| Co-Investigator(Kenkyū-buntansha) |
USUI Taiichi Shizuoka University, Faculty of Agriculture, Professor, 農学部, 教授 (50111802)
WATANABE Naoharu Shizuoka University, Faculty of Agric., Associate Professor, 農学部, 助教授 (90230979)
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| Research Abstract |
1) Study on the molecular basis of the alcoholic aroma formation mechanism in oolong tea :
We have isolated and identified aroma precursors in most of the alcoholic aroma of oolong tea (Camellia sinensis var.sinensis cv.Shuixian and Maoxie) as glycosides. Most of them were found to be beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides), but the aroma precursors of linalool oxides III and IV (cis-and trans-linalool 3,7-oxides) and (Z)-3-hexenol were exceptionally isolated as 6-O-beta-D-apiofuranosyl-beta-D-glucopyranosides and beta-D-glucopyranoside, respectively.
As a preliminary experiment, we have purified a beta-primeverosidase from fresh tea leaves (cv.Yabukita), which showed high substrate specificity toward beta-primeverosides, which are hydrolyzed into primeverose and the corresponding aglycon. As we succeeded in enzymatic synthesis of p-nitrophenyl beta-primeveroside as shown bellow, we tried to purify beta-primeverosidases from fresh tea leaves of green tea (c
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v.Yabukita) and oolong tea (cv.Shuixian). Both enzymes have been shown to be the same enzyme with very similar molecular weight (61KDa by SDS-PAGE analysis). On the basis of the foregoing most of the alcoholic tea aroma such as linalool, geraniol, etc.contributing to the floral aroma of oolong tea have been shown to be generated from their glycosidic aroma precursors (beta-primeverosides) by the action of an endogenous beta-primeverosidase during tea processing.
2) Study on the molecular basis of the alcoholic aroma formation mechanism in Jasmine tea : We have isolated, for the first time, aroma precursors of linalool, 2-phenylethanol, and benzyl alcohol as their disaccharide glycosides. We also tried to purify the glycosidases which are concerned with the hydrolysis of the aroma precursors to liberate floral aroma from the acetone powder prepared from flower buds of Jasminum sambac just after flowering. After chromatographic separation we found that three kinds of glycosidases are at least concerned with the hydrolysis of the aroma precursors and these enzymes hydrolyze the disaccharide aroma precursors into aglycon (alcoholic aroma) and disaccharide.
3) Enzymatic synthesis of p-nitrophenyl beta-primeveroside (pNP-Pri) : We found that a new enzyme, beta-primeverosidase, is one of the most important glycosidases in the alcoholic aroma formation in tea leaves as well as flowers. We tried to enzymatically synthesize pNP-Pri by transglycosylation reaction. We screened several kinds of commercially available enzymes for transxylosylation activity to find that a few enzymes can transfer a xylose moiety of xylobiose to 6'-position of pNP-beta-D-glucopyranoside. After partial purification of one (Pectinase G) of the enzymes and optimization of the reaction conditions we were able to obtain pNP-beta-primeveroside in an appreciable yield. This useful substrate has let us to effective purify the beta-primeverosidases from tea leaves and flower buds of J.sambac and determine their enzymatic properties. Less
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