阅读说明：本技术 一种表达腈水合酶突变体的重组菌及其制备方法和应用 (Recombinant bacterium for expressing nitrile hydratase mutant and preparation method and application thereof ) 是由 董亢 濮梦华 李文兵 夏华跃 夏斌 李习红 于 2019-08-01 设计创作，主要内容包括：本发明公开了一种表达腈水合酶突变体的重组菌及其制备方法和应用,制备方法包括易错PCR法构建基因随机突变质粒文库,转化阳性克隆的筛选,经过酶活、蛋白电泳检测,水合小试验证筛选等步骤最终成功获得了表达腈水合酶突变体蛋白的重组大肠杆菌。本发明以高产腈水合酶大肠杆菌的基因组为模板,采用易错PCR技术重组构建随机突变质粒文库,从库中筛选腈水合酶突变体基因,该重组基因转化进入工程菌中后可高效诱导表达腈水合酶突变体蛋白。以高表达腈水合酶突变体编码基因的重组工程菌在经诱导表达后获得的菌体为酶源,在适合条件下进行水合反应生产烟酰胺,副产物烟酸低90％以上。(The invention discloses a recombinant bacterium for expressing nitrile hydratase mutant and a preparation method and application thereof. The invention uses the genome of Escherichia coli with high nitrile hydratase yield as a template, adopts error-prone PCR technology to recombine and construct a random mutant plasmid library, screens nitrile hydratase mutant genes from the library, and can efficiently induce and express nitrile hydratase mutant proteins after the recombinant genes are transformed into engineering bacteria. The recombinant engineering bacteria of the high expression nitrile hydratase mutant coding gene are induced and expressed to obtain thallus which is used as an enzyme source, hydration reaction is carried out under a proper condition to produce nicotinamide, and the byproduct nicotinic acid is over 90 percent lower.)

1. A preparation method of a recombinant bacterium for expressing a nitrile hydratase mutant is characterized by comprising the following specific steps:

(1) the method is characterized in that pET28-NHase plasmid of escherichia coli with high nitrile hydratase yield is used as a template, and an error-prone PCR method is adopted to construct a gene random mutation plasmid library, and the specific method is as follows:

recovering DNA products from the amplified fragments by agarose gel electrophoresis;

the carrier pET-28(a) is digested with BamHI and HindIII, the recovered DNA product is digested with the same method, the digested DNA fragment and the carrier pET-28(a) are linked with T4DNA ligase, and the mixture is reacted at 16 ℃ overnight;

transforming the ligation product to DH10B competent cells by an electric converter (Bio-rad MicroPulser), coating the competent cells on an LB plate of 50mg/mL kanamycin sulfate, culturing overnight, collecting thalli and extracting plasmids to obtain a gene random mutation plasmid library;

(2) transferring the gene random mutation plasmid library into BL21 competent cells by a heat shock transformation method, coating the competent cells on an LB plate containing kanamycin with the final concentration of 50mg/ml, selecting a single clone for bacterial detection, wherein the target band is 1.9kb, and the bacterial sample system is as follows:

(3) randomly selecting bacteria to detect positive monoclonal, inoculating into 1mL liquid 48-pore plate LB culture medium containing 50mg/mL kanamycin at the final concentration, carrying out shake culture at 37 ℃ and 220rpm for 16h, inoculating the culture solution into 50mL liquid LB culture medium containing 50mg/mL kanamycin at 3% volume ratio, carrying out shake culture at 37 ℃ and 220rpm until OD is achieved₆₀₀When the concentration is 0.4-0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 100umol/L and cobalt chloride with the final concentration of 40mg/L, performing shake table induction culture at 18 ℃ and 220rpm for 24 hours to obtain fermentation liquor, directly adding 3-cyanopyridine with the final concentration of 10% into the fermentation liquor, reacting at 20 ℃ for 30 minutes, immediately placing on ice for 30 minutes, and eliminating colonies precipitated by crystals;

(4) the strains obtained by screening are shaken again according to the method, and the concentration of the 3-cyanopyridine is increased to 20 percent or 30 percent to obtain the high-activity strains.

2. A recombinant bacterium for expressing a nitrile hydratase mutant, which is characterized in that: the recombinant bacterium expressing a nitrile hydratase mutant according to claim 1.

