High-performance nitrilase mutant and application thereof in biocatalytic synthesis of organic acid

Abstract

The invention discloses a high-performance nitrilase mutant and application thereof in biocatalytic synthesis of organic acid, and belongs to the technical field of enzyme engineering. According to the method, an ancestor enzyme sequence-structure-molecular dynamics strategy (ASSMD strategy) is designed to obtain information for eradicating ancestor enzyme in an evolutionary landscape, and the ancestor nitrilase capable of tolerating 90 DEG C is obtained by applying the method to mining of extreme thermophilic ancestor nitrilase. Afterwards, the wild-type enzyme is used as a research object, and an evolutionary event causing the extreme heat resistance of the wild-type enzyme is researched. And finally, in combination with the trend that the thermal stability in an evolutionary event is increased firstly and then reduced, determining a thermal stability beneficial residue site, and carrying out independent or combined verification on the thermal stability beneficial residue site to obtain a high-performance mutant.

Description

[0001] This application is a divisional application with application number: 202411219712.2, application date: September 2, 2024, and application name: A method for mining a target ancestral enzyme and its application. Technical Field

[0002] The invention relates to a high-performance nitrilase mutant and application thereof in biocatalytic synthesis of organic acids, belonging to the technical field of enzyme engineering. Background Art

[0003] In recent decades, advances in genetic engineering and protein engineering have driven the development of biocatalysis, demonstrating its potential as an environmentally friendly alternative to traditional chemical reactions. However, enzymes from natural sources often require artificial enhancement to meet specific catalytic requirements. Among them, thermostable enzymes are of particular importance because they can tolerate high temperatures and extend reaction times, making them very popular in various applications. Since extreme enzymes from extreme organisms exhibit good heat resistance, exploring extreme enzyme resources is the most direct way to meet the needs of various high-performance catalytic applications. For example, Taq DNA polymerase from the thermophilic bacterium Thermus aquaticus has become a well-known thermostable DNA polymerase (Brock, TD et al. Journal of bacteriology 98 (1), 289-97). Thermoxylanases from the thermophilic bacterium Herbinix hemicellulosilytica have application potential in high-temperature processing of cellulose and wood products (Mechelke, M. et al. Journal of biotechnology 257, 122-130). In addition, Ni et al. used trans-feruloyl-CoA synthetase and enoyl-CoA hydratase from the thermophilic bacterium Amycolatopsis thermoflava N1165 to construct a whole-cell Escherichia coli biocatalyst to produce vanillin at high temperature (Ni et al. Angewandte Chemie-International Edition 57(5), 1214-1217). However, the living environment of its host extremophiles is very harsh and requires specific nutritional requirements or growth conditions, which are difficult to replicate in the laboratory. Therefore, it is relatively difficult to discover and cultivate them. Accordingly, as the natural environment changes, extremophiles may also become extinct in the process of evolution of extremophiles to adapt to milder environments.

[0004] With advances in fields like bioinformatics and computational biology, the use of enzyme ancestral sequence reconstruction methods has enabled us to use computer algorithms to derive the amino acid sequences of ancient enzymes from extinct organisms. Previous research has primarily focused on resurrecting the oldest ancestral forms at the root of the evolutionary tree. However, during enzyme evolution, from the oldest ancestors to their present-day forms, enzymes have experienced countless environments, including extreme conditions. These environmental challenges likely exerted selective pressures, potentially leading to the adaptation and development of favorable traits, such as enhanced thermostability, in ancestral enzymes. Therefore, within this evolutionary landscape, we have a high probability of identifying extremely thermophilic ancestral enzymes and potentially modifying them to optimize their performance, leading to the present invention. Summary of the Invention

[0005] To address the above problems, the present invention provides a workflow for constructing an ancestral enzyme sequence-structure-kinetic parameter library by revealing molecular-level information of the evolutionary landscape, namely the ASSMD strategy. This strategy was used to discover and mutate thermophilic nitrilase (EC 3.5.5.1). The mutant had improved thermal stability and enzyme activity, and could efficiently catalyze the hydrolysis of nitrile compounds to carboxylic acids.

