Nitrilase mutants and uses thereof

Abstract

The application provides a nitrilase mutant and its application in synthesis of p-cyanobenzoic acid. The application provides a nitrilase mutant with improved activity in catalytic synthesis of p-cyanobenzoic acid, wherein the mutant protein is a non-natural protein, and one or more substitution mutations exist in the amino acid sequence shown in SEQ ID NO:1, V65, H137, M193, I197, Q201, H208 and R281 are catalysts, and the mutant protein has significantly improved activity in catalyzing p-xylyl cyanide to generate p-cyanobenzoic acid, and the mutant protein is mutated in two or more core amino acids of wild-type nitrilase which are related to enzyme catalytic activity. In a preferred example, the conversion rate is 99% at a p-xylyl cyanide concentration of 600 g / L.

Description

Technical Field

[0001] This invention relates to the field of enzymes and enzyme engineering, specifically to the application of nitrile hydrolases and their mutants in the catalytic synthesis of p-cyanobenzoic acid from terephthalonitrile. Background Technology

[0002] Tranexamic acid and tranexamic acid are widely used hemostatic agents in clinical practice, with annual sales in China alone reaching billions of yuan. High-temperature nylon retains high rigidity, high strength, and excellent dimensional accuracy and stability even at high temperatures, making it widely used in high-end fields with stringent material performance requirements, such as aerospace, military industry, electronics, integrated circuits, new energy vehicles, and 5G communications. It is a strategic emerging material and an advanced basic material. 4-Cyanobenzoic acid is a key precursor to tranexamic acid, tranexamic acid, and high-temperature nylon.

[0003] p-Cyanobenzic acid is also one of the main raw materials for synthesizing liquid crystal materials. With the continuous development of display technology (such as LCD), the demand for high-performance liquid crystal materials continues to grow, which makes the application prospects of p-cyanobenzic acid in this field very broad. As a benzoic acid derivative, it can be used as a basic raw material to synthesize other organic compounds, such as preparing corresponding acyl chloride derivatives through acyl chloride reactions, or condensing with amine compounds to prepare amide compounds.

[0004] Currently, the main methods for producing p-cyanobenzoic acid are:

[0005] First, p-cyanobenzoic acid is synthesized using 4-chloromethylbenzonitrile as a raw material and concentrated nitric acid as an oxidant. Although this synthetic route is simple, it produces too many byproducts, causes serious pollution, and the waste is difficult to treat, making it unsuitable for industrial application.

[0006] Secondly, the methods for preparing p-aminobenzoic acid via diazotization and cyanidation are cumbersome and complex, and the diazotization process is a dangerous process with poor safety. Therefore, it is necessary to develop a simpler method for preparing p-cyanobenzoic acid.

[0007] Compared to chemical methods, biocatalysis offers advantages such as milder reaction conditions, environmental friendliness, and absence of heavy metal pollution. Previously, we reported a production strain capable of efficiently catalyzing the synthesis of p-cyanobenzoic acid from terephthalonitrile (CN 107641622 B, Process Biochemistry. 2018, 75, 152-156.), and further mutated it to obtain a mutant strain with improved catalytic performance (CN119823972B). To enhance the industrial properties of the engineered strain, we explored new enzymes, obtaining a nitrile hydrolase from Zobellia roscoffensis with higher activity and better stability. Further modification of this new enzyme resulted in enhanced activity. This route offers advantages such as being green and environmentally friendly, having mild reaction conditions, saving energy and reducing carbon emissions, high selectivity, and low cost. It achieves efficient and green bio-manufacturing of p-cyanobenzoic acid, solving the chemically challenging problem of selective hydrolysis of symmetrical dinitriles. Summary of the Invention

[0008] This invention provides a nitrile hydrolase and enables the efficient synthesis of p-cyanobenzoic acid.

[0009] This invention provides a nitrile hydrolase mutant with high catalytic activity for hydrolyzing terephthalonitrile to p-cyanobenzoic acid, wherein the mutant protein has a mutation in one or more core amino acids of the wild-type nitrile hydrolase corresponding to SEQ ID NO: 1 selected from the group consisting of V65, H137, M193, I197, Q201, H208 and R281, which are related to enzyme catalytic activity.

