Purification and characterization of extracellular β-glucosidase from Myceliophthora thermophila.

The extracellular β-glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH4)2SO4 precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl-β-D-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K m of the enzyme was 1.6MM for p-nitrophenyl-β-D-glucoside and 8.0MM for cellobiose. D-Glucose was a competitive inhibitor of the enzyme with a K of 22.5MM. Enzyme K activity was inhibited by HgCl2, FeSO4, CuSO4, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 M had no effect on the enzyme activity.