CN119823972B - Nitrile hydrolases and their application in the preparation of p-cyanobenzoic acid - Google Patents

Abstract

This invention provides a nitrile hydrolase mutant and its application in the synthesis of p-cyanobenzoic acid. The invention provides a nitrile hydrolase mutant that enhances the catalytic activity for the synthesis of p-cyanobenzoic acid. The mutant protein is a non-natural protein, and it exhibits significantly enhanced activity in catalyzing the conversion of terephthalonitrile to p-cyanobenzoic acid. Furthermore, the mutant protein contains mutations in two or more core amino acids related to the enzyme's catalytic activity in the wild-type nitrile hydrolase. The nitrile hydrolase mutant of this invention can significantly improve the yield of the synthesized product catalyzed by the nitrile hydrolase.

Description

Nitrilase and application thereof in preparation of p-cyanobenzoic acid

Technical Field

The invention relates to the field of enzymes and enzyme engineering, in particular to application of nitrilase and mutants thereof in catalyzing synthesis of p-cyanobenzoic acid from p-phthalonitrile.

Background

The p-aminomethylbenzoic acid can treat or prevent hyperfibrinolytic hemorrhage caused by excessive primary fibrinolysis, has remarkable effect on chronic bleeding, and is mainly prepared by catalytic hydrogenation of p-cyanobenzoic acid in industry.

The p-cyanobenzoic acid is an important intermediate compound for synthesizing the drug p-aminomethylbenzoic acid, and can also be used as an intermediate of dye and hemostatic aromatic acid for organic synthesis and liquid crystal polymer synthesis.

At present, the production method of the p-cyanobenzoic acid mainly comprises the following steps:

Firstly, 4-chloromethyl benzonitrile is used as a raw material, concentrated nitric acid is used as an oxidant to synthesize the p-cyanobenzoic acid, and the synthetic route is simple, but the byproducts are excessive, the pollution is serious, the three wastes are not easy to treat, and the method is not suitable for industrial application.

Secondly, the diazotization and cyanidation of the p-aminobenzoic acid are complicated and complex, and the diazotization process is a dangerous process, so that the safety is poor, and a simple and convenient preparation method of the p-cyanobenzoic acid is necessary to develop.

Compared with a chemical method, the biocatalysis method has the advantages of mild reaction conditions, environmental friendliness, no heavy metal pollution and the like. In the early stage, we report a production strain (CN 107641622 B,Process Biochemistry.2018,75,152-156) capable of efficiently catalyzing terephthalonitrile to synthesize p-cyanobenzoic acid, and on the basis of early stage work, we mutate nitrilase to obtain a mutant strain with greatly improved catalytic performance.

The industrial adaptability mutant enzyme is created, the substrate addition amount is further improved, the biocatalysis method using the nitrilase as a catalyst can adapt to the industrial production requirement, the production cost is reduced, the traditional production mode is changed, and a foundation is laid for the green production of the p-cyanobenzoic acid and the p-aminomethylbenzoic acid.

Disclosure of Invention

The invention provides a nitrilase and realizes efficient synthesis of p-cyanobenzoic acid.

In a first aspect the present invention provides a nitrilase, in particular a mutant protein, which is a non-native protein and which has a high catalytic activity for the hydrolysis of terephthalonitrile to p-cyanobenzoic acid and which is mutated at the four or more core amino acids of the wild-type nitrilase corresponding to the catalytic activity of the enzyme SEQ ID NO. 1, methionine 63 (M), threonine 131 (T), histidine 135 (H), glutamic acid 165 (E), leucine 191 (L), isoleucine 195 (I), glutamine 199 (Q), valine 202 (V) and histidine 206 (H).

In a further preferred embodiment, the mutein is mutated in the core amino acid corresponding to SEQ ID NO.1 of the wild-type nitrilase in histidine 135 (H), glutamic acid 165 (E), leucine 191 (L), isoleucine 195 (I), glutamine 199 (Q).

In a further preferred embodiment, the mutein is mutated in the core amino acid corresponding to SEQ ID No. 1 of the wild-type nitrilase in histidine 135 (H), leucine 191 (L) and isoleucine 195 (I) selected from the group of related to the catalytic activity of the enzyme.

In another preferred embodiment, methionine (M) at position 63 is mutated to alanine (A), valine (V), tyrosine (Y), preferably tyrosine (Y).

