ABSTRACT

Enzymes are considered environmentally friendly (green) chemicals that have the potential to completely replace or reduce the use of hazardous chemicals in industrial processes, thereby promising sustainable production and manufacturing. Proteases derived from microbes have almost all of the properties required for biotechnological applications. This study was conducted with the aim of screening for potent protease-producing bacteria from Bacillus subtilis, determining optimal production conditions and partially characterizing the stability of the protease with regards to some physicochemical parameters. Hence, identification of Bacillus subtilis at a molecular level and purification as well as detailed characterization of the types of the proteases are recommended for effective utilization in different area of applications as the purification of the extract from the study yielded 71.3%-fold with high specific activity of 2787.1U/g. Based on optimization of physicochemical parameters and nutritional parameters for the optimum production of protease, the optimum temperature of protease production was 40°C with protease concentration of 32.9 ±1.60U/g. pH 9 was the optimum for production of protease with protease concentration of (14.1±1.40U/g and 22.7±2.1U/g) for both phosphate and carbonate buffers respectively. Among the metallic ions, media containing Mn²⁺ performed better than Cu²⁺, Mg²⁺, Fe²⁺, and Zn²⁺ with a protease concentration of 24.6±2.45U/g and 23.3±1.31U/g at 5mM and 1mM respectively. Studies on the effect of concentration of divalent ions revealed that both the activity and stability of protease was better in 5mM than in 1mM concentration. Furthermore, evaluation of some agro-industrial wastes as potential substrates for protease production indicated that wheat bran was better for carbon agro-residues (3.9±0.30U/g), whereas soybean meal was better for nitrogen agro-residues (4.0±0.23U/g). Since protease was produced from readily available complex substrates and agro-residues, Bacillus subtilis appear to have substantial potential for application in various proteolytic processes. Also, from the study, it was observed that immobilized alkaline protease from Bacillus subtilis was most compatible with the laboratory-based detergent Tween 20 and consequently showed highest protease activity of 6.3±0.62U/g, and also the enzyme showed highest compatibility with the commercial detergent omo and ariel and consequently showed highest protease activity of 5.8±0.55U/g and 5.0±0.10U/g respectively. Finally, in this study, stain removal was very efficient and haemolysis of blood occurred within a minute of enzyme action on the blood stain. An addition of purified alkaline protease from Bacillus subtilis effectively removed the stain. It could therefore be used as an additive in laundry detergents.