A nitrile hydrolase mutant with substrate universality and its application

Abstract

This invention relates to a substrate-universal nitrile hydrolase mutant and its applications, belonging to the field of genetic engineering technology. This invention employs molecular dynamics simulations combined with Rosetta energy calculations to systematically analyze the Loop region (amino acids 190-203) near the active pocket of the nitrile hydrolase, identifying key sites G191, L194, V199, and A201 that control the binding state of the active pocket. Through evolutionary conservation analysis and enzyme activity assays, G191 and A201 were determined to be key regulatory sites affecting substrate transport, and precise regulation was achieved through saturation mutations. Experiments show that single mutations of G191S and A201G can significantly enhance the catalytic activity against aniline acetonitrile, and for combined mutations, the combined mutation of G191S and A201G can significantly increase the enzyme activity against aniline acetonitrile.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a nitrile hydrolase mutant with substrate universality and its application. Background Technology

[0002] Nitrilases (EC 3.5.5.1) are enzymes that catalyze the hydrolysis of nitrile compounds (RC≡N) to the corresponding carboxylic acids. They are the only enzymes in the 13 branches of the nitrile hydrolase superfamily with the ability to hydrolyze nitrile groups. Nitrilases can hydrolyze toxic nitrile compounds containing cyano groups into non-toxic carboxylic acids, making them highly valuable in the pharmaceutical industry. Nitriles are chemical substances containing cyano functional groups (RC≡N), and are important raw materials and intermediates in organic synthesis, capable of undergoing a series of derivatization transformations involving functional groups such as aldehydes, ketones, carboxylic acids, and amides. Traditional chemical hydrolysis reactions of nitrile compounds require relatively harsh conditions, while the biocatalysis of nitrile hydrolases enables a green and efficient conversion of this process.

[0003] Nitrile hydrolases have shown broad application prospects in the catalysis of aromatic nitrile compounds. Among the many substrates for nitrile hydrolases, aromatic nitrile compounds, due to their rigid molecular structure, high hydrophobicity, and large steric hindrance, often have difficulty binding efficiently to the enzyme's active site, resulting in low conversion and catalytic efficiency in the catalytic process. Aniline acetonitrile can be catalyzed by nitrile hydrolases to produce phenylglycine, an important non-natural α-amino acid intermediate widely used in the side-chain synthesis of β-lactam antibiotics, especially ampicillin, which, as a first-generation broad-spectrum penicillin antibiotic, is widely used to treat Gram-positive and some Gram-negative bacterial infections; cephalexin, as a first-generation cephalosporin drug, also relies on the supply of phenylglycine intermediates.

[0004] Currently, the main industrial methods for producing phenylglycine include chemical routes such as reduction after cyanation, asymmetric hydrogenation, or indirect hydrolysis. However, these methods generally suffer from problems such as cumbersome reaction steps, low raw material utilization, poor enantioselectivity, numerous byproducts, and difficulties in treating waste, making it difficult to meet the needs of green manufacturing and large-scale preparation of high-value-added pharmaceutical intermediates. Biosynthesis, on the other hand, is simple and efficient and is now widely used. Among these methods, using nitrile hydrolases to hydrolyze aniline acetonitrile to synthesize phenylglycine is a common biosynthetic method. However, existing nitrile hydrolases still suffer from low activity towards aniline acetonitrile. Furthermore, the substrate range of existing nitrile hydrolases is also relatively narrow. Therefore, there is an urgent need for a nitrile hydrolase with high activity towards aniline acetonitrile and a broad substrate range. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a nitrile hydrolase with high enzyme activity and substrate universality in the prior art.

[0006] To address the aforementioned technical problems, this invention provides a nitrile hydrolase mutant with substrate universality and its applications. This invention employs molecular dynamics simulations combined with Rosetta energy calculations to systematically analyze the Loop region (amino acids 190-203) near the active pocket of the nitrile hydrolase, identifying key sites G191, L194, V199, and A201 that control the binding state of the active pocket. Through evolutionary conservation analysis and enzyme activity assays, G191 and A201 were determined to be key regulatory sites affecting substrate transport, and precise regulation was achieved through saturation mutagenesis. Experiments show that the G191S and A201G mutations significantly enhance the catalytic activity towards aniline acetonitrile. Furthermore, the combined mutation of G191S and A201G significantly increases the enzyme activity towards aniline acetonitrile. In addition, the nitrile hydrolase mutant of this invention also exhibits substrate universality, possessing enzyme activity against numerous compounds in both aliphatic and aromatic nitrile compounds.

