Purification and Characterization of Alkaline Proteases from Aspergillus terreus

Abstract

Proteases belong to an important class of enzymes known as hydrolases and catalyze hydrolysis of proteins. They act primarily to degrade proteins that are used for energy production and as biosynthetic precursors. In the following study, protease produced from Aspergillus terreus was found to be thermostable and included in the category of alkaline serine and metalloprotease. During partial purification, presence of enzyme in 60% (NH₄)₂SO₄ indicated small molecular weight polypeptide; later purification with Sephadex G-75 fractionation yielded a single proteolytic active molecule. At final purification step, the increase in specific activity of the enzyme was 7.5 fold with 23% yield. SDS-PAGE analysis revealed that alkaline protease of Aspergillus terreus is a monomer with approximate molecular weight of 35 kDa. Optimum pH for protease activity was found in the range of 7.5-11.0 (maximum at pH 8.5), thus apparently classified as an alkaline protease. The enzyme was thermostable towards high temperature (60 °C), however it denatured irreversibly at 70 °C showing 80% loss of activity. The maximum proteolytic activity was found at 40 °C. The enzyme was effectively inhibited by PMSF, EDTA and urea whereas iodoacetamide and thiourea did not result in any loss in activity while cysteine was found to be activator molecule. The study with metal ions Mg⁺², Mn⁺² and Fe⁺³ (1 mM each) showed minute stimulatory effects on enzyme activity. Co⁺² and Ca⁺² (1 mM) had neither excitatory nor inhibitory effect while Hg⁺² and Cu⁺² (1 mM) slightly reduced the enzyme activity.