Method for enzymatic synthesis of indobufen intermediate 2-(4-nitrophenyl) butyric acid

Abstract

The invention belongs to the technical field of enzyme engineering and biology, and discloses a nitrilase mutant as well as a coding gene, a recombinant vector, a recombinant gene engineering bacterium, an enzyme preparation and application thereof. The nitrilase mutant is obtained by carrying out single-point mutation or multi-point mutation on 8th, 17th, 35th or 150th amino acids of an amino acid sequence of the nitrilase as shown in SEQ ID NO.2. The invention further discloses a preparation method of the nitrilase mutant. Compared with wild type Gi-Nit, the nitrilase mutant disclosed by the invention has the advantages that the activity is improved by 32 times, the yield is improved by 15.8 times, and a foundation is laid for synthesizing 2-(4-nitrophenyl) butyric acid by an industrial enzyme method.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and biotechnology, specifically relating to a nitrile hydrolase mutant derived from Gibberella fuciformis and its application in the catalytic synthesis of 2-(4-nitrophenyl)butyric acid. Background Technology

[0002] Indobufen is an antiplatelet aggregation drug that combines safety and reversibility. It has multi-target characteristics, reducing platelet adhesion through multiple factors including cyclooxygenase, adenosine diphosphate, and activating factors. Indobufen has few adverse reactions and is widely used to treat various cardiovascular diseases, lipid metabolism disorders, venous thrombosis, and diabetes, making it a first-line drug for antiplatelet aggregation.

[0003] Currently, the industrial synthesis of indobufen mainly relies on chemical methods, but these methods suffer from problems such as harsh reaction conditions, the use of toxic and hazardous raw materials, large emissions of waste, and difficulties in controlling impurities. For example, patent CN116283715A reports a synthetic route using phenylacetonitrile as a starting material, involving multiple steps such as alkylation, nitration, and hydrolysis to generate the key intermediate 2-(4-nitrophenyl)butyric acid, followed by condensation and reduction steps to obtain the target product, indobufen. This route requires the use of large amounts of highly corrosive raw materials such as concentrated sulfuric acid, and the strong acid hydrolysis process generates various byproducts, making separation and purification difficult. Therefore, developing a green and efficient new method for the synthesis of indobufen has significant application value.

[0004] Nitrile hydrolases catalyze the hydrolysis of various nitrile compounds to synthesize the corresponding carboxylic acids. Due to their high catalytic efficiency and strict chemoselectivity, they have become important tool enzymes for the industrial synthesis of carboxylic acids and their derivatives. 2-(4-nitrophenyl)butyric acid is a key intermediate of indobufen. The one-step hydrolysis of 2-(4-nitrophenyl)butyronitrile to synthesize 2-(4-nitrophenyl)butyric acid via nitrile hydrolases offers significant advantages such as high atom economy and environmental friendliness. However, wild-type nitrile hydrolases suffer from bottlenecks such as low catalytic efficiency and low product concentration when catalyzing the hydrolysis of 2-(4-nitrophenyl)butyronitrile. This invention constructs a highly efficient 2-(4-nitrophenyl)butyronitrile hydrolase using a semi-rational protein design method, laying the foundation for the industrial synthesis of indobufen. Summary of the Invention

[0005] The purpose of this invention is to provide a method for synthesizing the indobufen intermediate 2-(4-nitrophenyl)butyric acid via nitrile hydrolysis enzymes, particularly by modifying the protein derived from *Gibberellinia spp.* Gibberella intermedia The nitrile hydrolase Gi-Nit was improved to enhance its hydrolytic activity in the hydrolysis of 2-(4-nitrophenyl)butyronitrile, thereby facilitating its application in the industrial synthesis of indobufen and solving the problems of low activity and low product concentration when wild-type nitrile hydrolase catalyzes the hydrolysis of 2-(4-nitrophenyl)butyronitrile.

