Nitrilase and application thereof in preparation of p-cyanobenzoic acid

Abstract

The invention provides a nitrilase mutant and application of the nitrilase mutant in synthesis of p-cyanobenzoic acid. The invention provides a nitrilase mutant capable of improving the activity of catalytic synthesis of p-cyanobenzoic acid, the mutant protein is a non-natural protein, and the mutant protein can significantly improve the activity of catalyzing p-phthalonitrile to generate p-cyanobenzoic acid. And the mutant protein mutates in two or more core amino acids related to enzyme catalytic activity of the wild nitrilase. The nitrilase mutant provided by the invention can significantly improve the yield of a nitrilase catalytic synthesis product.

Description

Technical Field

[0001] The present invention relates to the field of enzyme and enzyme engineering, and in particular to the application of nitrilase and its mutant in catalyzing terephthalonitrile to synthesize p-cyanobenzoic acid. Background Art

[0002] Para-aminomethylbenzoic acid can treat or prevent hyperfibrinolytic bleeding caused by excessive primary fibrinolysis, and has a significant effect on chronic oozing blood. Para-aminomethylbenzoic acid is mainly prepared industrially by catalytic hydrogenation of para-cyanobenzoic acid.

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

[0004] At present, the production method of p-cyanobenzoic acid mainly includes:

[0005] First, 4-chloromethylbenzonitrile is used as raw material and concentrated nitric acid is used as oxidant to synthesize p-cyanobenzoic acid. Although this synthetic route is simple, it has too many by-products, causes serious pollution, and is difficult to handle the three wastes, making it unsuitable for industrial application.

[0006] Second, the method of preparing p-aminobenzoic acid by diazotization and cyanidation is cumbersome and complicated, and the diazotization process is a dangerous process with poor safety. It is necessary to develop a simple method for preparing p-cyanobenzoic acid.

[0007] Compared with chemical methods, biocatalysis has the advantages of mild reaction conditions, environmental friendliness, and no heavy metal pollution. Earlier, we reported a production strain that can efficiently catalyze terephthalonitrile to synthesize p-cyanobenzoic acid (CN 107641622 B, Process Biochemistry. 2018, 75, 152-156.). Based on the previous work, we mutated the nitrilase to obtain a mutant with greatly improved catalytic performance.

[0008] Creating industrially adaptable mutant enzymes and further increasing the amount of substrate added will enable the biocatalytic method using nitrilase as a catalyst to adapt to industrial production needs, reduce production costs, and transform traditional production methods, which will lay the foundation for the green production of p-cyanobenzoic acid and p-aminomethylbenzoic acid. Summary of the invention

[0009] The invention provides a nitrilase and realizes efficient synthesis of p-cyanobenzoic acid.

[0010] The first aspect of the present invention provides a nitrilase, in particular, a mutant protein is a non-natural protein, and the mutant protein has a higher catalytic activity of hydrolyzing terephthalonitrile to generate p-cyanobenzoic acid, and the mutant protein is mutated in four or more core amino acids related to the catalytic activity of the enzyme selected from the following group corresponding to SEQ ID NO.: 1 in the wild-type nitrilase: methionine (M) at position 63; threonine (T) at position 131; histidine (H) at position 135; glutamic acid (E) at position 165; leucine (L) at position 191; isoleucine (I) at position 195; glutamine (Q) at position 199; valine (V) at position 202; and histidine (H) at position 206.

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

[0012] In another preferred embodiment, the mutant protein is mutated in the core amino acids corresponding to SEQ ID NO.: 1 of the wild-type nitrilase and selected from the following group of core amino acids associated with enzyme catalytic activity: histidine (H) at position 135; leucine (L) at position 191; isoleucine (I) at position 195.

[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 131st threonine (T) 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 (E) at position 165 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 phenylephrine (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 199th glutamine (Q) 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%, 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 nitrilase is derived from Pantoeasp.

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

[0025] In another preferred embodiment, compared with the wild-type nitrilase, the yield of p-cyanobenzoic acid catalyzed by the mutant is ≥95%, preferably, ≥99%;

[0026] In another preferred embodiment, the polynucleotide further contains auxiliary elements selected from the following groups on the flank of the ORF of the mutant protein of nitrilase: a signal peptide, a secretory peptide, a tag sequence (such as 6His), or a combination thereof.

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

[0028] The second aspect of the present invention provides a host cell, wherein the host cell contains the vector of the present invention, or the polynucleotide of the present invention is 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] The third aspect of the present invention provides a method for producing a mutant protein of the nitrilase according to the first aspect of the present invention, comprising the steps of:

[0032] The host cell according to the second aspect of the present invention is cultured under conditions suitable for expression, thereby expressing a mutant protein of the nitrilase; and / or isolating the mutant protein of the nitrilase.

