A method for the enzymatic synthesis of 2-(4-nitrophenyl)butyric acid, an intermediate of indobufen.

Abstract

This invention belongs to the fields of enzyme engineering and biotechnology, and discloses a nitrile hydrolase mutant, its encoding gene, recombinant vector, recombinant genetically engineered bacteria, enzyme preparation, and applications. The nitrile hydrolase mutant is obtained by single-point or multi-point mutation of amino acids 8, 17, 35, or 150 of the nitrile hydrolase amino acid sequence shown in SEQ ID NO. 2. The nitrile hydrolase mutant of this invention exhibits 32-fold increased activity and 15.8-fold increased yield compared to the wild-type Gi-Nit, laying the foundation for the industrial enzymatic synthesis of 2-(4-nitrophenyl)butyric acid.

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 solution adopted in this invention is:

[0007] The present invention provides a nitrile hydrolase mutant, which is obtained by single-point or multi-point mutation of amino acids 8, 17, 35 or 150 of the amino acid sequence of the wild-type nitrile hydrolase shown in SEQ ID NO.2.

[0008] Preferably, the nitrile hydrolase mutant is one in which the amino acid shown in SEQ ID NO.2 (nucleotide sequence as shown in SEQ ID NO.1) is mutated to at least one of the following:

[0009] (1) The 8th alanine is mutated to aspartic acid (A8D, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4);

[0010] (2) The alanine at position 8 is mutated to glycine (A8G, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6);

[0011] (3) The alanine at position 8 is mutated to valine (A8V, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8);

[0012] (4) The asparagine at position 17 is mutated to glutamic acid (N17E, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10).

[0013] (5) The 17th position of asparagine is mutated to alanine (N17A, the nucleotide sequence of the encoded gene is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12).

[0014] (6) The lysine at position 35 is mutated to serine (K35S, the nucleotide sequence of the encoded gene is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14);

[0015] (7) The alanine at position 150 is mutated to serine (A150S, the nucleotide sequence of the encoded gene is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.16).

[0016] Furthermore, as a specific embodiment, it is preferred that the alanine at position 8 is mutated to glycine, while the asparagine at position 17 is mutated to glutamic acid (A8G / N17E, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.18).

[0017] Furthermore, as a specific embodiment, it is preferred that 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 encoding gene is shown in SEQ ID NO.19, and the amino acid sequence is shown in SEQ ID NO.20).

[0018] Any amino acid sequence shown in SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18 or 20 that has one or more amino acids deleted, inserted or replaced, and has the catalytic activity to prepare 2-(4-nitrophenyl)butyric acid from 2-(4-nitrophenyl)butyronitrile, is still within the scope of protection of this invention.

[0019] The nucleotide sequences provided by this invention can typically be obtained using polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities using recombination. The obtained nucleotide sequence is usually cloned into a vector, then transformed into genetically engineered bacteria, and then the relevant nucleotide sequence is isolated from the proliferated host cells using conventional methods.

[0020] In addition, known methods of artificial chemical synthesis can be used to synthesize the relevant nucleotide sequences.

[0021] This invention also relates to a recombinant vector for the encoding gene of the nitrile hydrolase mutant. The present invention can be constructed by linking the nucleotide sequence of the nitrile hydrolase mutant of the present invention to various vectors using conventional methods in the art. The recombinant vector of the present invention is not limited, as long as it can maintain its replication or autonomously replicate in various host cells of prokaryotic and / or eukaryotic cells. The vector can be any conventional vector in the art, such as various plasmids, bacteriophages, or viral vectors. The recombinant vector of the present invention is preferably based on pET28a.

[0022] In this invention, the "vector" used in the recombinant vector can be any vector known in the art, such as commercially available plasmids, granules, bacteriophages, and retroviruses. The preferred expression vector in this invention is the pET-28a plasmid. Exemplarily, the expression vector and the target fragment are ligated using seamless cloning to obtain the recombinant vector.

[0023] The present invention also relates to a recombinant engineered bacterium containing the gene as described above or the recombinant vector as described above.

