pH=7.0), and diatomaceous earth was added at a final concentration of 0.006g/mL and stirred at room temperature for 1 hour. Then, an aqueous solution of polyethyleneimine with a mass concentration of 5% was added and stirred at room temperature for 1 hour. Finally, adding glutaraldehyde water solution with the mass concentration of 25%, stirring for 0.5h, and vacuum filtering to obtain immobilized cells; wherein the volume addition of the polyethyleneimine aqueous solution is 3% based on the volume of the buffer solution, and the volume addition of the glutaraldehyde aqueous solution is 1% based on the volume of the buffer solution.

0.5G of the immobilized cells corresponding to the wet cells were suspended in 10mL of Na ₂HPO₄-NaH₂PO₄ buffer system (200 mM, pH=7.0), and 0.13g of 3,4,5, 6-tetrachloropyridine-2-carbonitrile (final concentration: 50 mM) was added thereto for reaction in a constant temperature water bath at 40 ℃. Wherein the immobilized cells prepared from the original strain E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1 are reacted for 7-8 hours per batch, and the immobilized cells prepared from E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-W166G/N164A are reacted for 4-6 hours per batch. After each batch of reaction is finished, the reaction solution is subjected to vacuum filtration and solid-liquid separation, the concentration of the product is analyzed by high performance liquid chromatography (see example 4), and the immobilized cells are put into the next batch of reaction. The results are shown in Table 9.

TABLE 9 transformation of 50mM 3,4,5, 6-tetrachloropyridine-2-carbonitrile using immobilized cells

Example 12: influence of the addition of organic solvents on the catalytic reaction

The reaction system was 10mL of Na ₂HPO₄-NaH₂PO₄ buffer (0.2M, pH 7.0), the catalyst (E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A wet cell obtained by the method of example 2) had a concentration of 10G/L, a substrate concentration of 10mM, and a reaction temperature of 40 ℃. Several miscible cosolvents were selected, including methanol, ethanol, isopropanol, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO) and the effect on the catalytic reaction, the cosolvent concentration gradient was (0%, 2%,5%,10%,20%, v/v), the amount of product was detected after 1h of reaction, and the remaining enzyme activity was calculated. As a result, as shown in FIG. 5, when the organic solvent was added in an amount of 2% (v/v), the yield of the enzyme-catalyzed hydrolysis reaction of 4-TCN was significantly reduced, and the yield of the product was slightly improved as compared with that of the pure water phase only when DMSO was used as a cosolvent. The effect of the addition of the co-solvent was examined by selecting DMSO and it was found that the reaction was inhibited from proceeding by further addition of DMSO, except that the yield was slightly increased at 2% (v/v).

Example 13: enzymatic reaction system in two-phase system

The reaction system was 10mL of Na ₂HPO₄-NaH₂PO₄ buffer (0.2M, pH 7.0), the catalyst (E.coli BL21 (DE 3)/pET 28b (+) -Nit-RR-J1-T-W171G/N169A wet cell obtained by the method of example 2) had a concentration of 10G/L, a substrate concentration of 10mM, and a reaction temperature of 40 ℃. The effect of organic solvents that partially form a two-phase system with water on cell catalysis was studied. Organic matters such as xylene, cyclohexane, n-octane, isooctane, ethyl acetate, isobutyl acetate, n-dodecane, methylene dichloride and the like and water phase (sodium phosphate buffer solution, 0.2M and pH 7.0) are selected to form a two-phase system, and the proportion is that the water phase: organic phase=9:1 (v/v), samples were taken at intervals for detection. As a result, the catalytic capacity of the two-phase system constructed by the alkane organic solvent and the water phase is greatly improved, and the final yield of the product after the reaction for 1h is improved by 180-210% compared with that of pure water phase, so that the product has better biocompatibility. The benzene and ester organic solvents have inhibitory effect on the reaction, and the cell catalyst is basically inactive in the two-phase systems.

