Optimization, Characterization, and Detergency Application of Alkaline Protease Produced by <i>Bacillus haynesii</i>-A6 Using Agrowastes

Abstract

Most industrial processes rely heavily on chemical reactions, which can be dangerous to human health and produce toxic byproducts that are released into the environment. Hence, there is a need for a healthier, more eco-friendly option, which microbial enzymes offer. Bacterial isolation was performed using the pour-plate method, and the isolates were screened for proteolytic activity. Identification of the selected isolate was carried out by cultural, biochemical, and molecular techniques. Isolate A6 was identified to be a species of Bacillus. The 16S rRNA gene sequence showed it to be closely related to B. haynesii. The best parameters for protease synthesis included pH 8.0, 40 °C, and plantain and soybean bran as carbon and nitrogen sources, respectively. Total protein content ranged from 39.7 mg/mL in crude to 7.05 in dialysis, while specific enzyme activity increased from 3.48 to 6.29 U/mL, a fold increase from 1.00 to 1.79 as purification increased. Total enzyme activity decreased from 412 mg/mL in crude enzyme to 132.0 mg/mL in dialysis. A molecular mass of 36.0 kDa was determined for the partially purified enzyme. The purified enzyme was active at 40 °C, pH 8, with Mg2+ and Na+ as metal ions, and was stable in the presence of surfactants, EDTA, organic solvents, and oxidizing agents. Our report showed that B. haynesii-A6 can be used to produce alkaline protease from cheaper, readily available agro-wastes. The protease obtained in this study can be used in detergent formulations and other biotechnological applications.