[0033] The fourth aspect of the present invention provides an enzyme preparation, which comprises a mutant protein of the nitrilase according to 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 the present invention provides the use of the nitrilase in the hydrolysis of terephthalonitrile to prepare p-cyanobenzoic acid.

[0036] The sixth aspect of the present invention provides a method for catalyzing the reaction of terephthalonitrile to produce p-cyanobenzoic acid by the mutant protein of the nitrilase, comprising the steps of:

[0037] (i) contacting the mutant protein of the nitrilase described in the first aspect of the present invention with a reaction substrate to carry out a catalytic reaction, thereby obtaining p-cyanobenzoic acid;

[0038] (ii) optionally, isolating and purifying the p-cyanobenzoic acid.

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

[0040] (ii) The reaction system solvent is no solvent, acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, ethyl acetate, methyl tert-butyl ether, dichloromethane, 1,4-dioxane, preferably no solvent, methanol, ethanol, N,N-dimethylformamide, acetone, more preferably methanol, ethanol.

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

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

[0043] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0045] Figure 2 The results after purification of PANIT mutant proteins are shown.

[0046] Among them, M represents Marker, 1 represents wild-type protein, and 2 represents representative mutant protein. DETAILED DESCRIPTION

[0047] Through extensive and in-depth research, the inventors screened a large number of key amino acid sites that can significantly change the activity of the mutant protein of nitrilase to generate products. The present invention found that after the key sites in the wild-type nitrilase of the present invention are modified, the catalytic activity of the nitrilase can be significantly changed. On this basis, the inventors completed the present invention.

[0048] the term

[0049] As used herein, the term "AxxB" means that the amino acid A at position xx is changed to amino acid B, for example, "Y66A" means that the amino acid Y at position 59 is mutated to A, and so on.

[0050] Mutant protein and its encoding nucleic acid of the present invention

[0051] As used herein, the terms "mutant protein", "mutant protein of the present invention", "mutant protein of nitrilase of the present invention", and "nitrilase mutant of the present invention" are used interchangeably, and all refer to non-naturally occurring nitrilase mutant proteins, and the mutant protein is an artificially modified protein based on the protein shown in SEQ ID NO.: 1, wherein the mutant protein contains core amino acids related to enzyme catalytic activity, and at least one of the core amino acids is artificially modified; and the mutant protein of the present invention has an enzyme activity of catalyzing terephthalonitrile to form p-cyanobenzoic acid.

[0052] The term "core amino acids" 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, wherein the corresponding positions are specific amino acids described herein, such as based on the sequence shown in SEQ ID NO.: 1, the core amino acids are:

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

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

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

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

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

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

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

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

[0061] Histidine (H) at position 206;

[0062] The mutant protein obtained by mutating the core amino acids has a higher activity of catalyzing the formation of terephthalonitrile.

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

[0064] Table 1

[0065] Location wild type The most preferred mutant Mutation site 1 (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

[0066] It should be understood that the amino acid numbering in the mutant protein of the present invention is based on SEQ ID NO.: 1. When the homology between a specific mutant protein and the sequence shown in SEQ ID NO.: 1 reaches 80% or more, the amino acid numbering of the mutant protein may be misplaced relative to the amino acid numbering of SEQ ID NO.: 1, such as misplacement of 1-5 positions to 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 misplacement is within a reasonable range, and mutant proteins with a homology of 80% (such as 90%, 95%, 98%) and the same or similar catalytic activity for producing p-cyanobenzoic acid should not be excluded from the scope of the mutant protein of the present invention due to the misplacement of amino acid numbering.

[0067] Mutants of the present invention are synthetic proteins or recombinant proteins, i.e., they can be the product of chemical synthesis, or they can be produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production scheme, the mutagen of the present invention can be glycosylated, or it can be non-glycosylated. The mutagen of the present invention may also include or not include 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 "analog" refer to proteins that substantially retain the same biological function or activity of the mutant protein.

[0069] The mutant protein fragment, derivative or analog of the present invention can be (i) a mutant protein with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a mutant protein with a substitution group in one or more amino acid residues, or (iii) a mutant protein formed by fusion of a mature mutant protein with another compound (such as a compound that prolongs the half-life of the mutant protein, such as polyethylene glycol), or (iv) a mutant protein formed by fusion of an additional amino acid sequence to the mutant protein sequence (such as a leader sequence or secretory sequence or a sequence or protein sequence used to purify the mutant protein, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0070] The active mutant protein of the present invention has the enzyme activity of catalyzing terephthalonitrile to form p-cyanobenzoic acid.