[0006] The technical scheme adopted by the present application is: The present application provides a nitrilase mutant, which is obtained by single-point mutation or multi-point mutation of amino acids at positions 8, 17, 35 or 150 in the wild-type nitrilase amino acid sequence shown in SEQ ID NO. 2.

[0007] Preferably, the nitrilase mutant is mutated to at least one of the following in the amino acid (nucleotide sequence as shown in SEQ ID NO. 2) shown in SEQ ID NO. 2: (1) the alanine at position 8 is mutated to aspartic acid (A8D, the encoding gene nucleotide sequence is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4); (2) the alanine at position 8 is mutated to glycine (A8G, the encoding gene nucleotide sequence is shown in SEQ ID NO. 5, and the amino acid sequence is shown in SEQ ID NO. 6); (3) the alanine at position 8 is mutated to valine (A8V, the encoding gene nucleotide sequence is shown in SEQ ID NO. 7, and the amino acid sequence is shown in SEQ ID NO. 8); (4) the asparagine at position 17 is mutated to glutamic acid (N17E, the encoding gene nucleotide sequence is shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO. 10); (5) the asparagine at position 17 is mutated to alanine (N17A, the encoding gene nucleotide sequence is shown in SEQ ID NO. 11, and the amino acid sequence is shown in SEQ ID NO. 12); (6) the lysine at position 35 is mutated to serine (K35S, the encoding gene nucleotide sequence is shown in SEQ ID NO. 13, and the amino acid sequence is shown in SEQ ID NO. 14); (7) the alanine at position 150 is mutated to serine (A150S, the encoding gene nucleotide sequence is shown in SEQ ID NO. 15, and the amino acid sequence is shown in SEQ ID NO. 16).

[0008] Further, as a specific embodiment, preferably the alanine at position 8 is mutated to glycine, and the asparagine at position 17 is mutated to glutamic acid (A8G / N17E, the encoding gene nucleotide sequence is shown in SEQ ID NO. 17, and the amino acid sequence is shown in SEQ ID NO. 18).

[0009] Further, as a specific embodiment, the alanine at position 8 is mutated to glycine, the asparagine at position 17 is mutated to glutamic acid, and the lysine at position 35 is mutated to serine (A8G / N17E / K35S, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 19, and the amino acid sequence is shown in SEQ ID NO. 20).

[0010] Any one or more amino acids in the amino acid sequence shown in SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, or 20 are deleted, inserted, or replaced, and the resulting amino acid sequence has the activity of catalyzing the preparation of 2-(4-nitrophenyl)butyric acid from 2-(4-nitrophenyl)butyronitrile, and still falls within the protection scope of the present application.

[0011] The nucleotide sequence provided in the present application can be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the nucleotide sequence is obtained, the amino acid sequence can be obtained in large quantities by recombination. The obtained nucleotide sequence is usually cloned into a vector and then introduced into a genetically engineered bacterium, and then the nucleotide sequence is separated from the proliferated host cell by a conventional method.

[0012] In addition, the nucleotide sequence can also be synthesized by a publicly known artificial chemical synthesis method.

[0013] The present application also relates to a recombinant vector of the coding gene of the nitrile hydrolase mutant. The nucleotide sequence of the nitrile hydrolase mutant of the present application can be linked to various vectors by conventional methods in the art to construct the recombinant vector of the present application. The recombinant vector of the present application is not limited as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and / or eukaryotic cells, and the vector can be various vectors in the art, such as various plasmids, bacteriophages, or viral vectors, etc. The recombinant vector of the present application preferably uses pET28a as the basic vector.

[0014] In the present application, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various plasmids, cosmids, bacteriophages, and retroviruses available on the market, and the preferred expression vector of the present application is pET-28a plasmid. Exemplarily, the expression vector is linked to the target fragment by seamless cloning to obtain the recombinant vector.

[0015] The present application also relates to a recombinant engineered bacterium containing the gene as described above or the recombinant vector as described above.

[0016] In the present application, the recombinant vector can be transformed, transduced, or transfected into a host cell (strain) by conventional methods in the art. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably an Escherichia coli, and more preferably an Escherichia coli BL21 (DE3).