[0006] The first object of the present invention is to provide an ancestral nitrilase mutant, wherein the ancestral nitrilase mutant is obtained by mutating the wild-type ancestral nitrilase shown in SEQ ID NO.1, wherein the mutation comprises:

[0007] Mutate asparagine at position 124 to histidine;

[0008] or mutating asparagine at position 124 to histidine and histidine at position 155 to tyrosine;

[0009] Alternatively, the serine at position 97 is mutated to glutamic acid, the serine at position 101 is mutated to alanine, and the asparagine at position 124 is mutated to histidine.

[0010] The second object of the present invention is to provide a polynucleotide encoding the ancestral nitrilase mutant.

[0011] The third object of the present invention is to provide a recombinant expression vector carrying the polynucleotide.

[0012] The fourth object of the present invention is to provide an expression cassette containing the polynucleotide.

[0013] Furthermore, the expression cassette includes elements necessary for gene expression and / or elements assisting expression, such as a promoter, a terminator, a signal peptide, an RBS, and the like.

[0014] A fifth object of the present invention is to provide a recombinant cell expressing the ancestral nitrilase mutant.

[0015] Furthermore, the host cell of the recombinant cell is a non-plant cell, including a microorganism, preferably Escherichia coli.

[0016] The sixth object of the present invention is to provide a method for producing an ancestral nitrilase mutant, comprising the step of fermentation production using the recombinant cell.

[0017] A seventh object of the present invention is to provide use of the ancestral nitrilase mutant, polynucleotide, recombinant expression vector, expression cassette or recombinant cell in the hydrolysis of nitrile compounds.

[0018] Furthermore, the application includes using the ancestral nitrilase, mutant or recombinant cell as a catalyst to catalyze the conversion of nitrile compounds into carboxylic acids and ammonia.

[0019] Beneficial effects of the present invention:

[0020] Based on the ASSMD (Ancestral Sequence-Structure-Molecular Dynamics Parameter Library) strategy, this study discovered a kinetically flexible depression in the nitrilase evolutionary landscape (the RMSD values ​​of ancestral nitrilase enzymes in this region are significantly lower than those in other regions). Consequently, these ancestral enzymes were experimentally characterized, leading to the discovery of a series of extremely thermophilic ancestors capable of withstanding temperatures as high as 90°C. Furthermore, to gain a deeper understanding of nitrilase evolution, the team combined energy calculations and laboratory evolution to further analyze its evolutionary events in an effort to enhance its thermostability. This led to the generation of a series of mutants with further improved enzyme activity and thermostability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flowchart of the ASSMD strategy.

[0022] Figure 2 Discovering kinetically flexible depressions in the evolutionary landscape for nitrilases.

[0023] Figure 3 Characterization of the activity and extreme thermophilicity of ASR135.

[0024] Figure 4 To analyze the evolutionary events of ASR135 and construct the evolutionary mutation library.

[0025] Figure 5 Construction and activity characterization of ASR135 combinatorial mutation library. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0027] The solution involved in the present invention is:

[0028] The wild-type ancestral nitrilase is obtained by mining the following method: collecting the sequence of nitrilase from a database, analyzing the evolutionary landscape of nitrilase through phylogenetic tree, obtaining the amino acid sequences of multiple ancestral enzymes located at the branch points of the phylogenetic tree, and constructing a primary structure library; performing tertiary structure prediction on the obtained multiple ancestral enzymes to construct a tertiary structure library; performing molecular dynamics simulation on the tertiary structure to obtain the root mean square deviation, screening out the ancestral enzyme with the lower root mean square deviation, and obtaining the ancestral enzyme of nitrilase. The method for obtaining the root mean square deviation is as follows: first performing a molecular dynamics simulation on the ancestral enzyme for 30ns, and after the molecular dynamics simulation is completed, collecting the root mean square deviation RMSD value during the dynamics simulation process (which measures the stability of the carbon atoms of the nitrilase during the molecular dynamics simulation process). In the present invention, the convergence value of RMSD is the average value after the molecular dynamics simulation RMSD is stabilized.

[0029] Based on the ASSMD strategy, we discovered the extremely thermophilic ancestral nitrilase and its thermostability-beneficial residue sites in the kinetic flexibility depression, and obtained a series of high-performance mutants by performing individual or combined mutations.

