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°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°C. The Michaelis constant (Km) for pullulan was 5.13mg·ml −1 (V max 1.0U · 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 α‐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 μM). The enzyme has been characterized as a glucoamylase (1,4‐α‐D‐glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.