3. A recombinant bacterium which expresses a nitrile hydratase mutant according to claim 2, wherein: containing a nitrile hydratase wild-type or mutant gene sequence including, but not limited to, one of SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO. 5.

4. A recombinant bacterium which expresses a nitrile hydratase mutant according to claim 3, wherein: the nitrile hydratase wild type or mutant gene is expressed to synthesize a nitrile hydratase wild type or mutant, and the amino acid sequence of the hydratase wild type or mutant includes, but is not limited to, one of SEQ NO.6, SEQ NO.7, SEQ NO.8, SEQ NO.9, and SEQ NO. 10.

5. A nicotinamide preparation method is characterized in that nicotinamide is produced by using the recombinant bacteria expressing the nitrile hydratase mutant as claimed in any one of claims 2-4 as an enzyme source, and the specific steps are as follows:

adding the thalli into pure water for resuspension, preserving heat at 20 ℃, using 70 wt% of 3-cyanopyridine aqueous solution as a reaction substrate, feeding at an initial flow rate of 40g/min, reducing the flow rate according to the content of 3-cyanopyridine in a reaction solution in the reaction process, continuously reacting at 20 +/-2 ℃ to obtain nicotinamide, and reducing the byproduct nicotinic acid by more than 90%.

6. A process for the preparation of nicotinamide according to claim 5, which comprises: the enzyme activity final concentration of the nitrile hydratase mutant in the reaction liquid is 70U/mL, the pH of pure water is adjusted to 8.0 +/-0.2 by adopting liquid caustic soda, the reaction substrate is prepared by double distilled water, the temperature is preserved by water bath at 50 ℃, and the 3-cyanopyridine final concentration is 30 wt% of the reaction substrate.

Technical Field

The invention relates to the technical field of microbial gene recombination, in particular to a recombinant bacterium for expressing a nitrile hydratase mutant and a preparation method and application thereof.

Background

Directed evolution belongs to irrational design, and refers to the technical scheme that a natural evolution process of Darwinian is simulated in a laboratory, a coding gene of a certain protease is modified by error-prone PCR, DNA recombination and other technologies, and then valuable mutant enzymes are screened according to specific modification purposes. In the last 10 years, directed evolution technology has been successful in the field of modification of relevant properties of enzymes, mainly focusing on improving catalytic activity, improving substrate specificity, improving thermal stability, and the like.

At present, the production of nicotinamide is commonly carried out by biocatalytic production by recombinant engineering bacteria for expressing nitrile hydratase protein, 3-cyanopyridine hydrate is used as a raw material, and nicotinamide is prepared by catalysis of nitrile hydratase-containing microorganisms.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: aiming at the defect of high content of byproducts in the existing nicotinamide microbe catalytic production process, the invention constructs the nitrile hydratase mutant coding gene, and thalli obtained by the recombinant engineering bacteria after induction expression are used as enzyme sources, so that the recombinant engineering bacteria can be applied to nicotinamide production and the generation of byproduct nicotinic acid is reduced.

In order to solve the technical problems, the invention provides the following technical scheme:

a preparation method of a recombinant bacterium for expressing a nitrile hydratase mutant comprises the following specific steps:

(1) the method is characterized in that pET28-NHase plasmid of escherichia coli with high nitrile hydratase yield is used as a template, and an error-prone PCR method is adopted to construct a gene random mutation plasmid library, and the specific method is as follows:

recovering DNA products from the amplified fragments by agarose gel electrophoresis;

carrying out enzyme digestion on pET-28(a) idle body by using BamHI and HindIII, carrying out enzyme digestion on the recovered DNA product by using the same method, connecting the DNA fragment after enzyme digestion and pET-28(a) vector by using T4DNA ligase, and carrying out reaction at 16 ℃ overnight;

transforming the ligation product to DH10B competent cells with an electric converter, coating the competent cells on an LB plate of 50mg/mL kanamycin sulfate, culturing overnight, collecting thalli and extracting plasmids to obtain a gene random mutation plasmid library;

(2) the plasmid library is transferred into BL21 competent cells by a heat shock transformation method, the competent cells are coated on an LB plate containing kanamycin with the final concentration of 50mg/ml, a single clone is selected for bacterial detection, the target band is 1.9kb, and the bacterial sample system is as follows: (screening results graph)

