A method for mining target ancestral enzymes and its applications

Abstract

The present invention discloses a method for mining target ancestral enzymes and its applications. The present invention designs an ancestral enzyme sequence-structure-molecular dynamics strategy to obtain information on extinct ancestral enzymes in the evolutionary landscape, and applies it to the mining of extremely thermophilic ancestral nitrilases to verify the feasibility of the method. Specifically, sequences of nitrilases are collected for phylogenetic tree analysis to obtain the evolutionary landscape, and the amino acid sequences of ancestral enzymes are obtained by combining the ancestral enzyme reconstruction algorithm to construct a library of primary structure sequences of ancestral enzymes; subsequently, the tertiary structures of all ancestral enzymes are predicted to obtain a structure library; finally, molecular dynamics simulations are performed on the tertiary structures of all ancestral enzymes to obtain a library of kinetic parameters, and screening is carried out through specific kinetic parameters. Based on this strategy, the present invention obtains an ancestral nitrilase that can tolerate 90 °C, and evolves its thermal stability, and the obtained mutants can exhibit nitrilase activity at 100 °C.

Description

Technical Field

[0001] The present invention relates to a method for mining target ancestral enzymes and its applications, belonging to the technical field of enzyme engineering. Background Art

[0002] In recent decades, the progress of genetic engineering and protein engineering has promoted the development of the biocatalysis field, 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 extremozymes from extremophiles exhibit good thermostability, exploring extremozyme resources is the most direct method 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, T.D. et al. Journal of bacteriology 98(1), 289-97). The thermophilic xylanase from the thermophilic bacterium Herbinix hemicellulosilytica has application potential in the high-temperature processing of cellulose and wood products (Mechelke, M. et al. Journal of biotechnology 257, 122-130). In addition, Ni et al. constructed a whole-cell Escherichia coli biocatalyst using trans-feruloyl-CoA synthase and enoyl-CoA hydratase from the thermophilic bacterium Amycolatopsis thermoflava N1165 to produce vanillin at high temperatures (Ni et al. Angewandte Chemie-International Edition 57(5), 1214-1217). However, the living environment of their host extremophiles is extremely harsh, requiring specific nutritional requirements or growth conditions, which are difficult to replicate in the laboratory, so they are relatively difficult to discover and culture. Correspondingly, with the change of the natural environment, during the evolution of extremophiles to adapt to a milder environment, extremozymes may also become extinct.

[0003] With the development of fields such as bioinformatics and computational biology, using the enzyme ancestral sequence reconstruction method, we can use computer algorithms to deduce the amino acid sequences of ancient enzymes from extinct organisms. Previous studies mainly focused on resurrecting the most ancient ancestral forms at the root of the evolutionary tree. However, during the evolution of enzymes, from the most ancient ancestors to the extant forms, enzymes have experienced numerous environments, including extreme conditions. These environmental challenges may have exerted selective pressures that could have led to the adaptation and development of favorable characteristics in ancestral enzymes, such as enhanced thermal stability. Therefore, in this evolutionary landscape, it is very likely that we can find extremely thermophilic ancestral enzymes, thus leading to the present invention. Summary of the Invention

[0004] To solve the above problems, the present invention provides a workflow for constructing a library of ancestral enzyme sequence-structure-kinetic parameters, namely the ASSMD strategy, by revealing molecular-level information of the evolutionary landscape, and uses this strategy to discover a thermophilic nitrilase (EC 3.5.5.1), which can catalyze the hydrolysis of nitrile compounds into carboxylic acids and is widely used in many fields such as food, medicine, and chemical engineering.