[0017] The nitrilase mutant of the present application can be prepared into a corresponding enzyme preparation, in particular, the enzyme preparation can exist in a solid, semi-solid or liquid form, and the enzyme preparation can contain a preparation aid or additive, etc., which can be selected by those skilled in the art as needed, and will not be described here.

[0018] Further, the present application also provides the use of the nitrilase mutant, the recombinant vector, the genetically engineered bacteria and the enzyme preparation in the preparation of 2-(4-nitrophenyl) butyric acid from 2-(4-nitrophenyl) butyronitrile.

[0019] The nitrilase mutant of the present application can be used in the form of whole cell of genetically engineered bacteria, in the form of crude enzyme without purification, or in the form of partially or completely purified enzyme protein. If necessary, the nitrilase mutant of the present application can also be prepared into immobilized enzyme or immobilized cell form by using immobilization technology known in the art.

[0020] Further, the preparation method of 2-(4-nitrophenyl) butyric acid of the present application is as follows: using 2-(4-nitrophenyl) butyronitrile as a substrate, and using the nitrilase mutant, the recombinant vector, the wet bacteria of the genetically engineered bacteria, the wet bacteria immobilized cell, or the enzyme preparation as a catalyst to obtain the reaction product.

[0021] Further, in the reaction system, the pH of the buffer is 7.0-8.0 (preferably pH 7.5).

[0022] Further, in the reaction system, the reaction temperature is 30-45 ℃.

[0023] Further, in the reaction system, the concentration of the substrate added is 10-180 g / L.

[0024] Further, as a specific embodiment, taking the bacterial cell as an example, the amount of the catalyst is 1-5 g / L (preferably 3 g / L) based on the dry weight of the bacterial cell.

[0025] Further, in the present application, the fermentation conditions of the recombinant strain are not particularly limited, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. As a specific embodiment, the wet bacteria are prepared as follows: the genetically engineered bacteria containing the nitrilase mutant encoding gene are inoculated into LB medium containing kanamycin with a final concentration of 50 mg / L, and cultured at 37 ℃ and 180 r / min for 12 h, and then inoculated into fresh LB medium containing kanamycin with a final concentration of 50 mg / L at a volume concentration of 2%, and cultured at 37 ℃ and 180 r / min until the bacterial concentration reaches OD 600The IPTG is added to the culture medium in a final concentration of 0.1-1 mM (preferably 0.1 mM), and the culture is induced at 28 DEG C and 180 r / min for 12 h, and then the culture is centrifuged to obtain the wet bacteria by collecting the precipitate.

[0026] Compared with the prior art, the beneficial effects of the present application mainly include: The activity of the nitrile hydrolase mutant is 32 times higher than that of the wild type Gi-Nit, and the yield is 15.8 times higher, which lays a foundation for the industrialized enzymatic synthesis of 2-(4-nitrophenyl) butyric acid. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 , Gi - Reaction process of the whole cell catalyzed synthesis of 2-(4-nitrophenyl) butyric acid by the mutants A8G, N17E, K35S and A150S.

[0028] Figure 2 Reaction process of the whole cell catalyzed synthesis of 2-(4-nitrophenyl) butyric acid by the mutant A8G / N17E.

[0029] Figure 3 Reaction process of the whole cell catalyzed synthesis of 2-(4-nitrophenyl) butyric acid by the mutant A8G / N17E / K35S. DETAILED DESCRIPTION

[0030] The application will be further described below in conjunction with specific embodiments, but the application is not limited to the following embodiments. The embodiments of the application are described in detail below, and examples of the embodiments are shown in the drawings. The embodiments described below with reference to the drawings are exemplary and are intended to explain the application, and cannot be understood as a limitation of the application.

[0031] In this document, the term "comprising" is an open term, i.e., including the indicated content but not excluding other aspects.

