Thermostable alkaline protease from Scytalidium thermophilum: production, purification, and biochemical characterization

Abstract An extracellular alkaline protease from Scytalidium thermophilum was produced in a glucose-containing medium supplemented with 5 mM NaCl for 3 days at pH 8.0 and 45 °C. The enzyme was 10-fold purified using ammonium sulfate precipitation followed by ion-exchange chromatography, and its molecular weight was calculated as 80 kDa from SDS-PAGE. The enzyme exhibited optimum activity at pH 8.0 and 60 °C. It was stable at pH and temperature range of 6.0–10.0 and 30–80 °C, respectively. Its half time was 30 h at pH 6.0, 7.0 and 8.0, while those were 22, 16, 8, and 3 h at 50 °C, 60 °C, 70 °C, and 80 °C, respectively. Kinetic parameters including Km (2 ± 0.02 mg/ml), Vmax (18.7 ± 1.5 µmole tyrosine ml−1 min−1), and kcat (2.5 x 103 min−1) were determined using casein. Ca2+ increased the enzyme activity, but it was slightly reduced by EDTA, Triton X-100, Tween 20, and Tween 80. It was active against reducing agents like β-mercaptoethanol but completely inhibited by phenyl methyl sulphonyl fluoride supporting the enzyme belonging to the serine protease family. Chloroform (143%), methanol (138%), and isopropanol (111%) increased the enzyme activity at 5% (v/v), while ethanol (71%) and acetone (81%) moderately reduced the proteolytic activity at the same concentration. Dimethyl sulfoxide (5%, v/v) did not significantly affect the enzyme. The enzyme was compatible with several detergents (1%, w/v), maintaining more than 90% of its original activity in almost all detergents tested. The stability of the enzyme presented against pH, temperature, organic solvents, and detergents indicates its potential use in various industrial applications, especially in peptide synthesis and the laundry industry.