CN114107269A

CN114107269A - Modification and application of nitrile hydratase amino acid motif

Info

Publication number: CN114107269A
Application number: CN202111395709.2A
Authority: CN
Inventors: 周哲敏; 程中一; 印猛; 马东; 刘中美; 崔文璟; 周丽; 韩来闯; 郭军玲
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2022-03-01
Anticipated expiration: 2041-11-23
Also published as: CN114107269B

Abstract

The invention provides modification and application of nitrile hydratase amino acid motifs, and belongs to the technical field of bioengineering. The motifs are highly conserved at sites 108, 111 and 113 of the enzyme active center on the alpha subunit, which form coordination bonds with cobalt ions, respectively. Nitrile hydratases from other sources are also highly conserved at these corresponding amino acid residue positions by sequence to structure alignment. According to the invention, the alanine at the 129 th site on the nitrile hydratase beta subunit is mutated, so that the mutant A129R has obviously improved specific enzyme activity compared with a wild type, has a broader substrate spectrum compared with the wild type, and has better catalytic activity on isobutyronitrile, n-valeronitrile, nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, 1-naphthonitrile, thiacloprid and the like.

Description

Modification and application of nitrile hydratase amino acid motif

Technical Field

The invention relates to modification and application of nitrile hydratase amino acid motif, belonging to the technical field of biological engineering.

Background

Nitrile hydratase (NHase for short, EC 4.2.1.84) is a metalloenzyme which can catalyze Nitrile substances to be converted into amide compounds with high added values through hydration reaction, and is widely applied to the industrial production of bulk chemical acrylamide. At present, nitrile hydratase gradually replaces the traditional chemical method with the advantages of environmental protection, mild reaction conditions, high safety factor and the like, so that the production of amides conforms to the sustainable development and green production concept.

Nitrile hydratases are generally composed of two subunits, namely alpha subunit and beta subunit, and researches show that most of the nitrile hydratases from prokaryotes reported at present have the problems of poor stability, low catalytic activity and narrow catalytic substrate spectrum. There have also been many studies attempting to modify them to improve their relevant properties. However, the problems of low catalytic efficiency, narrow substrate spectrum and the like at present still limit the further development and application of nitrile hydratase.

Disclosure of Invention

Aiming at the technical difficulties and problems in the prior art, the invention aims to broaden the substrate spectrum and improve the catalytic performance by modifying the amino acid of nitrile hydratase derived from Pseudonocardia thermophila (Pseudonocardia thermophila).

The present invention provides a nitrile hydratase mutant comprising an alpha subunit and a beta subunit; the mutant is a nitrile hydratase parent beta subunit 129 bit mutation; the amino acid sequence of the alpha subunit of the nitrile hydratase parent is shown as SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 2.

In one embodiment, the mutant is characterized by: the 129 th site of the beta subunit is mutated into arginine, and the mutant A129R is obtained.

The present invention provides a gene encoding the mutant.

The invention provides a recombinant plasmid carrying the gene.

In one embodiment, pET-24a (+) is used as an expression vector.

The invention provides host cells expressing the mutants, or containing the genes.

In one embodiment, the host cell comprises a prokaryotic or eukaryotic microorganism.

In one embodiment, the host cell is escherichia coli as a starting strain, and preferably, e.coli BL21(DE3) as an expression host.

The invention provides a method for producing amide substances, which takes nitrile substances as substrates and utilizes the mutant A129R to catalyze and generate the amide substances.

In one embodiment, the nitrile includes isobutyronitrile, n-valeronitrile, nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, 1-naphthonitrile, and thiacloprid.

In one embodiment, the amide species includes isobutyramide, valeramide, niacinamide, pyrazinamide, benzamide, cinnamamide, naphthalene-1-carboxamide, and thiacloprid amide.

The invention also provides the application of the mutant, the gene, the recombinant plasmid or the host cell in preparing amide substances.

In one embodiment, the gene on the expression vector is linked in the order of a gene encoding a β subunit gene, a gene encoding an α subunit gene, and a gene encoding a regulatory protein.

