CN112501151B

CN112501151B - Nitrile hydratase mutant and application thereof

Info

Publication number: CN112501151B
Application number: CN202011450515.3A
Authority: CN
Inventors: 周哲敏; 江诗进; 程中一; 刘中美; 崔文璟; 周丽; 韩来闯; 郭军玲
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2021-11-16
Anticipated expiration: 2040-12-09
Also published as: CN112501151A

Abstract

The invention discloses a nitrile hydratase mutant and application thereof, belonging to the technical field of biological engineering. The enzyme activities of single-point mutants L48D and L48H constructed on the No. 48 subunit in a nitrile hydratase amino acid motif are 750.26 +/-1.63U/mg and 820.01 +/-0.98U/mg respectively, and are obviously improved compared with the specific enzyme activity 220.30 +/-2.28U/mg of wild nitrile hydratase without modification. Wherein, the enzyme activities of the mutant L48D and the mutant L48H are respectively 3.4 times and 3.7 times of the wild type. The mutation of the two amino acid residues obviously improves the catalytic activity of nitrile hydratase, is beneficial to the production of fine chemicals such as nicotinamide in industry, improves the catalytic efficiency and reduces the production cost.

Description

Nitrile hydratase mutant and application thereof

Technical Field

The invention relates to a nitrile hydratase mutant and application thereof, 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 has huge application potential in the industrial production of fine chemical nicotinamide. The amide biological method production technology is a typical case of replacing a chemical method with a biological method, has the advantages of environmental protection, mild reaction conditions, high safety coefficient and the like, and accords with sustainable development and green production concepts.

Amide products have great application value in the fields of industry, agriculture, medicine and the like, and especially, the biocatalysis of nicotinamide and acrylamide is the most common. Among them, 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.

Nitrile hydratase not only plays an important role in the application, but also has attracted extensive attention from researchers as a metalloenzyme capable of binding cobalt ions or iron ions for its catalytic activity and stability. 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. Many research teams improve the stability of the polypeptide through constructing strategies such as covalent bond interaction, salt bond, Link addition, subunit fusion and the like, but reports that the enzyme activity and the stability are improved simultaneously through enzyme modification and even the substrate spectrum is widened are hardly available.

Nitrile hydratase derived from thermokalite bacillus thermonatrum (Caldalkalibacillus tam 2.A1) has good thermal stability, and based on the characteristic, the catalytic activity of the nitrile hydratase can be improved and the substrate spectrum can be widened. Current commercial bulk chemical production, such as acrylamide and nicotinamide, is not very catalytic efficient, except for being limited by thermal stability. Therefore, the method for improving the stability and 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 an amino acid motif of nitrile hydratase derived from Bacillus thermokali thermonatum (Caldalkalibacillus thermonatum TA2.A1) and application thereof in substrate catalytic production.

The first object of the present invention is to provide nitrile hydratase mutants comprising an alpha subunit, a beta subunit and a regulatory protein.

In one embodiment of the invention, the amino acid sequence of the α -subunit is as set forth in SEQ ID No. 1.

In one embodiment of the invention, the amino acid sequence of the beta subunit is as set forth in SEQ ID No.4 or SEQ ID No. 5.

In one embodiment of the invention, the amino acid sequence of the regulatory protein is as set forth in SEQ ID NO. 3.

It is a second object of the present invention to provide a gene encoding the mutant.

The third purpose of the invention is to provide a vector containing the gene.

It is a fourth object of the present invention to provide a microbial cell of the nitrile hydratase mutant.

In one embodiment of the present invention, the cell is a cell that uses escherichia coli BL21 as a host and uses a pET-series plasmid as a vector.

In one embodiment of the present invention, the plasmid is pET-24 (+).

The fifth purpose of the invention is to provide a method for improving the enzyme activity of nitrile hydratase, which is to mutate the 48 th leucine of the beta subunit of nitrile hydratase with the amino acid sequence shown as SEQ ID NO.2 into aspartic acid or histidine.

Sixth inventionIt is an object to provide a method for recombinantly expressing the nitrile hydratase mutant, comprising inoculating a microbial cell expressing the nitrile hydratase mutant into LB medium, culturing at 37 ℃ to OD₆₀₀When the temperature is 0.6-0.8 ℃, adding an inducer IPTG to induce for 12-16h at 24 ℃.

A seventh object of the present invention is to provide the use of the above nitrile hydratase mutant or a microbial cell containing the nitrile hydratase mutant for producing a nicotinamide-containing product.

Has the advantages that:

the invention provides an amino acid motif of Cal.t-NHase and an amino acid motif of a mutant, wherein the motif and nitrile hydratase motifs from other sources are highly conserved at sites 108, 111 and 113 of an enzyme active center on an alpha subunit, and the motif and the nitrile hydratase motifs form coordinate bonds with cobalt ions respectively. The enzyme activities of single-point mutants L48D and L48H constructed on the No. 48 subunit in a nitrile hydratase amino acid motif are 750.26 +/-1.63U/mg and 820.01 +/-0.98U/mg respectively, and are obviously improved compared with the specific enzyme activity 220.30 +/-2.28U/mg of wild nitrile hydratase without modification. The enzyme activity of the mutant L48D is about 3.4 times of that of the wild type, and the enzyme activity of the mutant L48H is about 3.7 times of that of the wild type. The mutation of the two amino acid residues obviously improves the catalytic activity of nitrile hydratase, is beneficial to the production of fine chemicals such as nicotinamide in industry, improves the catalytic efficiency and reduces the production cost.

