Purification and Characterization of a New Endoglucanase from Clostridiumthermocellum

摘要

An endoglucanase (1,4-Beta-D-glucan glucanohydrolase, EC 3.2.1.4) from thethermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 deg C and retains approx. 50 % of its activity after 48 h at 60 deg C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mMHg2+ and is inhibited by a thiol-blocking reagent.