Method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester through microorganism catalysis and bacterial strain

A pyrrolidine acetate, microorganism technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of pollution, many steps, low product purity and the like

Active Publication Date: 2013-03-27
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These traditional processes have problems such as low yield, high cost, numerous steps, low product purity, and serious pollution.

Method used

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  • Method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester through microorganism catalysis and bacterial strain
  • Method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester through microorganism catalysis and bacterial strain
  • Method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester through microorganism catalysis and bacterial strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Pick the slant colonies of Bacillus cereus WZZ001 and inoculate them into 50 mL of seed medium, and culture them at 30 °C and 200 r / min for 24 h to obtain seed liquid; under sterile conditions, take 3 mL of seed liquid to inoculate 50 mL of In the enzyme medium, culture at 30 °C and 200 r / min for 24 h to obtain the fermentation broth. After the cultivation, the fermentation broth was centrifuged at 6000 rpm for 15 min, and the supernatant was discarded to obtain the wet bacteria of Bacillus cereus WZZ001, and a small amount of pH 7.2 0.2 mol / L phosphate buffer was added to stir evenly, and freeze-dried at -40°C to obtain Dried bacteria. The specific enzyme activity of the dried bacteria was 5.0 U / g by enzyme activity determination.

[0039] The formula of seed medium is: peptone 5 g / L, beef extract 5 g / L, yeast extract 1 g / L, glucose 5 g / L, MgSO 4 5 g / L, K 2 HPO 4 1.5 g / L, KH 2 PO 4 0.5 g / L, the solvent is water, pH 7.0; the formula of the enzyme production med...

Embodiment 2

[0042] Add 0.1g of dry bacteria of Bacillus cereus WZZ001 to 10mL pH7.2 phosphate buffer solution containing 5g / L racemic α-ethyl-2-oxo-1-pyrrolidine acetate, and react at 30°C After 24 hours, the converted reaction solution was centrifuged, the supernatant was taken, and an equal volume of ethyl acetate was added for extraction to obtain (S)-α-ethyl-2-oxo-1-pyrrolidine acetate methyl ester. The e.e. 99.5% enantiomeric excess of (S)-α-ethyl-2-oxo-1-pyrrolidine acetate methyl ester was detected by the aforementioned method, and the yield was 48.9%.

Embodiment 3

[0044] Add 0.2g of dry bacteria of Bacillus cereus WZZ001 to 10mL pH7.2 phosphate buffer solution containing 10g / L racemic α-ethyl-2-oxo-1-pyrrolidine acetate, and react for 24h at 30°C Finally, the converted reaction solution was centrifuged, the supernatant was taken, and an equal volume of ethyl acetate was added for extraction to obtain (S)-α-ethyl-2-oxo-1-pyrrolidine acetate methyl ester. The enantiomeric excess value of (S)-α-ethyl-2-oxo-1-pyrrolidine acetate methyl ester was detected by the aforementioned method, e.e. 99.2%, and the yield was 46.8%.

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Abstract

The invention provides a cheap and efficient microorganism enzyme producing bacteria, namely, bacillus cereus (Bacillus cereus) WZZoo1, and provides a method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester by taking racemic alpha-ethyl-2-oxygen-1-pyrrolidine acetic acid ester as reaction substrate and catalyzing stereoselectivity hydrolysis(R)-alpha-ethyl-2-oxygen-1-pyrrolidine acetic acid ester through carboxylic ester hydrolase generated by the bacterium. The bacterial strain is preserved in China Center For Type Culture Collection (CCTCC) on October 14, 2012, the preservation serial number is CCTCC No: M2012403. The beneficial effect of the method mainly reflects in that the carboxylic ester hydrolase generated by the bacterial strain has strong area selectivity and high reaction conversion rate, downstream separation is simple, energy consumption is low, and environment pollution is light, so that the method is applicable to industrial production.

Description

(1) Technical field [0001] The present invention relates to Bacillus cereus (Bacillus cereus) WZZ001 and its preparation of levetiracetam intermediate by microorganism catalysis—(S)-α-ethyl-2-oxo-1-pyrrole represented by formula (II) Application in alkane acetates, and a method for preparing (S)-α-ethyl-2-oxo-1-pyrrolidine acetates shown in formula (II) by microbial catalysis, the intermediate can be passed through amine Decomposition reaction synthesis levetiracetam. (2) Background technology [0002] Levetiracetam (LEV, trade name Keppra) chemical name is (S)-α-ethyl-2-oxo-1-pyrrolidineacetamide, which is a derivative of piracetam and is a new type of anti-inflammatory drug. Epilepsy drugs (AEDs). Approved by the US FDA in 1999, it was initially used for partial seizures in adults. In June 2005, its oral tablet and solution were approved for adjuvant treatment of partial seizures in children over 4 years old (including 4 years old). It was launched in China on March 10...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P41/00C12P17/10C12R1/085
Inventor 汪钊郑建永鄢洪德章银军宣磊
Owner ZHEJIANG UNIV OF TECH
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