[0024] In this invention, the recombinant vector can be transformed, transduced, or transfected into host cells (strains) using methods conventional in the art. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably *Escherichia coli*, and more preferably *Escherichia coli* BL21(DE3).

[0025] The nitrile hydrolase mutant described in this invention can be formulated into corresponding enzyme preparations. Specifically, the enzyme preparations can exist in solid, semi-solid, or liquid form. The enzyme preparations can contain excipients or additives for preparing the enzyme preparations, which can be selected by those skilled in the art as needed, and will not be elaborated here.

[0026] Furthermore, the present invention also provides the application of the above-described nitrile hydrolase mutant, recombinant vector, genetically engineered bacteria, and enzyme preparation in the catalytic preparation of 2-(4-nitrophenyl)butyric acid from 2-(4-nitrophenyl)butyronitrile.

[0027] The nitrile hydrolase mutant described in this invention can be used in whole-cell engineered bacteria, in unpurified crude enzyme form, or in partially or completely purified enzyme protein form. If desired, the nitrile hydrolase mutant of this invention can also be used in immobilized enzyme or immobilized cell form using immobilization techniques known in the art.

[0028] Furthermore, the preparation method of 2-(4-nitrophenyl)butyric acid of the present invention specifically involves: using 2-(4-nitrophenyl)butyronitrile as a substrate, and using the nitrile hydrolase mutant, the recombinant vector, the wet cells of the genetically engineered bacteria, the wet cells immobilized with the bacterial cells, or the enzyme preparation as a catalyst to carry out the reaction.

[0029] Furthermore, the reaction system contains a buffer solution with a pH of 7.0 to 8.0 (preferably pH 7.5).

[0030] Furthermore, the reaction temperature in the reaction system is 30~45 ℃.

[0031] Furthermore, in the reaction system, the concentration of the substrate is 10-180 g / L.

[0032] Furthermore, as a specific embodiment, taking bacterial cells as an example, the amount of catalyst used is 1~5 g / L (preferably 3 g / L) based on the dry weight of bacterial cells.

[0033] Furthermore, in this invention, the fermentation conditions of the recombinant strain are not particularly limited, as long as the fermentation process allows the recombinant strain to proliferate in large quantities. As a specific embodiment, the wet bacterial cells are prepared as follows: Engineered bacteria containing the nitrile hydrolase mutant encoding gene are inoculated into LB medium containing a final concentration of 50 mg / L kanamycin and cultured at 37 ℃ and 180 r / min for 12 h. Subsequently, the inoculum is transferred at a volume concentration of 2% to fresh LB medium containing a final concentration of 50 mg / L kanamycin and cultured at 37 ℃ and 180 r / min until the bacterial cell concentration reaches OD0.05. 600 The initial concentration was 0.4–0.8. IPTG (preferably 0.1 mM) was then added to the culture medium to a final concentration of 0.1–1 mM. The culture was induced at 28 °C and 180 r / min for 12 h. The culture was then centrifuged, and the precipitate was collected to obtain wet cells. The composition of LB liquid medium (g / L) was: peptone 10, yeast extract 5, NaCl 10, water as solvent, and pH 7.0. The composition of LB plate medium (g / L) was: peptone 10, yeast extract 5, NaCl 10, agar 20, water as solvent, and pH 7.0.

[0034] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0035] The nitrile hydrolase mutant of this invention has 32 times higher activity and 15.8 times higher yield than the wild-type Gi-Nit, laying the foundation for the industrial enzymatic synthesis of 2-(4-nitrophenyl)butyric acid. Attached Figure Description

[0036] Figure 1 Gi - The reaction process of whole-cell catalytic synthesis of 2-(4-nitrophenyl)butyric acid by Nit and mutants A8G, N17E, K35S and A150S.

[0037] Figure 2 The reaction process of whole-cell catalytic synthesis of 2-(4-nitrophenyl)butyric acid by mutant A8G / N17E.

[0038] Figure 3 The reaction process of whole-cell catalytic synthesis of 2-(4-nitrophenyl)butyric acid by mutant A8G / N17E / K35S. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0040] In this document, the term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention but does not exclude other aspects.