[0007] The first object of the present invention is to provide a nitrile hydrolase mutant, said nitrile hydrolase mutant being one or more of the following mutations to the nitrile hydrolase with the amino acid sequence shown in SEQ ID NO.1:

[0008] (1) Glycine at position 191 is mutated to serine;

[0009] (2) The leucine at position 194 is mutated to tryptophan;

[0010] (3) The valine at position 199 is mutated to lysine;

[0011] (4) The alanine at position 201 is mutated to glycine.

[0012] Furthermore, SEQ ID NO.1:

[0013] MVAYTNKFRAATVQAEPVWFDAAATVEKSIGLIKEAASNDAQIIAFPEVFIPGYPYHIWLDSPFAAMGKFAVRYHEQSLPIDSPLIVRLQLAAERNSISVVMGFSERDGGTLYMSQIIINEHGNIVAHRRKLKPTHVERTVFGEGDGSDIAVYDMAVGRVGALNCWEHFQTL TKYAMYAMHEQIHIAAWPGMSLYQPEVYAFSSEAQSVATQMYAMEGQTFVLCATQVVGPSAHETFCLNDEQRALIGYGGGFAQIFGPDGRALADRLPGDGEGILYAEINLAEIAMAKAAADPVGHYARPDVTRLLLNRTERRPVEYATYAAPSAEHFDDASSVSQVTGPSQ.

[0014] Furthermore, compared with the amino acid sequence shown in SEQ ID NO.1, the nitrile hydrolase mutant has any of the following sets of mutations:

[0015] (1)G191S;

[0016] (2)L194W;

[0017] (3)V199K;

[0018] (4)A201G;

[0019] (5) G191S and L194W;

[0020] (6) G191S and V199K;

[0021] (7) G191S and A201G;

[0022] (8) L194W and V199K;

[0023] (9) L194W and A201G;

[0024] (10) V199K and A201G;

[0025] (11) G191S, L194W and V199K;

[0026] (12) G191S, L194W and A201G;

[0027] (13) G191S, V199K and A201G;

[0028] (14) L194W, V199K and A201G;

[0029] (15) G191S, L194W, V199K and A201G.

[0030] A second objective of this invention is to provide a gene encoding the aforementioned nitrile hydrolase mutant.

[0031] Furthermore, the gene is shown in SEQ ID NO.2:

[0032]

[0033] A third objective of this invention is to provide a recombinant plasmid carrying the aforementioned nitrile hydrolase mutant.

[0034] A fourth objective of this invention is to provide a recombinant cell expressing the above-described nitrile hydrolase mutant, wherein the host of the recombinant cell includes bacteria or fungi.

[0035] A fifth objective of this invention is to provide the application of the above-mentioned nitrile hydrolase mutant, the above-mentioned gene, the above-mentioned recombinant plasmid, or the above-mentioned recombinant cell in the hydrolysis of aniline acetonitrile.

[0036] The application involves adding a nitrile hydrolase mutant or an expression system containing the nitrile hydrolase mutant to a reaction system containing aniline acetonitrile.

[0037] A sixth objective of this invention is to provide a method for hydrolyzing aliphatic nitrile compounds, wherein the above-mentioned nitrile hydrolase mutant is added to a system containing aliphatic nitrile compounds to carry out the hydrolysis reaction.

[0038] Furthermore, the aliphatic nitrile compound is selected from one or more of methylaminoacetonitrile, aminoacetonitrile, iminoglycolic acid, 3-aminopropionitrile, 3-methylaminopropionitrile, 3-methoxypropionitrile, 3-hydroxypropionitrile, cyclopropionitrile, acrylonitrile, 2-butenonitrile, butyronitrile, valerate, 3-chloropropionitrile, 5-chlorovalerate, ethyl cyanoacetate, and citrile.

[0039] A seventh object of the present invention is to provide a method for hydrolyzing aromatic nitrile compounds by adding the above-mentioned nitrile hydrolase mutant to a substrate containing aromatic nitrile compounds to carry out the hydrolysis reaction.

[0040] Furthermore, the aromatic nitrile compound is selected from one or more of aniline acetonitrile, benzonitrile, phenylacetonitrile, p-hydroxyphenylacetonitrile, 3-cyanopyridine, 3-thiophene acetonitrile, 2-thiophene acetonitrile, 2-cyanopyrazine, 2-cyanopyrimidine, and 2-cyanofuran.

