Identification and Reaction Assay of Galloylated Catechins Hydrolase in Tea Plant

NIE Zhi-yin; LIU Ya-jun; LIU Li; GAO Li-ping; XIA Tao

doi:10.13305/j.cnki.jts.2011.05.014

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Journal of Tea Science ›› 2011, Vol. 31 ›› Issue (5) : 439-446. DOI: 10.13305/j.cnki.jts.2011.05.014

Identification and Reaction Assay of Galloylated Catechins Hydrolase in Tea Plant

NIE Zhi-yin¹, LIU Ya-jun², LIU Li², GAO Li-ping^1*, XIA Tao^2*

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Abstract

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.

Key words

galloylated catechins hydrolase / identification / reaction assay / tea plant 〔Camellia sinensis (L.) O. Kuntze〕

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NIE Zhi-yin, LIU Ya-jun, LIU Li, GAO Li-ping, XIA Tao. Identification and Reaction Assay of Galloylated Catechins Hydrolase in Tea Plant[J]. Journal of Tea Science. 2011, 31(5): 439-446 https://doi.org/10.13305/j.cnki.jts.2011.05.014

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