[0010] Specifically, the amino acid sequence corresponding to SEQ ID NO: 1 contains one of the following mutations: M193L, Q201V, H208A, R281G, M193L / Q201V, H208A / R281G, Q201V / H208A, M193L / H208A / R281G, M193L / Q201V / H208A, M193L / Q201V / H208A / R281G.

[0011] In another preferred embodiment, the homology with the sequence shown in SEQ ID NO:1 is at least 80%, more preferably at least 85% or 90%, more preferably at least 95%, and most preferably at least 98% or 99%.

[0012] In another preferred embodiment, the catalytic substrate of the nitrile hydrolase mutant is terephthalonitrile.

[0013] In another preferred embodiment, the yield of p-cyanobenzoic acid obtained by the mutant catalysis is ≥95% compared with that of the wild-type nitrile hydrolase, preferably ≥99%;

[0014] In another preferred embodiment, the polynucleotide flanking the ORF of the nitrile hydrolase mutant further contains an auxiliary element selected from the group consisting of: signal peptides, secretory peptides, tag sequences (such as 6His), or combinations thereof.

[0015] In another preferred embodiment, the carrier includes an expression carrier, a shuttle carrier, and an integration carrier.

[0016] A second aspect of the present invention provides a recombinant bacterium containing the vector described in the present invention, or having the polynucleotides described in the present invention integrated into its genome.

[0017] In another preferred embodiment, the recombinant bacteria are eukaryotic cells, such as yeast cells or plant cells.

[0018] In another preferred embodiment, the recombinant bacteria are prokaryotic cells, such as Escherichia coli.

[0019] A third aspect of the present invention provides a method for generating the nitrile hydrolase mutant described in the first aspect of the present invention, comprising the steps of:

[0020] Under suitable expression conditions, host cells as described in the second aspect of the present invention are cultured to express the mutant protein of nitrile hydrolase; and / or the nitrile hydrolase mutant is isolated.

[0021] A fourth aspect of the present invention provides an enzyme preparation comprising the nitrile hydrolase mutant described in the first aspect of the present invention.

[0022] In another preferred embodiment, the enzyme preparation includes an injection and / or a lyophilized preparation.

[0023] The fifth aspect of this invention provides the application of the nitrile hydrolase mutant in the preparation of p-cyanobenzoic acid by hydrolyzing terephthalonitrile.

[0024] The sixth aspect of this invention provides a method for the reaction of the nitrile hydrolase mutant with terephthalonitrile to produce p-cyanobenzoic acid, comprising the steps of:

[0025] (i) The nitrile hydrolase mutant described in the first aspect of the present invention is contacted with a reaction substrate to carry out a catalytic reaction, thereby obtaining p-cyanobenzoic acid;

[0026] (ii) Optionally, the p-cyanobenzoic acid is isolated and purified.

[0027] Wherein, (i) the pH of the reaction system is 6.0-10.0, preferably 6-8, more preferably 8;

[0028] (ii) The co-solvent of the reaction system is a solvent-free solvent, such as acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, ethyl acetate, methyl tert-butyl ether, dichloromethane, 1,4-dioxane, preferably a solvent-free solvent, methanol, ethanol, N,N-dimethylformamide, acetone, and more preferably methanol and ethanol.

[0029] (iii) The reaction time is 1-24 hours, preferably 8-16 hours, and more preferably 8-14 hours.

[0030] (iv) The temperature of the catalytic reaction is 20-60°C, preferably 25-50°C, and more preferably 25-32°C.

[0031] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0032] Figure 1 The chemical formulas for terephthalonitrile and p-cyanobenzoic acid are shown.

[0033] Figure 2 The results of purification of the ZrNIT mutant protein are shown.