[0041] The beneficial effects of this invention are:

[0042] This invention, through the investigation of mutation sites, discovered that a combined mutation of G191S and A201G in nitrile hydrolase can enhance the enzyme activity of nitrile hydrolase for aniline acetonitrile, achieving an enzyme activity of 6.26 U·mL. -1 The activity was increased by 7 times compared to the initial strain. In addition, the nitrile hydrolase mutant of this invention also exhibits substrate universality, possessing enzymatic activity for a wide range of aliphatic and aromatic nitrile compounds. Attached Figure Description

[0043] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0044] Figure 1 It is the binding state of fatty nitriles and aromatic nitriles to the enzyme activity pocket;

[0045] Figure 2 This is a saturated mutant library of G191, L194, V199 and A201 sites and enzyme activity characterization, where Bb represents wild type and N-Phenylglycinonitrile represents aniline acetonitrile;

[0046] Figure 3 It is a site-directed and combinatorial mutation of key sites and enzyme activity characterization. In the combinatorial mutation, S represents G191S, W represents L194W, K represents V199K, and G represents A201G.

[0047] Figure 4 The figure shows the results of a kinetic simulation of wild-type nitrile hydrolase and nitrile hydrolase mutants with combined mutations of G191S and A210G with the substrate aniline acetonitrile for 200 ns.

[0048] Figure 5 It is a substrate spectral network;

[0049] Figure 6 It is an enzyme activity characterization based on substrate profiles. Detailed Implementation

[0050] 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 and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0051] The experimental methods involved in the following embodiments are as follows:

[0052] (1) Recombinant expression of nitrile hydrolase in Escherichia coli host

[0053] Using the *E. coli* expression plasmid pET-3b as the expression vector, the recombinant plasmid pET-3b-BbNit, containing either a mutant or wild-type nitrile hydrolase gene, was transformed into the expression host *E. coli* BL21(DE3) via heat shock. The culture was carried out at 37°C for 12 h on LB agar plates containing 100 μg / mL ampicillin. Positive colonies were inoculated into 10 mL of LB medium containing 100 μg / mL ampicillin for seed culture and cultured for 10–12 h. Subsequently, a 1% inoculum was transferred to 30 mL of medium for fermentation to express the recombinant nitrile hydrolase.

[0054] (2) Methods for the isolation and purification of recombinant enzymes

[0055] The cultured bacterial cells were collected by low-temperature centrifugation and suspended in buffer A (50 mM sodium phosphate, 500 mM sodium chloride, pH 7.4). The bacterial cells were disrupted by ultrasonication or high-pressure homogenization. Cell debris was removed by filtration or ultra-high-speed centrifugation to prepare a cell-free extract. The extract was then 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 at a detection wavelength of 280 nm. Elution peaks at each stage were collected, and the molecular weight and purity of the purified fungal nitrile hydrolase were analyzed by SDS-PAGE.

[0056] (3) Method for determining the activity of nitrile hydrolase

[0057] Standard enzyme activity assays were performed by mixing the substrate solution (final concentration 50 mM) and nitrile hydrolase 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 12000 × g for 1 min. Enzyme activity was determined by measuring the ammonia produced in the reaction mixture. Enzyme activity assays were performed 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 nitrile hydrolase-catalyzed reaction were detected by HPLC using an Xterra MS C18 column at 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.

[0058] Example 1: Construction of a mutant library of nitrile hydrolases

[0059] like Figure 1 As shown, molecular docking of the substrate molecule with nitrile hydrolase was performed, and the most reasonable complex structure with the lowest docking binding energy was selected for analysis. The substrate surrounding... The residues within this range form the active pocket, and the substrate channel connecting the external environment to the active site was identified. Using loop engineering, molecular dynamics simulations combined with Rosetta energy calculations, we systematically analyzed the loop region (amino acids 190-203) at the entrance of the nitrile hydrolase substrate channel, identifying key sites G191, L194, V199, and A201 that control the channel opening and closing dynamics.

[0060] Example 2: Enzyme activity assay of nitrile hydrolase mutant

[0061] Through evolutionary conservation analysis and enzyme activity assays, G191, L194, V199, and A201 were identified as key regulatory sites affecting substrate transport, and precise regulation was achieved through saturation mutagenesis. Experiments showed that mutations in G191S, L194W, V199A, and A201G significantly enhanced the catalytic activity towards aniline acetonitrile (e.g., ...). Figure 2 and Figure 3 (As shown).