(3) Randomly selecting bacteria-detecting positive monoclonal, inoculating into 48-well plate, shake culturing at 37 deg.C and 220rpm in 1ml liquid LB culture medium containing 50mg/ml kanamycin at final concentration for 16 hr, inoculating the culture solution into 50ml liquid LB culture medium containing 50mg/ml kanamycin at 3% volume ratio, shake culturing at 37 deg.C and 220rpm to OD₆₀₀＝0.4～0.6, adding IPTG with the final concentration of 100umol/L and cobalt chloride with the final concentration of 40mg/L, performing shake culture at 18 ℃ and 220rpm for 24 hours to obtain fermentation liquor, directly adding 3-cyanopyridine with the final concentration of 10% into the fermentation liquor, reacting at 20 ℃ for 30 minutes, and immediately placing on ice for 30 minutes. Eliminating colonies with crystal precipitation;

(4) the strains obtained by screening are shaken again according to the method, and the concentration of the 3-cyanopyridine is increased to 20 percent or 30 percent to obtain the high-activity strains.

A recombinant bacterium for expressing a nitrile hydratase mutant is prepared by the preparation method of the recombinant bacterium for expressing the nitrile hydratase mutant.

Preferably, the recombinant bacteria contain a nitrile hydratase wild-type or mutant gene sequence including, but not limited to, one of SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO. 5.

Preferably, the nitrile hydratase wild type or mutant gene is expressed to synthesize a nitrile hydratase wild type or mutant, the amino acid sequence of which includes, but is not limited to, one of SEQ NO.6, SEQ NO.7, SEQ NO.8, SEQ NO.9, SEQ NO. 10.

A preparation method of nicotinamide uses the recombinant bacteria expressing the nitrile hydratase mutant as an enzyme source to produce nicotinamide, and comprises the following specific steps:

adding the thalli into pure water for resuspension, preserving heat at 20 ℃, using a 70 wt% concentration 3-cyanopyridine aqueous solution as a reaction substrate, feeding at an initial flow rate of 40g/min, reducing the flow rate according to the content of 3-cyanopyridine in a reaction solution in the reaction process, continuously reacting at 20 +/-2 ℃ to obtain nicotinamide, and reducing the byproduct nicotinic acid by more than 90%.

Preferably, the enzyme activity final concentration of the nitrile hydratase mutant in the reaction liquid is 70U/mL, the pure water is adjusted to pH8.0 +/-0.2 by using liquid alkali, the reaction substrate is prepared by double distilled water, the temperature of the reaction substrate is kept in a water bath at 50 ℃, and the 3-cyanopyridine final concentration is 30 wt% of the reaction substrate.

The invention has the following beneficial effects:

1. the invention uses the genome of Escherichia coli with high nitrile hydratase yield as a template, adopts error-prone PCR technology to recombine and construct a random mutant plasmid library, screens nitrile hydratase mutant genes from the library, and can efficiently induce and express nitrile hydratase mutant proteins after the recombinant genes are transformed into engineering bacteria.

2. The nitrile hydratase mutant protein is used as a catalytic enzyme, can efficiently catalyze a substrate 3-cyanopyridine to synthesize nicotinamide, and can remarkably reduce the generation of a byproduct nicotinic acid.

3. The recombinant engineering bacteria of the high expression nitrile hydratase mutant coding gene are induced and expressed to obtain thallus which is used as an enzyme source, hydration reaction is carried out under a proper condition to produce nicotinamide, and the byproduct nicotinic acid is over 90 percent lower.

Drawings

FIG. 1 is a diagram showing the amino acid sequence alignment of mutant strains and wild strains;

FIG. 2 shows the results of enzyme activity measurement of a nitrile hydratase mutant of a wild-type strain;

FIG. 3 shows the results of enzyme activity measurement of the nitrile hydratase mutant of the high-activity mutant strain 1;

FIG. 4 shows the results of enzyme activity measurement of the nitrile hydratase mutant of the high-activity mutant strain 2;

FIG. 5 shows the results of enzyme activity measurement of the nitrile hydratase mutant of the high-activity mutant strain 3;

FIG. 6 shows the results of enzyme activity measurement of the nitrile hydratase mutant of the high-activity mutant strain 4;

wherein, the peak appearing in 5.5-6 min is nicotinamide in the reaction solution, and the peak appearing in 11min is byproduct nicotinic acid.

Detailed Description

The following examples are included to provide further detailed description of the present invention and to provide those skilled in the art with a more complete, concise, and exact understanding of the principles and spirit of the invention.