Characterization of β-primeverosidase, being concerned with alcoholic aroma formation in tea leaves to be processed into black tea, and preliminary observations on its substrate specificity

A beta-primeverosidase has been, for the first time, purified from fresh leaves of a cultivar (Camellia sinensis var. assamica) for black tea in the same way as in the case of green and oolong tea leaves previously reported. The molecular weight was shown to be 60.3 kDa by MALDI-TOFMS analysis. Its pI, optimum temperature, and pH are 9.5, 45 degrees C, and 4, respectively. The enzyme is stable below 40 degrees C and between pH 4 and 5. These enzymic characteristics are very similar to those of the beta-primeverosidases from cvs. Yabukita and Shuixian which are exclusively processed into green and oolong tea, respectively. Each beta-primeverosidase from tea leaves for green, oolong, and black teas was further purified by HPLC (ODS) and digested by trypsin to be analyzed by HPLC (ODS capillary column). Their chromatograms were not identical but very similar to each other. The molecular weight differences among these enzymes (60.5, 60.2, and 60.3 kDa from the cultivars for green, oolong, and black teas, respectively) suggest that these beta-primeverosidases are enzymatically identical, but slightly different on their molecular basis. Next, several kinds of disaccharide glycosides which had been isolated as aroma precursors were reacted with endogenous beta-primeverosidase and beta-glucosidase fractions from fresh tea leaves (cv. Yabukita). The beta-primeverosidase hydrolyzed beta-primeverosides and 6-O-beta-D-apiofuranosyl-beta-D-glucopyranosides isolated as aroma precursors from tea leaves more effectively than other disaccharide glycosides to yield each disaccharide and aglycon. It also hydrolyzed both beta-vicianoside and 6-O-alpha-L-arabinofuranosyl-beta-D-glucopyranoside into each disaccharide and aglycon, but the amount of generated aroma was smaller than that produced by the beta-glucosidase fraction.