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Candida sorboxylosa and application thereof in preparation of (S)-4-chloro-3-hydroxybutanoate

A technology of ethyl hydroxybutyrate and Candida, applied in the direction of microorganism-based methods, fermentation, biochemical equipment and methods, etc., can solve the problems of high reactor requirements, expensive catalysts, high production costs, etc.

Active Publication Date: 2013-05-01
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical catalysis is the use of chiral ruthenium compounds (such as RuX 2 [(S)-BINAP]) is used as a catalyst for asymmetric hydrogenation reduction, and the e.e. value of the product can reach more than 97%. The cost is high; the biocatalyst used can be either purified enzymes or microbial cells

Method used

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  • Candida sorboxylosa and application thereof in preparation of (S)-4-chloro-3-hydroxybutanoate
  • Candida sorboxylosa and application thereof in preparation of (S)-4-chloro-3-hydroxybutanoate
  • Candida sorboxylosa and application thereof in preparation of (S)-4-chloro-3-hydroxybutanoate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: Screening, identification of microorganisms

[0064] (1) Screening of microorganisms

[0065] Strain enrichment medium formula: glucose 50g / L, soybean sprouts 100g / L, COBE 12g / L, solvent is water, natural pH.

[0066] Both plate medium and slant medium formula are: glucose 50g / L, soybean sprouts 100g / L, agar 20g / L, solvent is water, natural pH.

[0067] Fermentation enzyme production medium formula: glucose 50g / L, soybean sprouts 100g / L, solvent is water, pH 7.0.

[0068] More than 200 soil samples were collected from vegetable gardens and orchard plant clusters in Zhejiang, Sichuan, Yunnan and Jiangsu for strain screening. The specific process is as follows:

[0069]Take a little soil sample and add it to normal saline to make a soil suspension. Place it in the strain enrichment medium at 150r / min on a shaker at 30°C for two enrichment cultures. After dilution and coating, select a single colony for transfer. To the slant of the test tube and numbered...

Embodiment 2

[0079] Example 2 Fermentation of Candida sorboxylosa ZJB-12164

[0080] (1) Slant culture: Candida sorboxylosa ZJB-12164 was inoculated on the slant medium and cultured at 30°C for 24 hours to obtain slant cells. The slant medium is bean sprout juice agar medium (fresh soybean sprouts 100g / L, glucose 20g / L, agar 20g / L), natural pH.

[0081] (2) Seed culture: pick a ring of bacteria from the slant with an inoculation loop and inoculate it in the seed medium, and cultivate it at 30°C and 150r / min for 24 hours to obtain the seed liquid. The formula of the seed medium is: glucose 20g / L, yeast extract 20g / L, KH 2 PO 4 2.5g / L, NaCl 1g / L, solvent is water, initial pH is 6.0.

[0082] (3) Fermentation culture: Inoculate the cultured seed liquid into the fermentation medium at a volume ratio of 2%, and cultivate it for 24 hours at 28°C and a shaker rotation speed of 150r / min to obtain a fermentation liquid containing bacteria cells. The obtained fermentation broth containing bacte...

Embodiment 3

[0083] Example 3 Fermentation of Candida sorboxylosa ZJB-12164

[0084] (1) Incline cultivation: same as in Example 2.

[0085] (2) Seed culture: with embodiment 2.

[0086] (3) Fermentation culture: Inoculate the cultured seed liquid into the bacterial culture medium at a volume ratio of 2%, and cultivate it for 24 hours at 30°C and a shaker rotation speed of 150r / min to obtain a fermentation liquid containing bacteria cells. The fermented liquid containing bacteria cells obtained was centrifuged, the supernatant was discarded, the precipitate was washed twice with normal saline, and the precipitate was collected by centrifugation to obtain wet cells with a yield of 75 g / L and a water content of 80%. The formula of the bacterial culture medium is: glucose 20g / L, yeast extract powder 50g / L, K 2 HPO 4 2.5g / L, NaCl 1g / L, CuCl 2 0.02g / L, the solvent is water, and the initial pH is 5.0.

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Abstract

The invention discloses a Candida sorboxylosa and the application of the Candida sorboxylosa in preparation of (S)-4-chloro-3-hydroxybutanoate (CHBE). The Candida sorboxylosa ZJB-12164 is preserved in China Center for Type Culture Collection, the preservation address is Wuhan University in Wuhan, China, the postcode is 430072, the preservation number is CTCC No: M2012420, and the preservation date is Oct. 22, 2012. The invention provides a new strain of Candida sorboxylosa ZJB-12164 and the application of the new strain in bioconversion of (S)-CHBE, the synthetic reaction condition is in mild condition, is environmental-friendly, has a simple process, and is high in conversion rate and product optical purity (e.e.>99%).

Description

(1) Technical field [0001] The present invention relates to a new bacterial strain - Candida sorboxylosa ZJB-12164 (Candida sorbylosa ZJB-12164), and its biocatalytic synthesis of ethyl 4-chloroacetoacetate (S)-4-chloro-3- Application of ethyl hydroxybutyrate. (2) Background technology [0002] (S)-4-chloro-3-hydroxybutanoate (Ethyl (S)-4-chloro-3-hydroxybutanoate, (S)-CHBE) is a colorless transparent oily liquid with the molecular formula C 6 h 11 ClO 3 , molecular weight 166.6, relative density 1.19g / mL, boiling point 93-95℃ / 5mmHg, (S)-CHBE structure is shown in formula (I). (S)-CHBE, as an important chiral drug intermediate, can be used to synthesize statins—hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors, and its preparation Lipitor (atorvastatin calcium) It is the first drug with an annual sales volume of more than 10 billion US dollars in the United States, and it is currently the highest-selling drug in the world. In addition, (S)-CHBE can also be transf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12P7/62C12R1/72
Inventor 郑裕国孙丽慧舒学香柳志强沈寅初
Owner ZHEJIANG UNIV OF TECH
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