Optimization and Partial Characterization of <i>Bacillus</i> Protease Isolated from Soil and Agro-industrial Wastes

Abstract

Proteases from microbial sources possess almost all the characteristics desired for their biotechnological applications. This study was conducted with the aim of screening for potent protease-producing bacteria from soils and agro-industrial wastes, determining optimal production conditions and partially characterizing the stability of the protease with regards to some physicochemical parameters. Thus, the optimum protease production time for these 3 isolates, was found to be 36 h isolated from industrial waste, manure, and soil, respectively. The optimum temperature of protease production for both PS-3 and PI-3 was 40°C. Whereas 37°C was the optimum for PM-1. In all cases, pH 7 was the optimum for production of protease. <i>Furthermore, 0.6 M NaCl concentration was found to give better protease activity than the media containing no NaCl in all 3 isolates,. Among the metallic ions, media containing Mn²⁺ performed better than </i>Cu²⁺, Mg²⁺, Fe²⁺, and Zn²⁺<i> for PS-3 and PM-1, whereas Mg²⁺ was the best for PI-3. </i>Studies on the effect of pH on the stability of protease enzymes revealed that the crude enzyme had a maximum stability at pH 9.0 for isolates PS-3 (36.5 and 26.5 U/ml) and PI-3 (29.7 and 22.7 U/ml), while for isolate PM-1 maximum stability was achieved at pH 8 with values corresponding to 18.2 and 15.5 U/ml, respectively. These proteases are also stable at 75°C for PS-3 (42.9 and 33.4 U/ml) and PI-3 (46.8 and 42.1 U/ml), while they showed maximum activity and stability at 50°C for PM-1 (22.5 and 23.1 U/ml, respectively). Pre-incubation at temperatures above 70°C for PS-3 and PI-3 and 50°C for PM-1 resulted in reduction of enzyme activity, indicating that the proteases are thermally unstable. Studies on the effect of concentration of divalent ions revealed that both the activity and stability of protease were better in 1 mM than in 5 mM concentration. Furthermore, evaluation of some agro-industrial wastes as potential substrates for protease production indicated that wheat bran was better for PS-3 (4.3 U/ml) and PM-1 (3.0 U/ml), whereas human hair was better for PI-3 (3.9 U/ml). Since protease was produced from readily available complex substrates and agro-industrial wastes, the 3 Bacillus species appear to have substantial potential for application in various proteolytic processes. Thus, identification of the 3 Bacillus isolates 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.