Nitrilase combined mutant and application thereof in whole-cell biological catalysis

Abstract

The invention relates to a nitrilase combined mutant and application thereof in whole-cell biological catalysis, and belongs to the technical field of biological catalysis. Through semi-rational design, a double-combination mutant is constructed based on a loop engineering strategy, and the catalytic activity of nitrilase on methylaminoacetonitrile can be remarkably enhanced. The enzyme activity of the nitrilase combined mutant to methylaminoacetonitrile can reach 6.69 U.mL <-1 > and is increased by 8.5 times compared with that of an initial strain. Whole-cell catalysis conditions are further optimized, the nitrilase combined mutant has the highest activity under the conditions that the temperature is 30 DEG C and the pH is 7.2 through a PBS buffer solution, and 75 mM methylaminoacetonitrile can be efficiently converted at the cell concentration of 7.5 g.L <-1 >. The invention provides a novel enzyme tool and process support for efficient biological catalysis of methylaminoacetonitrile.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, in particular to nitrilase combination mutants and applications thereof in whole-cell biocatalysis. Background Art

[0002] Sarcosine is an N-methylated amino acid with broad application prospects in daily chemicals, aquaculture, food, healthcare, and medicine. Sarcosine has the potential to serve as a recovery drug for post-operative brain surgery and brain injury, as well as an antipsychotic. It can be developed as an ingredient in health supplements, sports drinks, and feed. Currently, industrial sarcosine synthesis primarily relies on sodium sarcosinate acidification, specifically using the chloroacetic acid method, the hydroxyacetonitrile method, and the methylethanolamine method. However, these methods are generally subject to challenges such as high raw material transportation costs, high toxicity, harsh reaction conditions, and excessive risk.

[0003] In contrast, biocatalysis offers advantages such as mild reaction conditions, environmental friendliness, high efficiency, and excellent stereoselectivity, aligning with the development of green chemistry and gaining increasing attention. Recent studies have demonstrated that the imine reductase DpkA can catalyze the synthesis of sarcosine using glyoxylate and methylamine as substrates, relying on the reducing power of NADPH. However, this pathway is expensive. Nitrilases, which catalyze the hydrolysis of methylaminoacetonitrile to produce sarcosine in a single step, have emerged as a promising alternative. Nitrilases, through the Cys-Glu-Lys catalytic triad structure in their active centers, directly hydrolyze the cyanide group of the substrate to the corresponding carboxyl group. This catalytic process eliminates the need for amide formation, resulting in lower costs, easier controllable conditions, and greater operability. These enzymes have shown promising application prospects in fields such as biopharmaceuticals, fine chemicals, and environmental protection. However, wild-type nitrilases often exhibit insufficient catalytic activity and selectivity for industrially important substrates, such as methylaminoacetonitrile, and suffer from a narrow substrate spectrum, low catalytic activity, and poor stability, making them inadequate for industrial production.

[0004] Flexible regions are often overlooked due to their high flexibility and diversity, but with the deepening of catalytic mechanisms, researchers have begun to pay attention to these regions far away from the active center. In recent years, modification of flexible regions has become an important strategy for regulating enzyme activity, selectivity and substrate range. For example, Liu et al. used computer-assisted methods to perform terminal loop truncation and His tag cleavage on 7α-hydroxysteroid dehydrogenase (Bm7α-HSDH) and found that C-terminal truncation changed the interaction of the substrate binding region, affected the stereoselective contact between the substrate and the active site, and revealed the importance of loop C in substrate binding and catalysis. Liu Song's team developed a Rosetta Cartesian_ddg-based script for the flexible region of the non-substrate binding pocket, predicted the folding free energy of transglutaminase (smTG), and obtained mutants with significantly enhanced specific activity and catalytic efficiency. Zhang et al. performed substitution mutations on the hypervariable loop region of levansucrase and successfully obtained a series of mutants with different product spectra. Heinemann et al. improved the activity and selectivity of cumene dioxygenase by identifying hotspot residues, remodeling the loop region, and linker-based intra-loop insertion (LILI) strategies. The catalytic activity and substrate specificity of nitrilase are highly dependent on its three-dimensional structure, particularly the flexible loop region connecting the active pocket to the external environment. Therefore, there is an urgent need to provide a new generation of biocatalysts that can overcome the efficiency bottleneck of natural nitrilase in the entire process of substrate transport, catalysis, and product release, thereby providing industrial-grade green sarcosine synthesis. Summary of the Invention

