Purification and characterisation of amylolytic enzymes from thermophilic fungus Thermomyces lanuginosus strain ATCC 34626

Amylolytic enzymes (alpha-amylase and glucoamylase) from Thermomyces lanuginosus ATCC 34626 were purified to electrophoretic homogeneity. The molecular mass of purified alpha-amylase and glucoamylase were 61 and 75 kDa, respectively. Their pI values were calculated to be 3.5-3.6 and 4.1-4.3. The amylolytic enzymes from T lanuginosus exhibit pH optima in the range 4.6-6.6 in the case of alpha-amylase and 4.4-5.6 in the case of glucoamylase. Both purified enzymes have temperature optima at 70degreesC. Zn2+ ions strongly inhibit both enzyme activities. Mn2+ and Fe2+ ions are activators in the case of glucoamylase; Ca2+ and Ba2+ are activators in the case of alpha-amylase. With half-life times longer than 1 day at 60degreesC both enzymes prove to be thermostable in the pH range 4.5-8.5. The amylolytic enzymes from T. lanuginosus loose activities rapidly when incubated at temperature higher than 80degreesC or at pH lower than 4.0. Both enzymes are found to be glycosylated; 8.5% carbohydrate in the case of alpha-amylase and 3.3% in the case of glucoamylase. The K-m and V-max of alpha-amylase on soluble starch were 0.68 mg/ml and 45.19 U/mg, respectively. The K-m values of glucoamylase on maltose, maltotriose, maltotetraose, maltopentose and soluble starch were 6.5, 3.5, 2. 1, 1.1 mM and 0.8 mg/ml, respectively. The first 37 residues of N-terminal of the purified alpha-amylase of T lanuginosus ATCC 34626 were sequenced. Almost complete homology with the a-amylase from Aspergillus oryzae and Emericella nidulans was observed. (C) 2002 Elsevier Science Inc. All rights reserved.