[0071] Preferably, the mutant protein of the present invention can also be modified. Modifications (usually without changing the primary structure) include: chemical derivatization forms of mutant proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those mutant proteins produced by glycosylation modification during the synthesis and processing of mutant proteins or in further processing steps. This modification can be accomplished by exposing the mutant protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are mutant proteins that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0072] The term "polynucleotide encoding a mutant protein" may include a polynucleotide encoding a mutant protein of the present invention, or may include a polynucleotide further including additional coding and / or non-coding sequences.

[0073] The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or mutant proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the mutant protein encoded by it.

[0074] The present invention also relates to polynucleotides that hybridize to the above-mentioned 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 can hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95%.

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

[0076] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0077] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0078] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is shorter. Usually, by synthesizing multiple small fragments first and then connecting them, very long fragments of the sequence can be obtained. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0079] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0080] In a preferred embodiment of the present invention, the preparation method of the recombinant nitrilase of the present invention is: culturing the recombinant expression transformant as described above to obtain the recombinant expression nitrilase. The culture medium used for culturing the recombinant expression transformant is any culture medium in the art that can grow the transformant and produce the recombinant nitrilase of the present invention. The culture method and culture conditions are not particularly limited and can be appropriately selected according to the conventional knowledge in the art according to factors such as the host cell type and the culture method, as long as the transformant can grow and produce the nitrilase.

[0081] In a preferred embodiment of the present invention, the preparation method of the nitrilase mutant of the present invention is as follows: Escherichia coli is used as the expression host.

[0082] Specifically, the preparation method comprises the following steps: (1) constructing the gene of the corresponding mutation site of PANIT into the pET-32a expression vector to obtain a recombinant plasmid carrying the target enzyme gene. (2) transferring the recombinant plasmid into a host bacterial cell (preferably Escherichia coli BL21 (DE3)) to obtain a corresponding engineered strain.

[0083] (3) The engineered strain was inoculated into LB medium, cultured at 37°C for 6 hours, 0.1 mM isopropylthiogalactoside (IPTG) was added, and cultured at 25°C for 12 hours. (4) The cells were collected by centrifugation.

[0084] The present invention also provides a method for converting dinitrile compounds using PANIT and mutant recombinant bacteria as biocatalysts. Specifically, a reaction system is constructed by using a substrate dinitrile compound, a recombinant bacterium or a broken bacterial solution, and a pure enzyme. The reaction system is a buffer solution of pH 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 amount of a conventional water-insoluble organic solvent in the art, such as ethyl acetate, butyl acetate, toluene, dichloromethane, chloroform, isopropyl ether, methyl tert-butyl ether, etc., and the extraction is repeated three times. The extracts are combined, and anhydrous sodium sulfate is added to dry overnight. The solvent is removed by rotary evaporation to obtain a product, and further purification by conventional methods, such as silica gel column separation, reduced pressure distillation, recrystallization, etc., can obtain a highly chemically pure and optically pure product.

[0085] Wild-type nitrilase

[0086] As used herein, "wild-type nitrilase" refers to a naturally occurring, unmodified nitrilase, whose nucleotide sequence can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and whose amino acid sequence can be derived from the nucleotide sequence. The amino acid sequence of the wild-type nitrilase is shown in SEQ ID NO.: 1.

[0087] The information of the wild protein and mutant protein of the present invention is shown in Table 2 (see Examples).

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

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

[0090] The sequence of SEQ ID No.1 was fully synthesized and connected to the pET32a empty plasmid, and then double-digested with restriction endonucleases NcoI and BamHI overnight, and then purified by agarose gel electrophoresis and recovered by a DNA kit. The recovered enzyme-cut target fragment and the empty vector were connected at 4°C for 12 hours under the action of T4-DNA ligase to obtain the recombinant plasmid pET32a-PANIT, which was further transformed into BL21 (DE3), and the positive clones were picked to obtain the recombinant expression transformant E. coli BL21 (DE3) / pET32a-PANIT.