[0005] The present invention addresses the technical bottlenecks of low substrate transfer efficiency, poor stability of catalytic intermediates, and limited product release in the process of catalyzing the hydrolysis of methylaminoacetonitrile to synthesize sarcosine by existing nitrilase. The present invention provides a nitrilase combination mutant and its application in whole-cell biocatalysis. The present invention transforms nitrilase based on a computationally guided loop engineering strategy to enhance the catalytic activity of methylaminoacetonitrile. By precisely regulating the dynamics of the loop conformation at the entrance of the substrate channel, balancing the contradiction between flexibility (promoting substrate entry and exit) and rigidity (maintaining stability), a synergistic improvement in enzyme catalytic efficiency and stability is achieved. Through rational design, the substrate specificity, thermal stability, and organic solvent tolerance of the enzyme can be precisely regulated, thereby adapting to specific reaction conditions (such as high substrate concentration, continuous catalytic process) to achieve green and efficient production.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a nitrilase combination mutant, which is a nitrilase with an amino acid sequence as shown in SEQ ID NO.1 as a parent and undergoes any of the following mutations:

[0008] (1) the 194th leucine is mutated into tryptophan, and the 199th valine is mutated into lysine;

[0009] (2) the 194th leucine is mutated into tryptophan, and the 201th alanine is mutated into glycine.

[0010] A second object of the present application is to provide a gene encoding the nitrilase combination mutant.

[0011] A third object of the present application is to provide an expression vector carrying the gene.

[0012] A fourth object of the present application is to provide a recombinant bacterium expressing the nitrilase combination mutant or carrying the expression vector.

[0013] A fifth object of the present application is to provide a method for synthesizing sarcosine, using methylaminoacetonitrile as a substrate, and using the nitrilase combination mutant or the whole cell of the recombinant bacterium as a catalyst to obtain sarcosine.

[0014] In an embodiment of the present application, the concentration of the substrate is 25mM-200mM.

[0015] In an embodiment of the present application, the catalytic conditions are: pH value is 5.0-9.0; temperature is 25℃-60℃.

[0016] In an embodiment of the present application, the reaction system further comprises a cosolvent and a metal ion.

[0017] In an embodiment of the present application, the cosolvent is selected from one or more of methanol, ethanol, propanol, acetone, ethylene glycol, DMSO, DMF, Tween80, TritonX100 and PEG200;

[0018] and / or, the metal ion is selected from one or more of K + , Ba 2+ , Na + , Al 3+ , Li + , Ni 2+ , Co 2+ , Ca 2+ , Mg 2+ , Zn 2+ , Cu 2+ and Fe 2+ ;

[0019] and / or, the final concentration of the metal ion is 1mM-5mM.

[0020] A sixth object of the present invention is to provide use of the nitrilase combination mutant or the recombinant bacterium in preparing sarcosine or products containing sarcosine.

[0021] The present invention uses molecular dynamics simulation combined with Rosetta energy calculation to identify the key sites G191, L194, V199, and A201 that control the dynamics of channel opening and closing. Through evolutionary conservation analysis and enzyme activity determination, L194 and V199 are identified as key regulatory sites affecting substrate transport, and saturation mutagenesis is performed to achieve precise regulation. The double combination mutation of L194W / V199K and L194W / A201G can significantly enhance the catalytic activity of methylaminoacetonitrile. Among them, the enzyme activity of the mutant L194W / V199K towards methylaminoacetonitrile reaches 6.69U·mL -1 , 8.5 times higher than the initial strain.

[0022] Based on the whole-cell catalytic system of the engineered strain, the present invention optimizes the conversion reaction conditions: pH 5.0-9.0, temperature 25°C-60°C, cell loading 0.75 g·L -1 -15.00g·L -1 (wet weight), substrate concentration 25mM-200mM. At the same time, the effects of cosolvents and metal ions on enzyme activity were further studied.

[0023] The above technical solution of the present invention has the following advantages over the prior art:

[0024] This invention provides nitrilase combination mutants and their application in whole-cell biocatalysis. This invention proposes, for the first time, to address the scientific challenge of the "flexibility-rigidity" balance of nitrilase by dynamically regulating the loop. The constructed double combination mutants, L194W / V199K and L194W / A201G, significantly enhance the catalytic activity of p-methylaminoacetonitrile. The accompanying whole-cell process enables the green and efficient synthesis of sarcosine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0026] Figure 1 This is a saturation mutation library of the L194 and V199 sites of the present invention;

[0027] Figure 2Site-directed and combinatorial mutation of the key sites of the application and characterization of enzyme activity; wherein, SW: double mutation G191S / L194W; SK: double mutation G191S / V199K; SG: double mutation G191S / A201G; WK: double mutation L194W / V199K; WG: double mutation L194W / A201G; KG: double mutation V199K / A201G; SWK: triple mutation G191S / L194W / V199K; SWG: triple mutation G191S / L194W / A201G; WKG: triple mutation L194W / V199K / A201G; SWKG: quadruple mutation G191S / L194W / V199K / A201G;