[0005] The first object of the present invention is to provide a method for mining target ancestral enzymes, including the following steps:

[0006] S1. Collect the sequences of the enzymes to be mined from the database, analyze the evolutionary landscape of the enzymes to be mined by phylogenetic tree, obtain the amino acid sequences of various ancestral enzymes located at the branch points of the phylogenetic tree, and construct a primary structure library;

[0007] S2. Predict the tertiary structures of the various ancestral enzymes obtained in S1 and construct a tertiary structure library;

[0008] S3. Perform molecular dynamics simulations on the tertiary structures in S2 to obtain the root mean square deviation (RMSD), screen out the ancestral enzymes with lower RMSD values, and obtain the target ancestral enzymes.

[0009] Furthermore, in step S3, the method for obtaining the root mean square deviation is as follows: First, perform a 30-ns molecular dynamics simulation on the ancestral enzyme. After the molecular dynamics simulation ends, collect the RMSD values (measuring the stability of the carbon atoms of the nitrilase during the molecular dynamics simulation) during the kinetic simulation process. In the present invention, the convergence value of RMSD is the average value after the RMSD of the molecular dynamics simulation stabilizes.

[0010] Furthermore, the enzyme to be mined includes nitrilase.

[0011] The second object of the present invention is to provide an ancestral nitrilase or its mutant, and the amino acid sequence of the ancestral nitrilase is the sequence shown in SEQ ID NO.1 or a sequence with a homology of not less than 80%, preferably not less than 90%.

[0012] Furthermore, the ancestral nitrilase mutant is obtained by mutating the ancestral nitrilase shown in SEQ ID NO.1, and the mutations include:

[0013] mutating serine at position 97 to glutamate;

[0014] or mutating serine at position 101 to alanine;

[0015] or mutating asparagine at position 124 to histidine;

[0016] or mutating histidine at position 155 to tyrosine;

[0017] or mutating serine at position 97 to glutamate and mutating serine at position 101 to alanine;

[0018] or mutating serine at position 97 to glutamate and mutating asparagine at position 124 to histidine;

[0019] or mutating serine at position 97 to glutamate and mutating histidine at position 155 to tyrosine;

[0020] or mutating serine at position 101 to alanine and mutating asparagine at position 124 to histidine;

[0021] or mutating serine at position 101 to alanine and mutating histidine at position 155 to tyrosine;

[0022] or mutating asparagine at position 124 to histidine and mutating histidine at position 155 to tyrosine;

[0023] or mutating serine at position 97 to glutamate, mutating serine at position 101 to alanine and mutating asparagine at position 124 to histidine;

[0024] or mutating serine at position 97 to glutamate, mutating serine at position 101 to alanine and mutating histidine at position 155 to tyrosine;

[0025] or mutating serine at position 97 to glutamate, mutating asparagine at position 124 to histidine and mutating histidine at position 155 to tyrosine;

[0026] or mutating serine at position 101 to alanine, mutating asparagine at position 124 to histidine and mutating histidine at position 155 to tyrosine;

[0027] Alternatively, serine at the 97th position is mutated to glutamate, serine at the 101st position is mutated to alanine, asparagine at the 124th position is mutated to histidine, and histidine at the 155th position is mutated to tyrosine.

[0028] The third object of the present invention is to provide a gene encoding the ancestral nitrilase or its mutant.

[0029] Furthermore, the sequence of the gene encoding the ancestral nitrilase is shown as SEQ ID NO.3.

[0030] The fourth object of the present invention is to provide a recombinant expression vector carrying the gene.

[0031] The fifth object of the present invention is to provide an expression cassette containing the gene.

[0032] Furthermore, the expression cassette includes the necessary elements and / or auxiliary expression elements for expressing the gene, such as promoters, terminators, signal peptides, RBS, etc.

[0033] The sixth object of the present invention is to provide a recombinant cell expressing the ancestral nitrilase or its mutant.

[0034] Furthermore, the host cell of the recombinant cell includes microorganisms, such as Escherichia coli.

[0035] The seventh object of the present invention is to provide a method for producing the ancestral nitrilase or its mutant, including the step of fermentative production using the recombinant cell.

