Coding gene of meso-2,3-butanediol dehydrogenase, recombinase and preparation method and application of recombinase

A technology of meso-2 and butanediol dehydrogenase, which is applied in the field of bioengineering technology, can solve the problems of narrow asymmetric reduction substrate spectrum, low enzyme yield, and low overall catalytic activity, and achieve substrate applicability wide, high catalytic efficiency, and strong stereoselectivity

Inactive Publication Date: 2015-12-30
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved in the present invention is aimed at the defects that when the whole cell of wild Bacillus licheniformis (Bacillus licheniformis) CICIMB0109 is used as a diacetyl asymmetric reduction catalyst, the enzyme yield is low so that the overall catalytic activity is not high and the asymmetric reduction substrate spectrum is narrow, To provide a meso-2,3-butanediol dehydrogenase with excellent asymmetric catalytic activity, wide substrate applicability, and environmental friendliness and its coding gene, as well as a recombinant expression vector containing the gene and a recombinant Expression transformant, preparation method of recombinant enzyme, and the application of the preparation (2S,3S)-2,3-butanediol

Method used

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  • Coding gene of meso-2,3-butanediol dehydrogenase, recombinase and preparation method and application of recombinase
  • Coding gene of meso-2,3-butanediol dehydrogenase, recombinase and preparation method and application of recombinase
  • Coding gene of meso-2,3-butanediol dehydrogenase, recombinase and preparation method and application of recombinase

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Experimental program
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Effect test

Embodiment 1

[0022] Cloning of embodiment 1 meso-2,3-butanediol dehydrogenase gene

[0023] The meso-2,3-butanediol dehydrogenase gene in the above Bacillus licheniformis was cloned by shotgun method.

[0024] (1) First, the above-mentioned Bacillus licheniformis was cultured overnight at 37° C. using LB medium. After the bacterial cells were obtained by centrifugation, the total genomic DNA was obtained using a conventional bacterial genome extraction kit.

[0025] (2) Digest the total DNA with restriction endonuclease Sau3AI to form GATC cohesive ends. By controlling the enzyme dosage and reaction time, the total DNase is cut into 2-6kb fragments and recovered. These fragments were ligated with the pET28a plasmid digested with BamHI (recognition sequence GGATCC, cohesive end GATC), and the ligated products were transformed into competent cells E.coliBL21 ((DE3) After culturing at 37°C for 12-16 hours on the LB solid plate, pick a single colony for the next step of activity screening. ...

Embodiment 2

[0032] Example 2 Construction and cultivation of recombinant Escherichia coli BL21(DE3) / pET28a-Blbdh

[0033] The gene Blbdh and plasmid pET28a recovered in Example 1 were double-digested with restriction endonucleases BamHI and EcoRI in a water bath at 37°C overnight, purified by agarose gel electrophoresis the next day, and the target fragment was recovered using an agarose recovery kit . At 4°C, use T4 DNA ligase to ligate the excised gene Blbdh with the plasmid pET28a overnight to obtain the recombinant expression vector pET28a-Blbdh. The constructed recombinant expression vector pET28a-Blbdh was thermally transferred into Escherichia coli BL21 (DE3) competent, coated with Kan-resistant LB solid plate, and colony PCR verification was carried out after overnight culture. The positive clone was the recombinant Escherichia coli BL21 ( DE3) / pET28a-Blbdh. Pick positive clones and culture them overnight in LB medium, then transfer them into fresh LB culture at 2% transfer amou...

Embodiment 3

[0034] Embodiment 3 meso-2, the separation and purification of 3-butanediol dehydrogenase

[0035] Suspension-cultured recombinant cells were placed in solution A (20mmol·L -1 Sodium phosphate, 500mmol·L -1 NaCl, 20mmol·L -1 imidazole, pH 7.4), the crude enzyme solution was obtained after sonication and centrifugation. The column used for purification is an affinity column HisTrapFFcrude (nickel column), which is accomplished by using the histidine tag on the recombinant protein for affinity binding. First, use solution A to equilibrate the nickel column, load the crude enzyme solution, continue to use solution A to elute the breakthrough peak, and after equilibrium, use solution B (20mmol L -1 Sodium phosphate, 500mmol·L -1 NaCl, 1000mmol·L -1 imidazole, pH 7.4) for gradient elution to elute the recombinant protein bound to the nickel column to obtain recombinant meso-2,3-butanediol dehydrogenase. Enzyme activity assay (diacetyl as substrate, NADH as coenzyme) and SDS-P...

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Abstract

The invention discloses a coding gene of meso-2,3-butanediol dehydrogenase, a recombinant expression vector and recombinant expression transformant with the gene, a preparation method of recombinase, and application of the recombinase as a catalyst in an asymmetrically reducing prochiral dicarbonyl compound for preparing optical activity chiral diols. The meso-2,3-butanediol dehydrogenase comes from Bacillus licheniformis CICIMB0109, can be used as a catalyst for efficiently synthesizing chiral diols, is high in catalytic activity and stereoselectivity, mild in suitable reaction condition and friendly to the environment, and has quite good application and development prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a meso-2,3-butanediol dehydrogenase derived from Bacillus licheniformis (Bacillus licheniformis) CICIMB0109 and its gene, as well as a recombinant expression vector containing the gene and recombinant expression transformation A body, its recombination enzyme and the preparation method of the recombination enzyme, and the application of the reductase or its recombination enzyme as a catalyst in the asymmetric reduction of prochiral dicarbonyl compounds to prepare optically active chiral alcohols. Background technique [0002] Chiral diols have become a very important chiral compound because they have two chiral centers, and they are chiral building blocks for the synthesis of fine chemicals, chiral drugs, and chiral reagents. Among them, (2S,3S)-2,3-butanediol plays an important role in the asymmetric synthesis of chiral compounds containing two ortho chiral centers. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/70C12N1/21C12P7/18C12R1/19
Inventor 倪晔许国超韩瑞枝董晋军边雅倩
Owner JIANGNAN UNIV
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