Purification and Substrate Specificity of Glucoamylase of Paecilomyces varioti AHU 9417

Abstract

A glucoamylase was purified from the culture broth of Paecilomyces varioti AHU 9417 by precipitation with ethanol, chromatography on DEAE-Sepharose CL-6B, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was estimated to be 6.9×104 by SDS-disc electrophoresis, and the optimum pH was 4.5. The enzyme preferentially hydrolyzes α-1,4-glucosidic linkages in a series of maltooligosaccharides, and is capable to slowly hydrolyzing nigerose, isomaltose, and panose, but is not active on kojibriose. Soluble starch, amylopectin, glycogen, and β-limit dextrin are rapidly degraded. Activity toward raw starches is very low, but rice and waxy corn raw starches are relatively subject to attack. The subsite affinities (Ai) of each subsite (i) in the active site of the enzyme were evaluated from the Michaelis constants (Km) and the molecular activities (k0) for a series of maltooligosaccharides according to the subsite theory. The active site was considered to be made up of about four subsites: A1≃0.46 kcal/mol, A2≃4.78 kcal/mol, A3≃1.76 kcal/mol and A4≃0.67 kcal/mol.