In another preferred embodiment, threonine (T) at position 131 is mutated to alanine (a), cysteine (C), glycine (G), preferably alanine (a).

In another preferred embodiment, histidine (H) at position 135 is mutated to alanine (a), valine (V), phenylalanine (F), preferably phenylalanine (F).

In another preferred embodiment, glutamic acid (E) at position 165 is mutated to alanine (a), valine (V), aspartic acid (D), arginine (R), preferably alanine (a).

In another preferred embodiment, leucine (L) at position 191 is mutated to phenylalanine (F), glycine (G), alanine (A), preferably to phenylalanine (A).

In another preferred embodiment, isoleucine (I) at position 195 is mutated to glycine (G), valine (V), alanine (A), or a combination thereof, preferably alanine (A).

In another preferred embodiment, glutamine (Q) at position 199 is mutated to cysteine (C), valine (V), alanine (a), preferably valine (V).

In another preferred embodiment, valine (V) at position 202 is mutated to tryptophan (W), aspartic acid (D), phenylalanine (F), alanine (a), preferably tryptophan (W).

In another preferred embodiment, histidine (H) at position 206 is mutated to cysteine (C), alanine (a), serine (S), isoleucine (I), glycine (G), valine (V), tyrosine (Y), preferably valine (V), serine (S), isoleucine (I), more preferably alanine (a).

In another preferred embodiment, the sequence shown in SEQ ID No. 1 has a homology of at least 80%, preferably at least 85% or 90%, more preferably at least 95%, most preferably at least 98% or 99%.

In another preferred embodiment, the nitrilase is derived from Pantoeasp.

In another preferred embodiment, the catalytic substrate of the nitrilase is terephthalonitrile.

In another preferred embodiment, the yield of p-cyanobenzoic acid obtained by catalysis of the mutant is greater than or equal to 95%, preferably greater than or equal to 99%, compared to the wild-type nitrilase;

in another preferred embodiment, the polynucleotide additionally comprises an auxiliary element selected from the group consisting of a signal peptide, a secretory peptide, a tag sequence (e.g., 6 His), or a combination thereof, flanking the ORF of the mutant protein of the nitrilase.

In another preferred embodiment, the vector comprises an expression vector, a shuttle vector, an integration vector.

In a second aspect, the invention provides a host cell comprising a vector according to the invention, or having integrated into its genome a polynucleotide according to the invention.

In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

In another preferred embodiment, the host cell is a prokaryotic cell, such as E.coli.

In a third aspect, the present invention provides a method for producing a mutein of a nitrilase according to the first aspect of the invention, comprising the steps of:

Culturing the host cell according to the second aspect of the invention under conditions suitable for expression, thereby expressing a mutant protein of a nitrilase, and/or isolating the mutant protein of a nitrilase.

In a fourth aspect, the invention provides an enzyme preparation comprising a mutein of a nitrilase according to the first aspect of the invention.

In another preferred embodiment, the enzyme preparation comprises an injectable preparation, and/or a lyophilized preparation.

In a fifth aspect, the invention provides the use of said nitrilase for the preparation of p-cyanobenzoic acid by the hydrolysis of p-phthalonitrile.

In a sixth aspect, the present invention provides a method for catalyzing a reaction of terephthalonitrile to produce p-cyanobenzoic acid by using a mutein of said nitrilase, comprising the steps of:

(i) Contacting the mutant protein of the nitrilase of the first aspect of the invention with a reaction substrate to perform a catalytic reaction, thereby obtaining p-cyanobenzoic acid;

(ii) Optionally, isolating and purifying the p-cyanobenzoic acid.

Wherein (i) the pH of the reaction system is from 6.0 to 10.0, preferably from 6 to 8, more preferably 7;

(ii) The cosolvent of the reaction system is no cosolvent, acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, N-dimethylformamide, tetrahydrofuran, ethyl acetate, methyl tertiary butyl ether, dichloromethane and 1, 4-dioxane, and preferably no cosolvent, methanol, ethanol, N-dimethylformamide, acetone, more preferably methanol and ethanol.

(Iii) The reaction time is 1 to 24 hours, preferably 8 to 16 hours, more preferably 8 to 14 hours.

