Modification method for improving specificity and catalytic activity of fatty nitrilase substrate and mutant obtained by modification

Abstract

The invention relates to a transformation method for improving substrate specificity and catalytic activity of fatty nitrilase and a mutant obtained by transformation, and belongs to the technical field of enzyme engineering. The method comprises the following steps: starting from to-be-modified parent nitrilase with an amino acid sequence shown as SEQ ID NO.1, combining the to-be-modified parent nitrilase with an aliphatic nitrile substrate, determining a to-be-modified site related to substrate combination in a protein recognition channel, synthesizing site-specific mutagenesis, saturated mutagenesis, combinatorial mutagenesis and iterative mutagenesis, and combining a semi-rational design and machine learning method, so as to obtain the amino acid sequence of the aliphatic nitrile. A series of nitrilase mutants with improved fatty nitrile substrate specificity and catalytic activity are screened by determining key sites related to substrate combination and adjacent sites synergistically playing a role with the key sites in parent nitrilase, the conversion rate of 3-chloropropionitrile substrates reaches 99% or above, the highest enzymatic activity is 58.35 U / mL, and the nitrilase mutants have a broad application prospect. After the 3-chloropropionitrile is added in five batches, the conversion is completed within 190 minutes, and the cumulative product concentration reaches 108.52 g / L, so that the efficient conversion of the aliphatic nitrile substrate is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, in particular to a method for modifying to improve the substrate specificity and catalytic activity of a fatty nitrile hydrolase and a mutant obtained by the modification. Background Art

[0002] Fatty nitrile hydrolase is an enzyme that can specifically catalyze the hydrolysis of fatty nitrile substrates to produce high-value carboxylic acids. Its reaction mechanism usually involves the electrophilic carbon atom in the nitrile group and the nucleophilic attack of a cysteine residue, and then water molecules participate in the reaction to complete the catalytic process. The cysteine-glutamic acid-lysine (Cys-Glu-Lys) catalytic triad in the active center of nitrile hydrolase plays a crucial role in substrate recognition, binding, and catalytic reactions. It can be widely used in the biocatalytic synthesis of pharmaceutical intermediates (atorvastatin, gabapentin, (R)-baclofen, and (S)-pregabalin), bioremediation of nitrile pollutants, safe degradation of food preservatives, fiber modification in the textile industry, etc. The substrate specificity of these enzymes is crucial for their catalytic performance and practical application effects. However, most reported fatty nitrile hydrolases have low catalytic activity and specificity for fatty nitrile substrates, thus limiting their industrial applications.

[0003] Nevertheless, nitrile hydrolases often show unlimited potential in terms of substrate preference, regioselectivity, and stereoselectivity. Obtaining excellent mutants through large-scale random mutagenesis and high-throughput screening is a classic protein engineering method. Sun et al. combined traditional random mutagenesis with site-directed mutagenesis and constructed two enantiocomplementary nitrile hydrolase mutants PpL19-LH and PpL19-GYY that showed high S- and R-selectivity for mandelonitrile, respectively, through error-prone PCR. Subsequently, researchers developed in vitro recombination technology and achieved a complete conversion of the substrate preference for 6-heptenenitrile and 3-butenenitrile by recombining enzyme fragments from different sources, and also obtained a chimeric nitrile hydrolase BaNit with high activity and enantioselectivity for isobutyl succinonitrile (IBSN). On this basis, researchers began to focus on the complex relationship between sequence, structure, and function, emphasizing higher modification accuracy and predictability through rational design. Based on the sequence analysis of two nitrile hydrolases, bll6402 and blr3397, Chen et al. screened key sites adjacent to the catalytic residue Cys, which can freely switch the regioselectivity for succinonitrile. The site-directed mutant Mut-F168V of the nitrile hydrolase derived from Acidovorax facilis ZJB09122 constructed by Wu et al. can meet the large-scale production of 1-(cyanocyclohexyl)acetic acid with low cost and almost 100% substrate conversion rate.

