Process for enzymatic preparation of chiral 3-hydroxytetrahydrofuran and alcohol dehydrogenase mutant

A technology of hydroxytetrahydrofuran and ketotetrahydrofuran, which is applied in the field of enzymatic preparation of chiral 3-hydroxytetrahydrofuran and alcohol dehydrogenase mutants, can solve the problem of high conversion rate and achieve low environmental pollution, high purity and conversion rate and efficiency high effect

Active Publication Date: 2017-03-22
洛阳华荣生物技术有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

It is also obtained by dynamic resolution of 3-acetate tetrahydrofuran with esterase ( S )-3-Hydroxytetrahydrofuran was reported ( FEBS Journal 2013, 280, 3084-3093.), but the highest conversion rate of this method is only 50%

Method used

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  • Process for enzymatic preparation of chiral 3-hydroxytetrahydrofuran and alcohol dehydrogenase mutant
  • Process for enzymatic preparation of chiral 3-hydroxytetrahydrofuran and alcohol dehydrogenase mutant
  • Process for enzymatic preparation of chiral 3-hydroxytetrahydrofuran and alcohol dehydrogenase mutant

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Experimental program
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Effect test

Embodiment 1

[0063] In the reaction system, the pH value is 7.0, the concentration is 50 mmol / L, phosphate buffer, 5mmol / L 3-ketotetrahydrofuran as the substrate and the final concentration is 10% isopropanol, 0.01mmol / L NAD + React with the crude enzyme solution of alcohol dehydrogenase mutant A85V / I86N / L294V / C295N at 30°C for 14-16 hours, extract the product and substrate with ethyl acetate and detect the reaction process with GC, measure the ee value and Conversion rate; the product is (R )-3-Hydroxytetrahydrofuran, the ee value is 97%, and the conversion rate reaches 97%.

Embodiment 2

[0065] In the reaction system, the pH value is 7.4, and the concentration is 100 mmol / L phosphate buffer solution, 5 mmol / L 3-ketotetrahydrofuran as the substrate and the final concentration is 10% isopropanol, 0.05 mmol / L L-NAD + As a cofactor and I86N / C295N alcohol dehydrogenase mutant crude enzyme solution, react at 25°C for 14-16 hours, extract the product and substrate with ethyl acetate and use GC to detect the reaction process, measure the ee value and conversion rate , the product is ( R )-3-hydroxytetrahydrofuran, the ee value is 99%, and the conversion rate is 99%.

Embodiment 3

[0067] In the reaction system, add phosphate buffer solution with a pH value of 7.4 and a concentration of 50mmol / L, 5mmol / L 3-ketotetrahydrofuran as a substrate and isopropanol with a final concentration of 10%, 0.01~0.1 mmol / LNADP + As a cofactor and I86V / W110L / L294Q alcohol dehydrogenase mutant lysate, react at 30°C for 14-16 hours, extract the product and substrate with ethyl acetate and use GC to detect the reaction process, determine the ee value and conversion rate; the product is ( S )-3-hydroxytetrahydrofuran, the ee value is 95%, and the conversion rate is 99%.

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Abstract

The invention relates to a process for enzymatic preparation of chiral 3-hydroxytetrahydrofuran and an alcohol dehydrogenase mutant. The process comprises the a step of, with 3-ketotetrahydrofuran as a substrate, subjecting the substrate, the alcohol dehydrogenase mutant, a cofactor and a cofactor regeneration system to a biological catalysis reaction in a buffer system so as to produce chiral 3-hydroxytetrahydrofuran, wherein for alcohol dehydrogenase used by the alcohol dehydrogenase mutant, the wild gene sequence of alcohol dehydrogenase is derived from thermophilic bacteria, the base sequence is as shown in SEQ ID No. 1, and the amino acid sequence is as shown in SEQ ID No. 2. Compared with chemical methods, the process using the alcohol dehydrogenase mutant for preparation of chiral 3-hydroxytetrahydrofuran has the advantages of small environmental pollution, high efficiency, high optical selectivity; and compared with enzyme resolution methods, the process provided by the invention has higher purity and conversion rate and has ee value and conversion rate of both 99%.

Description

technical field [0001] The invention relates to a process for preparing chiral 3-hydroxytetrahydrofuran, in particular to a process for preparing chiral 3-hydroxytetrahydrofuran by enzymatic method and an alcohol dehydrogenase mutant. Background technique [0002] Biocatalysis has the characteristics of mild reaction conditions, high efficiency, strong stereo- and regioselectivity, and environmental friendliness, so biocatalysis has been widely used in the research and development of new drugs. In addition, using rational design and directed evolution to modify and optimize biocatalysts, such as improving catalyst stability and substrate selectivity, will enable them to be better applied to production processes. [0003] Alcohol dehydrogenase (alcohol dehydrogenase, ADH) is mainly used as a biocatalyst for the synthesis of chiral alcohols ( Catal. Sci. Technol ., 2011, 1, 1311–1323) for the synthesis of chiral drugs. ( S )-3-Hydroxytetrahydrofuran and ( R )-3-Hydroxytetr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12P17/04C12R1/01
CPCC12N9/0006C12P17/04C12Y101/01001
Inventor 孙周通范文超
Owner 洛阳华荣生物技术有限公司
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