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

A mutant and butanediol technology, applied in the field of genetic engineering, can solve problems such as low accumulation concentration and refractory acetoin, and achieve the effect of improving catalytic efficiency

Active Publication Date: 2020-03-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the metabolic process of most strains, acetoin exists as a by-product of the metabolism of 2,3-butanediol and diacetyl, and the accumulation concentration is low, which directly leads to the difficulty of using these microbial strains for industrial fermentation production Acetoin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 Construction of the Bacillus subtilis expression vector containing BDH mutant

[0031] (1) Obtaining of BDH mutants: using the nucleotide sequence shown in SEQ ID NO.3 as a template, Fprimer (sequence shown in SEQ ID NO.5) and Rprimer (sequence shown in SEQ ID NO.6) are primers, PCR is performed to obtain the recombinant gene shown in SEQ ID NO.3.

[0032] (2) The recombinant gene and the Bacillus subtilis expression vector pMA5 were digested with BamH I and Mlu I respectively, and after purification, they were ligated with T4 DNA ligase overnight at 16°C. The ligation product was used to transform B. subtilis 168 competent cells by chemical method. The transformation solution was applied to an LB plate containing kanamycin (50mg / L), the plasmid was extracted, and the recombinant plasmid constructed was verified by double enzyme digestion, which was named pMA5-bdhA2. The sequencing work was completed by Shanghai Sangong.

Embodiment 2

[0033] Example 2 Construction of BDH Mutant Recombinant Bacillus subtilis Engineering Bacteria

[0034] The bacterial strain containing the correct recombinant plasmid pMA5-bdhA2 obtained in Example 1 is the recombinant genetically engineered bacterium B. subtilis 168 / pMA5-bdhA2 of the present invention.

Embodiment 3

[0035] Example 3 BDH Expression and Enzyme Activity Determination of Recombinant Bacteria B.subtilis 168 / pMA5-bdhA2

[0036] The recombinant strain B.subtilis168 / pMA5-bdhA2 constructed in Example 2 and the control strain B.subtilis168 / pMA5-bdhA expressing unmutated wild BDH (amino acid sequence shown in SEQ ID NO: 2) were respectively inoculated in 10 mL of card-containing In the LB medium of namycin, shake culture overnight at 37°C, transfer to fermentation medium at 4% inoculum the next day, and culture at 37°C for 24 hours. The cells were collected by centrifugation and broken, and the cell broken supernatant (crude enzyme solution) was used for the determination of enzyme activity. The obtained crude enzyme solution was purified to obtain a BDH mutant, and its specific enzyme activity was increased by 68% compared with that before the mutation.

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Abstract

The invention discloses an enzyme activity improved 2,3-butanediol dehydrogenase mutant and an establishment method thereof and belongs to the technical field of genetic engineering. According to themutant disclosed by the invention, the 52nd proline is mutated into lysine on the basis of amino acid as shown in SEQ ID No.2. The enzyme activity of the mutant disclosed by the invention is improvedby 68% when being compared with that before mutation, and the industrial application potential of the enzyme is improved.

Description

technical field [0001] The invention relates to a 2,3-butanediol dehydrogenase mutant with improved enzyme activity and stability and a construction method thereof, belonging to the technical field of genetic engineering. [0002] technical background [0003] Acetoin has a wide range of applications in food flavoring, biochemistry and pharmacology. At present, there are relatively in-depth researches on its synthesis and production at home and abroad. Some bacteria in nature have the ability to produce acetoin, mainly including Klebsiella (Klebisella), Enterobacter (Enterobacter), Bacillus (Bacillus), Serratia (Serratia) and milk Lactococcus and the like. However, in the metabolic process of most strains, acetoin exists as a by-product of the metabolism of 2,3-butanediol and diacetyl, and the accumulation concentration is low, which directly leads to the difficulty of using these microbial strains for industrial fermentation production Acetomarry. [0004] There is a stra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N1/21C12P7/26C12R1/125
CPCC12N9/0006C12P7/26C12Y101/01004
Inventor 饶志明张显杨套伟徐美娟韩如梦
Owner JIANGNAN UNIV
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