Preparation of two kinds of high optical activity enantiomer of ethyl 4-cyano-3-hydroxybutyrate by biological catalysis method

A technology of ethyl hydroxybutyrate and biocatalysis, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., to achieve the effects of less by-products, high optical purity, and strong reaction specificity

Inactive Publication Date: 2008-09-10
中科院嘉兴中心应用化学分中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] It is currently reported that its synthetic route mainly uses epichlorohydrin or (S)-3-hydroxy-γ-butyrolactone as the starting material, and also obtains it from 4-chloro-3-hydroxybutyrate ethyl ester through cyanation. However, direct enzymatic asymmetric catalytic reduction of ethyl 4-cyano-3-carbonyl butyrate has not been reported in the literature.

Method used

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  • Preparation of two kinds of high optical activity enantiomer of ethyl 4-cyano-3-hydroxybutyrate by biological catalysis method
  • Preparation of two kinds of high optical activity enantiomer of ethyl 4-cyano-3-hydroxybutyrate by biological catalysis method

Examples

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Embodiment 1

[0038] Example 1: plate culture.

[0039] The medium is configured with 1% peptone, 0.5% yeast extract, 1.0% sodium chloride, 1.5% agar, LB agar medium with pH 7.0 (the rest is water), sterilized at 121°C for 20 minutes, cooled, poured plate, respectively Inoculate Bacillus pumilus Phe-C3 and Klebsiella pneumoniaePhe-E4, culture at 28°C for 4-6 days, then transfer to shake flask culture.

Embodiment 2

[0040] Example 2: Shake flask culture.

[0041] Medium configuration Na 2 HPO 4 6.78‰, KH 2 PO 4 3.0‰, NaCl 0.5‰, NH 4 Cl 1.0‰, MgSO 4 0.24‰, CaCl 2 0.01‰, pH 7.4 M9 medium (the rest is water), put it into a Erlenmeyer flask (filling volume 500mL / 1000mL), sterilize at 121°C for 20 minutes, cool, divide into 250mL shake flasks, and add 2% Glucose was inoculated with the above-mentioned Bacillus pumilusPhe-C3 and Klebsiella pneumoniae Phe-E4, respectively, with an inoculation amount of 5-10%, at 28° C., and the rotation speed of the shaker flask was 220 rpm. Cultivate for 2-3 days, centrifuge, collect the bacteria, and save for later use.

Embodiment 3

[0042] Example 3: Fermenter cultivation of Bacillus pumilus Phe-C3.

[0043] The fermenter medium was the same as that used for shake flask culture. When Bacillus pumilusPhe-C3 grew to the late exponential growth stage in the shake flask, it was inoculated into a 5-liter fermenter. The fermenter was prefilled with 2 liters of medium (containing 2% glucose). Fermentation conditions: rotation speed 220rpm, temperature 30°C, air flow rate 1.5L / min. At 20 hours, 50% glucose was added to the fermenter at a rate of 25 g / h to maintain the glucose concentration of the system, while the rotating speed was increased to 400 rpm to increase the ventilation. At 40 hours, reaching its post-exponential growth phase, it was centrifuged at 4°C and stored in a refrigerator until use.

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Abstract

The invention provides a method for preparing two high optical activity enantiomers of 4-cyano-3-hydroxy ethyl butyrate by biocatalysis. The microbial strain adopted in the method is Klebsiella pneumoniae Phe-E4 or Bacillus pumilus Phe-C3 which is used for catalysis. The product obtained by the biocatalysis of the strain Klebsiella pneumoniae Phe-E4 is (S)-4-cyano-3-hydroxy ethyl butyrate, and the product obtained by biocatalysis of the strain Bacillus pumilus Phe-C3 is (R)-4-cyano-3-hydroxy ethyl butyrate. The substrate percent conversion of the product obtained by the method can reach more than 98 percent. Chiral products with high optical purity can be obtained by the method, and the reaction is strong in specificity, byproducts are fewer and the post processing is simple. Therefore, the method has good prospect of industrial application and development.

Description

technical field [0001] The present invention relates to the preparation method of (±)-4-cyano-3-hydroxybutyrate ethyl ester, more specifically the microbial conversion method of producing chiral β-hydroxybutyrate by catalyzing asymmetric reduction thereof. Background technique [0002] Optically active chiral secondary alcohols are important intermediates in the synthesis of many chiral drugs and chiral fine chemicals, and their synthesis methods usually include asymmetric reduction of ketones and asymmetric resolution of racemates. [0003] The preparation of optically active chiral secondary alcohols through the resolution of the racemate can use natural or synthetic chiral resolution reagents, or biological enzymatic methods, but the source of chiral resolution reagents is far less than the type of biological enzyme methods There are many, so the most common is to use commercial esterase or lipase for hydrolysis and splitting. However, for splitting, the maximum yield wil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/62C12P41/00C12R1/22C12R1/08
Inventor 何春茂张杰常东亮
Owner 中科院嘉兴中心应用化学分中心
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