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

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Identification and Reaction Assay of Galloylated Catechins Hydrolase in Tea Plant

NIE Zhi-yin1, LIU Ya-jun2, LIU Li2, GAO Li-ping1*, XIA Tao2*   

  1. 1. School of Biology Science, Anhui Agricultural University, Hefei 230036, China;
    2. Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei 230036, China
  • Received:2011-02-25 Revised:2011-05-24 Online:2011-10-15 Published:2019-09-09

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

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