[0030] The materials and methods involved in the following examples are as follows:

[0031] (1) Sequence:

[0032] Wild-type nitrilase amino acid sequence (SEQ ID NO.1):

[0033] MVTYTNKFKAATVQAEPVWFDAAATVEKTIGLIKEAASNNAQIIAFPEVFIPGYPYHIWLDSPFAAIGMGKFAVRYHEQSLSIDSPLITRIQEAARSNNISVVIGFSERDGGSLYMSQLIIDENGKIVAHRRKLKPTHVERTVFGEGDGSDIAVHDMPDIGRVG ALNCWEHFQTLTKYAMYAMHEQVHIAAWPGMSLYQPEVFAFSSEAQLVATQMYAMEGQTFVLCSTQVVGKAAHEFFCENEMHKKLIGYGGGFAQIFGPDGRPLAERLPADGEGILYAEIDLAQITMAKQAADPVGHGGPDPFPLPFWQGQSPVPRLGDTGGTESP

[0034] Highly specific fragment of nitrilase (SEQ ID NO. 2):

[0035] GGPDPFPLPFWQGQSPVPRLGDTGGTESP

[0036] Wild-type nitrilase nucleotide sequence (SEQ ID NO.3):

[0037] ATGGTTACTTATACCAATAAGTTTAAGGCAGCTACAGTACAGGCAGAGCCCGTATGGTTCGATGCAGCGGCAACTGTTGAGAAAACCATTGGTCTTATTAAAGAGGCTGCGTCAAACAACGCGCAAATTATTGCGTTTCCTGAAGTTTTTATCCCTGGGTACCCGTATCATATCTGGTTGGATAGTCCTTTCGCGGCGATCGGAATGGGGAAGTTCGCAGTACGTTATCATGAGCAATCACTTTCAATCGACAGCCCCCTTATTACCCGCATCCAGGAGGCCGCCCGCTCAAATAACATCAGTGTAGTTATTGGATTTTCTGAACGCGACGGCGGTTCACTGTACATGTCCCAATTAATTATTGATGAAAACGGTAAGATCGTGGCCCATCGTCGTAAGTTGAAGCCGACCCATGTGGAGCGCACTGTTTTCGGCGAAGGGGACGGATCTGACATCGCCGTACACGATATGCCGGATATTGGACGCGTAGGGGCTTTGAATTGTTGGGAGCATTTCCAAACTCTTACGAAGTACGCCATGTACGCTATGCACGAGCAGGTTCACATCGCAGCATGGCCGGGGATGTCTCTTTACCAACCTGAAGTGTTCGCTTTTAGCTCGGAGGCTCAGTTGGTCGCTACACAGATGTACGCGATGGAGGGTCAAACATTCGTGTTATGCAGTACGCAGGTCGTGGGCAAGGCTGCACATGAGTTTTTTTGTGAGAACGAAATGCACAAAAAATTGATCGGGTACGGTGGCGGCTTCGCGCAGATCTTCGGGCCAGATGGGCGTCCTTTAGCTGAACGTTTGCCTGCTGACGGAGAGGGAATCTTATATGCGGAAATTGACTTAGCTCAAATCACAATGGCAAAGCAAGCCGCAGATCCGGTGGGTCACGGCGGCCCCGACCCCTTCCCCTTACCCTTTTGGCAGGGCCAATCTCCAGTCCCCCGCTTAGGCGATACCGGAGGCACCGAGTCTCCCTAA

[0038] (2) Enzyme expression plasmid construction, heterologous expression and protein purification

[0039] The gene sequences of the ancestral enzyme and the existing nitrilase were synthesized with codon optimization and subsequently inserted into the pET-3b plasmid vector using the NdeI and BamHI restriction endonuclease sites. In addition, a 6×His tag was introduced at the N-terminus of the enzyme for purification purposes. The plasmids were transformed into Escherichia coli BL21 (DE3). The recombinant E. coli was cultured in 10 mL of LB medium at 37°C and 220 rpm for 8-12 hours. Subsequently, 300 μL of the culture was transferred to 30 mL of LB medium and cultured for an additional 7-10 hours to express the nitrilase.