[0032] In this document, the endpoints of the ranges and any values stated are not limited to the precise range or value stated, and should be interpreted as including values near the range or value stated. For numerical ranges, the endpoints of each range and the individual points within the ranges can be combined with one or more other points to form new ranges or sub-ranges, which are also contemplated herein.

[0033] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] Example 1: Expression of wild-type nitrile hydrolase Construction of wild-type nitrile hydrolase genetically engineered bacteria: bacteria derived from *Fusarium graminearum* (… Gibberella intermedia ) nitrile hydrolase gene (Gi - The nitrile hydrolase gene was artificially synthesized using Nit (Genbank: JN236216), and its nucleotide sequence is shown in SEQ ID NO.1, while its amino acid sequence is shown in SEQ ID NO.2. The nitrile hydrolase gene was ligated between the NcoI and HindIII sites of the pET-28a plasmid and transformed into competent cells. E. coli BL21 (DE3) was used to obtain a wild-type nitrile hydrolase genetically engineered bacterium. E. coli BL21 (DE3) / pET-28a- Gi- Nit.

[0035] Expression of wild-type nitrile hydrolase genetically engineered bacteria: 10 μL of wild-type nitrile hydrolase genetically engineered bacteria culture was inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37 ℃ and 200 rpm. The culture was then transferred to 100 mL of fresh LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 2% and cultured until OD500 was reached. 600 The concentration of the culture medium was 0.4–0.8, and IPTG was added to a final concentration of 0.1 mM. The cells were then induced and cultured at 28 °C for 12 h. After the culture was completed, the cells were centrifuged at 8000 rpm for 10 min at 4 °C, and the cells were collected. The cells were washed twice with 0.9% physiological saline to obtain wild-type nitrile hydrolase genetically engineered bacterial cells.

[0036] Example 2: Single-point saturation mutagenesis and screening of nitrile hydrolases 1. Site-directed saturation mutation Through homology modeling and molecular docking, key amino acid sites A8, N17, K35, and A150 that affect the activity of nitrile hydrolase in Example 1 were screened. Saturation mutation primers were designed for these sites in the amino acid sequence of the nitrile hydrolase shown in SEQ ID NO.2 (see Table 1 for details). Using the plasmid of the nitrile hydrolase Gi-Nit gene (nucleotide sequence SEQ ID NO.1) as a template, degenerate primers were designed to replace the original codons with NNK for the nucleotides corresponding to the above sites, and the whole plasmid was amplified.

[0037] Table 1 Design table of site saturation primer Note: N = A / G / C / T, K = G / T, M = A / C PCR reaction system (50 μL): 2 x phanta Max buffer 25 μL, dNTP mixture (10 mM) 1 μL, mutant primer 10 μM 2 μL each as shown in Table 1, plasmid pET28- Bd -Nit 1 μL, Phanta Max DNA polymerase 0.5 μL, ddH2O to 50 μL.

[0038] PCR conditions: (1) 95 ℃ pre-denaturation for 5 min; (2) 95 ℃ denaturation for 15 s, 59 ℃ annealing for 5 s, 72 ℃ extension for 30 s, step (2) for 30 cycles; (3) finally 72 ℃ extension for 3 min, 4 ℃ preservation. After the reaction, the PCR products were analyzed by 0.9 % agarose gel electrophoresis, and then Dpn I digestion of the original template, gel recovery, purification of PCR products, heat shock introduction E. coli In the BL21 (DE3) competent, spread on LB plate containing 50 ug / mL kanamycin, 30 ℃ culture overnight, screening positive clones, get saturated mutation library.

[0039] 2. Transformation of nitrilase mutants Take competent cells E.coli BL21 (DE3), add 10 μL of the above PCR product, stand on ice for 30 min, heat shock at 42 ℃ for 90 s, add 600 μL of non-resistant LB liquid medium, and culture at 37 °C, 180 rpm for 1 h. After the culture is completed, centrifuge at 4 ℃, 4000 rpm for 1 min, discard the supernatant, blow the remaining bacterial liquid evenly and spread on LB plate containing 50 mg / L kanamycin, 37 ℃ culture overnight.

