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

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

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] p-Aminomethylbenzoic acid can treat or prevent hyperfibrinolytic bleeding caused by excessive primary fibrinolysis, and has a significant effect on chronic bleeding. Industrially, p-aminomethylbenzoic acid is mainly prepared by the catalytic hydrogenation of p-cyanobenzoic acid.

[0003] p-Cyanobenzic acid is an important intermediate compound in the synthesis of the drug p-aminomethylbenzoic acid. It can also be used as an intermediate in dye synthesis and in the synthesis of hemostatic aromatic acids, and is used in organic synthesis and liquid crystal polymer synthesis.

[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 with chemical methods, biocatalysis has advantages such as mild reaction conditions, environmental friendliness, and no 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.). Building on this previous work, we mutated the nitrile hydrolase to obtain a mutant strain with significantly improved catalytic performance.

[0008] The creation of industrially adaptable mutant enzymes, which further increases the substrate dosage, will enable the biocatalytic method using nitrile hydrolase as a catalyst to adapt to industrial production needs, reduce production costs, and transform traditional production methods, laying the foundation for the green production of p-cyanobenzoic acid and p-aminomethylbenzoic acid. Summary of the Invention

[0009] This invention provides a nitrile hydrolase and enables the efficient synthesis of p-cyanobenzoic acid.

[0010] The first aspect of this invention provides a nitrile hydrolase, particularly a mutant protein that is a non-natural protein, and the mutant protein has high catalytic activity for hydrolyzing terephthalonitrile to p-cyanobenzoic acid. The mutant protein contains mutations in four or more core amino acids related to enzyme catalytic activity from the group consisting of SEQ ID NO.:1, corresponding to wild-type nitrile hydrolase: methionine at position 63 (M); threonine at position 131 (T); histidine at position 135 (H); glutamate at position 165 (E); leucine at position 191 (L); isoleucine at position 195 (I); glutamine at position 199 (Q); valine at position 202 (V); and histidine at position 206 (H).

[0011] In another preferred embodiment, the mutant protein is mutated in the wild-type nitrile hydrolase at the following core amino acid group corresponding to SEQ ID NO.:1, which is related to the enzyme's catalytic activity: histidine at position 135 (H); glutamate at position 165 (E); leucine at position 191 (L); isoleucine at position 195 (I); and glutamine at position 199 (Q).

[0012] In another preferred embodiment, the mutant protein is mutated in the wild-type nitrile hydrolase by selecting the following core amino acids related to enzyme catalytic activity: histidine at position 135 (H); leucine at position 191 (L); and isoleucine at position 195 (I).

[0013] In another preferred embodiment, the methionine (M) at position 63 is mutated to alanine (A), valine (V), or tyrosine (Y), preferably tyrosine (Y).

[0014] In another preferred embodiment, the threonine (T) at position 131 is mutated to alanine (A), cysteine ​​(C), or glycine (G), preferably alanine (A).

[0015] In another preferred embodiment, the histidine (H) at position 135 is mutated to alanine (A), valine (V), or phenylalanine (F), preferably phenylalanine (F).

[0016] In another preferred embodiment, the glutamic acid at position 165 (E) is mutated to alanine (A), valine (V), aspartic acid (D), or arginine (R), preferably alanine (A).

[0017] In another preferred embodiment, the leucine (L) at position 191 is mutated to phenylalanine (F), glycine (G), or alanine (A), preferably phenylalanine (A).

[0018] In another preferred embodiment, the isoleucine (I) at position 195 is mutated to glycine (G), valine (V), alanine (A), or a combination thereof, preferably alanine (A).

[0019] In another preferred embodiment, the glutamine (Q) at position 199 is mutated to cysteine ​​(C), valine (V), or alanine (A), preferably valine (V).

[0020] In another preferred embodiment, the valine (V) at position 202 is mutated to tryptophan (W), aspartic acid (D), phenylalanine (F), or alanine (A), preferably tryptophan (W).

[0021] In another preferred embodiment, the histidine (H) at position 206 is mutated to cysteine ​​(C), alanine (A), serine (S), isoleucine (I), glycine (G), valine (V), or tyrosine (Y), preferably valine (V), serine (S), or isoleucine (I), and more preferably alanine (A).

[0022] 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%.

[0023] In another preferred embodiment, the nitrile hydrolase is derived from Pantoesp.

[0024] In another preferred embodiment, the catalytic substrate of the nitrile hydrolase is terephthalonitrile.

[0025] In another preferred embodiment, the yield of p-cyanobenzoic acid obtained by the mutant catalysis is ≥95%, more preferably ≥99%, compared to the wild-type nitrile hydrolase;

[0026] In another preferred embodiment, the polynucleotide flanking the ORF of the mutant protein of the nitrile hydrolase further contains an auxiliary element selected from the group consisting of: signal peptides, secretory peptides, tag sequences (such as 6His), or combinations thereof.

[0027] In another preferred embodiment, the carrier includes an expression carrier, a shuttle carrier, and an integration carrier.

[0028] A second aspect of the present invention provides a host cell containing the vector described in the present invention, or having the polynucleotides described in the present invention integrated into its genome.

