阅读说明：本技术 一种高活性腈水合酶突变体及其应用 (High-activity nitrile hydratase mutant and application thereof ) 是由 周哲敏 程中一 崔文璟 浦卫峰 张广林 于 2021-09-17 设计创作，主要内容包括：本发明公开了一种高活性腈水合酶突变体及其应用,属于生物工程技术领域。本发明通过对来源于温泉热碱芽孢杆菌TA2.A1的腈水合酶的进行改造,具体为将其β亚基的第47位氨基酸进行突变,构建得到的突变体N47F的酶活为592.92±1.12U/mg,与未进行改造的野生型腈水合酶的比酶活165.30±1.21U/mg相比,有明显提高,约为野生型的3.6倍,腈水合酶催化活性得到显著改善,有利于应用于工业上酰胺类化学品如烟酰胺、丙烯酰胺的生产,提高催化效率,降低生产成本。(The invention discloses a high-activity nitrile hydratase mutant and application thereof, belonging to the technical field of biological engineering. The invention modifies nitrile hydratase derived from thermokalite bacillus spa TA2.A1, specifically, mutation is carried out on 47 th amino acid of beta subunit, the enzyme activity of the constructed mutant N47F is 592.92 +/-1.12U/mg, compared with the specific enzyme activity 165.30 +/-1.21U/mg of wild nitrile hydratase which is not modified, the enzyme activity is obviously improved and is about 3.6 times of that of the wild nitrile hydratase, the catalytic activity of the nitrile hydratase is obviously improved, and the nitrile hydratase is beneficial to the production of amide chemicals such as nicotinamide and acrylamide in industry, the catalytic efficiency is improved, and the production cost is reduced.)

1. A high-activity nitrile hydratase mutant, wherein the nitrile hydratase comprises an alpha subunit, a beta subunit and a regulatory protein; the mutant is obtained by substituting the amino acid at the 47 th position of the beta subunit with phenylalanine; the amino acid sequence of the alpha subunit is shown as SEQ ID NO.1, and the amino acid sequence of the regulatory protein is shown as SEQ ID NO. 3.

2.A gene encoding the nitrile hydratase mutant according to claim 1.

3. An expression vector comprising the gene of claim 2.

4. The expression vector of claim 3, wherein the expression vector comprises but is not limited to pET series, Duet series, pGEX series, pHY300PLK, pPIC3K or pPIC9K series vectors.

5. A microbial cell carrying the gene of claim 2 or the recombinant vector of claim 3 or 4.

6. The microbial cell of claim 5, wherein the microbial cell is a host escherichia coli.

7. A method for improving the enzyme activity of nitrile hydratase is characterized in that asparagine at the 47 th site of a beta subunit of nitrile hydratase with an amino acid sequence shown as SEQ ID NO.2 is mutated into phenylalanine; the nitrile hydratase further comprises an alpha subunit and a regulatory protein; the amino acid sequence of the alpha subunit is shown as SEQ ID NO.1, and the amino acid sequence of the regulatory protein is shown as SEQ ID NO. 3.

8. A method for producing a nitrile hydratase mutant, comprising inoculating the microbial cell according to any one of claims 5 to 6 into YT medium, culturing at 35 to 37 ℃ and 180 to 200rpm to OD₆₀₀When the concentration is 0.6-0.8, IPTG with the final concentration of 0.1-0.4 mM and CoCl with the final concentration of 0.0.5-0.2 g/L are added₂·6H₂And O, inducing for 12-16h at 22-24 ℃ to obtain the nitrile hydratase mutant-containing fermentation liquor.

9. Use of the nitrile hydratase mutant according to claim 1, the gene according to claim 2, or the microbial cell according to any one of claims 5 to 6 for producing amides.

10. The use according to claim 9, wherein the amide-based substance includes but is not limited to nicotinamide and derivatives thereof or acrylamide and derivatives thereof.

Technical Field

The invention relates to a high-activity nitrile hydratase mutant and application thereof, in particular to an amino acid motif of nitrile hydratase, a plasmid containing a gene for coding the amino acid sequence, a strain containing the plasmid after conversion and application thereof in improving the nitrile hydratase, and belongs to the technical field of biological engineering.

Background

Nitrile hydratase (Nitrile hydratase, NHase for short, EC 4.2.1.84) hydrates Nitrile substrates and is converted to the corresponding amide product. The enzyme derived from Rhodococcus rhodochrous J1 (Arthrobacter) is a nitrile hydratase that has been discovered and reported for the first time. Microorganisms that produce nitrile hydratase currently include Rhodococcus (Rhodococcus), Pseudomonas (Pseudomonas), Brevibacterium (Brevibacterium), and the like, and heterologous expression thereof has been achieved in model strains such as Escherichia coli (Escherichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Pichia pastoris (Pichia pastoris), and the like. With the research on nitrile hydratase, a biocatalysis method with mild conditions and strong selectivity is gradually replacing a chemical synthesis method, and is successfully applied to the industrial production of amide products such as acrylamide, nicotinamide, 5-cyano valeramide and the like.