Drawings

FIG. 1: SDS-PAGE electrophoresis of pure enzyme, M: protein Marker, 1: pET24a, 2: WT, 3: L48D, 4: L48H.

FIG. 2: cal. t NHase wild enzyme WT and mutant L48D, L48H, A41K, Y46E and V126K specific enzyme activity.

FIG. 3: cal.t NHase mutant L48H was used to produce nicotinamide.

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

A wild-type plasmid pET24a (+) -Cal.t WT (Zusanlan, litting, Zhongzhong, first-class, New-type heat-resistant nitrile hydratase heterologous expression and catalytic process research [ J ]. food and fermentation industry, 2020, volume 46 (14): pET24a (+) -Cal.t NHase in 108-113) is used as a template, mutation sites L48D and L48H are designed on a primer, a plasmid with a mutated base sequence is amplified through PCR, the sequence of the used primer is shown in Table 1, and an amplification system is shown in 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 plasmids, sequencing verification is carried out by Suzhou Jinzhi Biotech limited, and finally, reconstructed plasmids pET24a (+) -L48D and pET24a (+) -L48H are 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 plasmids pET24a (+) -L48D and pET24a (+) -L48H, respectively, and a single colony was picked up to 5mL LB medium and cultured at 37 ℃ and 200rpm for 7-8 hours. Transferring the seed solution to 100mL of 2 XYT medium at 1% (v/v), culturing at 37 deg.C and 200rpm until OD600 is 0.6-0.8, adding isopropyl thiogalactoside (IPTG) at final concentration of 0.4mM and CoCl at final concentration of 0.1g/L₂·6H₂O, changing the culture temperature to 24 ℃, and inducing expression for 12-16 h.

Step 2: wild-type WT and 2 mutants 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 220.30 + -2.28U/mg, and the enzyme activities of the mutant L48D and the mutant L48H are 750.26 + -1.63U/mg and 820.01 + -0.98U/mg respectively.

The enzyme activities of the two mutants are respectively improved by about 70 percent and 73 percent compared with the wild type, namely when the amino acid residue at the site is mutated, the specific enzyme activities of the nitrile hydratase are improved to different degrees, which indicates that the amino acid residue can be in the key structural domains of the two subunits and has important function on the catalytic activity of the nitrile hydratase.

Example 4: application of nitrile hydratase wild type and mutant in production of nicotinamide

The bacterial liquid BL21(DE3)/pET24a (+) -Cal.t NHase-L48H obtained in step 1 of example 2 was collected by centrifugation, washed with water and collected by centrifugation again, and 10mmol/L, pH was 7.4H₃PO₄Re-suspending the buffer solution, and adjusting to the OD of the bacterial liquid₆₀₀8. The temperature was adjusted to 30 ℃ and nicotinonitrile was added to the OD at a final concentration of 0.4mol/L₆₀₀To the bacterial solution (8), nicotinonitrile was added at intervals of 6min with stirring, in an amount of 0.4mol/(L · s). The amount of nicotinamide formed as a product in the reaction system was measured in the same manner as in example 3, and the concentration of nicotinamide calculated was 706g/L, as shown in FIG. 3. The wild-type nitrile hydratase reported in the literature had a nicotinamide yield of 495g/L (Zusanlan, litting, Zhongzhou-one. heterologous expression of novel heat-resistant nitrile hydratase and its catalytic process study [ J]The food and fermentation industry, 2020, volume 46 (14): 108-.

Comparative example 1: comparison of catalytic efficiency of mutant pET24a (+) -L48H and pET24a (+) -Y46E

The specific implementation manner is the same as that in example 1, except that tyrosine at position 46 is mutated into glutamic acid, an enzyme mutant is prepared according to example 2 and is used for catalyzing nicotinamide, and the result is shown in fig. 2, the enzyme activity of catalyzing nicotinonitrile is 375.29 +/-0.65U/mg, which is 54% lower than that of mutant L48H provided by example 1, and the enzyme activity of mutant pET24a (+) -L48H is obviously higher than that of pET24a (+) -Y46E.

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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Claims

1. A nitrile hydratase mutant comprising an alpha subunit, a beta subunit, and a regulatory protein;

the amino acid sequence of the alpha subunit is shown as SEQ ID NO. 1;

the amino acid sequence of the beta subunit is shown as SEQ ID NO.4 or SEQ ID NO. 5;

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. A recombinant vector comprising the gene of claim 2.

4. A microbial cell expressing the nitrile hydratase mutant according to claim 1.

5. The microbial cell of claim 4, wherein the microbial cell comprises Escherichia coli.

6. The microbial cell according to claim 4 or 5, wherein the cell is a host escherichia coli BL21 and a pET-series plasmid is a vector.

7. The microbial cell of claim 6, wherein the plasmid is pET-24 (+).

8. A method for improving the enzyme activity of nitrile hydratase is characterized in that the nitrile hydratase is derived from hot spring thermokalite bacillus (Bacillus (R) (R))Caldalkalibacillus thermarum) A1 nitrile hydratase beta subunit amino acid sequence of leucine amino acid mutation to aspartic acid or histidine at position 48; the amino acid sequence of the beta subunit is shown as SEQ ID NO. 2.

9. A method for producing nitrile hydratase, characterized in that microbial cells expressing the nitrile hydratase mutant according to claim 1 are inoculated into LB medium and cultured at 37 ℃ to OD₆₀₀When the temperature is 0.6-0.8 ℃, adding an inducer IPTG to induce for 12-16h at 24 ℃.

10. Use of a nitrile hydratase mutant according to claim 1 or a microbial cell according to claim 4 for the production of a nicotinamide-containing product.