Thermostableα-Amylase and Glucoamylase fromThermomyces lanuginosus F1

Abstract

An α-amylase and a glucoamylase produced by Thermomyces lanuginosus F1 were separated by ion-exchange chromatography on Q-Sepharose fast flow. The enzymes were further purified to electrophoretic homogeneity by chromatography on Sephadex G-100 and Phenyl-Sepharose CL-4B.The molecular weights and isoelectric points of the enzymes were 55,000 Da and pHi 4.0 for α-amylase and 70,000 Da and pHi 4.0 for glucoamylase, respectively. The optimum pH and temperatures for the enzymes were found to be 5.0 and 60 °C for α-amylase, and 6.0 and 70 °C for glucoamylase,respectively. Both enzymes were maximally stable at pH 4.0 and retained over 80% of their activity between pH 5.0 and 6.0 for 24 h. After incubation at 90 °C (1 h), the α-amylase and glucoamylase retained only 6% and 16% of their activity, respectively. The enzymes readily hydrolyzed soluble starch, amylose, amylopectin and glycogen but hydrolyzed pullulan very slowly. Glucoamylase and α-amylase had highest affinity for soluble starch with KM values of 0.80 mg/ml and 0.67 mg/ml, respectively. The α-amylase hydrolyzed raw starch granules with a predominant production of glucose and maltose. The activities of α-amylase and glucoamylase increased in the presence of Mn2+, Co2+, Ca2+, Zn2+ and Fe2+, but were inhibited by guanidine-HCl, urea and disodium EDTA. Both enzymes possess pH and thermal stability characteristics that may be of technological significance.