[0004] However, there are still more or less defects in these studies. For example, most nitrilases have low catalytic activity towards fatty nitriles, lack diverse modification strategies, and the potential of modifying residues outside the active site has not been fully explored. In addition, the research on fatty nitrilases is relatively scarce, lacking efficient mutants and systematic modification methods. Future research needs to further broaden the substrate spectrum of nitrilases, improve their catalytic activity and conversion rate towards fatty nitriles, and at the same time develop more strategies based on the modification of key residues and computer-aided design to enhance the activity and stability of the enzyme and meet the higher requirements of industrial applications. Summary of the Invention

[0005] To solve the above technical problems, the present invention starts from the parental nitrilase with the amino acid sequence shown in SEQ ID NO.1, binds it to the fatty nitrile substrate, determines the sites to be modified related to substrate binding in the protein recognition channel, combines site-directed mutagenesis, saturation mutagenesis, combinatorial mutagenesis and iterative mutagenesis, and combines semi-rational design and machine learning methods to determine the key sites related to substrate binding in the parental nitrilase and the adjacent sites that cooperate with it, and screens a series of nitrilase mutants with improved specificity and catalytic activity towards fatty nitrile substrates.

[0006] The first object of the present invention is to provide a nitrilase mutant, which is any one of the following mutations made to the starting sequence with the amino acid sequence shown in SEQ ID NO.1:

[0007] (1) Tyrosine at position 54 is mutated to phenylalanine;

[0008] (2) Proline at position 190 is mutated to cysteine;

[0009] (3) Leucine at position 194 is mutated to tryptophan;

[0010] (4) Phenylalanine at position 202 is mutated to glycine;

[0011] (5) Glutamine at position 207 is mutated to methionine;

[0012] (6) Tyrosine at position 54 is mutated to phenylalanine, and leucine at position 194 is mutated to tryptophan;

[0013] (7) Glycine at position 191 is mutated to alanine, and leucine at position 194 is mutated to tryptophan;

[0014] (8) Glutamine at position 207 is mutated to methionine, and leucine at position 194 is mutated to tryptophan;

[0015] (9) Leucine at position 194 is mutated to isoleucine, phenylalanine at position 202 is mutated to leucine, and glutamine at position 207 is mutated to methionine.

[0016] The second object of the present invention is to provide a gene encoding the above-mentioned nitrilase mutant.

[0017] The third object of the present invention is to provide an expression vector containing the above-mentioned gene.

[0018] The fourth object of the present invention is to provide a host cell containing the above-mentioned nitrilase mutant or the above-mentioned expression vector.

[0019] The fifth object of the present invention is to provide the application of the above-mentioned nitrilase mutant, the above-mentioned gene, the above-mentioned expression vector or the above-mentioned host cell in catalyzing a substrate.

[0020] Furthermore, the substrate is a fatty nitrile.

[0021] The sixth object of the present invention is to provide a method for producing 3-chloropropionic acid, which comprises adding the above-mentioned nitrilase mutant or an expression system containing the mutant to a reaction system containing a substrate for reaction, and the substrate is 3-chloropropionitrile.

[0022] The seventh object of the present invention is to provide a method for modifying a nitrilase to improve its specificity and catalytic activity towards a fatty nitrile substrate, comprising the following steps:

[0023] Step S1: Bind the parental nitrilase to be modified to a fatty nitrile substrate, determine the sites to be modified related to substrate binding in the protein recognition channel, perform site-directed mutagenesis on the obtained sites to be modified, and obtain a primary mutant library;

[0024] Step S2: Screen the sites to be modified in the primary mutant library that significantly improve the catalytic activity of the fatty nitrile substrate for saturation mutagenesis to obtain a secondary mutant library;