[0040] Escherichia coli cells were harvested by centrifugation at 5000 × g for 10 minutes at 4 ° C. The cell pellet was resuspended in PBS buffer (100mM, pH 7.2) and sonicated at 4 ° C for 45 minutes (10 seconds on / off cycle) to promote cell lysis. The cell lysate was then centrifuged at 6000 × g for 30 minutes at 4 ° C to separate cell debris. The supernatant was applied to a Ni-NTA agarose column equilibrated with dialysis buffer. After thoroughly washing the column with dialysis buffer, the target protein was eluted using elution buffer (100mM Tris-HCl, 200mM imidazole, pH 8.0). After protein purification, the purity of the protein was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie brilliant blue. Protein concentration was determined using a BCA protein assay kit.

[0041] (3) Protein denaturation temperature (Tm) detection

[0042] The Tm values ​​of nitrilase and mutants were determined using a circular dichroism (CD) spectrometer (JASCO-1700). The temperature was varied from 50°C to 90°C at a rate of 1°C / min. The pure enzyme was diluted to 0.2 mg mL with sodium phosphate buffer (100 mM, pH 7.2). -1 , used for Tm determination. The detection wavelength range was 180-260 nm. Protein circular dichroism (CD) spectra acquired at different temperatures were analyzed using Global 3 software to determine the calculated Tm value.

[0043] (4) Evolutionary landscape construction and ancestral sequence reconstruction

[0044] Nitrilase sequences from various species were obtained from databases. Multiple sequence alignments of the amino acid sequences were performed using MEGA-X. Evolutionary relationships among the sequences were determined using the maximum likelihood method and a JTT matrix-based model. A phylogenetic tree was again constructed using MEGA-X. Finally, the ancestral sequence of the nitrilase was reconstructed using the PAML 4.9j and EasyCodeML packages.

[0045] (5) Structural modeling, structural analysis, and interaction calculation

[0046] In the experiment, AlphaFold2 was used to predict the 3D structures of all ancestral enzymes, PpNit and BbNit. The AlphaFold2 package was deployed and installed on a cloud computing server for direct protein structure prediction. PyMol (https: / / pymol.org / 2 / ) was used for visual analysis of the structure files (PDB).

[0047] (6) Molecular dynamics simulation

[0048] Before molecular dynamics simulations, H++ (http: / / newbiophysics.cs.vt.edu / H++ / php) was used for protein protonation state analysis and protonation structure file generation. The GROMACS2021.3 software package was used for molecular dynamics (MD) simulations using AMBER99SB-ILDN (protein) at 300 K. The protein was placed in a cubic box and solvated with water (TIP 3-point), with the cubic box boundary distance set to 0.8 nm. Then, Na + and Cl - The charge of the system was neutralized to make it neutral (salt concentration was 0.15 M). Before the molecular dynamics simulation, the system was first energy minimized. The steepest descent method was used for energy minimization. When the potential energy gradient value was less than or equal to 1000.0 kJ·mol -1 nm -1 The simulation terminated at . The maximum step size along the gradient direction was 0.01 nm, and the maximum allowed step size was 50,000. Then, while maintaining the constraints, the entire system was simulated in the NVT ensemble for 50,000 steps. The time step was 2 fs and the temperature was 300 K. Subsequently, the entire system was simulated in the NPT ensemble for 50,000 steps with a time step of 2 fs and a temperature of 300 K. This was followed by a formal molecular dynamics simulation.

[0049] (7) Construction of nitrilase mutants

[0050] The recombinant plasmid pET-3b-Nit was used as a template for site-directed mutagenesis using Phanta DNA polymerase. A one-step inverse PCR was performed on the entire plasmid to introduce the corresponding mutations into the nitrilase DNA coding sequence. After PCR, the target fragment was isolated and purified by nucleic acid gel electrophoresis. The purified product was digested with DpnI to remove the template plasmid. After the digestion reaction, the product was cooled on ice and 10 μL of the digestion product was transformed into the cloning host Escherichia coli JM109. Transformants were grown and confirmed by sequencing. Once successful, the plasmid was extracted and transformed into Escherichia coli BL21(DE3) for recombinant expression of the mutant nitrilase.

