本试验利用体外酶学方法,结合薄层层析（TLC）、高效液相色谱（HPLC）和液相色谱-串联质谱（LC-MS/MS）分析,首次从茶树中检测到活性较高的酯型儿茶素水解酶（Galloylated Catechins Hydrolase,GCH）的存在。在酯型儿茶素水解酶催化下,酯型儿茶素发生水解反应,生成没食子酸（GA）和非酯型儿茶素。试验确立了酯型儿茶素水解酶的最适检测体系,在2.5mL反应体系中包含0.2mmol/L酯型儿茶素、2.4mmol/L抗坏血酸、粗酶提取液若干（含0.1mg酶蛋白）和0.1mol/L磷酸缓冲液（pH6.5）,在30℃下,反应30min。此外,试验利用硫酸铵分级沉淀、阴离子交换层析和凝胶过滤层析对该酶进行了初步纯化。
In this experiment, high activity of galloylated catechins hydrolase (GCH) was detected existing in tea plant 〔Camellia sinensis (L.) O. Kuntze〕 by enzymology analysis in vitro, combining thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. The galloylated catechins could be hydrolyzed to ungalloylated catechins and gallic acid (GA) with the GCH action. The optimum reaction assay of GCH has been established. The 2.5mL enzyme reaction assay included 0.1mol/L phosphate buffer (pH 6.5), 0.2mmol/L EGCG, 2.4mmol/L sodium ascorbate, crude enzyme extract (0.1mg total protein), and then it was incubated at 30℃ for 30min. Besides, the crude enzyme extract was partially purified via ammonium sulfate fractionation, anion exchange chromatography on Q Sepharose Fast Flow column and gel filtration chromatography on superdex 200 sequentially.
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