[0025] Step S3: Screen the sites to be modified in the secondary mutant library that significantly improve the catalytic activity of the fatty nitrile substrate for combinatorial mutagenesis to obtain a tertiary mutant library;

[0026] Step S4: Combine the results of multiple rounds of mutagenesis in Steps S1-S3, determine the key sites related to substrate binding, screen the neighboring sites that play a synergistic role with the key sites, and perform iterative combinatorial mutagenesis on the neighboring sites to obtain a quaternary mutant library;

[0027] Step S5: Combine the results of multiple rounds of mutagenesis in Steps S1-S4, and screen to obtain a nitrilase mutant with improved specificity and catalytic activity towards the fatty nitrile substrate.

[0028] Substrate channels determine the entry, exit, and orientation of substrates within the enzyme's active site and are key factors in determining substrate specificity. By manipulating the structure of these channels, the properties of the enzyme can be adjusted to favor specific catalytic reactions, enabling precise control of the substrate specificity of nitrilases. Understanding the molecular basis of substrate recognition and catalysis is crucial for optimizing these enzymes to meet specific substrate requirements.

[0029] In step S1, the parental nitrilase to be modified is derived from Pseudomonas putida (see Chinese published patent CN107254429A), and its nucleotide sequence is shown in SEQ ID NO.2. It has shown excellent thermal stability and catalytic activity in previous studies. Although PpNit exhibits higher activity towards aromatic nitriles, especially 3-cyanopyridine, it also has certain catalytic activity towards aliphatic nitriles. This dual activity, combined with its excellent performance, makes PpNit an ideal candidate for engineering modification.

[0030] Furthermore, in step S1, the aliphatic nitrile substrates include 3-chloropropionitrile, 4-chlorobutyronitrile, and 5-chlorovaleronitrile.

[0031] Furthermore, in step S4, alanine, leucine, and phenylalanine are respectively used to filter the key sites and the common neighboring residues around the aliphatic nitrile substrate molecules to obtain neighboring sites that play a synergistic role. within the range to obtain neighboring sites that play a synergistic role.

[0032] Advantages of the present invention:

[0033] The present invention provides a site-directed - saturation - combination - iterative semi-rational design workflow. By combining computational methods such as substrate channel modeling and molecular docking, the key amino acid residues related to substrate binding in nitrilases are systematically identified and optimized. The substrate specificity and catalytic activity of the screened nitrilase mutants towards aliphatic nitriles are significantly improved. The conversion rate of the 3-chloropropionitrile substrate reaches over 99%, the highest enzyme activity is 58.35 U / mL, and the conversion is completed within 190 minutes after adding 3-chloropropionitrile in 5 batches, with the cumulative product concentration reaching 108.52 g / L, achieving efficient conversion of aliphatic nitrile substrates. Description of the Drawings

[0034] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, where:

[0035] Figure 1It is the SDS-PAGE characterization of the purification of the nitrilase protein in the embodiments of the present invention. Among them, lane M is the protein Marker, lane 1 is the supernatant of the cell lysate of Escherichia coli BL21(DE3)-pET-3b, and lanes 2-4 are the initial strain PpNit, mutant G191A / L194W, mutant L194W, and mutant Y54F, respectively;

[0036] Figure 2 It is the structural analysis of the complex of nitrilase and substrate in Example 1 of the present invention. Among them, A is the complex of aromatic nitrile 3-cyanopyridine and the active pocket of nitrilase, B is the complex of aliphatic nitrile 3-chloropropionitrile and the active pocket of nitrilase, and C is the distance between the cyano C atom of 3-chloropropionitrile, 4-chlorobutyronitrile, and 5-chlorovaleronitrile and the S γ atom of Cys165 residue;

[0037] Figure 3 It is the construction of the mutant library of the substrate channel of nitrilase in Example 1 of the present invention. Among them, A is site-directed mutagenesis, B is saturation mutagenesis, and C is combinatorial mutagenesis;