[0040] 3. High-throughput screening and rescreening Picking single colony from step 2 into 96-deep well plate, each well added 600 mL LB liquid medium containing 50 μg / mL kanamycin, 37 ℃ incubated for 16 h as seed liquid. In a new sterile 96-deep well plate, added 600 μL LB liquid medium containing 50 μg / mL kanamycin and 0.1 mM IPTG with final concentration, and then added 200 μL seed liquid, incubated at 28 ℃ for 12 h. After 12 h expression, the bacterial liquid was centrifuged at 4000 r / min, 16 ℃ for 10 min, the supernatant was discarded, and the bacterial body was washed and resuspended in 200 μL KH2PO4-K2HPO4 buffer (100 mM, pH 7.5) in 96-deep well plate. Each well was added 200 μL 10 mM substrate 2-(4-nitrophenyl) butyronitrile dissolved in PB buffer, and reacted at 30 ℃ for 30 min. After the reaction was completed, the high-throughput screening method of nitrilase mutant was referred to the nitrilase high-throughput screening of patent CN202410304056.X.

[0041] The mutants with improved activity obtained by high-throughput screening were subjected to rescreening. The liquid phase rescreening reaction system (20 mL): 20 mM KH2PO4-K2HPO4 buffer (pH 7.5), 50 mM 2-(4-nitrophenyl) butyronitrile, 0.1 g wet bacterial body. After preheating at 30 ℃ for 10 min, the reaction solution was reacted at 600 rpm for 15 min. 500 μL sample was taken, 10 μL 6 M HCl was added to terminate the reaction, and the content of product 2-(4-nitrophenyl) butyric acid was detected by HPLC. The liquid chromatography detection conditions: the chromatographic column was Daicel-AD-H column, the liquid chromatography mobile phase was n-hexane: isopropyl alcohol 90:10 (v / v), the flow rate was 1 mL / min, and the detection wavelength was 210 nm.

[0042] The results showed that the activity of wild type Gi-Nit was 23.5 U / g, the activity of mutant A8D was 67 U / g, which was increased by 2.85 times; the activity of mutant A8G was 82.3 U / g, which was increased by 3.5 times; the activity of mutant A8V was 54.4 U / g, which was increased by 2.31 times; the activity of mutant N17E was 124 U / g, which was increased by 5.28 times; the activity of mutant N17A was 34 U / g, which was increased by 1.45 times; the activity of mutant K35S was 189 U / g, which was increased by 8.0 times; the activity of mutant A150S was 29.8 U / g, which was increased by 1.27 times (Table 2).

[0043] Table 2 Comparison of activities of nitrilase mutants Example 3, Iterative saturation mutation of nitrilase The obtained each advantageous mutant in Example 2 was subjected to iterative combinatorial mutation, i.e. on the basis of the A8 site optimal mutant A8G, saturation mutation of N17 site was carried out. The construction method of iterative mutant, the nitrilase activity detection method were the same as described in Example 2. The results showed that the activity of A8G / N17E was 324 U / g, which was 13.79 times higher than that of wild type Gi-Nit. Further, on the basis of mutant A8G / N17E, saturation mutation of K35 site was carried out. The construction method of iterative mutant, the nitrilase activity detection method were the same as described in Example 2. The results showed that the activity of A8G / N17E / K35S was 752 U / g, which was 32 times higher than that of wild type Gi-Nit (Table 3).

[0044] Table 3 Activity comparison of nitrilase mutants Example 4, nitrilase Gi - Reaction of 2-(4-nitrophenyl)butyronitrile catalyzed by nitrilase Gi-Nit whole cell The composition of the conversion system and the conversion operation were as follows: 20 mL of KH2PO4-K2HPO4 buffer solution (20 mM, pH 7.5) was added with the wild type recombinant nitrilase Gi-Nit whole cell obtained in Example 1, the bacterial cell addition amount was 3 g / L (dry weight), the substrate 2-(4-nitrophenyl)butyronitrile concentration was 150 g / L, and the reaction was carried out at 30 ℃, 600 rpm for 15 h. The reaction process was detected by HPLC, and the HPLC detection conditions were as shown in Example 2. The results showed that after 15 h of reaction, the product 2-(4-nitrophenyl)butyric acid concentration was 10.6 g / L (yield 7.07%). Figure 1 ).