(Iv) The temperature of the catalytic reaction is 20-60 ℃, preferably 25-50 ℃, more preferably 25-32 ℃.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the chemical formulas of terephthalonitrile and p-cyanobenzoic acid.

FIG. 2 shows the results of PANIT mutant proteins after purification.

Wherein M represents Marker,1 is wild type protein, 2 is representative mutant protein

Detailed Description

Through extensive and intensive studies, the present inventors have screened a number of critical amino acid sites that significantly alter the activity of the mutant protein catalytic production product of nitrilase. The invention discovers that the catalytic activity of the nitrilase can be obviously changed after the key sites in the wild type nitrilase are modified. On this basis, the present inventors have completed the present invention.

Terminology

As used herein, the term "AxxB" means that amino acid a at position xx is changed to amino acid B, e.g. "Y66A" means that amino acid Y at position 59 is mutated to a, and so on.

Muteins of the invention and nucleic acids encoding same

As used herein, the terms "mutein of the invention", "mutant of the nitrilase of the invention" and "mutant of the nitrilase of the invention" are used interchangeably and refer to non-naturally occurring mutant proteins of the nitrilase and are proteins which have been engineered based on the protein shown in SEQ ID NO. 1, wherein the mutein comprises core amino acids which are related to the catalytic activity of the enzyme and at least one of which has been engineered, and wherein the mutein of the invention has the enzymatic activity of catalyzing the formation of p-cyanobenzoic acid from p-phthalonitrile.

The term "core amino acid" refers to a sequence based on SEQ ID No.:1 and having at least 80%, such as 84%, 85%, 90%, 92%, 95%, 98% homology to SEQ ID No.:1, the corresponding site being a specific amino acid described herein, such as the sequence shown based on SEQ ID No.:1, the core amino acid being:

Methionine (M) at position 63, and/or

Threonine (T) at position 131, and/or

Histidine (H) at position 135, and/or

Glutamic acid (E) at position 165, and/or

Leucine (L) at position 191, and/or

Isoleucine (I) at position 195, and/or

Glutamine (Q) at position 199, and/or

Valine (V) at position 202, and/or

Histidine (H) at position 206;

And the mutant protein obtained by mutating the core amino acid has higher activity of catalyzing paracyanobenzoase formed by paraphthalonitrile.

Preferably, in the present invention, the core amino acid of the present invention is subjected to the following mutation as shown in table 1.

TABLE 1

Position of	Wild type	Most preferred mutants
			Mutation site1 (amino acid 63)	M	Y
Mutation site 2 (amino acid 131)	T	A
			Mutation site 3 (amino acid 135)	H	F
Mutation site 4 (amino acid 165)	E	A
			Mutation site 5 (amino acid 191)	L	A
Mutation site 6 (amino acid 195)	I	A
			Mutation site 7 (amino acid 199)	Q	V
Mutation site 8 (amino acid 202)	V	W
			Mutation site 9 (amino acid 206)	H	A

It will be appreciated that where the amino acid numbering in a mutein of the invention is based on SEQ ID NO. 1, when a particular mutein has 80% or more homology to the sequence shown in SEQ ID NO. 1, the amino acid numbering of the mutein may be shifted, e.g., 1-5 to the N-or C-terminus of the amino acid, relative to the amino acid numbering of SEQ ID NO. 1, and such shifting is generally understood by those skilled in the art to be within reasonable limits using conventional sequence alignment techniques and a mutein having 80% (e.g., 90, 95, 98%) homology due to amino acid numbering should not be within the scope of the mutein of the invention, which has the same or similar catalytic activity to cyanobenzoic acid.

The muteins of the present invention are synthetic or recombinant proteins, i.e., can be the product of chemical synthesis, or can be produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plants) using recombinant techniques. Depending on the host used in the recombinant production protocol, the muteins of the present invention may be glycosylated or may be non-glycosylated. The muteins of the present invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives and analogues of the muteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a protein that retains substantially the same biological function or activity of the mutein.

The mutein fragment, derivative or analogue of the present invention may be (i) a mutein having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a mutein having a substituent in one or more amino acid residues, or (iii) a mutein formed by fusion of a mature mutein with another compound, such as a compound that extends the half-life of the mutein, e.g. polyethylene glycol, or (iv) a mutein formed by fusion of an additional amino acid sequence to the mutein sequence, such as a leader or secretory sequence or a sequence used to purify the mutein or a pro-protein sequence, or a fusion protein formed with an antigen IgG fragment. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

The active mutant protein of the present invention has an enzymatic activity of catalyzing the formation of p-cyanobenzoic acid from p-phthalonitrile.

