Study On Production Of Theanine By Microbal Enzymatic Method

Theanine is the most important free amino acid in tea.Beside its graceful taste,theanine has many favorable physiological functions.This suggest that demand for theanine will become great as a supplement for improving and maintaining human health.Although several methods for preparing theanine have been reported,they are usually complicated and show unsatisfactory yield.So microbial enzymatic method to produce theanine is getting more and more attention.In this study,a sort of microbe-Bacillus licheniformis that can abundantly synthesize theanine was found. We have investigated theanine production from L-glutamine and ethylamin hydrochlorid as substrates by "coupled fermentation with energy transfer"using Bacillus licheniformisγ-glutamyltranspeptidase.This study has a better instruction for industrial producing theanine of microbial fermentation.The main results were follows:1.Enzymatic production strains:Bacillus licheniformisγ-glutamyltranspeptidase has a high ability of synthesis theanine. Enzymatic fermentation study shown thatγ-glutamyltranspeptidase activity could be achieved at 6.33U/ml.2.Theanine transformation test results:Bacillus licheniformisγ-glutamyltrans- peptidase has a good ability of synthesis theanine as a catalyst. The yield of theanine was 21g/L,The conversion rate of L-Gln as to theanine reach 35.2%.3. Enzyme promoters results: On the basis of conventional research about enzyme fermentation, the enzyme fermentation and theanine transformation test shown that some promoters were useful. The orthogonal test shown that,enzymatic fermentation medium with Mn2+,Mg2+,Tween-80,pyridoxal phosphate could increase the capacity of Bacillus licheniformis to produceγ-glutamyltranspeptidase. Theγ-GGT activity could be achieved at 7.42 U/ml, The yield of theanine was 26g/L,The conversion rate of L-Gln as to theanine reach 43.6%.4. Species coupled ATP regeneration study:In this study,a combination of Bacillus licheniformisγ-glutamyltranspeptidase system and Bacillus stearothermophilus ATP energy supply system could successfully synthesis theanine.The yield of theanine was 35g/L,The conversion rate of L-Gln as to theanine reach 59%.5.Immobilized cells to produce theanine results:Immobilized Bacillus licheniformisγ-glutamyltranspeptidase could synthesis theanine coupled with ATP regeneration. The yield of theanine was 30g/L,The conversion rate of L-Gln as to theanine reach 50.3%.Bacillus licheniformisγ-glutamyltranspeptidase has a good ability of synthesis theanine. It is a practical value to produce theanine to useγ-glutamyltranspeptidase because of its high activity .In this study, We have established the species coupled ATP regeneration tecnology.It could not only use to produce theanine,but also provide technical support to ferment other products.We also established immobilized cells tecnology to synthesis theanine.It is useful to handle industrial tecnology and improve product quality. In this study, all of tecnologies and results will provide a valuable line to produce theanine.