[0034] Where M represents Marker, 1 represents wild-type protein, and 2 represents representative mutant protein. Detailed Implementation

[0035] Through extensive and in-depth research, the inventors identified key amino acid sites in mutant proteins that can significantly alter the catalytic activity of nitrile hydrolases. This invention discovered that modifying these key sites in the wild-type nitrile hydrolase can significantly change its catalytic activity. Based on this, the inventors completed this invention.

[0036] the term

[0037] As used in this article, the term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, "Y59A" indicates that amino acid Y at position 59 is mutated to A, and so on.

[0038] The mutant protein and its encoded nucleic acid of this invention

[0039] As used herein, the terms "mutant protein," "mutant protein of the present invention," "mutant protein of the nitrile hydrolase of the present invention," and "nitrile hydrolase mutant of the present invention" are used interchangeably and all refer to nitrile hydrolase mutant proteins that are not naturally occurring. The mutant protein is a protein artificially modified based on the protein shown in SEQ ID NO: 1. The mutant protein contains core amino acids related to enzyme catalytic activity, and at least one of the core amino acids is artificially modified. Furthermore, the mutant protein of the present invention has enzymatic activity that catalyzes the formation of p-cyanobenzoic acid from terephthalonitrile.

[0040] Preferably, in this invention, the core amino acids of this invention are mutated as shown in Table 1.

[0041] Table 1

[0042]

[0043] It should be understood that the amino acid numbering in the mutant proteins of this invention is based on SEQ ID NO.: 1. When a specific mutant protein has 80% or more homology with the sequence shown in SEQ ID NO: 1, the amino acid numbering of the mutant protein may be misaligned relative to the amino acid numbering in SEQ ID NO: 1, such as misalignment by 1-5 positions towards the N-terminus or C-terminus of the amino acid. Using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such misalignment is within a reasonable range, and the misalignment of amino acid numbering should not exclude mutant proteins with 80% homology (such as 90%, 95%, 98%) and the same or similar catalytic activity for producing p-cyanobenzoic acid from the scope of the mutant proteins of this invention.

[0044] The mutant proteins of this invention are synthetic or recombinant proteins, meaning they can be chemically synthesized products or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production protocol, the mutant proteins of this invention can be glycosylated or non-glycosylated. The mutant proteins of this invention may also include or exclude an initial methionine residue.

[0045] The present invention also includes fragments, derivatives, and analogs of the mutant protein. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the mutant protein.

[0046] The mutant protein fragments, derivatives, or analogs of the present invention may be (i) mutant proteins in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) mutant proteins having substituent groups in one or more amino acid residues; or (iii) mutant proteins formed by fusing a mature mutant protein with another compound (e.g., a compound that extends the half-life of the mutant protein, such as polyethylene glycol); or (iv) mutant proteins formed by fusing an additional amino acid sequence to the mutant protein sequence (e.g., a leader sequence or secretory sequence, or a sequence used to purify the mutant protein, or a proteogenic sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0047] The active mutant protein of the present invention has enzymatic activity that catalyzes the formation of p-cyanobenzoic acid from terephthalonitrile.

[0048] Preferably, the mutant protein of the present invention can also be modified. Modifications (generally without altering the primary structure) include chemical derivatives of the mutant protein, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during the synthesis and processing of the mutant protein or in further processing steps. This modification can be accomplished by exposing the mutant protein to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Mutant proteins modified to improve their resistance to proteolysis or optimize their solubility are also included.

[0049] The term "polynucleotide encoding mutant protein" can include polynucleotides encoding the mutant protein of the present invention, or it can include polynucleotides with additional coding and / or non-coding sequences.

[0050] The present invention also relates to variants of the aforementioned polynucleotides that encode fragments, analogs, and derivatives of polypeptides or mutant proteins having the same amino acid sequence as those of the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is 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, but does not substantially alter the function of the mutant protein it encodes.

[0051] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0052] The mutant proteins and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, purified to homogenization.

