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Method for preparing chiral alcohol through ketoreductase continuous reaction

A reductase and chemical reaction technology, applied in the field of enzyme catalysis, can solve problems such as difficulty in continuous and industrialization, cofactor cannot be efficiently recycled and regenerated in the preparation process, ketone reductase activity loss, etc., and achieve the effect of simple operation

Pending Publication Date: 2022-04-12
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the problems of the loss of ketoreductase activity, the inability to efficiently regenerate the cofactor and the difficulty in continuous and industrialization of the preparation process in the prior art, and provides a pickering emulsion-based encapsulation of ketone reductase and cofactor for continuous fixed bed The new method realizes the non-destructive encapsulation of ketoreductase and cofactor and the efficient regeneration of cofactor, and the long-term continuous stable and efficient operation lays the foundation for industrialization

Method used

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  • Method for preparing chiral alcohol through ketoreductase continuous reaction
  • Method for preparing chiral alcohol through ketoreductase continuous reaction
  • Method for preparing chiral alcohol through ketoreductase continuous reaction

Examples

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Effect test

Embodiment 1

[0032] A method for preparing chiral alcohol by continuous reaction of ketoreductase, comprising the steps of:

[0033] Step 1: 1.0 g of SiO with a particle size of 60 nm was ultrasonically 2 Disperse the nanoparticles into 12mL of toluene, add 4.5mmol octyltrimethoxysilane and 4.5mmol triethylamine, stir and reflux for 4h at 110°C under nitrogen protection, and centrifuge the above mixed system after cooling. Wash with toluene 3-5 times and dry to obtain interface active nano-SiO 2 (Looks like figure 2 shown);

[0034] Step 2: Mix 0.36mL of ES-KRED-113 ketoreductase solution, 0.223mg of NADP + Add it to 3.2mL100mM PBS buffer solution, and mix uniformly by magnetic stirring to obtain an aqueous phase system; then add 0.072g of interface-active SiO 2 Ultrasonic dispersion was obtained in 25mL n-heptane to obtain an oil phase system; the oil-water two-phase system was mixed and stirred at a high speed of 5000rpm to form a Pickering emulsion of immobilized ketone reductase a...

Embodiment 2

[0037] A method for preparing chiral alcohol by continuous reaction of ketoreductase, comprising the steps of:

[0038] Step 1: 1.0 g of SiO with a particle size of 60 nm was ultrasonically 2 Disperse nanoparticles into 15mL toluene, add 4.5mmol dichlorodimethylsilane and 2.25mmol n-hexylamine, stir and reflux for 3 hours at 120°C under nitrogen protection, and centrifuge the above mixed system after cooling, and the obtained solid is washed with toluene Wash 3-5 times and dry to obtain interface active nano-SiO 2 ;

[0039]Step 2: Mix 0.72mL of ES-KRED-115 ketoreductase solution, 1mg NADP + Added to 6.4mL of 100mM PBS buffer solution, mixed uniformly by magnetic stirring to obtain an aqueous phase system; then 0.316g of interface-active SiO 2 Ultrasonic dispersion in 16mL of toluene to obtain an oil phase system; the oil-water two-phase system was mixed and stirred at a high speed of 10,000rpm to form a Pickering emulsion with immobilized ketone reductase and cofactor;

...

example 3

[0042] A method for preparing chiral alcohol by continuous reaction of ketoreductase, comprising the steps of:

[0043] Step 1: 1.2 g of SiO with a particle size of 60 nm was ultrasonically 2 Disperse the nanoparticles into 20mL toluene, add 1.5mmol dichlorodimethylsilane and 4.5mmol n-hexylamine, stir and reflux for 5h at 60°C under nitrogen protection, and centrifuge the above mixed system after cooling, and the obtained solid is washed with toluene Wash 3-5 times and dry to obtain interface active nano-SiO 2 ;

[0044] Step 2: Mix 0.05mL of ES-KRED-172 ketoreductase solution, 0.112mg NADP + Add it to 3.2mL100mM PBS buffer solution, and mix it uniformly by magnetic stirring to obtain an aqueous phase system; then add 0.158g of interface-active SiO 2 Ultrasonic dispersion in 15mL of ethyl acetate to obtain an oil phase system; the oil-water two-phase system was mixed and stirred at a high speed of 8000rpm to form a Pickering emulsion with immobilized ketone reductase and c...

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Abstract

The invention belongs to the field of enzyme catalysis, and particularly relates to a method for preparing chiral alcohol through ketoreductase continuous reaction. The method comprises the following steps: dispersing ketoreductase and cofactors in a water-phase PBS (Phosphate Buffer Solution) by taking interfacial active silicon dioxide nanoparticles as an emulsifier to form a water-in-oil type Pickering emulsion encapsulating the ketoreductase and the cofactors, and then filling the Pickering emulsion into a fixed bed reactor for continuous reverse preparation of the chiral alcohol. The water-in-oil type Pickering emulsion is used for realizing concerted catalysis of ketoreductase and cofactors in a confinement space, continuous reaction is carried out for more than 400 hours, the catalytic activity of the ketoreductase is basically kept unchanged, and the total conversion number of the cofactors can reach more than 60000.

Description

technical field [0001] The invention relates to the field of enzyme catalysis, in particular to a method for preparing chiral alcohol through continuous reaction of ketoreductase. Background technique [0002] Enzyme-catalyzed reactions have the advantages of mild conditions, high catalytic efficiency, and strong specificity. Their high-efficiency chemical, stereo, and regioselectivity can help reduce costs, improve reaction efficiency, avoid unnecessary side reactions, and reduce environmental pollution. Therefore, enzymes, as an important biocatalyst, have been widely used in the synthesis of various organic compounds such as drugs, chiral compounds, and optically active compounds, and have shown great application prospects. [0003] The production of chiral alcohol drug intermediates is a difficult point in the synthesis of chiral drugs, and it is also an indispensable raw material basis for the development of chiral drugs. At present, the industrial synthesis of chiral ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/04C12N9/02C12N9/96
Inventor 杨恒权卫伟张明
Owner SHANXI UNIV
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