Optimization of Culture Conditions and Reaction Parameters of β-glucosidase From a New Isolate of Bacillus subtilis (B1)

Introduction: The increased need for a considerable β-glucosidase activity, especially in the enzymatic saccharification of cellulose for bioenergy, has strongly stimulated the identification of effective β-glucosidase producing microbes. This study was conducted to optimize culture condition for β-glucosidase production from the identified new isolate of Bacillus subtilis (B1) and to find out an ideal condition for β-glucosidase activity. Materials and Methods: For β-glucosidase production, the bacterium was cultivated in a basal medium. The culture condition was optimized at several pH, different temperatures, varying cultivation periods, and various substrate concentrations. Finally, the activity of the β-glucosidase enzyme was investigated at different incubation periods, pH, temperatures, metal ions, and various percentages of methanol. The activity of β-glucosidase was measured by the capability of crude enzyme to convert pNPG (p-nitrophenyl-β-D glucopyranoside) into yellow product PNP (p-nitrophenol). Results: Cellulolytic bacterial strain B. subtilis (B1) showed high potentiality for β-glucosidase production at a pH of 7.0 after 24 hours incubation at 40°C. The highest level of enzyme production was achieved when 3% of CMC was provided in the culture medium. Optimum reaction conditions for β-glucosidase activity were shown to be 10 minutes, 60°C and at pH 7. Salts like Magnesium Sulfate (MgSO4), Calcium Chloride (CaCl2), and Manganese Sulfate (MnSO4) positively influenced the activity where NaCl and KCl had negative effects. The presence of methanol (80%) appreciably enhanced the activity of enzyme. Conclusions: Complete saccharification of different industrial processes can be augmented by using this novel β-glucosidase produced by B. subtilis strain isolated from effluent of biogas plant.

Keywords:

Saccharification , β-glucosidase , Bacillus subtilis , Cellobiose , Submerged culture