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Carbonyl reductase mutant, vector, engineering bacterium and application thereof

A technology of genetically engineered bacteria and mutants, applied in the direction of oxidoreductase, bacteria, and microorganism-based methods, can solve the problems of low catalytic efficiency, low concentration of catalytic substrate, low enzyme yield, etc., and achieve excellent catalytic activity, The effect of good app development prospects

Active Publication Date: 2018-01-16
杭州馨海生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

, 2010, 101). However, when the wild type is used as the catalyst, the enzyme yield is low, the concentration of the catalytic substrate is low, and the catalytic efficiency is not high, which limits the industrial application of this carbonyl reductase

Method used

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  • Carbonyl reductase mutant, vector, engineering bacterium and application thereof
  • Carbonyl reductase mutant, vector, engineering bacterium and application thereof
  • Carbonyl reductase mutant, vector, engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of mutants

[0037] Using the oligonucleotide fragment containing the mutation point as the primer (Table 1), the QuickChangeTM method (Stratagene, La Jolla, CA) was used to amplify the pET-30a recombinant plasmid containing the carbonyl reductase gene.

[0038] Table 1 Mutants to construct primers

[0039]

[0040] a The underline indicates the mutation site

[0041] PCR reaction system: 5×PrimerSTAR buffer(Mg 2+ plus), 5μL; dNTPs (2.5mM each), 2.0μL; upstream primer (10μM), 1.0μL; downstream primer (10μM), 1.0μL; recombinant plasmid template, 15ng; PrimerSTARpolymeraseTM HS (2.5U / μL), 0.5μL ; Add ddH 2 O to a total volume of 25μL.

[0042] PCR program: (1) 98°C, 1min; (2) 98°C, 10s; (3) 55°C, 10s; (4) 72°C, 7min. Steps (2)-(4) are cycled 20 times and then cooled to 4°C.

[0043] After the PCR product is washed, it is digested with the restriction enzyme DpnI that specifically recognizes the methylation site to degrade the template plasmid. Enzyme diges...

Embodiment 2

[0046] Example 2: Induced expression of carbonyl reductase mutants

[0047] The engineered bacteria constructed in Example 1 were inoculated into 50μg / mL kanamycin LB medium, cultured overnight at 37°C and 200 rpm, and then inoculated to 50μg / mL kanamycin at 1% inoculum amount (v / v) In LB medium, culture at 37°C and 200rpm until the cell concentration OD600 is about 0.6, add IPTG with a final concentration of 0.1mM, induce culture at 26°C for 6h, centrifuge at 4°C, 8000rpm for 10min to collect the cells, store at -80°C spare.

Embodiment 3

[0048] Example 3: Fermenter culture of carbonyl reductase mutant

[0049] The engineered bacteria constructed in Example 1 were inoculated into 50μg / mL kanamycin LB medium, cultured overnight at 37°C and 200 rpm, and then inoculated to 50μg / mL kanamycin at 2% inoculum (v / v) Cultivate in the medium at 37°C at 200 rpm, and inoculate 10% inoculum (v / v) into the fermentor containing 15L of 50μg / mL kanamycin fermentation medium in the middle logarithmic phase at 37°C. About 14h (in the late logarithmic period), add lactose for induction for 20h, and collect the bacteria by centrifugation in a tube centrifuge for use.

[0050] The fermentation medium used in Example 3 is a well-known medium in the art that can grow mutants and produce the carbonyl reductase mutant protein of the present invention.

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Abstract

The invention provides a carbonyl reductase mutant, a recombinant expression vector, a genetically engineered bacterium, a method for preparing the mutant, and the application of the mutant in preparing an optically pure chiral alcohol by asymmetric reduction of a series of prochiral ketones. A catalyst is easy to prepare, reaction conditions are mild, substrate adaptability is wide, environmentalfriendliness is high, recombinant cells can efficiently catalyze the asymmetric reduction of the high-concentration prochiral ketones in a reaction system containing dimethyl sulfoxide and xylose without addition of any coenzymes, and the produced chiral alcohol with high optical purity (ee larger than or equal to 99%) has good industrial application prospects.

Description

Technical field [0001] The invention belongs to the field of biochemical technology, and specifically relates to a Bacillus subtilis carbonyl reductase mutant, gene, and recombinant expression vector and recombinant expression transformant containing the gene, as well as a series of potential asymmetric reduction of recombinant cells containing the enzyme. Application of chiral ketones in the preparation of optically pure chiral alcohols. Background technique [0002] The two enantiomers of chiral drugs often have different efficacies or their effects are very different. Therefore, the synthesis of a single enantiomer has attracted more and more attention. As one of the most important chiral building blocks, optically active chiral alcohols are widely used in the synthesis of chiral drugs and fine chemicals. The asymmetric reduction of latent chiral ketones is an important method for preparing optically active chiral alcohols. In theory, 100% of substrate ketones can be converte...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N1/21C12P7/02C12R1/19
Inventor 于洪巍俞鑫焱张志强金樑波
Owner 杭州馨海生物科技有限公司
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