[0036] The eighth object of the present invention is to provide the application of the ancestral nitrilase or its mutant, gene, recombinant expression vector, expression cassette or recombinant cell in hydrolyzing nitrile compounds.

[0037] 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.

[0038] The ninth object of the present invention is to provide a specific fragment of nitrilase, and the amino acid sequence of the specific fragment is shown as SEQ ID NO.2. This specific fragment can be used for identification, mechanism research, etc.

[0039] The beneficial effects of the present invention:

[0040] The present invention discovers based on the ASSMD (ancestral enzyme sequence-structure-molecular dynamics parameter library) strategy that there are kinetic flexibility depressions in the evolutionary landscape of nitrilase (the RMSD value of the ancestral nitrilase in this region is significantly lower than that in other regions). Therefore, these ancestral enzymes were experimentally characterized, and a series of extremely thermophilic ancestors that can tolerate temperatures up to 90 °C were discovered. This strategy can be extended to the discovery of ancestral enzymes of other types of enzymes, providing a new strategy for enzyme design technology.

[0041] In order to gain a deeper understanding of the evolutionary information of nitrilase, the present invention further analyzed its evolutionary events by combining energy calculations and laboratory evolution in order to enhance its thermal stability, and thus obtained a series of mutants. The optimal quadruple mutant still exhibits hydrolysis activity even at the boiling temperature (100 °C). Brief Description of the Drawings

[0042] Figure 1 It is a schematic flow diagram of the ASSMD strategy.

[0043] Figure 2 It shows the discovery of kinetic flexibility depressions in the evolutionary landscape of nitrilase.

[0044] Figure 3 It shows the activity and extreme thermophilicity characterization of ASR135.

[0045] Figure 4 It shows the analysis of the evolutionary events of ASR135 and the construction of an evolutionary mutant library.

[0046] Figure 5 It shows the construction and activity characterization of the combinatorial mutant library of ASR135. Detailed Embodiments

[0047] The present invention will be further described below in conjunction with the 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 specific embodiments cited are not intended to limit the present invention.

[0048] The materials and methods involved in the following embodiments are as follows:

[0049] (1) Sequences:

[0050] Wild-type nitrilase amino acid sequence (SEQ ID NO.1):

[0051] MVTYTNKFKAATVQAEPVWFDAAATVEKTIGLIKEAASNNAQIIAFPEVFIPGYPYHIWLDSPFAAIGMGKFAVRYHEQSLSIDSPLITRIQEAARSNNISVVIGFSERDGGSLYMSQLIIDENGKIVAHRRKLKPTHVERTVFGEGDGSDIAVHDMPDIGRVGALNCWEHFQTLTKYAMYAMHEQVHIAAWPGMSLYQPEVFAFSSEAQLVATQMYAMEGQTFVLCSTQVVGKAAHEFFCENEMHKKLIGYGGGFAQIFGPDGRPLAERLPADGEGILYAEIDLAQITMAKQAADPVGHGGPDPFPLPFWQGQSPVPRLGDTGGTESP

[0052] Highly specific fragment in nitrilase (SEQ ID NO.2):

[0053] GGPDPFPLPFWQGQSPVPRLGDTGGTESP

[0054] Wild-type nitrilase nucleotide sequence (SEQ ID NO.3):