[0041] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0042] 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.

[0043] Example 1: Expression of wild-type nitrile hydrolase

[0044] 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.

[0045] 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.

[0046] Example 2: Single-point saturation mutagenesis and screening of nitrile hydrolases

[0047] 1. Site-directed saturation mutation

[0048] 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.

[0049] Table 1. Site-Specific Saturated Primer Design Table

[0050]

[0051] Note: N=A / G / C / T, K=G / T, M=A / C

[0052] PCR reaction system (50 μL): 25 μL 2× phanta Max buffer, 1 μL dNTP mixture (10 mM), 2 μL each of the 10 µM mutant primers shown in Table 1, plasmid pET28- Bd Add 1 μL of Nit, 0.5 μL of Phanta Max DNA polymerase, and ddH2O to a final volume of 50 μL.

[0053] PCR conditions: (1) Pre-denaturation at 95 ℃ for 5 min; (2) Denaturation at 95 ℃ for 15 s, annealing at 59 ℃ for 5 s, extension at 72 ℃ for 30 s, step (2) for a total of 30 cycles; (3) Final extension at 72 ℃ for 3 min, storage at 4 ℃. After the reaction, the PCR products were analyzed by 0.9% agarose gel electrophoresis, and then... Dpn I. Digest the original template, excise the gel, purify the PCR product, and introduce it via heat shock. E. coli BL21(DE3) competent cells were plated on LB plates containing 50 ug / mL kanamycin and incubated overnight at 30 ℃. Positive clones were screened to obtain a saturated mutant library.

[0054] 2. Transformation of nitrile hydrolase mutants

[0055] Take competent cells E. coliAdd 10 μL of the above PCR product to BL21(DE3), incubate on ice for 30 min, heat shock at 42°C for 90 s, add 600 μL of antibiotic-free LB liquid medium, and incubate at 37°C and 180 rpm for 1 h. After incubation, centrifuge at 4°C and 4000 rpm for 1 min, discard the supernatant, and spread the remaining bacterial culture evenly onto LB agar plates containing 50 mg / L kanamycin, and incubate overnight at 37°C.

[0056] 3. High-throughput screening and secondary screening

[0057] Pick single colonies from step 2 and transfer them to 96-well plates. Add 600 mL of LB liquid medium containing 50 μg / mL kanamycin to each well and incubate at 37°C for 16 h to prepare the seed culture. Add 600 μL of LB liquid medium containing 50 μg / mL kanamycin and 0.1 mM IPTG to a new sterile 96-well plate, then add 200 μL of the seed culture and incubate at 28°C for 12 h. Centrifuge the bacterial culture after 12 h of expression at 4000 r / min and 16°C for 10 min, discard the supernatant, and wash and resuspend the cells in 200 μL of KH₂PO₄-K₂HPO₄ buffer (100 mM, pH 7.5) in each well. Add 200 μL of 10 mM 2-(4-nitrophenyl)butyronitrile dissolved in PB buffer to each well and react at 30°C for 30 min. After the reaction was completed, the high-throughput screening method for nitrile hydrolase mutants was based on the high-throughput screening method for nitrile hydrolase described in patent CN202410304056.X.

[0058] The mutants with enhanced activity obtained from high-throughput screening were re-screened. The liquid chromatography re-screening reaction system (20 mL) consisted of: 20 mM KH₂PO₄-K₂HPO₄ buffer (pH 7.5), 50 mM 2-(4-nitrophenyl)butyronitrile, and 0.1 g of wet bacterial cells. The reaction solution was preheated at 30℃ for 10 min, then reacted at 600 rpm for 15 min. A 500 μL sample was taken, and 10 μL of 6 M HCl was added to terminate the reaction. The content of 2-(4-nitrophenyl)butyric acid was determined by HPLC. The HPLC detection conditions were: Daicel-AD-H column, mobile phase of hexane:isopropanol 90:10 (v / v), flow rate of 1 mL / min, and detection wavelength of 210 nm.