The invention has the beneficial effects that:the invention provides a Pt-NHase mutant, which mutates alanine at position 129 on beta subunit relative to wild type to obtain mutant A129R. The specific enzyme activity of the mutant is higher for catalysis of different substratesThe wild type is obviously improved, the highest improvement is more than 8 times, the substrate spectrum of the mutant is widened compared with that of the wild type, the mutant has good catalytic action on isobutyronitrile, n-valeronitrile, nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, 1-naphthonitrile, thiacloprid and the like, and the catalytic performance is also obviously improved compared with that of the wild type.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of pure enzyme, M: protein Marker, 1: WT, 2: a 129R.

FIG. 2 shows the specific enzyme activities for nicotinonitrile catalysis of the Pt NHase wild enzyme WT and the mutant A29R.

FIG. 3 shows the catalytic activity of Pt NHase wild enzyme WT and mutant A29R on 8 different nitrile substrates.

FIG. 4 shows the enzyme activity after mutation at position 129 to different amino acids.

Detailed Description

Enzyme activity (U) of nitrile hydratase: the unit enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1. mu. mol amide product from nitrile substrates per minute at 25 ℃.

Specific enzyme activity (U/mg) of nitrile hydratase: the enzyme activity per mg of nitrile hydratase.

LB culture medium: tryptone 10.0g/L, yeast extract 5.0g/L, NaCl 10.0g/L, kanamycin final concentration 50 u g/mL.

2 × YT medium: tryptone 16.0g/L, yeast extract 10.0g/L, NaCl 5.0g/L, kanamycin final concentration 50 u g/mL.

Wild-type WT and its A129R mutant were purified by affinity chromatography using a Streptrap HP 5mL column from GE. 100mL of the bacterial suspension cultured in 500mL Erlenmeyer flask was centrifuged at 10000rpm for 3min to collect the cells, resuspended in 20mL of binding buffer PBS (20mM Na2HPO4 & 12H2O, 280mM NaCl, 6mM KCl, pH 7.4), and sonicated in an ice-water mixture. The disruption solution was centrifuged at 12000rpm at 4 ℃ for 30min, and the supernatant was filtered through a 0.22 μm organic filter. The purification column was loaded after equilibration with the binding buffer, followed by washing with the binding buffer to remove the desired protein, which was eluted with 100% elution buffer (20mM Na2HPO4 & 12H2O, 280mM NaCl, 6mM KCl, 2.5mM d-Desthiobiotin, pH 7.4) and collected. Protein concentration was quantified using the Bradford protein concentration detection kit.

Example 1: construction of Each mutant of PtNHase

A mutant plasmid pET24a (+) -A129R was constructed by whole plasmid PCR based on the wild type plasmid pET24a (+) -PtNHaseWT of Pt NHase (this plasmid is described in Cheng et al, comparative Design of Ni trile hydrase from Pseudomonas pseudocardinalis thermophila JCM3095 for Improved thermosectivity, 2020). Firstly, plasmid pET24a (+) -PtNHase WT is taken as a template, a mutation sequence is designed on a primer, a DNA fragment with a mutated base sequence is amplified through PCR, the sequence of the used primer is shown in table 1, an amplification system is shown in table 2, the PCR amplification reaction conditions are pre-denaturation at 98 ℃ for 1min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min for 30s, and extension at 72 ℃ for 5min for 30 cycles. Digesting the PCR product for 2h by using DpnI digestive enzyme, transforming the digested product into E.coli DH5 alpha, performing LB plate culture, selecting a single clone, performing sequencing verification by using Tianlin biotechnology (tin-free) limited company to obtain a positive transformant, and extracting a plasmid from the positive transformant, namely the mutant plasmid pET24a (+) -A129R.

TABLE 1 primer sequences

TABLE 2 PCR amplification System

Example 2: catalytic efficiency of wild type and mutant of Pt NHase on nicotinonitrile

The plasmid pET24a (+) -PtNHaseWT of wild type WT of PtNHase and the reconstructed plasmid pET24a (+) -A129R were transformed into E.coli BL21(DE3), respectively, and a single colony was picked up to 5mL LB medium and cultured at 37 ℃ and 200rpm for 7-8 h. Transferring the seed solution to 100mL 2 XYT medium at 1% (v/v), culturing at 37 deg.C and 200rpm to OD₆₀₀To 0.6-0.8, isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.4mM and CoCl at 0.1g/L₂·6H₂O, changing the culture temperature to 24 ℃, and inducing expression for 16 h.