[0055] ATGGTTACTTATACCAATAAGTTTAAGGCAGCTACAGTACAGGCAGAGCCCGTATGGTTCGATGCAGCGGCAACTGTTGAGAAAACCATTGGTCTTATTAAAGAGGCTGCGTCAAACAACGCGCAAATTATTGCGTTTCCTGAAGTTTTTATCCCTGGGTACCCGTATCATATCTGGTTGGATAGTCCTTTCGCGGCGATCGGAATGGGGAAGTTCGCAGTACGTTATCATGAGCAATCACTTTCAATCGACAGCCCCCTTATTACCCGCATCCAGGAGGCCGCCCGCTCAAATAACATCAGTGTAGTTATTGGATTTTCTGAACGCGACGGCGGTTCACTGTACATGTCCCAATTAATTATTGATGAAAACGGTAAGATCGTGGCCCATCGTCGTAAGTTGAAGCCGACCCATGTGGAGCGCACTGTTTTCGGCGAAGGGGACGGATCTGACATCGCCGTACACGATATGCCGGATATTGGACGCGTAGGGGCTTTGAATTGTTGGGAGCATTTCCAAACTCTTACGAAGTACGCCATGTACGCTATGCACGAGCAGGTTCACATCGCAGCATGGCCGGGGATGTCTCTTTACCAACCTGAAGTGTTCGCTTTTAGCTCGGAGGCTCAGTTGGTCGCTACACAGATGTACGCGATGGAGGGTCAAACATTCGTGTTATGCAGTACGCAGGTCGTGGGCAAGGCTGCACATGAGTTTTTTTGTGAGAACGAAATGCACAAAAAATTGATCGGGTACGGTGGCGGCTTCGCGCAGATCTTCGGGCCAGATGGGCGTCCTTTAGCTGAACGTTTGCCTGCTGACGGAGAGGGAATCTTATATGCGGAAATTGACTTAGCTCAAATCACAATGGCAAAGCAAGCCGCAGATCCGGTGGGTCACGGCGGCCCCGACCCCTTCCCCTTACCCTTTTGGCAGGGCCAATCTCCAGTCCCCCGCTTAGGCGATACCGGAGGCACCGAGTCTCCCTAA

[0056] (2) Enzyme expression plasmid construction, heterologous expression, and protein purification

[0057] The gene sequences of the ancestral enzyme and the extant nitrilase were codon-optimized and synthesized, and then inserted into the pET-3b plasmid vector using the NdeI and BamHI restriction enzyme sites. In addition, a 6×His tag was introduced at the N-terminus of the enzyme for purification purposes. The plasmid was transformed into Escherichia coli BL21(DE3). The recombinant E. coli was cultured in 10 mL LB medium at 37 °C and 220 rpm for 8 - 12 hours. Subsequently, 300 μL of the culture was transferred to 30 mL LB medium and cultured for another 7 - 10 hours to express nitrilase.

[0058] Escherichia coli cells were harvested by centrifugation at 5000×g for 10 minutes at 4 °C. The cell pellet was resuspended in PBS buffer (100 mM, pH 7.2) and sonicated at 4 °C for 45 minutes (10-second on / off cycle) to facilitate cell lysis. Then the cell lysate was 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 (100 mM Tris-HCl, 200 mM imidazole, pH 8.0). After protein purification, the purity of the protein was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie Brilliant Blue. The protein concentration was determined using a BCA protein assay kit.

[0059] (3) Detection of protein denaturation temperature (Tm)

[0060] The Tm values of nitrilase and mutants were detected using a circular dichroism (CD) spectrometer (JASCO-1700). The temperature range was from 50 to 90 °C, and the temperature change rate was 1 °C / min. The pure enzyme was diluted to 0.2 mg·mL -1 using sodium phosphate buffer (100 mM, pH 7.2) for Tm determination. The detection wavelength range was 180 - 260 nm. The protein circular dichroism (CD) spectra obtained at different temperatures were analyzed using Global 3 software to determine the calculated Tm values.

[0061] (4) Construction of the evolutionary landscape and reconstruction of the ancestral sequence

[0062] Nitrile hydratase sequences from different species were obtained from the database. The above amino acid sequences were subjected to multiple sequence alignment using MEGA-X. The evolutionary relationships among the above sequences were based on the maximum likelihood method and the JTT matrix-based model. The phylogenetic tree was again constructed by MEGA-X. Finally, the ancestral sequences of nitrile hydratase were reconstructed using PAML 4.9j and the EasyCodeML software package.

