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

Amylolytic enzymes (α-amylase and glucoamylase) from Thermomyces lanuginosus ATCC 34626 were purified to electrophoretic homogeneity. The molecular mass of purified α-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 α-amylase and 4.4–5.6 in the case of glucoamylase. Both purified enzymes have temperature optima at 70 °C. 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 α-amylase. With half-life times longer than 1 day at 60 °C 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 80 °C or at pH lower than 4.0. Both enzymes are found to be glycosylated; 8.5% carbohydrate in the case of α-amylase and 3.3% in the case of glucoamylase. The Km and Vmax of α-amylase on soluble starch were 0.68 mg/ml and 45.19 U/mg, respectively. The Km 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 α-amylase of T. lanuginosus ATCC 34626 were sequenced. Almost complete homology with the α-amylase from Aspergillus oryzae and Emericella nidulans was observed.