CN108060145B

CN108060145B - 2, 3-butanediol dehydrogenase mutant with improved enzyme activity and construction method thereof

Info

Publication number: CN108060145B
Application number: CN201711324120.7A
Authority: CN
Inventors: 饶志明; 张显; 杨套伟; 徐美娟; 韩如梦
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2017-12-13
Filing date: 2017-12-13
Publication date: 2020-03-24
Anticipated expiration: 2037-12-13
Also published as: CN108060145A

Abstract

The invention discloses a2, 3-butanediol dehydrogenase mutant with improved enzyme activity and a construction method thereof, belonging to the technical field of genetic engineering. The mutant of the invention changes proline at position 52 into lysine on the basis of the amino acid shown in SEQ ID N0.2. The mutant enzyme activity obtained by the invention is improved by 68 percent compared with that before mutation, and the industrial application potential of the enzyme is enhanced.

Description

2, 3-butanediol dehydrogenase mutant with improved enzyme activity and construction method thereof

Technical Field

The invention relates to a2, 3-butanediol dehydrogenase mutant with improved enzyme activity and stability and a construction method thereof, belonging to the technical field of genetic engineering.

Technical Field

Acetoin has wide application value in aspects of food flavor blending, biochemistry and pharmacology, and the synthesis and production of acetoin are deeply researched at home and abroad at present. Some bacteria in nature have the ability to produce acetoin, and mainly include Klebsiella (Klebsiella), Enterobacter (Enterobacter), Bacillus (Bacillus), Serratia (Serratia), and Lactococcus (Lactococcus), among others. But in the metabolic process of most strains, acetoin exists as a byproduct of the metabolism of 2, 3-butanediol and butanedione, and the accumulated concentration is low, so that the problem that the acetoin is difficult to produce by industrial fermentation of the microbial strains is directly caused.

A bacillus subtilis B.subtiliss JNA (the preservation number is CCTCCM209309) capable of highly producing acetoin by taking glucose as a substrate is preserved in the laboratory, and the phenomenon of mutual transformation of the acetoin and 2, 3-butanediol in the fermentation process of the bacillus subtilis is found in research, and the mutual transformation process is regulated by the metabolism of acetoin reductase. Acetoin reductase, also known as 2, 3-butanediol dehydrogenase (BDH, E.C. 1.1.1.4). Is attached to an oxidoreductase and can catalyze the generation of 2, 3-butanediol from acetoin in the presence of NADH and at the same time can catalyze the generation of 2, 3-butanediol from NAD⁺Catalyzing 2, 3-butanediol to reversibly form acetoin in the presence of the catalyst. In this case, the activity of BDH is improved, and the control of cofactors is very critical to further improve the yield of the desired product. By passingThe cofactor regeneration technology biotransforms the synthetic product, which not only can reduce the synthesis cost, but also can simplify the separation of the product while driving the reaction to be completed, and has important significance.

Disclosure of Invention

The invention provides a BDH mutant with improved enzyme activity and a construction method thereof without solving the problems.

The first purpose of the invention is to provide a BDH mutant with improved enzyme activity, and the amino acid sequence of the BDH mutant is the sequence shown in SEQ ID NO. 1.

The amino acid sequence of the mutant is obtained by mutating amino acid at position 52 from proline to lysine on the basis of the amino acid with the amino acid sequence shown as SEQ ID NO. 2.

It is a second object of the invention to provide nucleotide sequences encoding said mutants.

In one embodiment of the invention, the nucleotide sequence encoding the mutant is the sequence shown in SEQ ID NO. 3.

In one embodiment of the present invention, the nucleotide sequence is obtained by mutating a codon encoding proline at position 52 to a codon encoding lysine on the basis of the nucleotide sequence shown in SEQ ID NO. 4.

It is a third object of the present invention to provide a recombinant expression vector comprising a nucleotide sequence encoding the mutant.

The fourth purpose of the invention is to provide a genetically engineered bacterium for expressing the BDH mutant.