Example 14: product extraction of 3,4,5, 6-tetrachloropyridine-2-carboxylic acid

1.245Kg of the conversion solution in example 9 (the concentration of the product is about 40mM, namely 10.43 g/L) is added with 1% polyaluminium chloride for flocculation for 4 hours, then 1% diatomite is added for adsorption for 2 hours, a filtrate is obtained by suction filtration through a Buchner funnel, a certain amount of hydrochloric acid is added for adjusting the pH to about 2.0, an equal volume of dichloromethane is added for three times of extraction, the mixture is stirred for 20 minutes on a magnetic stirrer, the mixture is transferred into a separating funnel for standing for about 10 minutes, a lower organic phase is collected, the mixture is distilled at 25 ℃ in a rotary manner, the mixture is dried at 37 ℃ until the mixture has constant weight, and finally 12.8g of solid 3,4,5, 6-tetrachloropyridine-2-carboxylic acid is obtained by drying the mixture in an oven.

Claims

1. A nitrilase mutant is characterized by comprising a mutant sequence obtained by single mutation or double mutation of the following sites of the amino acid sequence shown in SEQ ID NO. 2: (1) mutating tryptophan at position 166 to glycine; (2) mutation of asparagine 164 to alanine.

2. The nitrilase mutant of claim 1, wherein: the nitrilase mutant further comprises a polypeptide tag GKGKG connected to the N end of the mutant sequence.

3. The nitrilase mutant of claim 1, wherein: the nitrilase mutant consists of a mutant sequence and a polypeptide tag GKGKG connected to the N end of the mutant sequence, wherein the mutant sequence is obtained by double mutation of the following sites of the amino acid sequence shown in SEQ ID NO. 2: (1) mutating tryptophan at position 166 to glycine; (2) mutation of asparagine 164 to alanine.

4. A gene encoding a nitrilase mutant according to any of claims 1 to 3.

5. A biological material comprising the coding gene according to claim 4, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid, a viral vector or a cell.

6. The biomaterial of claim 5, wherein: the vector of the plasmid is pET28b; the host of the cell is E.coli BL21 (DE 3).

7. Use of a nitrilase mutant according to any of claims 1 to 3 for the catalysis of 3,4,5, 6-tetrachloropyridine-2-carbonitrile to 3,4,5, 6-tetrachloropyridine-2-carboxylic acid.

8. The application of claim 7, wherein the application is: the preparation method comprises the steps of taking wet thalli, wet thalli immobilized cells or pure enzymes extracted after ultrasonic crushing of the wet thalli, which are obtained by fermenting and culturing engineering bacteria containing nitrilase mutant encoding genes, as a catalyst, taking 3,4,5, 6-tetrachloropyridine-2-carbonitrile as a substrate, adding a reaction medium to form a reaction system, reacting at 25-50 ℃, and separating and purifying the obtained reaction solution after the reaction is completed to obtain the 3,4,5, 6-tetrachloropyridine-2-carboxylic acid.

9. The use according to claim 8, wherein: the reaction medium is buffer solution or mixed solution of buffer solution and organic solvent; pH of the reaction medium=7; the organic solvent is one or two of alkane solvent, n-octane, isooctane, cyclohexane and n-dodecane.

10. The use according to claim 8, wherein: the final concentration of the substrate in the reaction system is 10-50 mM, and when the catalyst is wet bacteria, the concentration of the catalyst is 10-50 g/L of the reaction system according to the mass of the wet bacteria; when the catalyst is immobilized cells, the concentration of the catalyst is 50g/L of the reaction system based on the mass of the immobilized cells; when the catalyst is crude enzyme liquid, the catalyst dosage is 3g/L of reaction system based on the protein quantity of the crude enzyme liquid; when the catalyst is pure enzyme, the catalyst dosage is 2g/L of the reaction system based on the mass of the pure enzyme.