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Method for improving asymmetric conversion efficiency of (R)-phenyl glycol by recombination strains through cosubstrate coupling

A phenylethylene glycol, recombinant bacteria technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms and other directions, can solve the problems of reduced catalytic efficiency, catalytic sustainability and stability, and achieves coenzyme regeneration. Bottleneck, effect of increasing substrate concentration and reaction efficiency

Active Publication Date: 2011-03-16
JIANGNAN UNIV
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, during the oxidoreductase catalyzed reaction, coenzyme is required to participate in the reaction as an electron transporter, and the non-renewable cycle of coenzyme is the main limiting factor for its industrial application.
Although the whole-cell system itself can provide a certain amount of coenzyme, as the reaction progresses or the concentration of the substrate increases, the supply rate of the coenzyme is far lower than the consumption rate, resulting in a decrease in catalytic persistence and stability, and ultimately leading to catalytic efficiency. reduction of

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 The cultivation of recombinant bacteria

[0026] LB medium was used, and its composition was: tryptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0. Ampicillin (100 μg / mL) was added during incubation. Add 1.5% agar powder to the solid medium. A single colony of the recombinant strain CCTCC NO: M 209289 was picked and inoculated in 4 mL LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C with shaking at 200 rpm for 12-16 h.

Embodiment 2

[0027] Example 2 Expression of target protein

[0028] Take 1 mL of the culture solution in Example 1 and transfer it to 100 mL LB liquid medium containing 100 μg / mL ampicillin, and culture it at 37°C with shaking at 200 rpm until OD 600 After the temperature was 0.6-0.8, transfer to 30°C for induction culture overnight, centrifuge at 8,000 g for 10 min to collect the bacterial cells, wash the bacterial cells twice with normal saline, and collect the recombinant bacterial cells.

Embodiment 3

[0030] The bacterium obtained in Example 2 was used for biotransformation test. In 5 mL 0.1 mol / L Tris-HCl (pH 8.5), add 5 mg / mL substrate 2-hydroxyacetophenone, 0.1 g / mL recombinant bacteria CCTCC NO: M 209289 wet cells, 6% glycerol, After mixing, the reaction was shaken on a constant temperature shaker at 30 °C for 48 h. After the reaction, the mixture was centrifuged, and the supernatant was extracted, and the product ( R )-phenylglycol with an optical purity of 100% e.e. and a yield of 80.2%.

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Abstract

The invention discloses a method for improving the asymmetric conversion efficiency of (R)-phenyl glycol by recombination strains through cosubstrate coupling, belonging to the technical field of biological catalysis asymmetric conversion. In the invention, a cosubstrate coupling system is built in rationality so as to realize good coenzyme regenerating cycle and greatly improve the preparation efficiency of the (R)-phenyl glycol; the recombinant Escherichia coli BL21 / pET32a-mrcr with the preservation number of CCTCCNO:M209289 serves as a biocatalyst after being optimized by the secondary structure of mRNA, and the cosubstrate glycerol and / or isopropanol with different ratios are added to improve the substrate dissolubility; and a substrate coupling coenzyme regenerating cycle system is built to shorten the reaction time from 48h to 1.5h and realize efficient conversion of the (R)-phenyl glycol under high substrate concentration. The economic and convenient whole cell coenzyme regenerating method provides an effective path for preparing the (R)-phenyl glycol with high efficiency and low cost and lays a solid foundation for industrial production of chiral alcohols.

Description

technical field [0001] Auxiliary substrate coupling improves the asymmetric transformation of recombinant bacteria ( R )-phenylethylene glycol efficiency method, the present invention greatly improves the asymmetric transformation efficiency of recombinant bacteria whole cells by constructing a substrate-coupled regenerative coenzyme system, that is, adding auxiliary substrates glycerol and / or isopropyl to the reaction system Alcohol, on the one hand, improves the solubility of the substrate, on the other hand, realizes the regeneration cycle of the coenzyme NADH, and efficiently prepares ( R )-phenyl glycol belongs to the technical field of biocatalytic asymmetric transformation. Background technique [0002] Chiral compounds have been more and more widely used in industries such as medicine, pesticides and food additives. Because different enantiomers have different pharmacology, toxicology and functional effects, the preparation of optically pure chiral compounds has be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P41/00C12P7/22C12R1/19
Inventor 徐岩张荣珍王珊珊
Owner JIANGNAN UNIV
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