[0038] Figure 4 It is the design of the active site network of nitrilase and the characterization of enzyme activity under the guidance of machine learning in Example 2 of the present invention;

[0039] Figure 5 It is the ALF scanning and iterative mutagenesis strategy for the 194th amino acid residue of the parental nitrilase in Example 3 of the present invention;

[0040] Figure 6 It is the application verification of the nitrilase mutant in Example 3 of the present invention. Among them, A is the substrate conversion rate of the nitrilase mutant and the initial strain to 3-chloropropionitrile, and B is the batch conversion process of the mutant G191A / L194W to the substrate 3-chloropropionitrile;

[0041] Figure 7 It is the substrate spectrum analysis of the mutant and the initial strain to 28 different nitriles in Example 3 of the present invention. Detailed implementation manners

[0042] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0043] The amino acid sequence of the parental nitrilase PpNit involved in the following embodiments is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0044] The methods for recombinant expression and separation and purification of nitrilase involved in the following embodiments are as follows:

[0045] Using the Escherichia coli expression plasmid pET-3b as the expression vector, the recombinant plasmid pET-3b-PpNit with the nitrilase gene ligated was transformed into the expression host Escherichia coli BL21(DE3) by heat shock method, and cultured on an LB agar plate containing 100 μg / mL ampicillin resistance at 37 °C for 12 h. The positive colonies were inoculated into 10 mL of LB medium containing ampicillin resistance (100 μg / mL) for seed culture for 10 - 12 h. Then, it was transferred to 30 mL of medium for fermentation culture at an inoculation amount of 1% for the expression of recombinant nitrilase.

[0046] Using the successfully constructed recombinant expression plasmid pET-3b-PpNit of nitrilase as the template, the corresponding mutations were introduced into the DNA coding sequence of nitrilase by one-step inverse PCR of the whole plasmid using the previously designed amino acid mutation primers; after the PCR was completed, the target fragment was separated and purified by nucleic acid gel electrophoresis. The purified and recovered product was digested with Dpn I enzyme to remove the template plasmid. After the digestion reaction was completed, it was cooled on ice, and 10 μL of the digested product was transformed into the cloning host Escherichia coli JM109. After the transformants grew out, the bacteria were picked for sequencing verification. After successful verification, the plasmid was extracted and transformed into Escherichia coli BL21(DE3) for the recombinant expression of mutant nitrilase.

[0047] The recombinant Escherichia coli fermentation broth after 8 h of enzyme-producing fermentation was collected by low-temperature centrifugation, suspended in buffer A (50 mM sodium phosphate, 500 mM sodium chloride, pH = 7.4), and the cells were disrupted by methods such as ultrasonic disruption or high-pressure homogenization. Cell debris was removed by filtration or ultra-high-speed centrifugation to prepare a cell-free extract, which was separated and purified using a Ni-NTA agarose gel column. The column was equilibrated with buffer A, and gradient elution was performed with different concentrations of imidazole. The elution process was monitored in real time, and the detection wavelength was 280 nm. The elution peaks at each stage were collected separately, and the molecular weight and purity of the purified nitrilase were analyzed by SDS-PAGE. The results are as Figure 1 shown (taking some mutants obtained in the following examples as an example).

[0048] The method for measuring the enzyme activity of nitrilase involved in the following examples is as follows:

[0049] The standard enzyme activity assay was carried out by reacting a substrate solution (final concentration of 50 mM) and nitrilase (200 μL of crude enzyme solution) in PBS (100 mM, pH = 7.2). After pre-incubating for 5 min in a metal bath at 30 °C, 1 mL of the suspension was taken and reacted at 30 °C and 1500 rpm for 10 min. The reaction was terminated by centrifuging at 12000×g for 1 min. The enzyme activity was determined by measuring the ammonia produced in the reaction mixture. This assay was based on the phenol-hypochlorite colorimetric method. One enzyme activity unit (1 U) was defined as the amount of enzyme that produced 1 μmol of ammonia per minute under the above standard conditions. All assays were performed in triplicate. The products and substrates of the nitrilase-catalyzed reaction were detected by HPLC using an Xterra MS C18 column at a wavelength of 205 nm. The mobile phase consisted of acetonitrile and ultrapure water, with the proportion of acetonitrile being 95%, the flow rate being 0.6 mL / min, and the detection temperature being 30 °C.

