Determination Of Theanine And Theanine Synthetase By Capillary Electrophoresis And Enzymatic Analysis

Theanine is a non-protein amino acid. It mainly exists in the Theacea plants, accounting for about 1.2%~2.0% of the dry weight of budlets of the tea Camellin sinensis. It is a secondary metabolite that provides a number of biological functions. Besides its important biological and biochemical roles, theanine is good for health from the pharmaceutical and physiological points of view. Therefore, Theanine not only can be used as an additive in food and health care products, but also has potential applications in pharmaceutical industry. There has been some significant progress on the determination, separation, extraction, and production of theanine. There were also some preliminary pharmaceutical applications. However, producing large amount of theanine from the natural extract or chemical reactions has been hindered by the difficulty in the recovery process and the low cost-effectiveness. Nevertheless, Abelian V. succeeded in the mass production of theanine through enzymatic fermentation in 1993. They had to settle with the excessive residual of ethylamide in the product. As early as in 1963, Sasaoka K. et al. discovered that the theanine synthetase was the key enzyme in the theanine biosynthesis pathway. They conducted the preliminary studies on the biochemical properties of theanine using the partially purified enzyme. There has been little progress reported since then because of the instability of theanine synthetase in vitro. Thus, the molecular basis of theanine synthetase remains unknown. The lack of understanding severely limited our ability to advance the theanine biosynthesis and genetic engineering. First part of this dissertation focuses on studying the cultivating conditions of tea seeds including the physical and chemical induction factors, and the theanine synthetase separation and extraction procedures from different tissues. The results suggested that the optimal condition to have the highest theanine synthetase activity in tea seedlings was to use sand as the carrier and to cultivate at 20℃ for a duration of two weeks. A practical purification protocol was established to retain the majority of theanine synthetase activity. The crude extract from acetone powder was prepared by acetone and ammonium sulfate. The theanine synthetase activity from roots of tea seedlings was 7.1 times of that from tea seedlings or fresh tea leaves. However, the content of the protein in roots of tea seedlings was 1/4 of that in tea seedlings or fresh tea leaves. Additional tea seeds were first treated using salicylic acid solution, then cultivated in the dark. Theanine synthetase activity in this case was 4.7 times of that tea seedlings cultivating in natural light. The results showed that both light and salicylic acid had induced effects on the expression of theanine synthetase. The second part of the thesis presents a method for rapid and accurate determination of theanine using micellar electrokinetic capillary chromatography (MECC). The derivative reaction was performed for 25min at 50℃ in the 0.5mol/L boric acid-NaOH buffer, pH 9.5, with 2,4-dinitrofluorobenzen as the derivation reagent. The derivative had an absorption peak at 360nm. The linearity of this assay method was 0.2-5.0mmol/L (r=0.995), and the minimal detection limit (S/N=2) was 0.05mmol/L. The capillary chromatography separation conditions were optimized, which contain 0.03mol/L borax buffer at pH 9.8, Brij35 and isopropanol. Samples were injected into the capillary under pressure for 5 seconds, with a separation voltage of 28kV and the temperature at 17℃. An open tubular fused-silica capillary was used throughout the experiments with an internal diameter of 50μm and a length of 65cm (60cm from autosampler to detector). The theanine content of different tea samples was determined, which confirmed the feasibility to determine theanine using capillary chromatography. The third part of the thesis establishes a novel method to analyze the theanine synthetase activity based on determination of theanine using MECC. In comparison with the isotopic label...