The amide product has high industrial value, wherein, the nicotinamide is an important chemical production raw material and has wide application. Nicotinamide is a coenzyme of vitamin B12, can be used as a vitamin supplement, can be used for synthesizing various vitamin derivatives, and has wide market prospect. However, the industrial application of nitrile hydratase still has a certain problem at present. Since the hydration of nitriles is an exothermic reaction, nitrile hydratases are required to have good thermal stability to maintain a high reaction rate at high temperatures. In contrast, most nitrile hydratases are rapidly inactivated at temperatures exceeding 50 ℃, and therefore, it is important to obtain nitrile hydratases having both high enzyme activity and good thermal stability.

The nitrile hydratase derived from the thermokalite bacillus spa (Caldalkalibacillus thermosmarum TA2.A1) has good thermal stability, the half-life period of the nitrilase at 65 ℃ is 3h, the nitrilase has excellent thermal stability, but the activity of the wild catalytic enzyme is low. Therefore, the method for improving the catalytic activity of the nitrile hydratase has great application value and wide application prospect.

Disclosure of Invention

Aiming at the technical difficulties and problems in the prior art, the invention provides a mutant amino acid motif of nitrile hydratase derived from Bacillus thermokalis spa (Caldalkalibacillus athermarum TA2.A1) and application thereof in substrate catalytic production.

The invention provides a high-activity nitrile hydratase mutant, wherein the nitrile hydratase consists of an alpha subunit, a beta subunit and a regulatory protein; the mutant is obtained by substituting the amino acid at the 47 th position of the beta subunit with phenylalanine; the amino acid sequence of the alpha subunit is shown as SEQ ID NO.1, and the amino acid sequence of the regulatory protein is shown as SEQ ID NO. 3.

The present invention provides a gene encoding the nitrile hydratase mutant.

The invention provides an expression vector containing the gene.

In one embodiment, the expression vector includes, but is not limited to, pET series, Duet series, pGEX series, pHY300PLK, pPIC3K, or pPIC9K series vectors.

Preferably, pET-24a (+) is taken as an expression plasmid vector

The present invention provides a microbial cell carrying the gene of claim 2 or the recombinant vector of claim 3 or 4.

In one embodiment, the microbial cell is a host escherichia coli.

In one embodiment, the microbial cell is a host of escherichia coli BL 21.

The invention provides a method for improving the enzyme activity of nitrile hydratase, which comprises the steps of mutating asparagine at the 47 th site of a beta subunit of nitrile hydratase with an amino acid sequence shown as SEQ ID NO.2 into phenylalanine; the nitrile hydratase further comprises an alpha subunit and a regulatory protein; the amino acid sequence of the alpha subunit is shown as SEQ ID NO.1, and the amino acid sequence of the regulatory protein is shown as SEQ ID NO. 3.

The invention provides a method for producing nitrile hydratase mutants, which is characterized in that microbial cells are inoculated in YT culture medium and cultured to OD at 35-37 ℃ and 180-200 rpm₆₀₀When the concentration is 0.6-0.8, IPTG with the final concentration of 0.1-0.4 mM and CoCl with the final concentration of 0.0.5-0.2 g/L are added₂·6H₂And O, inducing for 12-16h at 22-24 ℃ to obtain the nitrile hydratase mutant-containing fermentation liquor.

The invention provides the nitrile hydratase mutant, the gene and the application of the microbial cell in the production of amide substances.

In one embodiment, the amide includes, but is not limited to, nicotinamide and derivatives thereof or acrylamide and derivatives thereof.

Has the advantages that:

the invention modifies the nitrile hydratase derived from the thermokalite bacillus spa strain TA2.A1, in particular to mutate the 47 th amino acid of the beta subunit of the nitrile hydratase, so that the enzyme activity of the constructed mutant N47F is 592.92 +/-1.12U/mg, and compared with the specific enzyme activity 165.30 +/-1.21U/mg of wild nitrile hydratase which is not modified, the enzyme activity is obviously improved and is about 3.6 times of that of the wild nitrile hydratase, the catalytic activity of the nitrile hydratase is obviously improved, the nitrile hydratase is favorable for producing fine chemicals such as nicotinamide in industry, the catalytic efficiency is improved, and the production cost is reduced.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of pure enzyme, M: protein Marker, 1: WT, 2: N47F.

FIG. 2 shows the specific activities of Cal.t NHase wild enzyme WT and mutant N47F.

Detailed Description

1. Culture medium:

LB Medium (L)^-1):10 g of tryptone, 10g of NaCl, 5g of yeast extract and pH 7.0, and 20g of agar powder is added when preparing a solid culture medium.