[0045] Example 5, reaction of 2-(4-nitrophenyl)butyronitrile catalyzed by mutant A8G whole cell The composition of the conversion system and the conversion operation were as follows: 20 mL of KH2PO4-K2HPO4 buffer solution (20 mM, pH 7.5) was added with the wild type recombinant nitrilase mutant A8G whole cell obtained in Example 2, the bacterial cell addition amount was 3 g / L (dry weight), the substrate 2-(4-nitrophenyl)butyronitrile concentration was 150 g / L, and the reaction was carried out at 30 ℃, 600 rpm for 15 h. The reaction process was detected by HPLC, and the HPLC detection conditions were as shown in Example 2. The results showed that after 15 h of reaction, the product 2-(4-nitrophenyl)butyric acid concentration was 17.6 g / L (yield 11.73%). Figure 1 ).

[0046] Example 6, reaction of 2-(4-nitrophenyl)butyronitrile catalyzed by mutant N17E whole cell The transformation system composition and transformation operation are as follows: 20 mL of KH2PO4-K2HPO4 buffer solution (20 mM, pH 7.5) is added with the wild-type recombinant nitrilase mutant N17E whole cell obtained in Example 2, the cell addition amount is 3 g / L (dry weight), the substrate 2-(4-nitrophenyl) butyronitrile concentration is 150 g / L, and the reaction is carried out at 30 °C, 600 rpm for 15 h. The reaction process is detected by HPLC, and the HPLC detection conditions are shown in Example 2. The results show that after 15 h of reaction, the product 2-(4-nitrophenyl) butyric acid concentration is 41.2 g / L Figure 1 ).

[0047] Example 7, Mutant K35S Whole Cell Catalyzing 2-(4-nitrophenyl) butyronitrile Reaction The transformation system composition and transformation operation are as follows: 20 mL of KH2PO4-K2HPO4 buffer solution (20 mM, pH 7.5) is added with the wild-type recombinant nitrilase mutant K35S whole cell obtained in Example 2, the cell addition amount is 3 g / L (dry weight), the substrate 2-(4-nitrophenyl) butyronitrile concentration is 150 g / L, and the reaction is carried out at 30 °C, 600 rpm for 15 h. The reaction process is detected by HPLC, and the HPLC detection conditions are shown in Example 2. The results show that after 15 h of reaction, the product 2-(4-nitrophenyl) butyric acid concentration is 81.5 g / L Figure 1 ).

[0048] Example 8, Mutant A150S Whole Cell Catalyzing 2-(4-nitrophenyl) butyronitrile Reaction The transformation system composition and transformation operation are as follows: 20 mL of KH2PO4-K2HPO4 buffer solution (20 mM, pH 7.5) is added with the wild-type recombinant nitrilase mutant A150S whole cell obtained in Example 2, the cell addition amount is 3 g / L (dry weight), the substrate 2-(4-nitrophenyl) butyronitrile concentration is 150 g / L, and the reaction is carried out at 30 °C, 600 rpm for 15 h. The reaction process is detected by HPLC, and the HPLC detection conditions are shown in Example 2. The results show that after 15 h of reaction, the product 2-(4-nitrophenyl) butyric acid concentration is 13.4 g / L Figure 1 ).

