Amylases of the thermophilic fungus Thermomyces lanuginosus: Their purification, properties, action on starch and response to heat.

ABSTRACT

A thermophilic fungus Thermomyces la nuginosus, strain IISc 91, secreted one form each of α-amylase and glucoamylase during growth. Both enzymes were purified to homogeneity by ion-exchange and gel-filtration chromatography and obtained in mg quantities. α-Amylase was considered to be a dimeric protein of ~ 42 kDa and contained 5% (by mass) carbohydrate. It was maximally active at pH 5·6 and at 65°C. It had an activation energy of 44 kJ mol–1. The apparent Km for soluble starch was 2·5 mg ml–1. The enzyme produced exceptionally high levels of maltose from raw potato starch. At 50°C, the enzyme was stable for > 7h. At 65°C, α-amylase was nearly 8-times more stable in the presence of calcium. Addition of calcium increaed the melting temperature of α-amylase from 66°C to 73°C. Upon incubation at 94°C, α-amylase was progressively and irreversibly inactivated, and converted into an inactive 72 kDa trimeric species. Glucoamylase was a monomeric glycoprotein of ~ 45 kDa with a carbohydrate content of 11% (by mass). It effected up to 76% conversion of starch in 24 h producing glucose as the sole product. Its apparent Km for soluble starch was 0·04 mg ml–1 and Vmax was 660 μmol glucose min–1 mg protein–1. It also hydrolyzed maltose. Its activity on maltooligosaccharides increased with the chain length of the substrates. Glucoamylase was stable at 60°C for over 7h. Its activation energy was 61 kJ mol–1 Glucoamylase did not show synergistic effect with α- amylase. The properties of α-amylase and glucoamylase of Thermo my ces lanuginosus strain IISc 91 suggest their usefulness in the commercial production of maltose and glucose syrups.