Glycerol-free TB Medium (L)^-1): tryptone 12g, yeast extract 24g, KH₂PO₄ 2.31g，K₂HPO₄12.54g。

2. Buffer solution:

binding buffer: 20mM Na₂HPO₄·12H₂O，280mM NaCl，6mM KCl，pH 7.4

Elution buffer: 20mM Na₂HPO₄·12H₂O，280mM NaCl，6mM KCl，2.5mM d-Desthiobiotin，pH 7.4。

Enzyme activity (U) of nitrile hydratase: the specific enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1. mu. mol nicotinamide by nicotinonitrile per minute at 25 ℃.

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

Example 1: plasmid construction of nitrile hydratase mutant

According to the wild type plasmid pET24a (+) -Cal.t WT of existing Cal.t NHase in the laboratory (the specific construction steps are disclosed in Zhang Sailan, Li Ting, Cheng et ai, the heterogenous expression of novel heat-resistant nitrile hydratase and the catalytic process research [ J ]. food and fermentation industry, 2020, volume 46 (14): 108-. Firstly, plasmid pET24a (+) -Cal.t WT is taken as a template, a mutation sequence is designed on a primer, the plasmid with mutation of a base sequence is amplified through PCR, the sequence of the used primer is shown in a table 1, an amplification system is shown in a table 2,

the PCR amplification reaction conditions are pre-denaturation at 95 ℃ for 3min, denaturation at 98 ℃ for 15s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min45s and extension at 72 ℃ for 5min, and 30 cycles are total. The PCR product was digested with DpnI digestive enzyme for 2-3h, transformed E.coli DH5 α, plated on LB medium plate containing 50mg/L kanamycin, and cultured overnight by inversion at 37 ℃.

A single colony is selected and inoculated in 5mL LB culture medium, shaking culture is carried out at 37 ℃ and 200rpm overnight, a commercial plasmid extraction kit is used for obtaining recombinant plasmid, sequencing verification is carried out by Suzhou Jinzhi Biotech limited, and finally, the reconstructed plasmid pET24a (+) -N47F is obtained.

TABLE 1 primer sequences

(Note: F denotes an upstream primer, R denotes a downstream primer)

TABLE 2 PCR amplification System

Example 2: expression and purification of wild enzyme WT and each mutant

Step 1: e.coli BL21(DE3) was transformed with the wild-type pET24a (+) -Cal.t WT of Cal.t NHase obtained in example 1 and the reconstituted plasmid pET24a (+) -N47F, respectively, and a single colony was picked up in 5mL of LB medium and cultured at 37 ℃ and 200rpm for 7-8 hours. Inoculating the seed solution to 100mL 2 XYT medium at 1% (v/v) inoculum size, 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 12-16 h.

Step 2: wild-type WT and the N47F mutant were purified by affinity chromatography using a StrepTrap HP 1mL column from GE. The cells were collected by centrifugation at 10000rpm for 3min, resuspended in 20mL of binding buffer, 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. After the purification column is equilibrated with the binding buffer, the column is loaded, the binding buffer is used for washing the impure protein, and the target protein is eluted by 100% of the elution buffer and collected. Protein concentration was quantified using the Bradford protein concentration detection kit. SDS-PAGE is adopted to detect the purification quality of the target protein, and the detection is shown in figure 1, so that the protein expressed by the wild type and the mutant thereof has single protein band after purification and high purification quality.

Example 3: cal.t NHase wild type and mutant catalytic efficiency detection

The concentration of WT and its mutant pure enzyme was diluted to 0.5mg/mL with 10mM KPB (pH 7.4) solution, and 10. mu.L to 1.5mL of the centrifuge tube was 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 are shown in FIG. 2, the specific enzyme activity of the wild enzyme WT is 165.30 + -1.21U/mg, and the specific enzyme activity of the mutant N47F is 592.92 + -1.12U/mg.

The mutant enzyme activity is improved by about 259 percent compared with the wild type, namely when the amino acid residue at the site is mutated, the specific enzyme activity of the nitrile hydratase is improved to a great extent, which indicates that the amino acid residue can be in the key structural domains of two subunits and plays an important role in the catalytic activity of the nitrile hydratase.

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

<110> Sn-free Xinchenyu bioengineering Co., Ltd

<120> high-activity nitrile hydratase mutant and application thereof

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Gly Ile Leu Ser Ser Asp Ala Ile Asp Arg Val Val Gln His Tyr Glu

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His Glu Leu Gly Pro Met Asn Gly Ala Lys Val Val Ala Lys Ala Trp

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Thr Asp Pro Ala Phe Lys Gln Arg Leu Leu Glu Asp Pro Glu Thr Val

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Trp Asn Tyr Tyr Glu His Trp Leu Ala Ala Leu Glu Gln Leu Val Val

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Phe Leu Ser Gly Glu Arg Asp Glu Phe Phe

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