The wild WT and A129R mutant were purified, and SDS-PAGE was used to detect the purification quality of the target protein, as shown in FIG. 1, it can be seen that the protein expressed by the wild WT and its mutant had a single protein band after purification, and the purification quality was high.

With 10mM KPB (in K)₂HPO₄:KH₂PO₄Prepared as 4:1, pH 7.4) buffer the concentration of WT and a129R mutant pure enzyme was diluted to 0.5mg/mL, 10 μ L to 1.5mL centrifuge tubes were placed on a 25 ℃ metal bath. mu.L of substrate (200mM nicotinonitrile solution) was added to the centrifuge tube, vortexed thoroughly, reacted at 25 ℃ for 10min, and quenched by the addition of 500. mu.L of pure acetonitrile. The reaction solution was diluted with pure acetonitrile by an appropriate factor and passed through a 0.22 μm filter.

The liquid phase detection method comprises the following steps: the mobile phase composition is acetonitrile: water 1: 2(v/v), the flow rate is 0.6mL/min, the detection wavelength is 215nm, the column temperature is 40 ℃, and the generation amount of the product nicotinamide in the reaction system is measured.

The calculation results of the specific enzyme activities of WT and the mutant A129R are shown in FIG. 2, the specific enzyme activity of the wild enzyme WT is 70.8 +/-2.3U/mg, and the specific enzyme activity of the mutant A129R is 661.8 +/-69.0U/mg.

Namely, when the 129 th amino acid residue on the beta subunit is mutated, the specific enzyme activity of the nitrile hydratase is obviously improved, which indicates that the 129 th amino acid residue may be in the key structural domain of the subunit and has important function for the catalytic activity of the nitrile hydratase.

Example 3: substrate spectra of wild type Pt NHase and mutant A129R

The culture induction and reaction system was the same as in example 2. Carrying out different substrate catalytic reactions based on the pure enzyme, and respectively adding different substrates into a culture medium, wherein the concentration of the isobutyronitrile, the 2-cyanopyrazine, the benzonitrile, the cinnamonitrile, the 1-naphthonitrile and the thiacloprid for catalytic reactions is 0.05mg/mL, and the concentration of the valeronitrile and the nicotinonitrile for catalytic reactions is 0.5 mg/mL.

The liquid phase detection method comprises the following steps: the mobile phase composition is acetonitrile: water 1: 2 (v/v); flow rate: the isobutyramide, the valeramide, the nicotinamide, the pyrazinamide and the naphthalene-1-formamide are all 0.6mL/min except that the benzamide, the cinnamamide and the thiacloprid amide are 1 mL/min; detection wavelength: isobutyramide and valeramide are 202nm, nicotinamide, benzamide and naphthalene-1-carboxamide are 215nm, pyrazinamide and cinnamamide are 261nm, and thiacloprid amide is 242 nm; the column temperature was 40 ℃ and the amount of amide produced in the reaction system was determined.

The calculation results of the specific enzyme activities of WT and mutant A129R are shown in FIG. 3, and the specific enzyme activities of wild enzyme WT are 666.2 + -80.8U/mg, 14.4 + -0.9U/mg, 70.8 + -2.33U/mg, 203.2 + -8.1U/mg, 176.5 + -8.1U/mg, 45.8 + -1.0U/mg, 13.1 + -1.7U/mg and 21.4 + -0.3U/mg respectively for 8 nitrile substrates such as isobutyronitrile, n-valeronitrile, nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, 1-naphthonitrile and thiacloprid, and the specific enzyme activities of mutant A129R are 1498.9 + -89.6U/mg, 60.5 + -15.3U/mg, 661.8 + -69.0U/mg, 660.4 + -4.0U/mg, 1211.9 + -17.6U/mg, 379.1.5 + -15.3U/mg, 3652 + -69.0U/mg, and 368 + -2.8 + -0U/mg respectively for 8 nitrile substrates such as isobutyronitrile, 14.4 + -0.9U/mg, 13.1.7 + -1.7U/mg and 21.3U/mg. Wherein, the specific enzyme activities of A129R for catalyzing isobutyronitrile and n-valeronitrile are respectively 2.25 times and 4.2 times of WT; the specific enzyme activities of the catalytic nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, 1-naphthonitrile, thiacloprid and toyocamycin are respectively 9.35 times, 3.25 times, 6.87 times, 8.3 times, 1.6 times and 3.69 times of that of WT.

