PURIFICATION AND CHARACTERIZATION OF A BETA-GLUCOSIDASE (CELLOBIASE) FROM A MUSHROOM TERMITOMYCES-CLYPEATUS

A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65-degrees-C and was stable up to 60-degrees-C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. K(m) and V(m) values for these substrates were 0.5, 1.25 mM and 95, 91-mu-mol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucoside and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with K(i) of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a p(I) of 4.5 and appeared to be a glycoprotein.