Method for preparing (R)-phenylglycol from SD-AS sequence coupled (R)-carbonyl reductase and glucose dehydrogenase

A technology of phenylethylene glycol and hydroxyacetophenone, applied in the field of biocatalytic asymmetric transformation, can solve the problems of unstable chemical properties and expensive coenzymes

Inactive Publication Date: 2015-08-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because coenzyme is expensive and its chemical properties are not stable, it cannot be replaced by general synthetic substances.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] recombine E. coli JM109 / pET-RCR and recombination E. coli The medium composition of JM109 / pET-GDH in g / L: peptone 10, yeast extract 5, NaCl 10, pH 7.0, prepared with deionized water.

[0066]The two recombinant bacteria were respectively inoculated into test tubes with a liquid volume of 5 mL of medium, and shaken at 37 °C and 200 rpm for 8 h.

Embodiment 2

[0068] recombine E. coli JM109 / pET-RCR and recombination E. coli Extraction of JM109 / pET-GDH plasmid: Centrifuge the bacteria cultured in Example 1 at 12,000 rpm for 1 min to collect the cells, and use the plasmid extraction kit Mini-Plasmid Rapid Isolation Kit (Beijing Broadtech Biogene Technology Co., Ltd.) to extract Plasmids pET-RCR and pET-GDH.

Embodiment 3

[0070] RCR and GDH co-expression primer design:

[0071] R-SD-AS-G-f: 5'-ATCCT GCTAG C ATGTCAATT CCATCAAGCC AGTAC-3'( Nhe I),

[0072] RCR-r: 5'-CGGATACAT G GTATATCTCC TTC CTATGGATTAAAAACAA

[0073] CTCTACCTT-3', (the underlined part is the reverse mutual SD-AS sequence)

[0074] GDH-f: 5'-AATCCATAG G AAGGAGATAT ACC ATGTATC CGGATTTAAA AGGAAAAGTC-3', (the underlined part is SD-AS sequence)

[0075] R-SD-AS-G-r: 5'-TGACT CTCGA G ACCGCGGCC TGCCTG-3'( xho I).

[0076] The SD-AS sequences were introduced into the primers respectively.

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Abstract

The invention discloses a method for preparing (R)-phenylglycol from SD-AS sequence coupled (R)-carbonyl reductase and glucose dehydrogenase and belongs to the technical field of biological catalytic asymmetric transformation. The invention provides recombinant escherichia coli E. coli RIL/pET-R-SD-AS-G. The recombinant escherichia coli E. coli RIL/pET-R-SD-AS-G is preserved in the China center for type culture collection (CCTCC) and has a preservation number of CCTCC NO: M2015170. R-carbonyl reductase and glucose dehydrogenase are coupled by a coexpression way, and the coupled enzyme, a cultured recombinant strain and 2-hydroxyacetophenone as a substrate undergo a catalytic asymmetric transformation reaction under optimized biotransformation reaction conditions to produce (R)-phenylglycol with optical purity of 100% and a yield of 99.9%. The method utilizes a SD-AS sequence high efficiency coupling double-enzyme technology, solves the problem of limitation of chiral catalytic reaction coenzyme regeneration cycle and provides an effective approach for high efficiency bio-preparation of the (R)-phenylglycol.

Description

technical field [0001] Utilize the SD-AS serial connection ( R )-carbonyl reductase and glucose dehydrogenase, asymmetric conversion preparation ( R )-phenylglycol method, the present invention relates to genetic engineering technology to make two kinds of enzyme co-expression, construct recombinant escherichia coli, be used for high-efficiency preparation ( R The method of )-phenylethylene glycol and application thereof belong to the technical field of biocatalytic asymmetric conversion. Background technique [0002] Chiral compounds are widely used in the preparation of fine chemicals such as medicine and pesticides. For example, optically pure phenylethylene glycol is an important intermediate for preparing optically active medicines, pesticides and functional materials, and is also an indispensable and important hand in liquid crystal materials. sexual additives. [0003] Oxidoreductases can catalyze asymmetric reduction reactions to prepare chiral compounds. These r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N9/04C12P7/22C12R1/19
CPCC12N1/20C12P7/22C12N1/205C12R2001/19
Inventor 张荣珍徐岩周晓天
Owner JIANGNAN UNIV
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