[0051] (8) Nitrilase enzyme activity determination method

[0052] A standard enzyme activity assay was performed by mixing a substrate solution (3-cyanopyridine, 50 mM final concentration) and a nitrilase reaction in PBS (100 mM, pH 7.2). After preincubation in a metal bath at 30°C for 5 min, 1 mL of the suspension was reacted at 30°C, 1500 rpm for 10 min. The reaction was terminated by centrifugation at 12,000 g for 1 min. Enzyme activity was determined by measuring the ammonia produced in the reaction mixture. The assay is based on a phenol-hypochlorite colorimetric method. One unit of enzyme activity (1 U) is defined as the amount of enzyme that produces 1 μmol of ammonia per minute under the above-mentioned standard conditions. All assays were performed in triplicate. The products and substrates of the nitrilase-catalyzed reaction were detected by HPLC using an XterraMS C18 column at a wavelength of 254 nm. The mobile phase was acetonitrile and 0.01% formic acid (gradient elution) at a flow rate of 1 mL / min and a temperature of 30°C.

[0053] Example 1: Designing an ASSMD strategy to mine molecular information of ancestral enzymes in the evolutionary landscape

[0054] Enzymes co-evolve with their hosts, adapting to a wide variety of environments during evolution. These enzymes have undergone a long evolutionary journey, encountering a wide variety of environments. Extremophiles favored for acquisition may face the risk of extinction during evolution. Therefore, we hypothesized that the landscape of enzyme evolution from ancient times to the present day might reveal extremely thermophilic ancestral enzymes. Numerous ancestral enzymes exist across multiple branches of this landscape. Collecting as much molecular information as possible about these ancestral enzymes could significantly improve the efficiency of discovering these ancestral enzymes. Therefore, we developed a workflow to collect molecular information about all ancestral enzymes in this landscape to guide the discovery of new enzymes.

[0055] Initially, evolutionary trees proved effective in depicting the evolutionary landscape of enzymes, with each ancestral enzyme located at a branch point on the tree ( Figure 1.A). Over time, these ancestral enzymes may have become extinct today. Fortunately, we can use ancestral enzyme reconstruction techniques to infer the amino acid sequences of all ancestral enzymes in the evolutionary landscape, thereby gaining structural insights into these ancestral enzymes ( Figure 1 .B). However, the information we have at the primary structure (amino acid sequence) level is inherently limited. To overcome these limitations, we utilized the highly accurate protein structure prediction program AlphaFold 2.0. This enabled us to obtain the 3D structures of the ancestral enzymes and to establish a comprehensive protein structure library for these ancestral enzymes ( Figure 1 .C). Finally, in order to simulate the working environment of the ancestral enzyme, molecular dynamics simulations were then performed on each ancestral enzyme structure. In addition, the dynamic trajectories and parameters of the ancestral enzyme were collected to establish a dynamic parameter library for revealing the biochemical characteristics of the ancestral enzyme ( Figure 1 Overall, the workflow we developed enables the creation of an ancestral enzyme-sequence-structure-molecular dynamics parameter library based on the evolutionary landscape. This library is used to systematically reveal the physicochemical properties of each ancestral enzyme, thereby mining ancestral enzymes of extreme thermophiles. This workflow is called the ASSMD strategy.

[0056] Example 2: Discovery of an extremely thermophilic ancestral nitrilase in a kinetically flexible depression based on the ASSMD strategy

[0057] Given the powerful application characteristics of nitrilase in catalyzing nitrile hydrolysis, it was selected as a research template in the present invention. 71 nitrilase sequences from different species were screened from the database to construct an evolutionary landscape. The evolutionary landscape is represented by a circular evolutionary tree ( Figure 1 ). Subsequently, based on the above ASSMD strategy ( Figure 1 ), we established an ancestral enzyme sequence-structure-molecular dynamics parameter library of nitrilase (69 ancestral enzymes).

