GLYCOSIDASES FROM TEA-LEAF (CAMELLIA-SINENSIS) AND CHARACTERIZATION OF BETA-GALACTOSIDASE

A wide range of glycosidase activities could be detected in the aceton e powder extract of tea leaves of Assam variety (Camellia sinensis). b eta-galactosidase (EC3.2.1.23) was found to be the most active among t hese enzymes at fermentation temperature needed for black tea processi ng and at acidic pH. Substrate staining showed only one detectable ban d for this enzyme. A 137-fold purification over acetone powder extract through gel filtration, DEAE-cellulose chromatography, centripep, and HPLC resulted in a preparation that was electrophoretically homogeneo us. Substrate staining revealed the absence of any isozymic form of th is enzyme. Determination of molecular mass by two independent methods (i.e., by gel filtration and by HPLC) showed the enzyme to be 60 kDa. SDS-PAGE data along with molecular mass data showed the active enzyme to be a monomer of 60 kDa. The enzyme was optimally active at 50 degre es C and at pH 4.0. K-m for lactose was 4.2 mmol/L. Monovalent and div alent cations had no effect on the activity of the enzyme. Galactose, a hydrolytic product of this enzyme was found to be a competitive inhi bitor with a K-i of 2.4 mmol/L. Galactose (5 mmol/L) could provide par tial, but significant, protection against thermal inactivation. Severa l thiol modifying agents at low concentrations could rapidly inactivat e the enzyme, inactivation could be reversed with dithiothreitol. Sign ificant protection against inactivation by methyl methane thiosulphona te (MMTS) could be observed in presence of galactose indicating the pr esence of an essential thiol at the active site of the enzyme. Presenc e of a wide range of glycosidases in tea leaves is indicative of their role in generating the characteristic flavor of black tea. (C) Elsevi er Science Inc. 1997.