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Application of carbonyl reductase ChKRED20 in catalyzing carbonyl substrate

A carbonyl compound and reductase technology, applied in the field of biocatalysis, can solve the problems of limited expansion range and narrow application range, and achieve the effect of high stereoselectivity and high activity

Active Publication Date: 2014-06-04
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the substrate spectrum of carbonyl reductase can be expanded by protein engineering techniques, the scope of expansion is limited, which makes the application range of the enzyme relatively narrow

Method used

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  • Application of carbonyl reductase ChKRED20 in catalyzing carbonyl substrate
  • Application of carbonyl reductase ChKRED20 in catalyzing carbonyl substrate
  • Application of carbonyl reductase ChKRED20 in catalyzing carbonyl substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Carbonyl Reductase ChKRED20 Reduction of Carbonyl Compounds Shown in Chemical Formula 1

[0023]

[0024] (1) R1 for H, R 2 for CH 3 :

[0025]

[0026] Carbonyl reductase ChKRED20 biocatalysis: reaction system 10ml, including potassium phosphate buffer (pH6.0, 0.1M), carbonyl reductase ChKRED20 pure enzyme 0.1mg / ml, NADH 20mM, substrate 10mM (prepared by dissolving in isopropanol) 10% mother liquor). Reaction conditions: 30°C, 150rpm, 24h.

[0027] After the reaction was finished, 10ml of ethyl acetate was used for extraction. The conversion and the ee value of the product were determined by chiral gas chromatography. Gas chromatography measurement conditions: Fuli Fuli GC9790 gas chromatograph, hydrogen ion flame FID detector, chiral column CP-Chirasil-DEX CB column (Varian, USA), inlet temperature 260 ° C, detector 280 ° C, column temperature 115°C, t R =9.3min,t S =10.1min. The conversion rate of the substrate was 83%, the product was in the...

Embodiment 2

[0050] Embodiment 2 Carbonyl reductase ChKRED20 catalyzes the carbonyl compound shown in chemical formula 2

[0051]

[0052] R 3 and R 4 Both are F, and respectively in the 3rd and 5th positions, R 5 for CH 3 :

[0053]

[0054] (a) Using the crude enzyme solution of carbonyl reductase ChKRED20 as a catalyst:

[0055] The reaction system and conditions are the same as in Example 1 (6), and the product treatment method is the same as in Example 1 (6). Gas chromatography measurement conditions: column temperature 90°C, other conditions are the same as in Example 1 (1). t R =46.2min,t S =50.5min. The conversion rate of the substrate was 100%, the product was in the R configuration, and the ee value was >99%. The product recovery yield was determined to be 93%.

[0056] (b) Using carbonyl reductase ChKRED20 recombinant bacterial dormant cells as a catalyst:

[0057] Biocatalytic system 10ml, including potassium phosphate buffer (pH7.0, 0.1M), carbonyl reductase C...

Embodiment 3

[0059] Embodiment 3 carbonyl reductase ChKRED20 catalyzes the carbonyl compound shown in chemical formula 3

[0060]

[0061] Using carbonyl reductase ChKRED20 lyophilized crude enzyme powder as catalyst:

[0062] Biocatalytic system 10ml, including potassium phosphate buffer (pH8.0, 0.1M), carbonyl reductase ChKRED20 crude enzyme powder 10g / l, isopropanol 40% (v / v), NAD + 0.2g / l, substrate 20mM (dissolved in isopropanol to make 30% mother solution). Reaction conditions: 30°C, 120rpm, 10h. The product treatment method is the same as in Example 1 (1). Gas chromatography measurement conditions: column temperature 100°C, other conditions are the same as in Example 1 (1). t R =30.1min,t S =32.2min. The conversion rate of the substrate was 100%, the product was in the R configuration, and the ee value was >99%.

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Abstract

The invention provides application of carbonyl reductase ChKRED20 serving as a biocatalyst in asymmetric catalysis of multiple prochiral carbonyl substrates to generate corresponding chiral alcohols. The carbonyl reductase ChKRED20 comes from Chryseobacterium sp. CA49 and has high activity and enantioselectivity.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and specifically relates to the use of a carbonyl reductase ChKRED20 in asymmetrically catalyzing carbonyl substrates to synthesize chiral alcohols. Background technique [0002] Carbonyl reductase is a class of important enzymes widely used in the synthesis of chiral alcohols. Using carbonyl reductase as a catalyst to reduce prechiral carbonyl compounds can directly construct the chiral center of optically active alcohols, thereby obtaining the possibility of chemical reactions in one step. Chiral compound building blocks that require multi-step reactions to obtain. Currently, more and more carbonyl reductases have been used in the industrial production of chiral alcohols (Nature, 2012, 485:185-194). Nevertheless, many carbonyl reductases catalyze a narrow spectrum of substrates and are often the most suitable biocatalysts for specific reactions, so high enantiomeric excess values ​​can on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22
Inventor 吴中柳汤脱险刘艳
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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