[0059] The results showed that the activity of wild-type Gi-Nit was 23.5 U / g, while the activity of mutant A8D was 67 U / g, an increase of 2.85 times; the activity of mutant A8G was 82.3 U / g, an increase of 3.5 times; the activity of mutant A8V was 54.4 U / g, an increase of 2.31 times; the activity of mutant N17E was 124 U / g, an increase of 5.28 times; the activity of mutant N17A was 34 U / g, an increase of 1.45 times; the activity of mutant K35S was 189 U / g, an increase of 8.0 times; and the activity of mutant A150S was 29.8 U / g, an increase of 1.27 times (Table 2).

[0060] Table 2 Comparison of the activities of nitrile hydrolase mutants

[0061]

[0062] Example 3: Iterative saturation mutation of nitrile hydrolase

[0063] Iterative combination mutations were performed on the superior mutants obtained in Example 2, specifically, a saturation mutation was performed at the N17 site based on the optimal mutant A8G at the A8 site. The construction method of the iterative mutants and the method for detecting nitrile hydrolase activity 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. Furthermore, a saturation mutation was performed at the K35 site based on the mutant A8G / N17E. The construction method of the iterative mutants and the method for detecting nitrile hydrolase activity 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).

[0064] Table 3 Comparison of the activities of nitrile hydrolase mutants

[0065]

[0066] Example 4, Nitrile hydrolase Gi - Nit whole-cell catalytic reaction of 2-(4-nitrophenyl)butyronitrile

[0067] The transformation system and operation were as follows: Wild-type recombinant nitrile hydrolase Gi-Nit whole cells obtained in Example 1 were added to 20 mL of KH₂PO₄-K₂HPO₄ buffer (20 mM, pH 7.5). The cell count was 3 g / L (dry weight), and the substrate concentration of 2-(4-nitrophenyl)butyric acid was 150 g / L. The reaction was carried out at 30 °C and 600 rpm for 15 h. The reaction progress was monitored by HPLC under the conditions shown in Example 2. The results showed that after 15 h of reaction, the concentration of the product 2-(4-nitrophenyl)butyric acid was 10.6 g / L. Figure 1 ).

[0068] Example 5: Whole-cell catalysis of 2-(4-nitrophenyl)butyronitrile by mutant A8G

[0069] The transformation system and operation were as follows: Whole cells of the wild-type recombinant nitrile hydrolase mutant A8G obtained in Example 2 were added to 20 mL of KH₂PO₄-K₂HPO₄ buffer (20 mM, pH 7.5). The cell count was 3 g / L (dry weight), and the concentration of the substrate 2-(4-nitrophenyl)butyric acid was 150 g / L. The reaction was carried out at 30 °C and 600 rpm for 15 h. The reaction progress was monitored by HPLC under the conditions shown in Example 2. The results showed that after 15 h of reaction, the concentration of the product 2-(4-nitrophenyl)butyric acid was 17.6 g / L. Figure 1 ).

[0070] Example 6: Whole-cell catalysis of 2-(4-nitrophenyl)butyronitrile by mutant N17E

[0071] The transformation system and operation were as follows: Whole cells of the wild-type recombinant nitrile hydrolase mutant N17E obtained in Example 2 were added to 20 mL of KH₂PO₄-K₂HPO₄ buffer (20 mM, pH 7.5). The cell count was 3 g / L (dry weight), and the concentration of the substrate 2-(4-nitrophenyl)butyric acid was 150 g / L. The reaction was carried out at 30 °C and 600 rpm for 15 h. The reaction progress was monitored by HPLC under the conditions shown in Example 2. The results showed that after 15 h of reaction, the concentration of the product 2-(4-nitrophenyl)butyric acid was 41.2 g / L. Figure 1 ).

[0072] Example 7: Whole-cell catalysis of 2-(4-nitrophenyl)butyronitrile by mutant K35S

[0073] The transformation system and operation were as follows: Whole cells of the wild-type recombinant nitrile hydrolase mutant K35S obtained in Example 2 were added to 20 mL of KH2PO4-K2HPO4 buffer (20 mM, pH 7.5). The cell count was 3 g / L (dry weight), and the concentration of the substrate 2-(4-nitrophenyl)butyric acid was 150 g / L. The reaction was carried out at 30 °C and 600 rpm for 15 h. The reaction progress was monitored by HPLC under the conditions shown in Example 2. The results showed that after 15 h of reaction, the concentration of the product 2-(4-nitrophenyl)butyric acid was 81.5 g / L. Figure 1 ).