[0050] Example 1: Construction of a nitrilase mutant library

[0051] The aliphatic nitrile model substrates 3-chloropropionitrile, 4-chlorobutyronitrile, 5-chlorovaleronitrile and the aromatic nitrile model substrate 3-cyanopyridine were docked with the nitrilase PpNit, and the complex structure with the lowest docking binding energy was selected for analysis. The results are as Figure 2 shown. Define the residues within the range around the substrate as the active pocket and confirm the substrate channel connecting the external environment and the active site. By analyzing the binding state of the substrate and the enzyme, it was observed that aliphatic nitrile substrates such as 3-chloropropionitrile, 4-chlorobutyronitrile and 5-chlorovaleronitrile have a linear and relatively flexible molecular structure. This structural flexibility allows them to freely swing and adjust their positions within the active site of the enzyme, so the binding tightness is relatively low, showing a trend that as the carbon chain of the substrate lengthens, the distance between the S γ atom of the Cys165 residue and the carbon atom of the nitrile group of the substrate becomes larger. In contrast, aromatic nitrile substrates, such as 3-cyanopyridine, contain a rigid aromatic ring structure, and its large steric hindrance limits the rotation and swing of the molecule, prompting the substrate to form a closer contact with the active site of the enzyme. This tight binding mode helps to improve the catalytic activity. It is hypothesized that by optimizing the structure and physicochemical properties of the substrate channel to limit the residence time of the elongated aliphatic nitrile substrate in the nitrilase active pocket, the binding efficiency and catalytic conversion rate can be improved, thereby affecting the substrate preference for different aromatic or aliphatic nitriles.

[0052] Based on the above inferences, a semi-rational design strategy was adopted to regulate the selectivity of nitrilase for aliphatic and aromatic nitrile substrates by systematically identifying and optimizing the key residues in the substrate channel.

[0053] First, 18 protein sequences of nitrilases from different sources were screened in the NCBI database for multiple sequence alignment. The selected nitrilases all had known substrate spectrum characteristics, especially the hydrolysis activity towards aliphatic nitriles. Although these enzymes were from different species, they had high similarity in amino acid sequences. The conserved regions and variable regions in the primary structure were determined through multiple sequence alignment, thus providing key information for subsequent protein engineering strategies.

[0054] Subsequently, based on the alignment of different protein three-dimensional structures, the HotSpot Wizard server was used to integrate databases and computational tools to identify hot-spot residues related to activity in the substrate channel and the entrance loop region. Through phylogenetic analysis of PROSS and Rosetta computational optimization of the energy of the native state, it was ensured that the mutations would not disrupt the stability and activity of the enzyme. At the same time, distal sites near the substrate channel were included in the scope of modification. The constructed primary mutant library involved 28 sites and 46 single-point mutants. The results of enzyme activity assays are shown as Figure 3 shown in A of

[0055] Five sites (Y54F, L194, Q207, P190, F202) with more than twice the increase in nitrilase activity were selected for saturation mutagenesis as the secondary mutant library, and double enzyme activity assays (enzyme activity towards aliphatic nitrile substrates and enzyme activity towards aromatic nitrile substrates) were performed on all the constructed saturation mutants in the secondary mutant library. The results are shown as Figure 3 shown in B of . The mutations Y54F, L194W, Q207M, P190C, and F202G significantly enhanced the preference for aliphatic nitrile substrates. The enzyme activities towards 3-chloropropionitrile were 5.2-fold, 5.1-fold, 4.4-fold, 3.3-fold, and 3.3-fold higher than that of nitrilase PpNit, respectively. In addition, although the catalytic activity of the L194W mutant towards 3-chloropropionitrile was comparable to that of Y54F, it completely lost the catalytic activity towards aromatic nitriles.