Preferably, the muteins of the present invention may also be modified. Modified (typically without altering the primary structure) forms include chemically derivatized forms of the muteins in vivo or in vitro such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the mutein or during further processing steps. Such modification may be accomplished by exposing the mutein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are muteins modified to enhance their proteolytic resistance or to optimize their solubility properties.

The term "polynucleotide encoding a mutein" may include polynucleotides encoding the muteins of the present invention, as well as polynucleotides further comprising additional coding and/or non-coding sequences.

The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polypeptides or muteins having the same amino acid sequence as the invention. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the mutein encoded thereby.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions (or stringent conditions) to the polynucleotides of the invention. In the present invention, "stringent conditions" means (1) hybridization and elution at a relatively low ionic strength and a relatively high temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃, or (2) hybridization with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃, etc., or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95%.

The muteins and polynucleotides of the invention are preferably provided in isolated form, and more preferably purified to homogeneity.

The full-length polynucleotide sequence of the present invention can be obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining polynucleotides of the invention. In particular, when it is difficult to obtain full-length cDNA from a library, it is preferable to use RACE method (RACE-cDNA end rapid amplification method), and primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

In a preferred embodiment of the present invention, the recombinant nitrilase of the present invention is produced by culturing the recombinant expression transformant as described above to obtain the recombinant expression nitrilase. The medium used for culturing the recombinant expression transformant is any medium in the art that allows the transformant to grow and produce the recombinant nitrilase of the invention. The culture method and the culture conditions are not particularly limited, and may be appropriately selected according to the conventional knowledge in the art, depending on the type of host cell and the culture method, so long as the transformant is allowed to grow and produce the nitrilase.

In a preferred embodiment of the present invention, the nitrilase mutants of the present invention are prepared by using E.coli as an expression host.

Specifically, the preparation method comprises the following steps of (1) constructing a gene of a PANIT corresponding mutation site on a pET-32a expression vector to obtain a recombinant plasmid with a target enzyme gene. (2) And transferring the recombinant plasmid into a host bacterial cell (preferably escherichia coli BL21 (DE 3)) to obtain a corresponding engineering strain.

(3) The engineering strain was inoculated into LB medium, cultured at 37℃for 6 hours, added with 0.1mM isopropyl thiogalactoside (IPTG), and cultured at 25℃for 12 hours. And (4) centrifugally collecting thalli.

The invention also provides a method for converting dinitriles by using PANIT and mutant recombinant bacteria as biocatalysts. Specifically, a substrate dinitrile compound, recombinant bacteria or bacteria breaking liquid and pure enzyme are used for constructing a reaction system, wherein the reaction system is buffer solution with pH of 6.0-9.0, and the reaction temperature is 20-50 ℃. After the hydrolysis reaction is completed, the reaction solution is extracted with an equal amount of a water-insoluble organic solvent such as ethyl acetate, butyl acetate, toluene, methylene chloride, chloroform, isopropyl ether, methyl tert-butyl ether, etc., which are conventional in the art, and the extraction is repeated three times, the extracts are combined, and dried overnight by adding anhydrous sodium sulfate. The solvent is removed by rotary evaporation, and the product is obtained, and is further purified by conventional methods such as silica gel column separation, reduced pressure distillation, recrystallization and the like, so that the highly chemically pure and optically pure product can be obtained.

Wild type nitrilase

As used herein, "wild-type nitrilase" refers to a naturally occurring, non-engineered nitrilase whose nucleotides can be obtained by genetic engineering techniques, such as genomic sequencing, polymerase Chain Reaction (PCR), etc., and whose amino acid sequences can be deduced from the nucleotide sequences. The amino acid sequence of the wild-type nitrilase is shown in SEQ ID NO. 1.

The information on the wild-type proteins and muteins of the present invention as described above is shown in Table 2 (see examples).

Reagents and materials in the examples of the present invention are commercially available products unless otherwise specified.

