Study On Microbial Enzymatic Synthesis Of The Chiral Intermediate Of Levetiracetam

Levetiracetam is a novel antiepileptic mechanism which is well known at its obvious curative effect, strong safety and great potential value in the research of new drugs. One of the key intermediates for the preparation of Levetiracetam is (S)-α-Ethyl-2-oxo-1-pyrrolidineacetic acid methyl ester. A strain which could resolve (R,S)-alpha-Ethyl-2-oxo-1-pyrrolidineacetic acid methyl ester well was isolated from soil. The strain was identified as Bacillus cereus by 16S rDNA sequence analysis, named as Bacillus cereus WZZ001, and preserved in China Center for Type Culture Collection.The condition for enzyme production of Bacillus cereus WZZ001 was studied. The appropriate culture medium ingredients were maltose 5 g/L, peptone 5 g/L, yeast extract 1 g/L, MgSO4 5mmol/L. The initial pH was 7.5. The optimized condition for enzyme production was temperature 30℃, inoculation 5%(v/v). Cultivated in this condition for 24 h, the enzymatic activity of Bacillus cereus WZZ001 was 163.59 U/L, and the bio-mass increased from 3.78 g/L to 7.29 g/L compared with the data before optimization.The condition for hydrolysis of α-Ethyl-2-oxo-1-pyrrolidineacetic acid methyl ester was also optimized. An optimal reacting condition was substrate concentration 2%(v/v), freezing-dried powder of Bacillus cereus WZZ0012% (w/v), reacting temperature37.5℃, in PBS buffer (0.4M, pH7.8). After 13 h reaction in such condition, the conversion and e.e.s were 54.04% and more than 99.9% respectively.