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 K_m (2 ± 0.02 mg/ml), V_max (18.7 ± 1.5 µmole tyrosine ml⁻¹min⁻¹), and k_cat (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.

Keywords:

• Proteolytic enzyme
• thermophilic fungus
• fermentation
• chromatography
• organic solvent

Acknowledgments

The authors also thank Dr. Arda Acemi for linguistic improvement of the manuscript.

Disclosure statement

The authors declare that they have no conflict of interest.

Author contribution

GSI produced the alkaline protease enzyme under optimized conditions and purified it using column chromatography. GSI, EKB & BM conducted biochemical characterization studies of the purified enzyme. EKB performed all statistical analyses. YYK conceived the project and wrote the paper.

Additional information

Funding

The authors would like to thank Kocaeli University for funding (Project No: 2019/048).