[0062] Using the successfully constructed recombinant expression plasmid pET-BbNit (pET-BbNit is a gene encoding wild-type nitrile hydrolase linked to plasmid pET), recombinant plasmids pET-G191S, pET-L194W, pET-V199K, and pET-A201G were constructed using pre-designed amino acid mutation primers. One-step inverse PCR was performed on the entire plasmid to introduce corresponding mutations into the nitrile hydrolase DNA coding sequence. The PCR program was as follows: 95℃ for 10 min, 95℃ for 30 s, 60℃ for 30 s, 72℃ for 5 min, 34 cycles, with a final extension time of 10 min. After PCR, the target fragment was isolated and purified by nucleic acid gel electrophoresis. The purified product was digested with DpnI enzyme to remove the template plasmid. After digestion, the product was cooled on ice, and 10 μL of the digestion product was transformed into the cloning host *E. coli* JM109. After transformants grew, bacteria were selected for sequencing verification. After successful validation, plasmids were extracted and transformed into *E. coli* BL21(DE3) for recombinant expression of nitrile hydrolase mutants. After isolation and purification, the recombinant bacteria yielded three nitrile hydrolase mutants: G191S (glycine at position 191 mutated to serine), L194W (leucine at position 194 mutated to tryptophan), V199K (valine at position 199 mutated to lysine), and A201G (alanine at position 201 mutated to glycine). Enzyme activity was measured using aniline acetonitrile as a substrate. Experimental results showed that G191S, V199K, and A201G all exhibited good enzyme activity with aniline acetonitrile (see [link to relevant documentation]). Figure 3 ).

[0063] Table 1. Primers used for mutation of G191, L194, V199, and A201.

[0064] G191S-F GCATGGCCCTCGATGAGTCTTTATCAACCGGAAG G191S-R AAGACTCATCGAGGGCCATGCTGCGATGTGAATTTG L194W-F GGCATGAGTTGGTATCAACCGGAAGTATATGCATTCTCC L194W-R CGGTTGATACCAACTCATGCCAGGCCATGCAG V199K-F CAACCGGAAAAGTATGCATTCTCCTCGGAGGCAC V199K-R GAATGCATACTTTTCCGGTTGATAAAGACTCATGCCAGG A201G-F GAAGTATATGGTTTCTCCTCGGAGGCACAGAGTG A201G-R CGAGGAGAAACCATATACTTCCGGTTGATAAAGACTCATG G191S / L194W-F GCATGGCCCTCGATGAGTTGGTATCAACCGGAAGTATATGCATTCTC G191S / L194W-R CGGTTGATACCAACTCATCGAGGGCCATGCTGCGATGTGAATTTG L194W / V199K-F GGCATGAGTTGGTATCAACCGGAAAAGTATGCATTCTCCTCGGAGG L194W / V199K-R GAATGCATACTTTTCCGGTTGATACCAACTCATGCCGGGCCATGC L194W / A201G-F ATGAGTTGGTATCAACCGGAAGTATATGGTTTCTCCTCGGAGGCACAGAG L194W / A201G-R CGAGGAGAAACCATATACTTCCGGTTGATACCAACTCATGCCGGGCCATG V199K / A201G-F CAACCGGAAAAGTATGGTTTCTCCTCGGAGGCACAGAGTGTTGCGACTC V199K / A201G-R CGAGGAGAAACCATACTTTTCCGGTTGATAAAGACTCATGCCGGGCCATG

[0065] Example 3: Construction and optimization of nitrile hydrolase co-mutants

[0066] To investigate the synergistic effects at different sites, we optimized the dominant mutants (G191S, L194W, V199K, and A201G) on the flexible loop using two, three, and four combinations. The optimal synergistic mutant was G191S / A201G. This mutant exhibited an enzyme activity of 6.26 U·mL⁻¹ against aniline acetonitrile. -1 It increased by 7 times compared to the initial strain (e.g. Figure 3 (As shown). In addition, among these fourteen mutants, L194W, WG, and SWG showed a sharp decrease in the enzyme activity of aniline acetonitrile, while the remaining mutants showed an increase in the catalytic activity of aniline acetonitrile.