[0056] To explore the synergistic effects of the above five sites, 20 combinatorial mutations were constructed and their enzyme activities were detected. The results are shown as Figure 3 shown in C of . The enzyme activity of the Y54F / L194W double mutant towards 3-chloropropionitrile increased slightly, but the enzyme activity towards 3-cyanopyridine decreased to half of that of the original strain PpNit. Although the triple mutants significantly reduced the activity towards aromatic nitrile substrates, they did not show an obvious preference for aliphatic nitrile substrates, indicating that there was no additive effect among these mutations.

[0057] Example 2: Active site network design and key residue filtering of nitrilase

[0058] Use machine learning for structure prediction and simulation, design mutation combinations at five sites of Y54, L194, Q207, P190, and F202 using the FuncLib calculation tool, construct five triple-combination mutants with lower Rosetta energy than the original strain and measure their enzyme activities. The results are as Figure 4 shown. The enzyme activity of the triple mutant ILM (L194I / F202L / Q207M) towards 3-chloropropionitrile is increased to 6.4 times that of PpNit, and the enzyme activity towards 3-cyanopyridine is decreased by about 1 / 3.

[0059] Analysis of the above mutants shows that residue 194 is a key "gating" site at the junction of the substrate channel and the active pocket, playing an important role in the binding and catalysis of fatty nitrile substrates. Using an Ala-Leu-Phe scanning (ALF-scanning) strategy (see Chinese published patent CN116004593A), preliminary filtering was performed on tryptophan at position 194 and the common neighboring residues within the range around the 3-chloropropionitrile substrate molecule. The results are as Figure 5 shown. Glycine at position 191 is an important residue that works synergistically with tryptophan at position 194, and its mutation has a particularly significant effect on enzyme activity. Further iterative saturation mutagenesis screening of G191 was carried out to obtain the G191A / L194W mutant with the highest catalytic activity. The enzyme activity of this mutant towards 3-chloropropionitrile is increased to 25.88 U / mL, which is 7.8 times that of the initial strain PpNit; while the enzyme activity towards 3-cyanopyridine is decreased to 4.25 U / mL, only 0.2 times that of PpNit. On this basis, to explore the combination potential of the G191A / L194W mutant with other beneficial mutations (Y54F, P190C, F202G, Q207M), iterative combinatorial mutagenesis was carried out. The results show that the synergistic effect between the G191A site and the L194W site cannot be further enhanced, and the highest increase in the enzyme activity of the constructed better combination mutant towards 3-chloropropionitrile is only 3.8 times.

[0060] Example 3: Exploration of the application of nitrilase mutants

[0061] Evaluate the conversion efficiency of the mutants screened in the above examples towards 3-chloropropionitrile and their performance in practical applications through biotransformation experiments (referring to the above enzyme activity detection system, with the difference that the reaction time is extended to 60 min). The results are as Figure 6As shown by A in [reference], the transformation efficiency of the initial strain PpNit was approximately 65%, while the conversion rates of the five mutants Y54F, L194W, L194W / Q207M, G191A / L194W, and ILM (L194I / F202L / Q207M) to 3-chloropropionitrile reached over 99% within 60 min. In contrast, the conversion rates of F202G, P190C, and Y54F / L194W were 96%, 85%, and 78% respectively, and the Q207M mutant was the lowest, only about 46%. Although some mutants did not completely convert the substrate, no by-products were detected in the experiment. Continuous conversion experiments were carried out on the optimal mutant G191A / L194W at a substrate concentration of 200 mM, and the results are as Figure 6 shown by B in [reference]. Five batches of 3-chloropropionitrile (added every 30 minutes) were completely converted within 190 minutes, and the cumulative concentration of 3-chloropropionic acid product was 108.52 g / L.