In one embodiment of the invention, the genetically engineered bacterium is obtained by connecting a nucleotide sequence shown in SEQ ID NO.3 to an expression vector to obtain a recombinant plasmid, and then transforming the recombinant plasmid to a host bacterium.

In one embodiment of the present invention, the genetically engineered bacterium is a recombinant bacillus subtilis genetically engineered bacterium.

In one embodiment of the invention, the expression vector is pMA 5.

In one embodiment of the invention, the host bacterium is b.

In one embodiment of the invention, the preparation method of the genetically engineered bacterium is that on the basis of the nucleotide sequence shown in SEQ ID No.4, a codon for encoding proline at position 52 is mutated into a codon for encoding lysine to obtain a recombinant gene, the recombinant gene is connected to an expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into B.subtiliss 168 host bacteria to obtain the genetically engineered bacterium.

In an embodiment of the present invention, the preparation method specifically includes:

(1) PCR is carried out by taking the nucleotide sequence shown by SE QID NO.4 as a template, Flprimer (shown by SEQ ID NO. 5) and Rlprimer (shown by SEQ ID NO. 6) as primers to obtain the recombinant gene bdhA2 shown by SEQ ID NO. 3.

(2) And connecting the recombinant gene sequence obtained in the last step to a pMA5 expression vector to obtain a recombinant plasmid pMA5-bdhA2, transforming B.subtilis168 through recombinant plasmid transformation to obtain a recombinant engineering strain named B.subtilis168/pMA5-bdhA 2.

The fifth purpose of the invention is to provide the mutant, the nucleotide sequence for coding the mutant, a carrier containing the nucleotide sequence for coding the mutant and the application of the genetic engineering bacteria for expressing the mutant.

In one embodiment of the invention, the application comprises the BDH mutant and corresponding NADH oxidase to construct a coenzyme NADH circulation system for converting 2, 3-butanediol to produce acetoin.

In one embodiment of the invention, the BDH mutant can be used for constructing a coenzyme NADH cycle system.

The invention has the beneficial effects that:

(1) according to the invention, on the basis of natural BDH, the BDH molecular structure is reconstructed through site-directed mutagenesis, and the specific activity of the mutant enzyme is improved by 68%. The invention shows that the catalytic efficiency of the enzyme can be improved by mutating the 52 th amino acid residue of BDH into lysine.

(2) The BDH mutant enzyme obtained by the invention can be combined with corresponding NADH oxidase to construct an NADH coenzyme circulation system for producing acetoin, and the BDH mutant enzyme can also be applied to the construction of a coenzyme NADH regeneration system.

Detailed Description

LB culture medium: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl.

BDH enzyme activity definition: enzyme activity units (IU) are defined as 1. mu. mol NAD reduced per minute at 25 deg.C⁺The required enzyme amount is one enzyme activity unit U. The specific activity of the enzyme is defined as the unit enzyme activity U/mg of the protein.

BDH activity determination method: the enzyme reaction system was 1mL, containing 0.1M2, 3-butanediol, 50mM sodium phosphate buffer pH8.0, 5mM NAD⁺(ii) a The enzymatic reaction starts immediately after a certain amount of enzyme solution is added, and the concentration of NADH is calculated according to the change of the absorbance value of the reaction solution at 340nm, and the enzyme activity is calculated.

Example 1 construction of Bacillus subtilis expression vectors containing BDH mutants

(1) Acquisition of BDH mutants: taking the nucleotide sequence shown in SEQ ID NO.3 as a template, and taking Fprimer (shown in SEQ ID NO. 5) and Rpcr primer (shown in SEQ ID NO. 6) as primers, and carrying out PCR to obtain the recombinant gene shown in SEQ ID NO. 3.

(2) The recombinant gene and a bacillus subtilis expression vector pMA5 are subjected to double enzyme digestion by BamH I and Mlu I respectively, and are connected by T4DNA ligase at 16 ℃ overnight after purification. Ligation product chemistry transformed b.subtilis168 competent cells. The transformation liquid is coated on LB plate containing kanamycin (50mg/L), plasmid is extracted, and the constructed recombinant plasmid is verified by double enzyme digestion and named as pMA5-bdhA 2. The sequencing work is completed by Shanghai worker.

