Production of chiral 1,2-diol compounds by biocatalysis of carbonyl reductase

A technology of biocatalysis and reductase, which is applied in the direction of chemical recovery and fermentation, can solve the problems of being unable to meet the needs of industrialization, low substrate input and catalytic efficiency, and affecting the stereoselectivity of catalytic products, etc., to achieve a simple reaction system, The effect of mild reaction conditions

Active Publication Date: 2018-11-06
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0014] These reported biocatalytic reactions are all catalyzed by yeast cells, the amount of substrate input and catalytic efficiency are low (substrate concentration 2-4g / l, reaction time 2 days), and other proteins contained in the cells will also affect the catalytic product. Stereoselectivity (ee value>99%, de value>80%), unable to meet the needs of industrialization (Tetrahedron:Asymmetry, 1997.8(14): 2355-2359; Cheminform, 1986.18(9): 1367-1370.)
There is no literature report on the preparation of optically pure 1,2-diol compounds by carbonyl reductase catalytic reduction

Method used

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  • Production of chiral 1,2-diol compounds by biocatalysis of carbonyl reductase
  • Production of chiral 1,2-diol compounds by biocatalysis of carbonyl reductase
  • Production of chiral 1,2-diol compounds by biocatalysis of carbonyl reductase

Examples

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Embodiment 1

[0040] Example 1: Screening of carbonyl reductase

[0041] In a method well known in the art, connect the carbonyl reductase gene into the pET28a(+) vector, transfer it into Escherichia coli BL21-DE3 for heterologous expression, centrifuge at 6,000rpm, 4°C for 10min to obtain wet cells of recombinant bacteria, resuspend in phosphoric acid Potassium buffer solution (0.1M, pH 7.0), break the cells with a homogenizer, centrifuge at 13,00 rpm, 4°C for 20 minutes, and the obtained supernatant is the crude enzyme solution, which is used as a biocatalyst. We screened our lab's toolbox of carbonyl reductases. When screening, the biocatalysis and system are: potassium phosphate buffer (0.1M, pH 7.0), carbonyl reductase crude enzyme solution 10g / l, substrate (1a) 1g / l, NAD + / NADP + Concentration 0.6 g / l, glucose dehydrogenase 3 g / l and glucose 10% (w / v). The reaction time is 12 hours, the reaction temperature is 30° C., and the rotation speed is 200 rpm.

[0042] At the same time, ...

Embodiment 2

[0044] Example 2: HPLC detection conditions of product standards of different α-alkoxy-β-keto esters

[0045] The chiral HPLC detection conditions of the anti-configuration racemate of the product are shown in Table 1. ChKRED12 catalyzed the substrate 1a to obtain the (2S,3S)-configuration product. The configurations of the corresponding products in the following examples are all the same and will not be repeated here.

[0046] Table 1 Substrates and assay conditions

[0047]

Embodiment 3

[0048] Example 3: Carbonyl reductase ChKRED12 crude enzyme solution catalyzes 4g / l substrate 1a

[0049] Take freshly cultured carbonyl reductase ChKRED12 recombinant bacteria and resuspend them in potassium phosphate buffer (0.1M, pH8.0), break the cells with a homogenizer, centrifuge at 13,000rpm, 4°C for 20min, and the supernatant obtained is the crude enzyme liquid.

[0050] Potassium phosphate buffer (0.1M, pH 8.0), carbonyl reductase ChKRED12 concentration 5g / l (crude enzyme solution), substrate 1a concentration 4g / l, substrate 1g / l, NADP + Concentration 0.6 g / l, glucose dehydrogenase 2 g / l and glucose 10% (w / v). The reaction temperature is 40°C, and the reaction time is 12h. An equal volume of methyl tert-butyl ether was extracted to terminate the reaction, and the sample processing method and detection method were the same as in Example 1. The results show that 4g / l substrate can be completely converted within 12h to obtain (2S,3S)-configuration alcohol, the ee valu...

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Abstract

The invention discloses a method for preparing chiral monohydroxy derivatized 1,2-alcohol compounds by biocatalysis of a carbonyl reductase. Specifically, the carbonyl reductase ChKRED12, a substrate,a cofactor, glucose dehydrogenase, glucose and a buffer solution are mixed in a certain proportion and subjected to a reaction at pH of 7-8 and temperature of 20-40 DEG C, and (2S, 3S)-configurationproducts can be obtained. Compared with the prior art, the adopted method has the advantages of easy-to-prepare catalyst, mild reaction conditions, simple reaction system, no side reaction and high optical purity of the obtained products, and has great industrial application potential.

Description

technical field [0001] The invention belongs to the field of enzymes and biocatalysis, in particular to a method for producing optically pure 1,2-diol derivatives by biocatalysis using carbonyl reductase. Background technique [0002] Chiral 1,2-diols and their derivatives are important building blocks for many natural compounds, such as polysaccharides, polyketides, alkaloids, etc. They are also often used in asymmetric synthesis as chiral ligands or chiral auxiliary reagents. At present, the methods for preparing chiral monohydroxyl derivatized 1,2-alcohol compounds mainly include aldol condensation synthesis of glycolic acid, epoxidation-reduction method and transition metal asymmetric catalytic reduction method, and yeast cell A method for catalytic asymmetric reduction. [0003] Aldol condensation synthesis of glycolic acid, as follows: [0004] [0005] The chiral pure syn-aldol product is obtained mainly by using aldehydes and enol silyl ethers as starting mater...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/18
CPCC12P7/18Y02P20/584
Inventor 吴中柳李超刘艳
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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