[0053] The full-length polynucleotide sequences of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0054] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0055] Furthermore, the relevant sequences can be synthesized artificially, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing multiple small fragments and then ligating them. Currently, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0056] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE (RACE-cDNA end amplification) method is preferred. Primers used for PCR can be appropriately selected based on the sequence information disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0057] In a preferred embodiment of the present invention, the method for preparing the recombinant nitrile hydrolase of the present invention is as follows: culturing the recombinant expression transformant as described above to obtain the recombinant expressed nitrile hydrolase. The culture medium used for culturing the recombinant expression transformant is any culture medium in the art that can enable the transformant to grow and produce the recombinant nitrile hydrolase of the present invention. There are no special limitations on the culture method and conditions; appropriate selections can be made according to the host cell type and culture method, based on conventional knowledge in the art, as long as the transformant can grow and produce the nitrile hydrolase.

[0058] In a preferred embodiment of the present invention, the method for preparing the nitrile hydrolase mutant of the present invention is as follows: Escherichia coli is used as the expression host.

[0059] Specifically, the preparation method includes the following steps: (1) The gene of the corresponding mutation site of ZrNIT is constructed into the pET-21a expression vector to obtain a recombinant plasmid carrying the target enzyme gene. (2) The recombinant plasmid is transformed into a host bacterial cell (preferably Escherichia coli BL21(DE3)) to obtain the corresponding engineered strain.

[0060] (3) Inoculate the engineered strain into LB medium and incubate at 37°C for 6 hours. Add 0.1 mM isopropyl thiogalactoside (IPTG) and incubate at 25°C for 12 hours. (4) Collect the bacterial cells by centrifugation.

[0061] This invention also provides a method for converting dinitrile compounds using ZrNIT and mutant recombinant bacteria as biocatalysts. Specifically, a reaction system is constructed by combining the substrate dinitrile compound with recombinant bacteria or bacterial lysate and purified enzyme. The reaction system is a buffer solution with a pH of 6.0-9.0, and the reaction temperature is 20°C to 50°C. After the hydrolysis reaction is completed, the reaction solution is extracted with an equal volume of a conventional water-insoluble organic solvent, such as ethyl acetate, butyl acetate, toluene, dichloromethane, chloroform, isopropyl ether, methyl tert-butyl ether, etc. The extraction is repeated three times, and the extracts are combined and dried overnight with anhydrous sodium sulfate. The solvent is removed by rotary evaporation to obtain the product, which is further purified by conventional methods, such as silica gel column chromatography, vacuum distillation, recrystallization, etc., to obtain a highly chemically and optically pure product.

[0062] Wild-type nitrile hydrolases

[0063] As used herein, "wild-type nitrile hydrolase" refers to a naturally occurring, unmodified nitrile hydrolase whose nucleotides can be obtained through genetic engineering techniques, such as genome sequencing and polymerase chain reaction (PCR), and whose amino acid sequence can be deduced from the nucleotide sequence. The amino acid sequence of the wild-type nitrile hydrolase is shown in SEQ ID NO:1.

[0064] Information on the wild-type proteins and the mutant proteins of the present invention mentioned above is shown in Table 1 (see Examples).

[0065] Unless otherwise specified, the reagents and materials used in the embodiments of this invention are all commercially available products.

[0066] Example 1: Preparation of ZrNIT nitrile hydrolase recombinant expression plasmid and recombinant expression transformant

[0067] The codons of SEQ ID No. 1 from *Zobellia roscoffensis* (WP_194524916.1) were optimized to obtain the nucleic acid sequence SEQ ID No. 2. After total synthesis, the sequence was ligated into the empty plasmid pET21a and digested with restriction endonucleases NdeI and XhoI. The resulting fragment was then purified by agarose gel electrophoresis and recovered using a DNA ligation kit. The recovered digested target fragment and the empty vector were ligated at 4°C for 12 hours using T4-DNA ligase to obtain the recombinant plasmid pET21a-ZrNIT. This plasmid was further transformed into BL21(DE3), and positive clones were selected to obtain the recombinant expression transformant *E. coli* BL21(DE3) / pET21a-ZrNIT.

[0068] Example 2: Construction of ZrNIT mutant nitrile hydrolase

[0069] Using pET21a-ZrNIT as a template, a two-step PCR method was employed, with PCR performed using the high-fidelity polymerase PrimerSTAR MAX.

