PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR BETA-GLUCOSIDASE FROM THE RUMEN FUNGUS NEOCALLIMASTIX-FRONTALIS EB188

An extracellular beta-glucosidase (E3.2.1.21) from the rumen fungus Neocallimastix frontalis EB188 was purified. The purification protocol included hydroxylapatite, gel filtration, and ion exchange chromatographies and native polyacrylamide gel electrophoresis. The enzyme readily hydrolysed p-nitrophenyl (pNP) beta-D-glucopyranoside, and cellobiose, but not a variety of other substrates. Activity was inhibited by glucose (K(i) = 10.5 mM) and glucono-delta-lactone (K(i) = 0.24 mM). Activity was inhibited in the presence of certain metal ions and stimulated by dithiothreitol. The enzyme had a denatured molecular weight of 125,500 daltons and possessed an isoelectric focusing point of 7.10. Using pNP beta-D-glucopyranoside as substrate, the K(m) and V(max) values of the enzyme were 2.5 mM and 5.02 IU mg-1, respectively. The temperature optimum of the activity was 45-degrees-C. The enzyme was rapidly inactivated at 65-degrees-C. The pH activity optimum occurred broadly between 5.5 and 7.0.