[0028] Figure 3 Characterization of L194W / V199K enzyme activity at different temperatures of the application;

[0029] Figure 4 Characterization of L194W / V199K enzyme activity at different pH of the application;

[0030] Figure 5 Characterization of L194W / V199K enzyme activity at different cell concentrations of the application;

[0031] Figure 6 Characterization of L194W / V199K enzyme activity at different substrate concentrations of the application;

[0032] Figure 7 Characterization of L194W / V199K enzyme activity at different organic solvents of the application;

[0033] Figure 8 Characterization of L194W / V199K enzyme activity at different metal ions of the application. DETAILED DESCRIPTION

[0034] The application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the application and implement it, but the examples are not limiting to the application.

[0035] The experimental methods used in the following examples are conventional methods, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0036] The culture medium used in the following examples is as follows:

[0037] (1) LB medium (g / L): peptone 10, yeast powder 5, NaCl 10.

[0038] (2) LB solid medium (g / L): peptone 10, yeast powder 5, NaCl 10, agar powder 20.

[0039] The primer sequences involved in the following examples are shown in Table 1:

[0040] Table 1

[0041]

[0042] Example 1: Recombinant expression of nitrilase in Escherichia coli host

[0043] The E. coli expression plasmid pET-3b was used as the expression vector, and the recombinant plasmid pET-3b-BbNit with the wild nitrilase gene (nucleotide sequence as shown in SEQ ID NO.2) was transformed into the expression host E. coli BL21 (DE3) by heat shock method. The cells were incubated at 37°C in the presence of 100 μg mL -1 Culture on LB agar plates containing ampicillin resistance for 12 h. Inoculate the positive colonies into 10 mL of ampicillin resistance (100 μg mL -1 ) in LB medium for 10-12 hours. Then, the inoculum size was transferred to 30 mL of culture medium for fermentation to express the recombinant nitrilase.

[0044] Example 2: Nitrilase enzyme activity determination method

[0045] The standard enzyme activity assay was performed by mixing the substrate solution (50 mM final concentration) and the nitrilase reaction in PBS (100 mM, pH 7.2). After pre-incubation in a metal bath at 30°C for 5 min, 1 mL of the suspension was reacted at 30°C and 1500 rpm for 10 min. The reaction was terminated by centrifugation at 12,000 × g for 1 min. The enzyme activity was determined by measuring the ammonia produced in the reaction mixture. The assay was based on the phenol-hypochlorite colorimetric method. One unit of enzyme activity (1 U) was defined as the amount of enzyme that produces 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 an acetonitrile ratio of 95% and a flow rate of 0.6 mL min. -1 , the detection temperature is 30℃.

[0046] Example 3: Construction and transformation of recombinant plasmids of nitrilase combination mutants

[0047] Using the successfully constructed nitrilase recombinant expression plasmid pET-3b-BbNit as a template, a one-step inverse PCR was performed on the entire plasmid using pre-designed amino acid mutation primers 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. After the transformants emerged, they were selected for sequencing verification. After successful verification, the plasmid was extracted and transformed into E. coli BL21(DE3) for recombinant expression of the mutant nitrilase.

[0048] Example 4: Isolation and purification method of recombinant enzyme

[0049] The cells were collected by low-temperature centrifugation after being cultured at 37°C and 220 rpm for 8 hours, suspended in buffer A (50 mM sodium phosphate, 500 mM sodium chloride, pH 7.4), disrupted by ultrasonication or high-pressure homogenization, and cell debris was removed by filtration or ultracentrifugation to prepare a cell-free extract. The extract was separated and purified using a Ni-NTA agarose gel column. The column was balanced with buffer A and gradient eluted with different concentrations of imidazole. The elution process was monitored in real time at a detection wavelength of 280 nm. The elution peaks at each stage were collected, and the molecular weight and purity of the purified nitrilase were analyzed by SDS-PAGE.

[0050] Example 5: Construction of a nitrilase mutant library

[0051] The substrate small molecule was docked with nitrilase, and the most reasonable complex structure with the lowest docking binding energy was selected for analysis. The residues in the range are active pockets, and the substrate channel connecting the external environment and the active site is confirmed. Using loop engineering, molecular dynamics simulation combined with Rosetta energy calculation, the loop region (amino acids 190-203) at the entrance of the nitrilase substrate channel was systematically analyzed, and the key sites G191, L194, V199 and A201 that control the dynamics of channel opening and closing were identified. Through evolutionary conservation analysis and enzyme activity determination, L194 and V199 were identified as key regulatory sites affecting substrate transport, and saturation mutations were performed to achieve precise regulation. The saturation mutation library of L194 and V199 sites is as follows. Figure 1 As shown, the experiments showed that the L194W and V199K mutations can significantly enhance the catalytic activity of methylaminoacetonitrile.