[0029] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

[0030] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0031] A third aspect of the present invention provides a method for generating a mutant protein of the nitrile hydrolase described in the first aspect of the present invention, comprising the steps of:

[0032] 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 mutant protein of said nitrile hydrolase is isolated.

[0033] A fourth aspect of the present invention provides an enzyme preparation comprising a mutant protein of the nitrile hydrolase described in the first aspect of the present invention.

[0034] In another preferred embodiment, the enzyme preparation includes an injection and / or a lyophilized preparation.

[0035] The fifth aspect of this invention provides the application of the nitrile hydrolase in the preparation of p-cyanobenzoic acid by hydrolyzing terephthalonitrile.

[0036] The sixth aspect of this invention provides a method for the reaction of terephthalonitrile to p-cyanobenzoic acid catalyzed by a mutant protein of the nitrile hydrolase, comprising the steps of:

[0037] (i) The mutant protein of the nitrile hydrolase 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;

[0038] (ii) Optionally, the p-cyanobenzoic acid is isolated and purified.

[0039] Wherein, (i) the pH of the reaction system is 6.0-10.0, preferably 6-8, more preferably 7;

[0040] (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.

[0041] (iii) The reaction time is 1-24 hours, preferably 8-16 hours, and more preferably 8-14 hours.

[0042] (iv) The temperature of the catalytic reaction is 20-60°C, preferably 25-50°C, and more preferably 25-32°C.

[0043] 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

[0044] Figure 1 The chemical formulas for terephthalonitrile and p-cyanobenzoic acid are shown.

[0045] Figure 2 The results show the purified PANIT mutant protein.

[0046] Where M represents Marker, 1 is the wild-type protein, and 2 is the representative mutant protein. Detailed Implementation

[0047] 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.

[0048] the term

[0049] As used in this article, the term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, "Y66A" indicates that amino acid Y at position 59 is mutated to A, and so on.

[0050] The mutant protein and its encoded nucleic acid of this invention

[0051] 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 non-naturally occurring nitrile hydrolase mutant proteins. The mutant protein is an artificially modified protein 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 possesses enzymatic activity for catalyzing the formation of p-cyanobenzoic acid from terephthalonitrile.

[0052] The term "core amino acid" refers to a sequence based on SEQ ID NO.:1, and sharing at least 80% homology with SEQ ID NO.:1, such as 84%, 85%, 90%, 92%, 95%, or 98%, where the corresponding site is the specific amino acid described herein. For example, based on the sequence shown in SEQ ID NO.:1, the core amino acid is:

[0053] Methionine at position 63 (M); and / or

[0054] Threonine (T) at position 131; and / or

[0055] Histidine (H) at position 135; and / or

[0056] Glutamic acid at position 165 (E); and / or

[0057] Leucine at position 191 (L); and / or

[0058] Isoleucine at position 195 (I); and / or

[0059] Glutamine (Q) at position 199; and / or

[0060] Valine at position 202 (V); and / or

[0061] Histidine (H) at position 206;

[0062] Furthermore, the mutant protein obtained by mutating the aforementioned core amino acids exhibits high p-cyanobenzoic acid catalysis activity for the formation of terephthalonitrile.

[0063] Preferably, in this invention, the core amino acids of this invention are mutated as shown in Table 1.

[0064] Table 1

[0065] Location wild type Optimal Mutant Mutation site 1 (amino acid at position 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 at position 202) V W Mutation site 9 (amino acid 206) H A

[0066] 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% (e.g., 90%, 95%, 98%) homology and the same or similar catalytic activity for producing p-cyanobenzoic acid from the scope of the mutant proteins of this invention.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] The active mutant protein of the present invention has enzymatic activity that catalyzes the formation of p-cyanobenzoic acid from terephthalonitrile.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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%, preferably at least 95%.

[0075] The mutant proteins and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, purified to homogenization.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] Specifically, the preparation method includes the following steps: (1) The gene of the corresponding mutation site of PANIT is constructed into the pET-32a 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.

[0083] (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.

[0084] This invention also provides a method for converting dinitrile compounds using PANIT 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.

[0085] Wild-type nitrile hydrolases

[0086] 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.

[0087] Information on the wild-type proteins and the mutant proteins of the present invention mentioned above is shown in Table 2 (see Examples).

[0088] Unless otherwise specified, the reagents and materials used in the embodiments of this invention are all commercially available products.

[0089] Example 1: Preparation of PANIT nitrile hydrolase recombinant expression plasmid and recombinant expression transformant

[0090] The sequence of SEQ ID No. 1 was fully synthesized and ligated into the empty pET32a plasmid. The plasmid was then digested overnight with restriction endonucleases NcoI and BamHI, followed by purification by agarose gel electrophoresis and recovery using a DNA 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 pET32a-PANIT. This plasmid was further transformed into BL21(DE3), and positive clones were selected to obtain the recombinant expression transformant E. coli BL21(DE3) / pET32a-PANIT.

