Purification and properties of cellulase enzymes from Robillarda sp. Y-20

One endo-β-1,4-glucanase (EC 3.2.1.4) and two unique β-glucosidases (EC 3.2.1.21) have been isolated from culture filtrates Robillarda sp. Y-20 by combinations of DEAE A-50 column chromatography and isoelectric focusing. These enzymes were homogeneous on gel filtration, isoelectric focusing and polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate (SDS). The molecular weights of endoglucanase, and the two β-glucosidases, I and II by SDS-polyacrylamide gel electrophoresis were 59000, 76000 and 54000, respectively. The pI values were 3.5, 7.5, and 3.8 for endoglucanase, β-glucosidase I and II, respectively. The major β-glucosidase I was a glycoprotein, but the endoglucanase and β-glucosidase II were not. The endoglucanase rapidly reduced the viscosity of carboxymethyl (CM) cellulose with concomitant production of reducing sugar. The enzyme had very low activity with crystalline cellulose such as insoluble acid treated cellulose, Avicel and filter paper. The endoglucanase attacked celloheptaose to cellotetraose more readily than cellotriose, but did not hydrolyze cellobiose. Both β-glucosidases attacked celloheptaose to cellotetraose more readily than cellotriose and cellobiose, but did not hydrolyze CM-cellulose and insoluble acid treated cellulose. Strong synergism was observed for hydrolysis of CM-cellulose by the endoglucanase and β-glucosidases.