Synthesis Of Methylated Epigallocatechin Gallate (EGCG) By O-methyltransferase From Camellia Sinensis

Methylated EGCG, a kind of special catechin in tea, has stronger effect than EGCG in many aspects such as antiallergic. Methylated EGCG is much more stable than EGCG in the animal blood, and its oral absorption rate is also higher than that of EGCG, so it has great value in pharmacology and has promising application prospect in food and beverage field. Nevertheless, the content of methylated EGCG (mainly EGCG3"Me) in tea leaves is very low, and very little tea germplasm contains more than 1% of EGCG3"Me in its leaves, it is quite difficult to extract EGCG3"Me directly from the tea leaves. Therefore, it has important theoretical significance and application value to produce a great deal of methylated EGCG through enzymatic synthesis or chemical synthesis methods. The purpose of this study is through the recombinant DNA technology and fermentation cultivation techniques to obtain a lot of O-methyltransferase, and using the enzyme to synthesis the methylated EGCG. It could provide theoretical basis for the enzymatic synthesis of methylated EGCG in the future. The main research results are as follows:1. The induced expression condition of O-methyltransferase in recombinant Escherichia coli is. optimized. When the concentration of the inducer IPTG is 0.05mmol/L, the induction time is 20 hours, the initial pH of culture medium is 7.0, and the induction temperature is 20℃, O-methyltransferase from the tea leaves (Camellia Sinensis ) can achieve the best expression effect.2. To obtain maximum yield of the methylated EGCG, the optimum reaction conditions of the O-methyltransferase is investigated. When the initial concentration of EGCG is 300μmol/L, pH of the buffer solution is 7.5, the reaction temperature is 35℃, the concentration of Mg²⁺ is 2mmol/L, and the concentration of dithiothreitol is 2mmol/L, the production of methylated EGCG is highest, including 386.39μg/mL of EGCG3′′Me, 23.40μg/mLof EGCG4′′Me and 107.01μg/mL of EGCG3′Me.3. The mass spectrometric behavior of EGCG3''Me, EGCG4''Me and EGCG3'Me in positive ion mode are analyzed. The fragmentation is formed by Rretro-Diels-Aider(RDA) reaction on the C ring of methylated EGCG. The possible nucleo plasmic relation of those fracture fragments are speculated.4. The mass spectrum of the enzymatic reaction product are analyzed according to the mass spectrometric behavior of methylated EGCG.The result shows that 10 kinds of methylated EGCG are detected.