Study On Microbial Enzymatic Synthesis Of L-Theanine

L-Theanine is one of the tea quality and functional components, it has a sedative sedative, immune regulation, and a variety of physiological effects, widely used in areas such as food, medicines and feed additives. Preparation of L-theanine directly isolated from the tea leaves are several methods of purification, chemical synthesis and biosynthesis, processing costs from the product, product safety and product quality considerations, biosynthesis is the preparation of L-theanine development trend, and in suspension cell culture, tissue culture and microbial enzymatic synthesis, such as bio-synthesis method, microbial enzymatic synthesis is the most promising method. However, due to the limitations of research methods and technical means, the high yield of L-theanine synthetase strains screening, culture and immobilized enzymatic synthesis of L-theanine key technical difficulties at home and abroad has yet to make a breakthrough sexual progress.In this study, based on the biochemical mechanisms of tea in vivo synthesis of L-theanine, and infer tea rhizosphere soil may exist based on the production of L-theanine synthetase strains to carry out the screening of L-theanine from tea rhizosphere soilsynthase strains, synthetic product identification, the dominant strain identification based on the innovative research of high yield L-theanine synthetase strains enzymatic synthesis of L-theanine, the efficient preparation of L-theanine in order to provide a theoretical support. The main findings are as follows:1. The high yield L-theanine strains of chitin synthase screening and product identificationFrom tea rhizosphere soil sampling, species domestication and purpose of isolation of strains,42microbial strains able to use methylamine methyl nutrition. Identification and screening for production of L-theanine strains by TLC thin layer chromatography and HPLC high performance liquid chromatography confirmed that the fermentation product is indeed L-theanine, and based on the detected level of product content screening predominant strains of C2has a high yield of L-theanine capacity, output reached2.454g/L.2. High yield L-theanine strains of chitin synthase strains identifiedIdentification of strains C2filter out the high yield of L-theanine ability. Observation of colony morphology and scanning electron microscopy images, being thrown from a preliminary classification to Sporidiobolus.sp. Molecular biological identification, access to555BP sequence, after BLAST than on analysis found that C2and Sporidiobolus pararoseus strain closest. Last routine biochemical tests, test and urease test positive for glucose fermentation strains, nitrate reduction and starch substances generated negative, also consistent with Sporidiobolus pararoseus strain is a characteristic. To sum up, determine the C2test screening of strains Sporidiobolus pararoseus strain.3. Based on high yield L-theanine synthase strains enzymatic synthesis L-theanine researchSuccessively by single factor experiments and orthogonal test effects of reaction temperature, the reaction system, pH value, reaction time, the amount of crude enzyme solution, the concentration of glutamate, the ethylamine hydrochloride salt and ATP concentration on the strains C2fermentation system, L-theanine yield, the best fermentation system was35℃, pH9, reaction12h, the crude enzyme amount70mL glutamate concentration of30mmoI/L, ethylamine hydrochloride salt150mmol/L, ATP concentrationfor15mmol/L,. After optimization of the fermentation system of L-theanine production is2.945g/L, higher than that of each single factor experiments and orthogonal test results.In summary, this study confirmed before the study can be screened to the production of L-theanine synthetase strains speculated that found that the predominant strains of high yield of L-theanine synthetase from tea rhizosphere soillock for Sporidiobolus pararoseus strain, the predominant strains, as well as optimized synthetic enzymatic fermentation conditions for the biosynthesis of L-theanine, L-theanine in the fermentation system to yield up to2.945g/L, is a highly efficient preparation of L-theanine, the new method, but the predominant strains of conditions for enzyme production optimization, product analysis as well as L-theanine product separation and purification of enzymes immobilized fermentation systemhas yet to be studied further.