Namely, when alanine at the 129 site on the beta subunit is mutated into arginine, the specific enzyme activities of nitrile hydratase to other different nitrile substrates are improved to different degrees, and especially the activity improvement to aromatic and heterocyclic substrate molecules with larger steric hindrance is more obvious.

TABLE 3 comparison of the enzyme activities of the wild type and the mutant on different substrates

Example 4

See example 1 for a difference that for wild-type PtNHase a single point saturation mutation was made to replace the alanine at position 129 with the other 19Amino acids (mutated to C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, respectively). After the mutant is cultured and induced to express, the cells are regulated to the same OD₆₀₀And measuring the enzyme activity of the mutant to 0.5, wherein the result shows that the enzyme activity is highest when the mutant is arginine.

TABLE 4 comparison of Whole cell catalytic Activity on nicotinonitrile between wild type and mutant

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Met Thr Glu Asn Ile Leu Arg Lys Ser Asp Glu Glu Ile Gln Lys Glu

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Ile Thr Ala Arg Val Lys Ala Leu Glu Ser Met Leu Ile Glu Gln Gly

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Ile Leu Thr Thr Ser Met Ile Asp Arg Met Ala Glu Ile Tyr Glu Asn

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Glu Val Gly Pro His Leu Gly Ala Lys Val Val Val Lys Ala Trp Thr

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Lys Glu Leu Gly Ile Gly Gly Leu Gln Gly Glu Asp Met Met Trp Val

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Glu Asn Thr Asp Glu Val His His Val Val Val Cys Thr Leu Cys Ser

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Ala Phe Ala Met Phe Pro Ala Thr Phe Arg Ala Gly Phe Met Gly Leu

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Asp Glu Phe Arg Phe Gly Ile Glu Gln Met Asn Pro Ala Glu Tyr Leu

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Glu Ser Pro Tyr Tyr Trp His Trp Ile Arg Thr Tyr Ile His His Gly

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Val Arg Phe Ser Thr Ala Ser Pro Lys Gly His Ala Arg Arg Ala Arg

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Tyr Val Arg Gly Lys Thr Gly Thr Val Val Lys His His Gly Ala Tyr

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Ile Tyr Pro Asp Thr Ala Gly Asn Gly Leu Gly Glu Cys Pro Glu His

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Leu Tyr Thr Val Arg Phe Thr Ala Gln Glu Leu Trp Gly Pro Glu Gly

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Claims

1. A nitrile hydratase mutant comprising an alpha subunit and a beta subunit; the mutant is a nitrile hydratase parent beta subunit 129 bit mutation; the amino acid sequence of the alpha subunit of the nitrile hydratase parent is shown as SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 2.

2. The mutant according to claim 1, wherein the mutant is obtained by mutating 129 th position of beta subunit into arginine.

3. A gene encoding the mutant of claim 1 or 2.

4. A recombinant plasmid carrying the gene of claim 3.

5. A host cell expressing the mutant of claim 1 or 2, or containing the gene of claims 3 and 4.

6. The host cell of claim 5, wherein the host cell comprises a prokaryotic or eukaryotic microorganism.

7. A method for producing amides, characterized in that nitriles are used as a substrate, and amides are produced by the catalysis of the mutant according to claim 1 or 2.

8. The method according to claim 7, wherein the nitrile comprises isobutyronitrile, n-valeronitrile, nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, 1-naphthonitrile and thiacloprid.

9. The method of claim 8, wherein the amide-based substance comprises isobutyramide, valeramide, nicotinamide, pyrazinamide, benzamide, cinnamamide, naphthalene-1-carboxamide, and thiaclopramide.

10. Use of the mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant plasmid according to claim 4, or the host cell according to claim 5 or 6 for the preparation of amides.