EXAMPLE 1 preparation of PANIT nitrilase recombinant expression plasmid and recombinant expression transformant

The sequence of SEQ ID No.1 was completely synthesized and ligated to pET32a empty plasmid while digested with restriction enzymes NcoI and BamHI overnight, and then purified by agarose gel electrophoresis and recovered using DNA kit. And (3) connecting the recovered enzyme-cut target fragment and the empty vector for 12 hours at 4 ℃ under the action of T4-DNA ligase to obtain recombinant plasmids pET32a-PANIT, further converting the recombinant plasmids pET 21 (DE 3), and selecting positive clones to obtain recombinant expression transformant E.coliBL21 (DE 3)/pET 32a-PANIT.

EXAMPLE 2 construction of the nitrilase PANIT mutant

PCR was performed using pET32a-PANIT as template, using a two-step method, using high fidelity polymerase PRIMERSTAR MAX. The PCR reaction conditions were round 1, 50 to 100ng of the template, 25. Mu.L of 2X PRIMERSTAR MAX (mix), 1. Mu.L (10. Mu.M) of each pair of mutation primers, and sterile distilled water to 50. Mu.L were added to a PCR reaction system having a total volume of 50. Mu.L. The PCR reaction procedure (1) denaturation at 98℃for 10sec, (2) annealing at 58℃for 30sec, and (3) extension at 72℃for 8sec, and the steps (1) to (3) were carried out for 30 cycles in total. Round 2 to 50. Mu.L of total volume of PCR reaction system, 50-100 ng of template, 25. Mu.L of 2X PRIMERSTAR MAX (mix), 1. Mu.L of mutation primer (Round 1 product) and distilled water were added to 50. Mu.L. The PCR reaction procedure (1) denaturation at 98 ℃ for 10sec, (2) annealing at 58 ℃ for 30sec, and (3) extension at 72 ℃ for 2min, and 25 cycles of steps (1) - (3) were performed. The product was stored at 4 ℃. The PCR product was verified by agarose gel electrophoresis analysis and digested with the endo-enzyme DpnI at 37℃for 2h. The digestions were transferred to E.coli BL21 (DE 3) competent cells and plated onto plates containing ampicillin and placed in a 37℃incubator for a resting culture for about 12h. The obtained monoclonal colonies were picked into 96-well plates for induction culture. The locus of the library-building mutation is F58、M63、K129、T131、P132、T133、Y134、H135、E136、R137、A162、W164、E165、F187、P188、G189、L191、V192、G193、I195、F196、A197、Q199、V202、H206、M282、M283、R254. for detecting the activity of the expressed protein, the substrate is terephthalonitrile, the screening method is phenol sodium hypochlorite method, and the gene of the mutant with higher activity is sequenced. And (3) carrying out activity detection on the expressed protein to obtain single mutation sites with more improved mutant activity, namely 63, 135, 191 and 195, namely mutants 1,2,3 and 4, and the results are shown in Table 2.

Construction of a combination mutation of nitrilase PANIT A combination mutation was constructed based on the result of saturation mutation. The obtained monoclonal colony is picked into a test tube containing 4ml of LB culture medium for culture, and the activity of the expressed protein is detected, wherein the substrate is terephthalonitrile. Preferred combination mutants selected are shown in Table 2 below.

TABLE 2 Single mutation sites with more improved mutant Activity and relative Activity thereof

Example 3 induced expression and purification of the nitrilase PANIT mutant

50ML of seed solution is prepared, the culture medium is LB liquid culture medium (peptone 10g/L, yeast powder 5g/L and NaCl 10 g/L), and single colony of the genetically engineered bacterium is picked up by an inoculating loop and inoculated into the culture medium for culture at 37 ℃ and 200rpm for overnight. The seed solution cultured overnight is transferred to a fermentation medium (LB medium) with an inoculum size of 1%, cultured at 37 ℃ and 200rpm until OD ₆₀₀ is about 0.6-1.0, added with 0.1mM IPTG, and placed at 30 ℃ and induced at 200rpm for 10-12 h. And centrifuging at 4 ℃ and 6000rpm to collect thalli, washing twice with sodium phosphate buffer (100 mM, pH 7.0), crushing with a high-pressure homogenizer, centrifuging at 13000rpm to obtain supernatant, purifying and recovering target protein by adopting a metal affinity chromatography (nickel column) method, and removing imidazole from the target protein by dialysis to obtain PANIT mutant pure enzyme solution. SDS-PAGE electropherograms showed single bands of the purified protein, as shown in FIG. 2.