[0091] Example 2: Construction of PANIT mutant nitrile hydrolase

[0092] Using pET32a-PANIT as a template, a two-step PCR method was adopted, and PCR was performed using the high-fidelity polymerase PrimerSTAR MAX. The PCR reaction conditions were 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), 1 μL (10 μM) of each of the two mutant primers, 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. Round 2: In a PCR reaction system with a total volume of 50 μL, add 50-100 ng of template, 25 μL of 2×PrimerSTARMAX (mix), 1 μL of mutant primer (round 1 product), and add sterile distilled water to 50 μL. PCR reaction procedure: (1) denaturation at 98℃ for 10 seconds, (2) annealing at 58℃ for 30 seconds, (3) extension at 72℃ for 2 minutes. Steps (1) to (3) were repeated for a total of 25 cycles. The product was stored at 4℃. After verification by agarose gel electrophoresis, the PCR product was digested with the limiting enzyme DpnI at 37℃ for 2 hours. The digested product was transferred into E. coli BL21(DE3) competent cells and plated on plates containing ampicillin. The plates were then incubated at 37℃ for about 12 hours. The obtained single colonies were picked and induced in 96-well plates. The mutation sites used for library construction were 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, and R254. The expressed proteins were tested for activity using terephthalonitrile as the substrate and the phenol-sodium hypochlorite method. Genes of mutants with high activity were sequenced. The single mutation sites with significantly increased mutant activity were identified as 63, 135, 191, and 195, corresponding to mutants 1, 2, 3, and 4, respectively. The results are shown in Table 2.

[0093] Constructing PANIT 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.

[0094] Table 2. Single mutation sites with significantly increased mutant activity and their relative activity.

[0095]

[0096] Example 3: Induction, expression and purification of PANIT mutant nitrile hydrolase

[0097] Prepare 50 mL of seed culture in LB liquid medium (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 medium. Incubate overnight at 37°C and 200 rpm. Transfer the overnight cultured seed culture to fermentation medium (LB medium) at a 1% inoculation rate and incubate at 37°C and 200 rpm until OD reaches [the desired growth rate]. 600 The pH was approximately 0.6-1.0. 0.1 mM IPTG was added, and the mixture was incubated at 30°C and 200 rpm for 10-12 hours. The cells were collected by centrifugation at 4°C 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 PANIT mutant enzyme solution was obtained. SDS-PAGE electrophoresis showed that the purified protein had a single band, as shown in the image. Figure 2 As shown.

[0098] The results show that the method of this embodiment can obtain relatively pure protein mutants with a single subunit protein molecular weight of 37 kDa and a purity of >95%.

[0099] Example 4: Method for catalyzing terephthalonitrile by recombinant bacteria with PANIT mutant nitrile hydrolase

[0100] The wild-type and mutant PANIT of the present invention were induced to express according to the method of Example 3, and the bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst.

[0101] (1) Wild-type PANIT nitrile hydrolase cells were resuspended in 200 mL of sodium phosphate buffer (pH 7.0, 100 mM) to a cell concentration of 30 g / L. Terephthalonitrile substrate was added to a final concentration of 100 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%.

[0102] (2) Take mutant strain 2 cells for transformation reaction, add substrate terephthalonitrile to a final concentration of 200 g / L, other conditions are the same as above, yield 96%.

[0103] (3) Take 5 mutant strains for transformation reaction, add substrate terephthalonitrile to a final concentration of 300 g / L, and the reaction conditions are the same as above. The yield is 98%.

[0104] (4) Take 5 mutant strains for transformation reaction, add substrate terephthalonitrile to a final concentration of 400 g / L, other conditions are the same as above, yield is 89%.

[0105] (5) Take mutant strain 6 cells for transformation reaction, add substrate terephthalonitrile to a final concentration of 500 g / L, other conditions are the same as above, yield 85%.

[0106] (6) Take mutant strain 7 cells for transformation reaction, add substrate terephthalonitrile to a final concentration of 500 g / L, other conditions are the same as above, yield is 99%.

[0107] (7) Take 8 mutant strains for transformation reaction, add substrate terephthalonitrile to a final concentration of 400 g / L, other conditions are the same as above, yield is 91%.

[0108] 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.

[0109] 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 corresponds to the mutation of methionine at position 63 of the amino acid sequence described in SEQ ID NO: 1 to 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 also includes coding sequences for signal peptides, secreted peptides, tag sequences, or combinations thereof.

4. A recombinant vector containing the nitrile hydrolase gene as described in claim 3.

5. The recombinant vector as described in claim 4, characterized in that, 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 as described in claim 6, characterized in that, 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 in that, Including the following steps: (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 as described in claim 9, characterized in that, The nitrile hydrolase is obtained by culturing and expressing the recombinant host cells as described in claim 6.

11. The method as described in claim 9, characterized in that, The pH of the reaction system is 6.0-10.0; (ii) the co-solvents of the reaction system are acetone, dimethyl sulfoxide, N,N-dimethylformamide, and tetrahydrofuran; the reaction time is 1-24 hours.

12. The method according to any one of claims 9 to 11, characterized in that, Also includes: (ii) Isolate and purify the p-cyanobenzoic acid.

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