[0063] (5) Structure modeling, structure analysis, and interaction calculations

[0064] In the experiment, Alphafold2 was used to predict the 3D protein structures of all ancestral enzymes PpNit and BbNit. The AlphaFold2 package was deployed and installed in the cloud computing server and directly used for protein structure prediction. PyMol (https: / / pymol.org / 2 / ) was used for visual analysis of the structure file (PDB).

[0065] (6) Molecular dynamics simulation

[0066] Before molecular dynamics simulation, H++ (http: / / newbiophysics.cs.vt.edu / H++ / php) was used for protein protonation state analysis and the generation of protonated structure files. The GROMACS2021.3 software package was used for molecular dynamics (MD) simulation, using AMBER99SB-ILDN (protein) at 300K. The protein was placed in a cubic box and solvated with water (TIP 3-point), and the cubic box boundary distance was set to 0.8 nm. Then, + and Cl - were used to neutralize the charge of the system to make it neutral (salt concentration of 0.15 M). Before molecular dynamics simulation, the system was first energy minimized. The steepest descent method was used for energy minimization, and the termination occurred when the potential energy gradient value was less than or equal to 1000.0 kJ·mol -1 ·nm -1 At that time. The maximum step size along the gradient direction was 0.01 nm, and the maximum allowed step size was 50000. Then, with constraints maintained, a 50000-step NVT ensemble simulation of the entire system was performed. The time step was 2 fs, and the temperature was 300K. Subsequently, a 50000-step NPT ensemble simulation of the entire system was performed with a time step of 2 fs and a temperature of 300K. Subsequently, the formal molecular dynamics simulation was carried out.

[0067] (7) Construction of nitrile hydratase mutants

[0068] The recombinant plasmid pET-3b-Nit was used as a template for site-directed mutagenesis using Phanta DNA polymerase. The corresponding mutations were introduced into the DNA coding sequence of nitrilase by one-step inverse PCR of the whole plasmid; 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 DpnI 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 taken and 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 recombinant expression of the mutant nitrilase.

[0069] (8) Method for determining nitrilase enzyme activity

[0070] The standard enzyme activity assay was carried out by mixing the substrate solution (3-cyanopyridine, final concentration 50 mM) and nitrilase in PBS (100 mM, pH 7.2). After pre-incubating in a metal bath at 30 °C for 5 min, 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 254 nm. The mobile phase was acetonitrile and 0.01% formic acid (gradient elution), the flow rate was 1 mL / min, and the temperature was 30 °C.

[0071] Example 1: Design of the ASSMD strategy to mine molecular information of ancestral enzymes in the evolutionary landscape

[0072] Enzymes co-evolve with their hosts and have adapted to a variety of environments during the evolutionary process. These enzymes have experienced a long evolutionary history and encountered various environments. The extreme enzymes that people favor may have faced the risk of extinction during the evolutionary process. Therefore, we hypothesized that it is possible to mine extreme thermophilic ancestral enzymes in the enzyme evolutionary landscape from ancient times to the present. There are a large number of ancestral enzymes in multiple branches of the evolutionary landscape. Collecting as much molecular information as possible about these ancestral enzymes can significantly improve the efficiency of discovering extreme thermophilic ancestral enzymes. Therefore, we developed a workflow to collect the molecular information of all ancestral enzymes in the evolutionary landscape to guide the discovery of new enzymes.

[0073] At first, phylogenetic trees were proven to be effective in depicting the evolutionary landscape of enzymes, and each ancestral enzyme was 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 obtaining structural insights into these ancestral enzymes ( Figure 1 .B). However, the information we obtain at the primary structure (amino acid sequence) level is limited in itself. To overcome these limitations, we use the AlphaFold 2.0 algorithm of a high-precision protein structure prediction program. These enable us to obtain the 3D structures of ancestral enzymes and establish a comprehensive protein structure library of these ancestral enzymes ( Figure 1 .C). Finally, to simulate the working environment of ancestral enzymes, molecular dynamics simulations are then performed on each ancestral enzyme structure. In addition, the dynamic trajectories and parameters of ancestral enzymes are collected to establish a kinetic parameter library for revealing the biochemical characteristics of ancestral enzymes ( Figure 1 ). Overall, the workflow we developed can establish an ancestral enzyme-sequence-structure-molecular dynamics parameter library based on the evolutionary landscape, which is used to systematically reveal the physicochemical properties of each ancestral enzyme to mine the ancestral enzymes of extreme thermophiles. This workflow is called the ASSMD strategy.