[0074] Example 8: Whole-cell catalysis of 2-(4-nitrophenyl)butyronitrile by mutant A150S

[0075] The transformation system and operation were as follows: Whole cells of the wild-type recombinant nitrile hydrolase mutant A150S obtained in Example 2 were added to 20 mL of KH₂PO₄-K₂HPO₄ buffer (20 mM, pH 7.5). The cell count was 3 g / L (dry weight), and the concentration of the substrate 2-(4-nitrophenyl)butyric acid was 150 g / L. The reaction was carried out at 30 °C and 600 rpm for 15 h. The reaction progress was monitored by HPLC under the conditions shown in Example 2. The results showed that after 15 h of reaction, the concentration of the product 2-(4-nitrophenyl)butyric acid was 13.4 g / L. Figure 1 ).

[0076] Example 9: Whole-cell catalysis of 2-(4-nitrophenyl)butyronitrile by mutant A8G / N17E

[0077] The transformation system and operation were as follows: Whole cells of the wild-type recombinant nitrile hydrolase mutant A8G / N17E obtained in Example 3 were added to 20 mL of KH2PO4-K2HPO4 buffer (20 mM, pH 7.5). The cell count was 3 g / L (dry weight), and the concentration of the substrate 2-(4-nitrophenyl)butyric acid was 150 g / L. The reaction was carried out at 30 °C and 600 rpm for 15 h. The reaction progress was monitored by HPLC under the conditions shown in Example 2. The results showed that after 15 h of reaction, the concentration of the product 2-(4-nitrophenyl)butyric acid was 120.7 g / L. Figure 2 ).

[0078] Example 10: Whole-cell catalysis of 2-(4-nitrophenyl)butyronitrile by mutant A8G / N17E / K35S

[0079] The transformation system and operation were as follows: Whole cells of the wild-type recombinant nitrile hydrolase mutant A8G / N17E / K35S obtained in Example 3 were added to 20 mL of KH2PO4-K2HPO4 buffer (20 mM, pH 7.5). The cell count was 3 g / L (dry weight), and the concentration of the substrate 2-(4-nitrophenyl)butyric acid was 150 g / L. The reaction was carried out at 30 °C and 600 rpm for 15 h. The reaction progress was monitored by HPLC under the conditions shown in Example 2. The results showed that after 15 h of reaction, the concentration of the product 2-(4-nitrophenyl)butyric acid was 164.5 g / L. Figure 3 ).

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. Any simple or equivalent changes or modifications made to the content of the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A nitrilase mutant, characterized by: The amino acid sequence of the nitrile hydrolase mutant is shown below: SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18 or 20.

2. A gene encoding the nitrile hydrolase mutant of claim 1.

3. A recombinant vector, characterized by: The recombinant vector contains the gene as described in claim 2.

4. A genetically engineered bacterium, characterized by: The genetically engineered bacteria contain the gene as described in claim 2, or contain the recombinant vector as described in claim 3.

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

6. The use of the nitrile hydrolase mutant of claim 1, the recombinant vector of claim 3, the genetically engineered bacteria of claim 5, or the enzyme preparation of claim 5 in the catalytic preparation of 2-(4-nitrophenyl)butyronitrile from 2-(4-nitrophenyl)butyric acid.

7. A method for preparing 2-(4-nitrophenyl)butyric acid, characterized in that: It is obtained by reacting 2-(4-nitrophenyl)butyronitrile as a substrate with the nitrile hydrolase mutant of claim 1 or the enzyme preparation of claim 5 as a catalyst.

8. The method as described in claim 7, characterized in that: The reaction medium is a buffer solution with a pH of 7.0 to 8.

0.

9. The method as described in claim 7, characterized in that: The reaction temperature in the reaction system is 30~45℃.

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