[0091] Example 2: Construction of nitrilase PANIT mutants

[0092] Using pET32a-PANIT as a template, two-step PCR was performed using 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 of each of a pair of mutant primers (10 μM), and add sterile distilled water to 50 μL. PCR reaction program: (1) denaturation at 98°C for 10 sec, (2) annealing at 58°C for 30 sec, (3) extension at 72°C for 8 sec, and steps (1) to (3) were performed 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 primers (round 1 product), and add sterile distilled water to 50 μL. PCR reaction procedure: (1) denaturation at 98°C for 10 sec, (2) annealing at 58°C for 30 sec, (3) extension at 72°C for 2 min, steps (1) to (3) for a total of 25 cycles. The product was stored at 4°C. After the PCR product was verified by agarose gel electrophoresis analysis, the restriction enzyme DpnI was added and digested at 37°C for 2 h. The digestion product was transferred into E. coli BL21 (DE3) competent cells and spread on a plate containing ampicillin antibiotics, and placed in a 37°C incubator for static culture for about 12 h. The obtained monoclonal colony was picked into a 96-well plate for induction culture. The sites of library mutation are 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. The expressed protein is tested for activity, the substrate is terephthalonitrile, the screening method is phenol sodium hypochlorite method, and the gene of the mutant with higher activity is sequenced. The expressed protein is tested for activity, and the single mutation sites with more mutant activity improvement are 63, 135, 191 and 195, which are mutants 1, 2, 3, 4, respectively. The results are shown in Table 2.

[0093] Construction of nitrilase PANIT combination mutation: Combination mutation was constructed based on the saturation mutation results. The obtained monoclonal colonies were picked into a test tube containing 4 ml of LB medium for culture, and the activity of the expressed protein was tested. The substrate was terephthalonitrile. The better combination mutants screened are shown in Table 2 below.

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

[0095]

[0096] Example 3: Inducible expression and purification of nitrilase PANIT mutant

[0097] Prepare 50 mL of seed solution, the culture medium is LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L), pick a single colony of genetically engineered bacteria with an inoculation loop and inoculate it into the culture medium, and culture it at 37°C, 200 rpm overnight. Transfer the overnight cultured seed solution to the fermentation medium (LB medium) at a 1% inoculation amount, and culture it at 37°C, 200 rpm until OD 600 The pH value was about 0.6-1.0, 0.1mM IPTG was added, and the mixture was induced at 30℃ and 200rpm for 10-12h. The cells were collected by centrifugation at 4℃ and 6000rpm, washed twice with sodium phosphate buffer (100mM, pH 7.0), crushed with a high-pressure homogenizer, and centrifuged at 13000rpm to retain the supernatant. The target protein was purified and recovered by metal affinity chromatography (nickel column). After the target protein was dialyzed to remove imidazole, the pure enzyme solution of PANIT mutant was obtained. The SDS-PAGE electrophoresis pattern showed that the purified protein band was single, as shown in Figure 2 shown.

[0098] The results showed that the method of this example could obtain a relatively pure protein mutant, with a single subunit protein molecular weight of 37 kDa and a purity of >95%.

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

[0100] According to the method of Example 3, the wild type and mutants of PANIT of the present invention were induced to express, and the bacterial cells were collected by centrifugation (6000 rpm) and used as biocatalysts.

[0101] (1) The wild-type nitrilase PANIT was resuspended in 200 mL sodium phosphate buffer (pH 7.0, 100 mM) to a cell concentration of 30 g / L. The substrate terephthalonitrile was added to a final concentration of 100 g / L. The reaction was carried out on a shaker at 30°C and 200 r / min for 18 h. After the reaction, 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 detection showed a yield of 86%.

[0102] (2) The mutant 2 bacteria were used for transformation reaction, and the substrate terephthalonitrile was added to a final concentration of 200 g / L. Other conditions were the same as above, and the yield was 96%.

[0103] (3) The mutant 5 bacteria were taken for transformation reaction, and the substrate terephthalonitrile was added to a final concentration of 300 g / L. The reaction conditions were the same as above, and the yield was 98%.

[0104] (4) The mutant 5 bacteria were used for transformation reaction, and the substrate terephthalonitrile was added to a final concentration of 400 g / L. Other conditions were the same as above, and the yield was 89%.

[0105] (5) The mutant 6 bacteria were used for transformation reaction, and the substrate terephthalonitrile was added to a final concentration of 500 g / L. Other conditions were the same as above, and the yield was 85%.

[0106] (6) The mutant 7 bacteria were used for transformation reaction, and the substrate terephthalonitrile was added to a final concentration of 500 g / L. Other conditions were the same as above, and the yield was 99%.

[0107] (7) The mutant strain 8 was used for transformation reaction. The substrate terephthalonitrile was added to a final concentration of 400 g / L. Other conditions were the same as above. The yield was 91%.

