Study On The Biosynthesis Of Theanine

Theanine is a special amino acid in tea plants, possessing high medical benefits and economic values. It is very difficult and more expensive to extract a high purity theanine directly from tea leaves. So the biosynthesis of theanine is getting more and more attention. In this study, a sort of microbe- Pseudomonas nitroreducens that can abundantly synthesize theanine was found. The culture condition of Pseudomonas nitroreducens, the biosynthesis of theanine by Pseudomonas nitroreducens cells, the gene clone of glutaminase and Gltamyltranspeptidase were studied. The main results were as follows:1. Among six microbes that contain glutaminase, Pseudomonas nitroreducens was a only microbe which can abundantly synthesis theanine under appropriate culture condition.2. The optimum condition for Pseudomonas nitroreducens culture was medium with pH7.0, 30℃,l% glucose for C substance, 0.6% glutamate for N substance, and nature oxygen quantity.3. The optimum condition of synthesis theanine by Pseudomonas nitroreducens cells was substance solution containing 0.3M Glutamine and l.SM ethylamine with pH 9.5, wet cells content 70mg/ml, culture temperature 30℃ and culture for 24hours.The maximum theanine yield was 24. 10g/L. And The optimum condition of synthesis theanine by Pseudomonas nitroreducens cells was substance solution containing 0.7M Glutamine and 1.4M ethylamine with pH 9.5, wet cells content 70mg/ml, culture temperature 30℃ and culture for 24hours.The maximum theanine yield was 41.38g/L. A further fermentation trial showed that under the same culture condition, the maximum theanine yield was 22.52g/L. The final average concentration of theanine in fermented liquid was 15.01g/L after 60 hours.4. The clone of glutaminase gene obtained from Pseudomonas nitroreducens was not success in this study and need a further research.5. A gltamyltranspeptidase gene obtained from E. coli was cloned and expressed successfully. Comparing its nucleotide sequence with other gltamyltranspeptidase genes from microbe reported in literature in Genebank, considerable homology (more than 99.9%) was observed. It can produce theanine with a yield of 9.00g/L.