PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR GLUCOAMYLASE FROM THE THERMOPHILIC FUNGUS HUMICOLA-GRISEA VAR THERMOIDEA

Humicola grisea var. thermoidea mycelium grown on maltose as the main source of carbon produced at least two amylases. The major amylolytic component was purified to homogeneity and classified as a glucoamylase. The apparent molecular mass of the purified enzyme was estimated to be 63 000 Da by SDS-PAGE and 65 000 Da by Bio-Gel P-100 filtration. The purified enzyme was a glycoprotein with 1.8% carbohydrate content and pH and temperature optima of 5.0 and 55-degrees-C, respectively. The purified glucoamylase was thermostable at 60-degrees-C with a half-life of 16 min at 65-degrees-C. In the presence of starch the purified enzyme retained 75% of its thermostability at 65-degrees-C, while the addition of maltose failed to protect the activity. The purified enzyme hydrolyzed branched substrates more efficiently than linear substrates. Starch and amylopectin were the best substrates utilized and amylose was hydrolyzed faster than maltopentaose, maltotetraose, and maltotriose. Kinetic experiments suggested that maltose and starch were hydrolyzed at the same catalytic site.