107(P-64) 茶の香気生成酵素β-プリメベロシダーゼの基質の合成および酵素学的特性の解明(ポスター発表の部)

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Many kinds of floral aroma such as linalool, geraniol, 2-phenylethanol, etc., have been reported to be present as glycosides in tea leaves and liberated by the action of endogenous enzymes during tea manufacturing process, so-called fermentation process. for oolong tea and black tea. In the course of our study on the molecular basis of the aroma formation during tea processing, we have isolated several kinds of glycosidic aroma precursors from tea leaves, and found that most of aroma precursors were present as β-primeverosides (6-O-β-D-xylopyranosyl-β-D-glucopyranosides). We also purified β-primeverosidase, the specific glycosidase that hydrolyzes the disaccharide glycosidic aroma precursors to liberate aroma compounds, from fresh leaves of a few kinds of tea cultivars. In order to know the substrate specificity of this enzyme, were tested nine kinds of diglycosides, a monoglycoside of 2-phenylethanol and a β-primeveroside of p-nitrophenol. 2-Phenylethyl β-glycosides (4-9) of commercially available disaccharides, gentiobiose (4a), lactose (6a), cellobiose (7a), maltose (8a) and melibiose (9a), were synthesized by the conventional Koenigs-Knorr reaction. However, this method was not applicable to synthesize 2-phenylethyl and p-nitrophenyl β-primeverosides (1 and 11), because the conventional bromination reaction on peracyl primeverose (12) did not yield 1-bromo α-primeveroside but 1-bromo α-xylopyranoside. Then, β-Primeverosides (1 and 11) were synthesized via an imidate and 1-chloro β-primeveroside from peracyl β-primeverose (12), respectively. β-Primeverosidase showed very narrow substrate specificity with respect to the glycon moiety, especially prominent specificity for the β-primeverosyl (6-D-β-D-xylopyranosyl-β-D-glucopyranosyl) moiety (1: 100%). The enzyme hydrolyzed other naturally occurring β-diglycosides such as β-vicianoside (2; 2.97%), β-acuminoside (3; 0.77%), β-gentiobioside (4; 0.25 %) and 6-O-α-L-arabinofuranosyl-β-D-glucopyranoside (5; 0.07%) in some extent, but were unable to hydrolyze synthetic unnatural β-diglycosides and 2-phenylethyl β-D-glucopyranoside. Kinetic parameters of the two kinds of b-diglycosides (1 and 2) for the β-primeverosidase were as follows: K_m=2.0mM, K_<cat>=43.7 S^<-1>,k_<cat>/K_m=21.9 s^<-1>mM^<-1> for 1 ; K_m=14.9mM, k_<cat>=5.85 s^<-1>,k_<cat>/K_m=0.391 s^<-1>mM^<-1> for 2. The enzyme was confirmed to hydrolyze each of the diglycosides into the corresponding disaccharide and 2-phenylethanol. These results indicate that the β-primeverosidase, a diglycosidase, is a key enzyme in aroma formation during the tea manufacturing process.
天然有機化合物討論会の論文
2001-09-01

107(P-64) 茶の香気生成酵素β-プリメベロシダーゼの基質の合成および酵素学的特性の解明(ポスター発表の部)

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