[0070] The PCR reaction conditions are as follows: Round 1: In a PCR reaction system with a total volume of 50 μL, add 50-100 ng of template, 25 μL of 2×primerSTAR MAX (mix), 2 μL (10 μM) of each of a pair of mutant primers (one primer carries the mutation at this site, and the other primer is a universal primer), and add sterile distilled water to 50 μL. PCR reaction program: (1) denaturation at 98℃ for 10 sec, (2) annealing at 58℃ for 30 sec, (3) extension at 72℃ for 8 sec, and perform steps (1) to (3) for a total of 30 cycles.

[0071] Round 2: In a PCR reaction system with a total volume of 50 μL, add 50-100 ng of template, 25 μL of 2×PrimerSTAR MAX (mix), and 1 μL of the first-round PCR product as primer for the second-round PCR. Add sterile distilled water to a final volume of 50 μL. PCR reaction procedure: (1) denaturation at 98℃ for 10 sec, (2) annealing at 58℃ for 30 sec, (3) extension at 72℃ for 2 min. Perform 25 cycles of steps (1) to (3). Store the product at 4℃. After verification by agarose gel electrophoresis, add the limiting enzyme DpnI and digest at 37℃ for 2 h. Transform the digested product into E. coli BL21 (DE3) competent cells and plate them on plates containing ampicillin antibiotics. Incubate at 37℃ for about 12 h. Pick the obtained single colonies into 96-well plates for induction culture.

[0072] The activity of the expressed protein was measured. The substrate was terephthalonitrile, and the product after the reaction was p-cyanobenzoic acid. Figure 1 As shown, the screening method was the phenol-sodium hypochlorite method. Under alkaline conditions, sodium nitrosoferricyanide catalyzes the reaction of ammonia with phenol and sodium hypochlorite to produce a blue soluble substance, indophenol, the concentration of which can be determined using a spectrophotometer. Genes of mutants with high activity were sequenced. The activity of the expressed proteins was detected, and the single mutation sites with significantly increased mutant activity were identified as 193, 197, 201, 208, and 281, as shown in Table 2.

[0073] Constructing ZrNIT combinatorial mutants: Combinatorial mutants were constructed based on the results of saturation mutagenesis. The obtained single-clone colonies were picked and cultured in test tubes containing 4 ml of LB medium. The activity of the expressed protein was detected. The substrate was terephthalonitrile. The better combinatorial mutant strains screened are shown in Table 2 below.

[0074] Table 2. Single mutation sites with significantly increased mutant activity and their relative activity

[0075]

[0076] Example 3: Induction, expression, and purification of the ZrNIT mutant nitrile hydrolase

[0077] Prepare 50 mL of seed culture in LB broth (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl). Use an inoculation loop to pick a single colony of the genetically engineered bacteria and inoculate it into the broth. Incubate overnight at 37°C and 200 rpm. Transfer the overnight cultured seed culture to fermentation medium (LB broth) at a 1% inoculation rate and incubate at 37°C and 200 rpm until OD (Organic Degrees Per Minute) is reached.600 The pH was approximately 0.6-1.0. 0.1 mM IPTG was added, and the mixture was incubated at 30℃ and 200 rpm for 10-12 h. The cells were collected by centrifugation at 4℃ and 6000 rpm, washed twice with sodium phosphate buffer (100 mM, pH 7.0), and homogenized using a high-pressure homogenizer. The supernatant was collected by centrifugation at 13000 rpm, and then purified and recovered using metal affinity chromatography (nickel column). After dialysis to remove imidazole, the ZrNIT mutant enzyme solution was obtained. SDS-PAGE electrophoresis showed a single band in the purified protein, as shown in Figure 2.

[0078] The results show that the method in this embodiment can obtain relatively pure protein mutants with a single subunit protein molecular weight of 37 kDa and a purity of >95%.

[0079] Example 4: Method for catalyzing terephthalonitrile by recombinant bacteria with nitrile hydrolase ZrNIT mutant

[0080] ZrNIT wild-type and mutant strains of the present invention were induced to express the method of Example 3, and the bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst.

