PURIFICATION AND CHARACTERIZATION OF A PULLULAN-HYDROLYZING GLUCOAMYLASE FROM SCLEROTIUM-ROLFSII

The pullulan-hydrolyzing enzyme from the culture filtrates of Sclerotium rolfsii grown on soluble starch as a carbon source has been purified by ultrafiltration (Amicon, PM-10), ion-exchange chromatography (DEAE-Cellulose DE-52) and gel filtration chromatography (Bio-Gel P-150). The enzyme moved as a single band in non-denaturing polyacrylamide gel electrophoresis carried out at pH 2.9 and 7.5. The relative molecular mass of the enzyme was estimated to be 64.000 D by SDS-PAGE and 66.070 D by gel filtration on Bio-Gel P150. The enzyme hydrolyzed pullulan optimally at 50-degrees-C between pH 4.0-4.5, whereas, soluble starch was optimally hydrolyzed at a pH of between 4.0-4.5 and at 65-degrees-C. The Michaelis constant (Km) for pullulan was 5.13 mg . ml-1 (V(max) 1.0 U . mg-1) and for soluble starch, it was 0.6mg . ml-1 (V(max) 8.33 U . mg-1). The enzyme was observed to be a glycoprotein (12-13% carbohydrate by weight) and had a strong affinity for Concanavalin A. The enzyme hydrolyzed alpha-D-glucans in an exo-manner, which resulted in the release of glucose as the sole product of hydrolysis. Acarbose, a maltotetraose analog, was found to be a potent inhibitor of both pullulan and starch hydrolysis (100% inhibition at 0.06 muM). The enzyme has been characterized as a glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.