[0067] To further investigate the mechanism by which the G191S / A210G combined mutant enhances the activity of aniline acetonitrile enzyme, this invention performed 200 ns kinetic simulations on BbNit and the G191S / A210G mutant with the substrate aniline acetonitrile, respectively, and obtained the RMSD values ​​of the BbNit and G191S / A210G mutant systems with aniline acetonitrile in the 200 ns molecular dynamics simulations. Figure 4 A); and RMSF value analysis of G191S / A210G mutants ( Figure 4 C); the distance between the Sγ atom of C165 and the C atom of the substrate cyano group ( Figure 4 B) To gain a deeper understanding of the molecular mechanisms underlying the enhanced activity of nitrile hydrolases.

[0068] Example 4: Substrate profile of nitrile hydrolase mutant

[0069] In addition, the present invention is also based on Figure 5 The substrates were analyzed, and the enzyme activities (e.g., substrates) of nitrile hydrolases obtained by combined mutations of G191S, L194W, V199K, A201G, G191S and A201G, and L194W and V199K on fatty nitrile and aromatic nitrile substrates were determined. Figure 6 (As shown).

[0070] The results showed that wild-type BbNit exhibited detectable enzyme activity for only 19 of the 26 tested substrates. Site modification significantly broadened the substrate spectrum and improved the catalytic activity of some mutants. Specifically, the G191S mutant catalyzed 24 of the 26 substrates, demonstrating the broadest substrate adaptability. The A201G mutant catalyzed 22 substrates. Notably, the L194W single-site mutant generally showed increased catalytic activity for aliphatic nitrile substrates, but significantly decreased or even lost activity for aromatic nitrile substrates.

[0071] All tested mutants collectively covered 24 out of 26 substrates. These results indicate that the L194 site is one of the key residues for BbNit nitrile hydrolases in catalyzing aromatic nitrile substrates. Mutations targeting this site (such as L194W) significantly weaken or even eliminate the enzyme's catalytic ability for aromatic nitriles. Therefore, if the modification target requires maintaining or enhancing the enzyme's activity for aromatic nitriles, the L194 site mutation strategy should be carefully considered.

[0072] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A nitrile hydrolase mutant, characterized in that, The nitrile hydrolase mutant is any one of the following mutations performed on the nitrile hydrolase with the amino acid sequence shown in SEQ ID NO. 1: (1) Mutate glycine at position 191 to serine and alanine at position 201 to glycine; (2) Mutate valine at position 199 to lysine and alanine at position 201 to glycine.

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

3. A recombinant plasmid carrying the gene described in claim 2.

4. A recombinant cell expressing the nitrile hydrolase mutant of claim 1, characterized in that, The host of the recombinant cells includes bacteria or fungi.

5. The application of the nitrile hydrolase mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3, or the recombinant cell of claim 4 in the hydrolysis of aniline acetonitrile.

6. The application according to claim 5, characterized in that, The application involves adding a nitrile hydrolase mutant or an expression system containing the nitrile hydrolase mutant to a reaction system containing aniline acetonitrile.

7. A method for hydrolyzing aliphatic nitrile compounds, characterized in that, The nitrile hydrolase mutant was added to a system containing aliphatic nitrile compounds to carry out the hydrolysis reaction. The aliphatic nitrile compound is selected from one or more of methylaminoacetonitrile, aminoacetonitrile, iminoglycolic acid, 3-aminopropionitrile, 3-methylaminopropionitrile, 3-methoxypropionitrile, acrylonitrile, 2-butenonitrile, pentonitrile, 3-chloropentonitrile and ethyl cyanoacetonitrile; The nitrile hydrolase mutant is a nitrile hydrolase with the amino acid sequence shown in SEQ ID NO.1 that has undergone the following mutations: glycine at position 191 is mutated to serine and alanine at position 201 is mutated to glycine.

8. A method for hydrolyzing aromatic nitrile compounds, characterized in that, The nitrile hydrolase mutant was added to a system containing aromatic nitrile compounds to carry out the hydrolysis reaction. The aromatic nitrile compound is selected from one or more of aniline acetonitrile, benzonitrile, p-hydroxyphenylacetonitrile, 3-cyanopyridine, 3-thiophene acetonitrile, 2-thiophene acetonitrile, 2-cyanopyrazine, and 2-cyanofuran; The nitrile hydrolase mutant is a nitrile hydrolase with the amino acid sequence shown in SEQ ID NO.1 that has undergone the following mutations: glycine at position 191 is mutated to serine and alanine at position 201 is mutated to glycine.

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