Example 2 construction of BDH-producing mutant recombinant Bacillus subtilis engineering bacteria

The strain containing the correct recombinant plasmid pMA5-bdhA2 obtained in example 1 is the recombinant gene engineering bacterium B.subtilis168/pMA5-bdhA 2.

Example 3 recombinant bacteria B.subtilis168/pMA5-bdhA2 expression BDH and enzyme activity determination

The recombinant strain B.subtiliss 168/pMA5-bdhA2 constructed in example 2 and a control strain B.subtiliss 168/pMA5-bdhA expressing non-mutated wild BDH (amino acid sequence shown in SEQ ID NO: 2) were inoculated into l0mL LB medium containing kanamycin, respectively, cultured overnight with shaking at 37 ℃ and transferred to a fermentation medium the next day in an inoculum size of 4%, and cultured at 37 ℃ for 24 hours. The cells were collected by centrifugation and disrupted, and the cell disruption supernatant (crude enzyme solution) was used for the measurement of the enzyme activity. The obtained crude enzyme solution is purified to obtain the BDH mutant, and the specific enzyme activity of the BDH mutant is improved by 68 percent compared with that before mutation.

Example 4 application of recombinant BDH mutant in conversion of 2, 3-butanediol to produce acetoin

The recombinant strain B.subtiliss 168/pMA5-bdhA2 cell disruption solution obtained in example 3 is applied to the conversion of 2, 3-butanediol to produce acetoin, and NAD is added⁺Recycling of regenerated enzyme or sufficient NAD⁺In the case of (3), 40g/L of 2, 3-butanediol can be converted within 2h to generate 37.8g/L of acetoin; 120g/L of 2, 3-butanediol can be converted into about 98.5g/L of acetoin within 30 hours by feeding 2, 3-butanediol

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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<120> 2, 3-butanediol dehydrogenase mutant with improved enzyme activity and construction method thereof

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1 ATGAAGGCAG CAAGATGGCA TAACCAAAAG GATATCCGTA TTGAACATAT CGAAGAGCCA

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ACCGACGCGTTTAGTTAGGTCTAACAAGG

Claims

1. The mutant of 2, 3-butanediol dehydrogenase is characterized in that the amino acid sequence of the mutant is shown as SEQ ID No. 1.

2. A nucleotide sequence encoding the mutant of claim 1.

3. The nucleotide sequence of claim 2, wherein the nucleotide sequence is represented by SEQ ID No. 3.

4. A recombinant expression vector comprising the nucleotide sequence of claim 2 or 3.

5. A genetically engineered bacterium expressing the 2, 3-butanediol dehydrogenase mutant of claim 1.

6. The genetically engineered bacterium of claim 5, wherein the genetically engineered bacterium is obtained by connecting a nucleotide sequence shown by SEQ ID No.3 to an expression vector to obtain a recombinant plasmid, and then transforming the recombinant plasmid to a host bacterium.

7. The genetically engineered bacterium of claim 5, wherein the genetically engineered bacterium is a recombinant Bacillus subtilis genetically engineered bacterium.

8. The mutant of claim 1, wherein the mutant is used for improving the capability of converting BDH into 2, 3-butanediol to generate acetoin and constructing a coenzyme cycle system with other enzymes for NAD + regeneration.

9. Use of a nucleotide sequence encoding the mutant of claim 1 to improve the ability of BDH to convert 2, 3-butanediol to acetoin and to use BDH in construction of a coenzyme cycling system with other enzymes for NAD + regeneration.

10. Use of a vector comprising a nucleotide sequence encoding a mutant according to claim 1 to improve the ability of BDH to convert 2, 3-butanediol to acetoin and to use BDH in construction of a coenzyme cycling system with other enzymes for NAD + regeneration.

11. The application of the genetically engineered bacteria expressing the mutant of claim 1 in improving the capability of converting BDH into 2, 3-butanediol to generate acetoin and constructing a coenzyme circulation system by using BDH and other enzymes for NAD + regeneration.