[0058] Among them, the root mean square deviation (RMSD) value of the molecular dynamics simulation of the ancestral enzyme is particularly noteworthy. The root mean square deviation (RMSD) can provide insight into the motion of backbone atoms, which largely reflects the structural rigidity of protein dynamics. In the molecular dynamics simulation of the ancestral enzyme in the evolutionary landscape, the overall convergence of the RMSD value tends to be close to about 0.76nm ( Figure 2 .B). However, within specific clade clusters ( Figure 2 .A), the RMSD convergence values ​​of the seven ancestral enzymes were significantly lower than the overall trend (ASR129: 0.47, ASR130: 0.41, ASR131: 0.39, ASR132: 0.28, ASR133: 0.30, ASR134: 0.47, ASR135: 0.34). This observation in the evolutionary landscape indicates the existence of a dynamic flexibility depression ( Figure 2.C). This suggests that the seven ancestral enzymes within this kinetic flexibility depression exhibit significant structural rigidity. We speculate that they may have experienced extreme environmental conditions. Therefore, extreme thermophilic enzymes may have been discovered within this kinetic flexibility.

[0059] These seven ancestral enzymes were successfully heterologously expressed in Escherichia coli and their enzymatic activities were characterized. The results showed that except for ASR131 and ASR133, all five ancestral enzymes showed enzymatic activity ( Figure 3 Specifically, ASR134 and ASR135 showed 4.18 U·mL -1 and 3.66 U·mL -1 It is noteworthy that after ASR135 was incubated at 90°C for 10 min, the enzyme activity of ASR135 retained 11.69%, while the other six ancestral enzymes lost all their enzyme activities under the same conditions ( Figure 3 This indicates that ASR135 has extremely high heat resistance. In addition, the half-life of ASR135 at 80°C reaches 100.25 min ( Figure 3 .B).

[0060] Based on its high temperature tolerance and enzyme activity, ASR135 was selected as the research object. Subsequently, we investigated the evolutionary events that led to the extreme heat tolerance of ASR135. We traced the evolutionary path from ASR134 (the ancestor of ASR135) to ASR135 and then to the current nitrilase BbNit (from β-Proteobacterium MOLA81453) and PpNit (from Pseudomonas putida CGMCC3830) as a single evolutionary event ( Figure 4 .A). During evolution, the sequences of the four nitrilases accumulated many mutations at three different times, resulting in an overall sequence identity of only 89%. This evolutionary event was studied to understand the evolution of thermostability of ASR135. The results showed that the ancestor of ASR135, ASR134, exhibited poor thermostability, with a half-life of only 16.86 hours at 40°C. The half-life of ASR135 at 40°C was 13.01 times that of ASR134, reaching 219.39 hours ( Figure 3 .C). As natural evolution progressed, ASR135 evolved into the current forms of nitrilase, BbNit, and PpNit. The thermal stability of BbNit and PpNit was significantly reduced, with half-lives at 40°C being only 23% and 11% of that of ASR135, respectively. Figure 3.C). In addition, the denaturation temperature (Tm) of ASR135 is 80.57°C, which is significantly higher than that of its ancestor ASR134 (68.03°C) and its descendants BbNit (74.06°C) and PpNit (71.41°C). This suggests that ASR135 evolved from the low-thermostability ASR134, gradually losing its extreme thermophilic properties over time and evolving into its current forms (BbNit, PpNit). Therefore, it is reasonable to assume that ASR135 may have evolved extreme thermostability due to the extreme high temperature environment experienced by its host species. In other words, ASR135 is an extreme thermophilic enzyme derived from an extreme microorganism. Subsequently, the optimal reaction temperatures of these four nitrilases showed that ASR134 (50°C), BbNit (60°C), and PpNit (60°C) all exhibited mesophilic properties. In contrast, ASR135 showed a significantly higher optimal reaction temperature of 80°C, and the enzyme activity of ASR135 at 80°C was found to be 12 times higher than that at 30°C, indicating that it has obvious thermophilic properties ( Figure 3 These experimental observations further support the above hypothesis. Furthermore, the C-terminus of the amino acid sequence of ASR135 (SEQ ID NO. 2), which plays an important role in the multimeric assembly of nitrilase, contains a highly specific sequence segment.