[0108] The results show that compared with the wild-type nitrilase, the mutant protein of the nitrilase of the present invention significantly improves the catalytic efficiency and can efficiently catalyze terephthalonitrile to generate p-cyanobenzoic acid.

[0109] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A nitrilase, characterized in that The amino acid sequence is shown in SEQ ID NO: 1, or a mutant having at least 90% identity with the sequence shown in SEQ ID NO: 1 and still having or improving the nitrilase function.

2. The nitrilase according to claim 1, wherein The mutant corresponds to a mutant obtained by mutation in one or more of positions 63, 131, 135, 165, 191, 195, 199, 202, and 206 in positions 1 to 232 of the amino acid sequence of SEQ ID NO:

1.

3. The nitrilase according to claim 2, wherein The mutant corresponds to the combined mutations at positions 63 and 135; the combined mutations at positions 191 and 195; and the combined mutations at positions 63, 191 and 195 of the amino acid sequence of SEQ ID NO:

1.

4. The nitrilase according to claim 2 or 3, wherein The mutant corresponds to the mutation of methionine (M) at position 63 of the amino acid sequence of SEQ ID NO: 1 to alanine (A), valine (V), or tyrosine (Y); Threonine (T) at position 131 was mutated to alanine (A), cysteine ​​(C), or glycine (G); The histidine (H) at position 135 was mutated to alanine (A), valine (V), or phenylalanine (F); The glutamic acid (E) at position 165 was mutated to alanine (A), valine (V), aspartic acid (D), and arginine (R); Leucine (L) at position 191 mutated to phenylalanine (F), glycine (G), and alanine (A); Isoleucine (I) at position 195 was mutated to glycine (G), valine (V), or alanine (A); Glutamine (Q) at position 199 was mutated to cysteine ​​(C), valine (V), or alanine (A); The valine (V) at position 202 mutated to tryptophan (W), aspartic acid (D), phenylalanine (F), and alanine (A); The histidine (H) at position 206 was mutated to cysteine ​​(C), alanine (A), serine (S), isoleucine (I), glycine (G), valine (V), and tyrosine (Y).

5. The nitrilase according to claim 4, wherein The mutant corresponds to the mutation of methionine (M) at position 63 of the amino acid sequence of SEQ ID NO: 1 to tyrosine (Y); Threonine (T) at position 131 was mutated to alanine (A); The histidine (H) at position 135 was mutated to phenylalanine (F); Glutamic acid (E) at position 165 was mutated to alanine (A); Leucine (L) at position 191 was mutated to phenylalanine (A); Isoleucine (I) at position 195 was mutated to alanine (A); Glutamine (Q) at position 199 was mutated to valine (V); The valine (V) at position 202 was mutated to tryptophan (W); The histidine (H) at position 206 is preferably mutated to valine (V), serine (S), isoleucine (I), and more preferably alanine (A).

6. A nitrilase gene, characterized in that It encodes the nitrilase according to any one of claims 1 to 5; specifically, it also includes a coding sequence of a signal peptide, a secretory peptide, a tag sequence, or a combination thereof.

7. A recombinant vector or recombinant host cell containing the nitrilase gene according to claim 6; the recombinant expression vector is an expression vector, a shuttle vector, or an integration vector; the recombinant host cell is, for example, a eukaryotic cell, such as a yeast cell or a plant cell, or a prokaryotic cell, such as Escherichia coli.

8. Use of the nitrilase according to any one of claims 1 to 5 in the hydrolysis of terephthalonitrile to prepare p-cyanobenzoic acid.

9. A method for preparing p-cyanobenzoic acid by hydrolyzing terephthalonitrile, characterized in that: Includes steps: (i) contacting the nitrilase according to any one of claims 1 to 5 with a reaction substrate terephthalonitrile to carry out a catalytic reaction, thereby obtaining the p-cyanobenzoic acid; Preferably, the pH of the reaction system is 6.0-10.0, preferably, pH 6-8, more preferably, pH 7; (ii) the reaction system co-solvent is no co-solvent, acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, N, N-dimethylformamide, tetrahydrofuran, ethyl acetate, methyl tert-butyl ether, dichloromethane, 1,4-dioxane, preferably, no co-solvent, methanol, ethanol, N, N-dimethylformamide, acetone, more preferably, methanol, ethanol; the reaction time is 1-24 hours, preferably, 8-16 hours, more preferably, 8-14 hours; (ii) optionally, isolating and purifying the p-cyanobenzoic acid.

10. The method according to claim 9, characterized in that The nitrilase is obtained by culturing the recombinant strain according to claim 7 for expression.

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