Detergent stable thiol-dependant alkaline protease produced from the endophytic fungus Aspergillus ochraceus BT21: Purification and kinetics

Abstract

Proteases have different industrial applications thus to increase their yield and reduce the production cost; a low cost fermentation medium was designed for large scale production. From seven tested agro-wastes, wheat bran (1.0 g%) was found to increase the overall yield of alkaline protease from Aspergillus ochraceus BT21 more than 2 fold (7359.8 U/mg) compared to the control (optimized fermentation conditions obtained from our previous study). The enzyme was purified to homogeneity using ammonium sulphate precipitation, Sephacryl S-200, followed by DEAE-Sepharose and CM-Sepharose chromatography. The efficiency of the purification steps was confirmed by SDS–PAGE where, the molecular mass of the enzyme was 59.0 KDa. The purification fold was 15.13 with specific activity reached 111379.5 U/mg and 59.7% recovery. The enzyme was alkaline thermo-tolerant with optimum values of temperature and pH at 50 °C and 8.0, respectively. The purified enzyme was stable in a wide range of temperature (20–60 °C) and pH (5.0–11.0), and its maximum activity was observed with casein substrate. The inhibition studies showed that it is a thiol-dependant serine protease. The enzyme exhibited good stability with the commercial detergents. The specific properties of the purified enzyme suggested it is suitable for future industrial applications especially in detergent formulations.