The results show that the method of this example is capable of obtaining purer protein mutants with a single subunit protein molecular weight of 37kDa and a purity of >95%.

Example 4 method for catalyzing terephthalonitrile by nitrilase PANIT mutant recombinant bacterium

The wild type PANIT and the mutant PANIT of the present invention were induced and expressed as in example 3, and the cells were collected by centrifugation (6000 rpm) and used as biocatalysts.

(1) The nitrilase PANIT wild type strain was resuspended in 200mL sodium phosphate buffer (pH 7.0,100 mM), the cell concentration was 30g/L, and the substrate terephthalonitrile was added to a shaker at a final concentration of 100g/L,30℃and 200r/min to react, and after 18 hours the reaction was stopped. After the reaction, HCl was adjusted to pH 1-2, the reaction mixture was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC detection shows 86% yield.

(2) And (3) taking the mutant strain 2 to perform a transformation reaction, adding a substrate paraphthalonitrile to a final concentration of 200g/L, and carrying out the other conditions under the same conditions, wherein the yield is 96%.

(3) And (3) taking the mutant strain 5 thalli for conversion reaction, adding a substrate paraphthalonitrile to the final concentration of 300g/L, and carrying out the reaction under the same conditions, wherein the yield is 98%.

(4) And (3) taking the mutant strain 5 thalli for conversion reaction, adding a substrate paraphthalonitrile to the final concentration of 400g/L, and carrying out the other conditions as above, wherein the yield is 89%.

(5) And (3) taking the mutant strain 6 to perform a conversion reaction, adding a substrate paraphthalonitrile to a final concentration of 500g/L, and carrying out the same conditions as above, wherein the yield is 85%.

(6) And (3) taking the mutant strain 7 to perform a conversion reaction, adding a substrate paraphthalonitrile to a final concentration of 500g/L, and carrying out the same conditions as above, wherein the yield is 99%.

(7) The mutant strain 8 is taken for transformation reaction, and the substrate terephthalonitrile is added to the final concentration of 400g/L, and the yield is 91% under the same conditions.

The result shows that compared with the wild type nitrilase, the mutant protein of the nitrilase has obviously improved catalytic efficiency and can efficiently catalyze the terephthalonitrile to generate the p-cyanobenzoic acid.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (12)

1. A nitrile hydrolase mutant, characterized in that the mutant corresponds to a methionine mutation at position 63 of the amino acid sequence described in SEQ ID NO: 1, which is replaced by tyrosine. 2. A nitrile hydrolase gene, characterized in that it encodes the nitrile hydrolase mutant as described in claim 1. 3. The nitrile hydrolase gene as described in claim 2, characterized in that it further comprises a coding sequence of a signal peptide, a secretory peptide, a tag sequence, or a combination thereof. 4. A recombinant vector containing the nitrile hydrolase gene as described in claim 3. 5. The recombinant vector as described in claim 4, wherein the recombinant vector is an expression vector, a shuttle vector, or an integration vector. 6. A recombinant host cell containing the nitrile hydrolase gene as described in claim 3. 7. The recombinant host cell according to claim 6, wherein the recombinant host cell is a yeast cell or a prokaryotic cell. 8. The application of the nitrile hydrolase mutant as described in claim 1 in the preparation of p-cyanobenzoic acid by hydrolysis of terephthalonitrile. 9. A method for preparing p-cyanobenzoic acid by hydrolyzing terephthalonitrile, characterized by comprising the steps of: (i) The nitrile hydrolase mutant of claim 1 is contacted with the reaction substrate terephthalonitrile to carry out a catalytic reaction, thereby obtaining the p-cyanobenzoic acid. 10. The method of claim 9, wherein the nitrile hydrolase is obtained by culturing and expressing the recombinant host cells as described in claim 6. 11. The method according to claim 9, wherein the pH of the reaction system is 6.0-10.0; (ii) the co-solvent of the reaction system is acetone, dimethyl sulfoxide, N,N-dimethylformamide, or tetrahydrofuran; and the reaction time is 1-24 hours. 12. The method according to any one of claims 9 to 11, characterized in that it further comprises: (ii) Isolate and purify the p-cyanobenzoic acid.