Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus

A major extracellular endoglucanase purified to homogeneity from Thermoascus aurantiacus had a M r of 34 kDa and a p I of 3.7 and was optimally active at 70–80 °C and pH 4.0–4.4. It was stable at pH 2.8–6.8 at 50 °C for 48 h and maintained its secondary structure and folded conformation up to 70 °C at pH 5.0 and 2.8, respectively. A 33-amino acid sequence at the N terminus showed considerable homology with 14 microbial endoglucanases having highly conserved 8 amino acids (positions 10–17) and Gly, Pro, Gly, and Pro at positions 8, 22, 23, and 32, respectively. The enzyme is rich in Asp (15%) and Glu (10%) with a carbohydrate content of 2.7%. Polyclonal antibodies of endoglucanase cross-reacted with their own antigen and with other purified cellulases from T. aurantiacus. The endoglucanase was specific for polymeric substrates with highest activity toward carboxymethyl cellulose followed by barley β-glucan and lichenan. It preferentially cleaved the internal glycosidic bonds of Glc n and MeUmbGlc n and possessed an extended substrate-binding site with five subsites. The data indicate that the endoglucanase from T. aurantiacus is a member of glycoside hydrolase family 5.