Microbial Production Of 5-Methylpyrazine-2-Carboxylic Acid

5-Methylpyrazine-2-carboxylic acid is an important chiral synthon for synthesis of various optical active compounds such as glypizide,acipimox,5-methylpyrazine-2-carboxylic acid methyl ester.More and more attentions were focused on efficient production of 5-Methylpyrazine-2-carboxylic acid with high stereselectivity,high activity by biotransformation.In this paper,the microbial synthesis of 5-methylpyrazine-2-carboxylic acid from 2,5-dimethyl prazine was investigated.Firstly we established a liquid chromatography analysis of 2,5-dimethyl prazine and 5-methylpyrazine-2-carboxylic acid.Then strain ZJB-LLJ,a bacterial with high stereselectivity and activity toward 2,5-dimethyl prazine was isolated from soil samples.Based on morphology,physiological characteristics and 16S rDNA analysis,ZJB-LLJ was identified as Pseudomonas putida.The reaction conditions of batch biotransformation for the production of 5-methylpyrazine-2-carboxylic acid from 2,5-dimethyl pyrazine were optimized.p-xylene was the suitable carbon source.The highest product rate was obtained when Pseudomonas putida was cultured in 100 mL medium(pH7-8)in a 500 mL flask at 30? and 2,5-dimethyl pyrazine was added after 12 h.In fed-batch biotransformation,2,5-dimethyl pyrazine was added to the reaction system continuously,the 5-methylpyrazine-2-carboxylic acid could reach 20.41 g/L with a yield of 75.6%at 528 h.Under the above conditions,adding 0.004mol/L glycerol can increase the production rate by 20%.Simultaneously,the monooxygenase reaction of 2,5-dimethyl pyrazine by resting cells of ZJB-LLJ were optimized.The data showed that the optimal reaction temperature were 30?,respectively.The optimal buffer was pH 8.0 Tris-HCl.Under 10 ml of the conversion system,after 12 hour-reaction with 23mM 2,5-dimethyl pyrazine under the 0.01 g/L bacteria concentration,the value and yield of 5-methylpyrazine-2-carboxylic acid reached 67%and 2.21 g/L,respectively.