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Arthrobacterium ZJB-09277 and application thereof in preparing (S)-3-cyan-5-methyl caproic acid

A technology of ZJB-09277 and methylhexanoic acid, applied in the field of Arthrobacter ZJB-09277 and its application in the preparation of (S)-3-cyano-5-methylhexanoic acid, can solve chiral resolution Expensive reagents, lengthy process steps, harsh reaction conditions, etc., to achieve high industrial application potential, high atom economy, and mild conditions

Active Publication Date: 2014-05-14
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lengthy process steps above, expensive chiral resolution reagents, harsh reaction conditions, and a large amount of toxic and harmful solvents, it is impossible to achieve large-scale production

Method used

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  • Arthrobacterium ZJB-09277 and application thereof in preparing (S)-3-cyan-5-methyl caproic acid
  • Arthrobacterium ZJB-09277 and application thereof in preparing (S)-3-cyan-5-methyl caproic acid
  • Arthrobacterium ZJB-09277 and application thereof in preparing (S)-3-cyan-5-methyl caproic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Isolation and identification of bacterial strain

[0038] (1) Separation

[0039] Isolation of ester hydrolase-producing bacteria: Add 1g of soil samples collected from Hangzhou and surrounding areas to 9mL of 0.85% normal saline, mix thoroughly on a vortex shaker to make a uniform soil suspension; draw 1.0mL of The soil suspension was inoculated into a 250mL Erlenmeyer flask containing 49mL enrichment medium, placed in a shaker at 30°C and 150rpm for 72 hours, and after the enrichment solution became turbid, transfer 1.0mL to a fresh enrichment medium. Collected in the culture medium, continue to cultivate for 72h; carry out three rounds of enrichment in this way, dilute the enriched culture solution in multiple gradients and spread it on the separation plate to obtain a single colony;

[0040] The enrichment medium uses 2g / L (R,S)-3-cyano-5-methylhexanoic acid ethyl ester as the sole carbon source, and the composition is as follows (final concentration)...

Embodiment 2

[0057] Embodiment 2: the cultivation of bacterial strain

[0058] Slant medium composition (final concentration): glucose 10g / L, yeast extract 5g / L, (NH 4 ) 2 SO 4 3g / L, MgSO 4 ·7H 2 O0.5g / L, NaCl1g / L, K 2 HPO 4 2g / L, FeSO 4 ·7H 2 O0.01g / L, agar 20g / L, prepared with distilled water, natural pH value;

[0059] A single colony of Arthrobacter ZJB-09277 was picked, inoculated into the slant medium, and cultured in a constant temperature incubator at 30°C for 48 hours to obtain slant bacteria, which were stored in a 4°C refrigerator for later use.

Embodiment 3

[0060] Embodiment 3: the acquisition of the bacterial suspension containing wet thalli

[0061] Medium preparation: sodium citrate 10g / L, yeast extract powder 40g / L, Na 2 HPO 4 1g / L, KH 2 PO 4 2g / L, MgSO 4 ·7H 2 O0.5g / L, FeSO 4 ·7H 2 O0.03g / L, NaCl1g / L, prepared with distilled water, pH6.0;

[0062] In a 250mL shake flask with 20% liquid volume, inoculate a ring of Arthrobacter ZJB-09277 obtained in Example 2, and shake and culture at 30°C and 150rpm for 72h; Bacterial cells (wet bacterial cells with a water content of 70%) are stored at -20°C for later use or suspended in 100mM, pH 7.0 phosphate buffer to obtain a bacterial suspension with a wet bacterial cell content of 0.4g / mL for later use .

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Abstract

The invention discloses arthrobacterium ZJB-09277 (Arthrobacter sp. ZJB-09277) and an application thereof in preparing (S)-3-cyan-5-methyl caproic acid. The arthrobacterium is preserved in the Chinese Typical Culture Collection Center which is located in Wuhan College, Wuhan, China, the preservation number is CCTCC NO: M 2012419, and the preservation date is October 22nd, 2012. The invention further provides a novel strain for preparing the (S)-3-cyan-5-methyl caproic acid in a stereoselectivity hydrolysis mode. An important chiral intermediate of pregabalin, namely, the (S)-3-cyan-5-methyl caproic acid can be prepared from the strain. The preparation method of the (S)-3-cyan-5-methyl caproic acid disclosed by the invention has the advantages of high atom economy, mild condition, environmental-friendliness, and has higher industrial application potentials.

Description

(1) Technical field [0001] The present invention relates to a new bacterial species - Arthrobacter sp. ZJB-09277 (Arthrobacter sp. ZJB-09277), and the intermediate preparation of pregabalin by using the microorganism to selectively resolve 3-cyano-5-methylhexanoate method for the body of (S)-3-cyano-5-methylhexanoic acid. (2) Background technology [0002] (S)-3-cyano-5-methylhexanoic acid is an important chiral intermediate in the synthesis of pregabalin. Pregabalin, whose chemical name is (S)-3-(aminomethyl)-5-methylhexanoic acid, is the 3-position isobutyl substituent of γ-aminobutyric acid developed by Pfizer (Angew Chem. Int Ed, 2008, 47(19):3500-3504). Because pregabalin can effectively inhibit neuronal hyperexcitability and increased neurotransmitter release associated with neuropathic pain and epilepsy (Curr Opin Pharmacol, 2006,6(1):108-113), it has been approved by the FDA for the treatment of Post-herpetic neuralgia, diabetic peripheral neuralgia, epilepsy, fib...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/00C12R1/06
Inventor 郑裕国郑仁朝李爱朋沈寅初
Owner ZHEJIANG UNIV OF TECH
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