[0061] Example 3: Laboratory evolution to further improve the thermal stability of ASR135

[0062] The experimental results and analysis showed that during the evolutionary events of ASR135, the thermal stability showed a trend of first increasing and then decreasing ( Figure 4 A). It is speculated that during the evolution of the ancestral enzyme ASR135 to its current form, changes in the host's habitat may have led to a loss of extreme environmental stress, thus reducing its extreme thermostability. Generally speaking, regardless of the evolutionary period, although ASR135 is significantly more thermostable than ASR134, PpNit, and BbNit, the latter three nitrilases may have had residues that could have further improved ASR135's thermostability during this evolutionary event. These sites are defined as ASR135 thermostability-beneficial residues (TBRS).

[0063] To identify these TBRS, we divided it into three steps, namely multiple sequence alignment, energy calculation, and mutation experimental verification. Initially, based on the multiple sequence alignment of ASR135 with ASR134, BbNit, and PpNit, the non-conserved amino acid residues in this evolutionary event were identified. Then, based on the different non-conserved amino acid residues, we constructed 55 virtual single-point mutants using a computational algorithm (RosettaCartesian_ddg) and calculated the ΔΔGfold values ​​( Figure 4.B). In theory, negative ΔΔGfold values ​​indicate a beneficial effect on protein folding and stability. Among the 55 virtual single mutants, 40 mutation sites with negative ΔΔGfold were selected. Subsequently, these 40 mutation sites were introduced into ASR135 for experimental verification. In addition, considering the limitations of computational accuracy, continuous multi-point mutations and amino acid insertions and deletions were directly verified experimentally. Therefore, a total of 48 mutants were constructed in this experiment ( Figure 4 .C, D). The thermal stability of ASR135 mutants was characterized by the residual enzyme activity after incubation at 90°C for 10 min ( Figure 4 To obtain high-performance mutants, the relative enzyme activity of wild-type ASR135 (ASR135-WT) was also characterized ( Figure 4 Among these mutants, four mutants showed improved thermostability and relative enzyme activity, namely S97E, S101A, N124H and H155Y ( Figure 5 .A). Subsequently, in order to achieve synergistic enhancement, 6 double mutants, 2 triple mutants and 1 quadruple mutant were constructed based on these 4 mutants. The experimental results showed that there was a significant synergistic effect between these mutants. Among them, the residual enzyme activity of the single mutant N124H was better than that of the wild-type enzyme in the thermal stability experiment. The residual enzyme activity of the double mutant N124H / H155Y was further improved compared with the single mutant, and the triple mutant S97E / S101A / N124H was even better ( Figure 5 .B).

[0064] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An ancestral nitrilase mutant, characterized in that The ancestral nitrilase mutant is obtained by mutating the ancestral nitrilase with an amino acid sequence as shown in SEQ ID NO.1, wherein the mutation comprises: Mutate asparagine at position 124 to histidine; or mutating asparagine at position 124 to histidine and histidine at position 155 to tyrosine; Alternatively, the serine at position 97 is mutated to glutamic acid, the serine at position 101 is mutated to alanine, and the asparagine at position 124 is mutated to histidine.

2. A polynucleotide encoding the ancestral nitrilase mutant of claim 1.

3. A gene expression cassette carrying the polynucleotide of claim 2.

4. The gene expression cassette according to claim 3, characterized in that The expression cassette includes elements necessary for gene expression and / or elements assisting expression.

5. A recombinant expression vector carrying the polynucleotide according to claim 2.

6. A recombinant cell expressing the ancestral nitrilase mutant of claim 1.

7. The recombinant cell according to claim 6, characterized in that The host cell of the recombinant cell is a non-plant cell; the host cell is Escherichia coli.

8. A method for producing an ancestral nitrilase mutant, characterized in that The method comprises the steps of using the recombinant cell according to claim 6 or 7 for fermentation production.

9. Use of the ancestral nitrilase mutant according to claim 1, the polynucleotide according to claim 2, the gene expression cassette according to claim 3 or 4, the recombinant expression vector according to claim 5, or the recombinant cell according to claim 6 or 7 in hydrolyzing nitrile compounds.

10. The use according to claim 9, characterized in that The application includes: using the ancestral nitrilase mutant or recombinant cell as a catalyst to catalyze the conversion of nitrile compounds into carboxylic acids and ammonia.

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