Enzymatic Production Of L-theanine And Its Crystallization And Detection

L-theanine a amino acid found exclusively in tea,is tea exceptional taste substances.It is widely used in food,medical health care fields.In recent years,with the development of the study about L-theanine,it has been found that theanine has many effects such as anti-cancer,lower blood pressure,neuroprotection,etc.Therefore,the market of L-theanine is more and more big.Currently,the main preparation methods of L-theanine includes separation and extraction from tea,plant tissue culture method,chemical synthesis and biosynthesis.However,the former three methods have obvious flaw rn the cost of production,production cycle and product quality.So biosynthesis has gradually become the most potential way.Now,there are seven kinds of enzymes which catalyze the biosynthesis of theanine:?-glutamyl transpeptidase,theanine synthetase,glutaminase,glutamine synthetase and y-glutamine methylamine synthetase.This experiment uses the y-glutamyl transpeptidase.At the present stage,the main method of separating L-theanine is ion-exchange resin method.This study attempts to separate the theaniene from the fermented liquid by a chemical reaction and then filtering.The detection methods of L-theanine include filter paper chromatography,HPLC,capillary electrophoresis,etc.The experiment tests preliminarily using the paper chromatography,and then uses the HPLC to test the purity of the crystallization product.1.Lactose induces the expression of y-glutamyl transpeptidase and then getting the thick enzyme fluid.The best condition of production L-theanine by enzymatic fermentation is at 37? for 24h in 1L reaction system(0.2M L-glutamine,1.0M ethylamine,0.2M phosphoric acid,thick enzyme fluid 70ml,pH 10.0).The output of theanine is about 25g tested by the filter paper chromatography and the percent conversion of L-glutamine is about 85%.2.Add 0.25M Magnesium carbonate basic in the processed fermented liquid,making it react with the phosphoric acid root and generate precipitation.Suction filter to remove sediment and take the supernatant,thus removing the phosphoric acid root.The supernatant is rotary steam condensed and the ethylamine is removed,then crystallized with ethanol.The phosphoric acid residue in the product is 0.9mM and the quality of crystallization is 26.1g.3.Test the purity of product by the method of high performance liquid chromatography(HPLC).The purity of the sample is 82.3%.