[0081] (1) Wild-type ZrNIT nitrile hydrolase cells were resuspended in 200 mL of sodium phosphate buffer (pH 8.0, 100 mM) to a cell concentration of 30 g / L. Terephthalonitrile substrate was added to a final concentration of 300 g / L. The reaction was carried out at 30 °C on a shaker at 200 r / min for 18 h, after which the reaction was stopped. After the reaction was complete, the pH was adjusted to 1-2 with HCl, and the reaction solution was extracted several times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC analysis showed a yield of 86%.

[0082] (2) Wild-type cells were used for transformation reaction. The substrate terephthalonitrile was added to a final concentration of 600 g / L. Other conditions were the same as above. The yield was 25%.

[0083] (3) Five mutant strains were used for transformation reaction. The substrate terephthalonitrile was added to a final concentration of 600 g / L. Other conditions were the same as above. The yield was 52%.

[0084] (4) Take mutant strain 6 cells for transformation reaction, add substrate terephthalonitrile to a final concentration of 600 g / L, other conditions are the same as above, yield 73%.

[0085] (5) Take mutant strain 7 cells for transformation reaction, add substrate terephthalonitrile to a final concentration of 600 g / L, other conditions are the same as above, yield 99%.

[0086] (6) Take 8 mutant strains for transformation reaction, add substrate terephthalonitrile to a final concentration of 600 g / L, other conditions are the same as above, yield 84%.

[0087] The results showed that, compared with the wild-type nitrile hydrolase, the mutant protein of the nitrile hydrolase of the present invention significantly improved the catalytic efficiency and could efficiently catalyze the production of p-cyanobenzoic acid from terephthalonitrile.

[0088] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A nitrile hydrolase mutant, characterized in that, The mutant is obtained by mutation at one or more of the following sites: V65, H137, M193, I197, Q201, H208 and R281, in the amino acid sequence of SEQ ID NO: 1 from position 1 to 335.

2. The nitrile hydrolase mutant according to claim 2, characterized in that, The amino acid sequence corresponding to SEQ ID NO: 1 contains any of the following mutations: M193L, Q201V, H208A, R281G, M193L / Q201V, H208A / R281G, Q201V / H208A, M193L / H208A / R281G, M193L / Q201V / H208A, M193L / Q201V / H208A / R281G.

3. The encoding gene of the nitrile hydrolase mutant as described in claim 1 or 2.

4. A recombinant expression vector containing the encoding gene as described in claim 3.

5. A recombinant bacterium containing the coding gene as described in claim 3, or the recombinant expression vector as described in claim 4, specifically a eukaryotic cell, such as a yeast cell or a plant cell; or a prokaryotic cell, such as Escherichia coli.

6. The use of the nitrile hydrolase mutant according to any one of claims 1 to 2 in the preparation of p-cyanobenzoic acid using terephthalonitrile as a substrate.

7. A method for preparing p-cyanobenzoic acid by hydrolyzing terephthalonitrile, characterized in that, The method includes the following steps: contacting the nitrile hydrolase mutant according to any one of claims 1 to 2 with the reaction substrate terephthalonitrile to carry out a catalytic reaction, thereby obtaining the p-cyanobenzoic acid.

8. The method as described in claim 7, characterized in that, The pH of the reaction system is 6.0-10.0, preferably 6-8, and more preferably 8. The reaction system may or may not include a co-solvent, specifically the co-solvent being selected from acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, ethyl acetate, methyl tert-butyl ether, dichloromethane, and 1,4-dioxane; preferably, the co-solvent is selected from methanol, ethanol, N,N-dimethylformamide, and acetone, and more preferably, methanol and ethanol; The reaction time is 1-24 hours, preferably 8-16 hours, and even better, 8-14 hours.

9. The method as described in claim 8, characterized in that, The nitrile hydrolase mutant was obtained by culturing and expressing the recombinant bacteria as described in claim 5.

10. The method as described in claim 8 or 9, characterized in that, It further includes separating and purifying the p-cyanobenzoic acid.

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