[0074] Example 2: Discovery of an extremely thermophilic ancestral nitrilase in the kinetic flexibility depression based on the ASSMD strategy

[0075] Given the strong application characteristics of nitrilase in catalyzing nitrile hydrolysis, it was selected as the research template in this invention. 71 nitrilase sequences from different species were screened from the database to construct the evolutionary landscape. The evolutionary landscape is represented by a circular phylogenetic 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).

[0076] Among them, the root mean square deviation (RMSD) values of the molecular dynamics simulations of ancestral enzymes are particularly worthy of attention. The root mean square deviation (RMSD) can provide insights into the movement of backbone atoms, which largely reflects the structural rigidity of protein dynamics. In the molecular dynamics simulations of ancestral enzymes in the evolutionary landscape, the overall convergence of RMSD values tends to approach approximately 0.76 nm ( Figure 2 .B). However, within a specific evolutionary branch cluster ( Figure 2 .A), the RMSD convergence values of 7 ancestral enzymes are 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 shows the existence of kinetic flexibility depressions ( Figure 2.C). This indicates that these 7 ancestral enzymes within this kinetic flexibility depression exhibit obvious structural rigidity. We speculate that they may have experienced extreme environmental conditions. Therefore, extreme thermophilic enzymes may be discovered within this kinetic flexibility.

[0077] These 7 ancestral enzymes were successfully heterologously expressed in Escherichia coli, and their enzyme activities were characterized. The results showed that except for ASR131 and ASR133, five ancestral enzymes exhibited enzyme activities ( Figure 3 ). Specifically, ASR134 and ASR135 showed relatively high enzyme activities of 4.18 U·mL -1 and 3.66 U·mL -1 , respectively. Notably, after ASR135 was incubated at 90 °C for 10 min, 11.69% of the enzyme activity of ASR135 was retained, while the other 6 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 reached 100.25 min ( Figure 3 .B).

[0078] Based on the high temperature resistance and enzyme activity, ASR135 was selected as the research object. Subsequently, we studied the evolutionary events that led to the extreme heat resistance of ASR135. We traced the evolutionary path from ASR134 (the ancestor of ASR135) to ASR135, and then to the existing nitrilases BbNit (derived from β-proteobacterium MOLA81453) and PpNit (derived 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, and the overall sequence identity was only 89%. This evolutionary event was studied to understand the evolution of the thermal stability of ASR135. The results showed that the ancestor of ASR135, ASR134, exhibited poor thermal stability, and its half-life at 40 °C was only 16.86 h. The half-life of ASR135 at 40 °C was 13.01 times that of ASR134, reaching 219.39 h ( Figure 3 .C). As natural evolution proceeded, ASR135 evolved into the existing forms of nitrilases BbNit and PpNit. The thermal stabilities of BbNit and PpNit were significantly reduced, and their half-lives at 40 °C were 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 indicates that ASR135 evolved from ASR134 with low thermal stability, gradually lost its extreme thermophilic characteristics over time, and evolved into the existing forms (BbNit, PpNit). Therefore, it is reasonable to assume that ASR135 may have evolved extreme thermal stability due to its host species experiencing extreme high-temperature environments. In other words, ASR135 is an extremely thermophilic enzyme derived from extremophiles. Subsequently, the optimal reaction temperatures of these 4 nitrilases showed that ASR134 (50 °C), BbNit (60 °C), and PpNit (60 °C) all exhibited mesophilicity. 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 its obvious thermophilic characteristics ( Figure 3 .D). These experimental observations further supported the above hypothesis. Moreover, the C-terminal of the amino acid sequence of ASR135 (SEQ ID NO.2), which plays an important role in the multimer assembly of nitrilase, has a highly specific sequence fragment.

