Plant Physiol, December 2002, Vol. 130, pp. 2164-2176

Cloning of -Primeverosidase from Tea Leaves, a Key Enzyme in Tea Aroma Formation¹

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan (M.M., H.N., J.-i.E., S.-J.M., E.N., K.S.); and Institute of Fundamental Research, Research Center, Suntory Ltd., Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan (M.I.-O., M.F.-M., M.N.)

A -primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of -primeverosides (6-O--D-xylopyranosyl--D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The -primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The -primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The -primeverosidase protein sequence was 50% to 60% identical to -glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the -primeverosidase expressed in Escherichia coli was able to hydrolyze -primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl -D-glucopyranoside. These results indicate that the -primeverosidase selectively recognizes the -primeverosides as substrates and specifically hydrolyzes the -glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl -primeveroside by the -primeverosidase was followed by ¹H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the -primeveroside by a retaining mechanism. The roles of the -primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.