PARTIAL PURIFICATION AND CHARACTERIZATION OF β-GLUCOSIDASE FROM TEA SHOOT

Abstract

β-glucosidase was purified from the shoots of Camellia sinensis cv. Asha from Kangra valley following acetone precipitation, hydroxyapatite, gel filtration and ultrafiltration to apparent homogeneity. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of purified enzyme indicated a dimer with a larger subunit of ∼63 kD and a smaller subunit of ∼36 kD, molecular mass of ∼99 kD. The native form was confirmed by nondenaturating polyacrylamide gel electrophoresis. Optimum pH was 6.2, optimum temperature within 45–50C. β-glucosidase exhibited Km value of 0.16 mM and kcat of 11,000/min with the substrate p-nitrophenyl-β-D-glucopyranoside (pNpGlc) at 37C, pH 6.2. Substrate staining with 6-bromo-2-napthyl-β-D-glucopyranoside ruled out the possibility of any isozymic forms. The purified enzyme showed specificity toward pNpGlc and equivalent activity toward p-nitrophenyl-β-D-fucopyranoside. The enzyme liberated geraniol, linalool, phenylethanol, benzyl alcohol on incubation with tea shoot aqueous glycosidic extract. Highest activity was observed toward pNpGlc (100%), followed by p-nitrophenyl-β-D-mannopyranoside (79%) and p-nitrophenyl-β-D-fucopyranoside (75%), p-nitrophenyl-β-D-galactopyranoside (64%) etc., implying highly active β-glucosidase among other glycosidases. PRACTICAL APPLICATIONS β-glucosidases are reported to have transglycosylation activity. Conjugation of flavor volatiles as water-soluble glycosides provides an alternate means of biochemical trapping and storage of the volatile flavor compounds. The purified enzyme can be used for exogenous addition of glycosides and enzyme preparation to stimulate liberation of flavor volatiles for improving flavor quality of foods including tea, beverages and wine. Structural and functional analysis of protein gives a better understanding of catalytic mechanism and aglycone substrate specificity of β-glucosidase.