[0049] Example 9, Mutant A8G / N17E Whole Cell Catalyzing 2-(4-nitrophenyl) butyronitrile Reaction The transformation system composition and transformation operation are as follows: 20 mL of KH2PO4-K2HPO4 buffer solution (20 mM, pH 7.5) is added with the wild-type recombinant nitrilase mutant A8G / N17E whole cell obtained in Example 3, the bacterial cell is added in an amount of 3 g / L (dry weight), the substrate 2-(4-nitrophenyl) butyronitrile is in a concentration of 150 g / L, and the reaction is carried out at 30 °C, 600 rpm for 15 h. The reaction process is detected by HPLC, and the HPLC detection conditions are shown in Example 2. The results show that after 15 h of reaction, the concentration of the product 2-(4-nitrophenyl) butyric acid is 120.7 g / L Figure 2 ).

[0050] Example 10, Mutant A8G / N17E / K35S Whole Cell Catalyzing Reaction of 2-(4-nitrophenyl) butyronitrile The transformation system composition and transformation operation are as follows: 20 mL of KH2PO4-K2HPO4 buffer solution (20 mM, pH 7.5) is added with the wild-type recombinant nitrilase mutant A8G / N17E / K35S whole cell obtained in Example 3, the bacterial cell is added in an amount of 3 g / L (dry weight), the substrate 2-(4-nitrophenyl) butyronitrile is in a concentration of 150 g / L, and the reaction is carried out at 30 °C, 600 rpm for 15 h. The reaction process is detected by HPLC, and the HPLC detection conditions are shown in Example 2. The results show that after 15 h of reaction, the concentration of the product 2-(4-nitrophenyl) butyric acid is 120.7 g / L Figure 3 ).

[0051] The above is only the preferred embodiment of the present application, and is not intended to limit the scope of the present application. The above examples of the present application can also be variously changed. Any simple, equivalent changes or modifications made to the content of the claims and the specification of the present application fall within the scope of the claims of the present patent. The present application is not described in detail, and is a routine technical content.

Claims

1. A nitrile hydrolase mutant, characterized in that: The nitrile hydrolase mutant is obtained by single-point or multi-point mutation of the 8th, 17th, 35th or 150th amino acids in the nitrile hydrolase amino acid sequence shown in SEQ ID NO.

2.

2. The mutant as described in claim 1, characterized in that: The nitrile hydrolase mutant is formed by mutating the amino acid shown in SEQ ID NO.2 to at least one of the following: (1) The alanine at position 8 is mutated to aspartic acid; (2) The alanine at position 8 is mutated to glycine; (3) The alanine at position 8 is mutated to valine; (4) The asparagine at position 17 is mutated to glutamic acid; (5) The 17th position of asparagine is mutated to alanine; (6) The lysine at position 35 is mutated to serine; (7) The alanine at position 150 is mutated to serine.

3. A gene encoding a nitrile hydrolase mutant as described in claim 1 or claim 2.

4. A recombinant vector, characterized in that: The recombinant vector contains the gene as described in claim 3.

5. A genetically engineered bacterium, characterized in that: The host cell of the genetically engineered bacteria contains the gene as described in claim 3, or contains the recombinant vector as described in claim 4.

6. An enzyme preparation, characterized in that, It contains the nitrile hydrolase mutant of claim 1.

7. The use of the nitrile hydrolase mutant of claim 1, the recombinant vector of claim 4, the genetically engineered bacteria of claim 5, or the enzyme preparation of claim 6 in the catalytic preparation of 2-(4-nitrophenyl)butyronitrile from 2-(4-nitrophenyl)butyric acid.

8. A method for preparing 2-(4-nitrophenyl)butyric acid, characterized in that: The reaction is carried out using 2-(4-nitrophenyl)butyronitrile as a substrate and the nitrile hydrolase mutant of claim 1, the recombinant vector of claim 4, the wet cells of the genetically engineered bacteria of claim 5, the wet cells immobilized, or the enzyme preparation of claim 6 as a catalyst.

9. The method as described in claim 8, characterized in that: The reaction medium is a buffer solution with a pH of 7.0 to 8.

0.

10. The method as described in claim 8, characterized in that: The reaction temperature in the reaction system is 30~45℃.

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