Purification and properties of a thermophilic amyloglucosidase from Aspergillus niger

Abstract

A thermophilic amyloglucosidase (EC 3.2.1.3) from a strain of Aspergillus niger was purified and separated from contaminating α-amylase (EC 3.2.1.1) and transglucosidase (EC 2.4.1.24) by ammonium sulphate fractionation, acetone precipitation and CM-BIO-GEL A chromatography. A 61-fold purification was achieved. The enzyme had highest affinity for starch (100), maltotriose (68) and maltose (31) and Km values of 0.025% and 1.42 mM with starch and maltose, respectively. It had a molecular weight of 63,000. The enzyme operated most efficiently on starch and maltose at pH 4.5 and surprisingly at the high temperature of 70°C. It possessed considerable pH stability, with 79% and 50% activity retained at pH 2.0 and pH 11.0, respectively, after 30 min at 40°C. The enzyme was 100% stable up to 50°C and 90% stable at 60°C for 30 min; above this latter temperature activity was rapidly destroyed. The presence of starch or glycerol improved the thermal stability of the enzyme. Polyvalent anions stimulated activity while the cations Cu2+ and Ag+ and to a lesser extent Ni2+ and Co2+ caused notable inhibitory effects. When incubated with high concentrations of glucose the enzyme formed small amounts of isomaltose as a reversion product.