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

Abstract

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 1 H-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.

Expression and Biochemical Characterization of β-Primeverosidase and Application of β-Primeverosylamidine to Affinity Purification

“…The N-terminal sequence of native PD isolated from tea leaves was identical to that of the expressed PD, 12) suggesting that the N-terminal secretion signal of PD was correctly cleaved during the posttranslational processing in High Five cells.…”

“…The molecular mass of the recombinant PD was similar to that of native PD from tea leaves, and was higher than the calculated molecular mass of the mature form (54 kDa). 12) These results suggest that post-transcriptional modification occurred in the recombinant PD. The medium and cells were separated by centrifugation.…”

“…We have reported the purification and cloning of three diglycosidases, -primeverosidase (PD) from tea plants (Camellia sinensis), 12) furcatin hydrolase from Viburnum furcatum, 11) and vicianin hydrolase from Vicia augustifolia. 13) Among these three diglycosidases, PD (EC 3.2.1.149) from tea plants has been characterized in detail.…”

“…Tel: +81-774-38-3232; Fax: +81-774-38-3229; E-mail: mizutani@scl.kyoto-u.ac.jp Abbreviations: PD, -primeverosidase; FH, furcatin hydrolase; VH, vicianin hydrolase; pNP, para-nitrophenyl; 2-PE, 2-phenylethyl; pAP, paraallylphenyl; U, unit; HPLC, high performance liquid chromatography; Endo, endo-N-acetylglucosaminidase; Abs, absorbance encodes a mature protein of 507 amino acid residues, including the secretion signal peptide of 28 amino acid residues at the N-terminal. 12) PD is classified into family 1 of the glycosyl hydrolases, and thus shares greater than 50% amino acid sequence identity with various plantglucosidases of family 1. It hydrolyzes the -glycosidic bond of -primeveroside between primeverose and aglycones by a retaining mechanism, and shows high substrate specificity for -primeveroside in terms of the disaccharide-glycone moiety.…”

Chemical Profiling and Gene Expression Profiling during the Manufacturing Process of Taiwan Oolong Tea “Oriental Beauty”

“…6,7) These glycosides are hydrolyzed to release alcoholic aroma compounds by the action of endogenous enzymes ( -glucosidase and -primeverosidase) during the tea manufacturing process, suggesting that the hydrolysis of glycosides is one of the key factors in alcoholic aroma formation during the processing of oolong y To whom correspondence should be addressed. Tel: +81-774-38-3230; Fax: +81-774-38-3229; E-mail: mizutani@scl.kyoto-u.ac.jp Abbreviations: ABA, abscisic acid; diol, 2,6-dimethylocta-3,7-diene-2,6-diol; EST, expressed sequencing tags; NCED, 9-cis-epoxycarotenoid dioxygenase; AMI, 2-O-( -L-arabinopyranosyl)-myo-inositol tea [6][7][8][9][10] as well as black tea. 11,12) Furthermore, the cells and tissues of tea leaves are still alive before panning (Fig.…”