Method for biologically preparing (S)-4-chloro-3-hydroxy butyric acid ethyl ester with recombinant escherichia coli expressed ketoreductase

A technology of recombinant Escherichia coli and ethyl hydroxybutyrate, applied in the biological field, can solve the problems of low optical purity of products and low conversion rate of substrates, achieve high optical purity and reduce production costs

Inactive Publication Date: 2013-06-26
JIANGXI NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, in the current technology of preparing (S)-CHBE by asymmetric reduction of COBE by biological methods, problems such as

Method used

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  • Method for biologically preparing (S)-4-chloro-3-hydroxy butyric acid ethyl ester with recombinant escherichia coli expressed ketoreductase
  • Method for biologically preparing (S)-4-chloro-3-hydroxy butyric acid ethyl ester with recombinant escherichia coli expressed ketoreductase
  • Method for biologically preparing (S)-4-chloro-3-hydroxy butyric acid ethyl ester with recombinant escherichia coli expressed ketoreductase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Acquisition of ketoreductase gene

[0024] according to Candida magnoliae Amino acid sequence of ketoreductase, using E.coli The codon preference was optimized to obtain a new gene sequence (Genbank: KC426948), which was sent to Shanghai Shenggong for synthesis, and NcoI and BamHI restriction sites were added to both ends of the gene. NcoI restriction site: CCATGG, BamHI restriction site: GGATCC.

Embodiment 2

[0025] Example 2 Acquisition of Glucose Dehydrogenase Gene

[0026] Bacillus subtilis ( Bacillus subtilis ) LB liquid medium: tryptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0.

[0027] Bacillus subtilis was inoculated into 5mL LB liquid medium at 37°C and cultivated to the logarithmic growth phase, and the genome was extracted using a genomic DNA extraction kit (product of TaKaRa).

[0028] The primers used to construct the expression vector are equipped with restriction sites, and the primer sequence is as follows:

[0029] GDH-F1: 5'-CGAATTCATGTATCCGGATTTAAAAGG-3' EcoRI restriction site

[0030] GDH-R1: 5'-GAAGCTTTTATGTTTAACCGCGGCCTG -3' HindIII restriction site

[0031] The primers were synthesized by Shanghai Biological Engineering Co., Ltd.

[0032] Gene PCR (50ul reaction system) conditions are as follows:

[0033] Pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 45s, annealing at 52°C for 45s, extension at 68°C for 1 min, 30 cycles; extension at 68°C for 10 minute...

Embodiment 3

[0034] Example 3 Expression of ketoreductase gene

[0035] Use EcoRI and HindIII to digest pET-28a (pET-28a purchased from Novagen (Merck China)) and the synthesized target gene containing two restriction sites, respectively, and recover the double digested target fragment and expression vector. , Connect the double digested expression vector pET-28a and the target gene with T4 ligase overnight, add 10μL of the ligation product pET-28a-ketoreductase to 100μL of BL21(DE3) competent cells, and place on ice 30min, heat shock at 42°C for 90 s. Place on ice for 2 minutes. Add pre-warmed 0.45mL medium. Incubate at 200rpm 37°C for 1h. Add 200μL of bacterial solution to LB plates containing 50μg / mL of kanamycin and chloramphenicol, and incubate overnight at 37°C for 12-16h. Build the map see figure 1 .

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Abstract

The invention discloses a method for biologically preparing (S)-4-chloro-3-hydroxy butyric acid ethyl ester with recombinant escherichia coli expressed ketoreductase and application thereof in preparing (S)-4-chloro-3-hydroxy butyric acid ethyl ester through asymmetric reduction of 4-chloroacetoacetic acid ethyl ester. The recombinant escherichia coli for expressing the gene of the ketoreductase (KRED) is coupled with glucose dehydrogenase (GDH) so that the escherichia coli cell is recombined into a catalyst; efficient preparation of (S)-4-chloro-3-hydroxy butyric acid ethyl ester through asymmetric reduction of 4-chloroacetoacetic acid ethyl ester in a single water phase is realized no matter in the presence of or in the absence of coenzyme NAD(P)H; and the molar transformation rate is higher than 92% and the enantiomer excess (e.e.) of the product is 100%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the application of a recombinant Escherichia coli expressing ketoreductase, especially the recombinant bacterium of ketoreductase ketoreductase synthesized by introducing the whole gene sequence, and using ethyl 4-chloroacetoacetate as the base Asymmetric reduction method for preparing (S)-4-chloro-3-hydroxybutyric acid ethyl ester. Background technique [0002] 4-Chloro-3-hydroxybutyrate ethyl ester (ethyl 4-chloro-3-hydroxyrate, CHBE) is an important organic intermediate, its main use is to synthesize the precursor compound of the cholesterol-lowering drug atorvastatin . Its chiral single enantiomer S-4-chloro-3-hydroxybutyrate ethyl ester ((S)-CHBE) can be used in the synthesis of many active drugs, such as hydroxymethylglutaryl CoA (HMG-CoA) reductase Inhibitors and 1,4-dihydropyridine β-blockers, etc. [0003] The latent chiral substance 4-chloroacetoacetate (ethyl 4-chloro-3-o...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12R1/19
Inventor 张志斌吴小芳朱笃颜日明汪涯
Owner JIANGXI NORMAL UNIV
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