[0062] The substrate spectra of the mutants obtained above were determined. Twenty-eight nitrile compounds with different structures were selected and the catalytic activities of different mutants towards them were verified. The results are as Figure 7 shown. It can be seen that these mutants have an extended substrate specificity for some long-chain aliphatic nitriles or aliphatic nitrile substrates that are difficult to catalyze, and at the same time have increased catalytic activities towards a variety of different aliphatic nitrile substrates. This not only promotes the diversification of enzyme functions and the improvement of catalytic efficiency, but also brings new possibilities for the application prospects of enzymes, and has important industrial, environmental, and scientific research values.

[0063] Obviously, the above embodiments are only examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A nitrilase mutant, characterized in that The nitrilase mutant is a mutant of the amino acid sequence of SEQ ID NO.1 with any of the following mutations: (1) Tyrosine at position 54 mutated to phenylalanine; (2) mutation of proline at position 190 to cysteine; (3) leucine at position 194 mutated to tryptophan; (4) phenylalanine at position 202 mutated to glycine; (5) glutamine at position 207 was mutated to methionine; (6) tyrosine at position 54 mutated to phenylalanine, and leucine at position 194 mutated to tryptophan; (7) Glycine at position 191 mutated to alanine, and leucine at position 194 mutated to tryptophan; (8) Glutamine at position 207 mutated to methionine, and leucine at position 194 mutated to tryptophan; (9) Leucine at position 194 mutated to isoleucine, phenylalanine at position 202 mutated to leucine, and glutamine at position 207 mutated to methionine.

2. A gene encoding the nitrilase mutant according to claim 1.

3. An expression vector comprising the gene according to claim 2.

4. A host cell comprising the nitrilase mutant of claim 1 or the expression vector of claim 3.

5. Use of the nitrilase mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3 or the host cell according to claim 4 in catalyzing a substrate.

6. The use according to claim 5, characterized in that: The substrate is a fatty nitrile.

7. A method for producing 3-chloropropionic acid, characterized in that: The nitrilase mutant according to claim 1 or an expression system containing the mutant is added to a reaction system containing a substrate for reaction, wherein the substrate is 3-chloropropionitrile.

8. A method for improving the specificity and catalytic activity of nitrilase for fatty nitrile substrates, characterized in that: The following steps are involved: Step S1, combining the parent nitrilase to be modified with the fatty nitrile substrate, determining the sites to be modified related to substrate binding in the protein recognition channel, performing site-directed mutagenesis on the obtained sites to be modified, and obtaining a primary mutation library; Step S2, screening the sites to be modified that significantly improve the catalytic activity of the fatty nitrile substrate in the primary mutation library for saturation mutation to obtain a secondary mutation library; Step S3, screening the sites to be modified that significantly improve the catalytic activity of the fatty nitrile substrate in the secondary mutation library for combined mutation to obtain a tertiary mutation library; Step S4, combining the results of multiple rounds of mutations in steps S1-S3, determining the key sites related to substrate binding, screening the adjacent sites that play a synergistic role with the key sites, performing iterative combination mutations on the adjacent sites, and obtaining a four-level mutation library; Step S5: combining the results of multiple rounds of mutations in steps S1-S4, screening and obtaining nitrilase mutants with improved substrate specificity and catalytic activity for fatty nitrile.

9. The method according to claim 8, characterized in that: In step S1, the fatty nitrile substrate includes 3-chloropropionitrile, 4-chlorobutyronitrile and 5-chlorovaleronitrile.

10. The transformation method according to claim 8, characterized in that: In step S4, alanine, leucine and phenylalanine are used to target key sites and the surrounding of the fatty nitrile substrate molecule. The common neighboring residues within the range are filtered to obtain the adjacent sites that play a synergistic role.

Images