[0079] Example 3: Laboratory evolution to further improve the thermal stability of ASR135

[0080] The experimental results and analysis showed that during the evolution of ASR135, the thermal stability showed a trend of increasing first and then decreasing ( Figure 4 .A). It is speculated that during the evolution of the ancestral enzyme ASR135 to the existing form, it may be due to the change in the living environment of the host, losing the pressure of the extreme environment, thus reducing the extreme thermal stability. Generally speaking, regardless of the evolutionary period, although the thermal stability of ASR135 is significantly higher than that of ASR134, PpNit, and BbNit, the latter 3 nitrilases may have residue sites, which can further improve the thermal stability of ASR135 in this evolutionary event. These sites are defined as the beneficial residue sites for the thermal stability of ASR135 (TBRS).

[0081] To identify these TBRS, we divided it into three steps, namely multiple sequence alignment, energy calculation, and mutant experiment 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 determined. Then, based on different non-conserved amino acid residues, we constructed 55 virtual single-point mutants using a computational algorithm (RosettaCartesian_ddg) and calculated the ΔΔGfold values of the 55 single-point mutations ( Figure 4.B). Theoretically, a negative ΔΔGfold value indicates 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-site mutations as well as amino acid insertions and deletions were directly verified through experiments. 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 .C). To obtain high-performance mutants, the relative enzyme activity of wild-type ASR135 (ASR135-WT) was also characterized ( Figure 4 .D). Among these mutants, four mutants showed improved thermal stability and relative enzyme activity, namely S97E, S101A, N124H, and H155Y ( Figure 5 .A). Subsequently, 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 among these mutants. The mutant S97E / S101A / N124H / H155Y (ASR135-M4) showed very high performance ( Figure 5 .B). Compared with ASR135-WT, the residual enzyme activity of ASR135-M4 increased from 11.69% to 43.23% ( Figure 5 .B) In addition, compared with ASR135-WT, the Tm value of ASR135-M4 increased by 6.2 °C, reaching 86.77 °C.

[0082] It is worth noting that ASR135-M4 can tolerate boiling water. We first heated the substrate solution to 100 °C. Under the condition of 100 °C, ASR135-M4 still retained catalytic activity, and the enzyme activity was 5.82 U·mL -1 ( Figure 5 .C-F). As Figure 5 shown, at 100 °C, ASR135-M4 could detect nicotinic acid in the 3-cyanopyridine conversion solution by HPLC. However, ASR135-WT could not carry out the enzymatic reaction under the reaction conditions of 100 °C ( Figure 5 .D).

[0083] In summary, by integrating sequence differences, energy calculations, and mutant library construction, the beneficial residue sites for thermal stability in the ASR135 evolution event can be further explored. This is of great significance for the protein engineering of enzymes.

[0084] Obviously, the above embodiments are merely 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 enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. An ancestral nitrilase mutant, characterized in that The ancestral nitrilase mutant is obtained by constructing an ancestral enzyme sequence and a structure-kinetic parameter library method. The ancestral nitrilase mutant is obtained by mutating the ancestral nitrilase whose amino acid sequence is shown in SEQ ID NO.

1. The mutation includes: The serine at position 97 was mutated to glutamic acid, the serine at position 101 was mutated to alanine, the asparagine at position 124 was mutated to histidine, and the histidine at position 155 was mutated to tyrosine.

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

3. A recombinant expression vector carrying the gene according to claim 2.

4. An expression cassette containing the gene according to claim 2.

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

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

7. The use according to claim 6, 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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