[0052] Example 6: Construction and optimization of nitrilase cooperative mutants

[0053] In order to explore the synergistic effect of different sites, the dominant mutations (G191S, L194W, V199K and A201G) on the flexible loop were optimized in two, three and four combinations. Figure 2 As shown in the figure, it can be seen that the double combination mutations of L194W / V199K and L194W / A201G can significantly enhance the catalytic activity towards methylaminoacetonitrile. The best synergistic mutant obtained is L194W / V199K. The enzyme activity of this mutant towards methylaminoacetonitrile reaches 6.69 U·mL -1 , 8.5 times higher than the initial strain.

[0054] Example 7: Whole-cell catalytic process optimization

[0055] In the catalytic process study of recombinant nitrilase, the method for determining the enzyme activity of nitrilase is shown in Example 2. The following experiment was based on the culture conditions of 37°C and 220 rpm for 8 h, using the controlled variable method to systematically explore the effect of conversion reaction conditions on enzyme activity.

[0056] First, the effect of reaction temperature on enzyme activity was investigated by setting a temperature gradient of 25-60°C. Second, three buffer systems, citric acid-sodium citrate, PBS sodium phosphate, and Tris-HCl, were selected to investigate the effect of different pH on enzyme activity. -1 , 1.50g·L -1 , 3g·L -1 4.50g·L -1 , 7.5g·L -1 、10.50g·L -1 , 15.0g·L -1 ) and substrate concentrations (25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM) were used to investigate the effects of cell concentration and substrate concentration on enzyme activity. In addition, cosolvents (methanol, ethanol, propanol, acetone, ethylene glycol, DMSO, DMF, Tween80, TritonX100, and PEG200) and metal ions (K + 、Ba 2+ 、Na + 、Al 3+ 、Li + 、Ni 2+ 、Co 2+ , Ca 2+ Mg 2+ 、Zn 2+ 、Cu 2+ 、Fe 2+) on enzyme activity. The effects of 1% by volume of a cosolvent and metal ions at final concentrations of 1 mM and 5 mM were analyzed in the reaction system. Figure 3-Figure 8 As shown in the figure, the enzyme has the highest activity at 30°C and pH 7.2 (PBS buffer), 7.5 g·L -1 The cell concentration was sufficient to efficiently convert 75 mM methylaminoacetonitrile. The addition of cosolvents and low concentrations of metal ions maintained the stability of the nitrilase or slightly promoted its activity without compromising enzyme activity, demonstrating its practical application.

[0057] 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. A nitrilase combination mutant, characterized in that: The nitrilase with the amino acid sequence shown in SEQ ID NO.1 is used as a parent, and any of the following mutations is performed: (1) Mutate leucine at position 194 to tryptophan and valine at position 199 to lysine; (2) Mutate the leucine at position 194 to tryptophan and the alanine at position 201 to glycine.

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

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

4. A recombinant bacterium expressing the nitrilase combination mutant of claim 1 or carrying the expression vector of claim 3.

5. A method for synthesizing sarcosine, characterized in that: Sarcosine is obtained by using methylaminoacetonitrile as a substrate and the nitrilase combination mutant according to claim 1 or the recombinant bacterial whole cell according to claim 4 as a catalyst.

6. The method according to claim 5, characterized in that The concentration of the substrate is 25 mM-200 mM.

7. The method according to claim 5, characterized in that The catalytic conditions are: pH value of 5.0-9.0; temperature of 25° C.-60° C.

8. The method according to claim 5, characterized in that The reaction system also includes a cosolvent and metal ions.

9. The method according to claim 5, characterized in that The cosolvent is selected from one or more of methanol, ethanol, propanol, acetone, ethylene glycol, DMSO, DMF, Tween80, TritonX100 and PEG200; And / or, the metal ion is selected from K + 、Ba 2+ 、Na + 、Al 3+ 、Li + 、Ni 2+ 、Co 2+ , Ca 2+ Mg 2+ 、Zn 2+ 、Cu 2+ and Fe 2+ One or more of; And / or, the final concentration of the metal ion is 1 mM-5 mM.

10. Use of the nitrilase combination mutant according to claim 1 or the recombinant bacterium according to claim 4 in the preparation of sarcosine or a product containing sarcosine.

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