Bacillus thuringiensis aizawai HD283 Partial Purified Thermo-Alkaline Metalloprotease Characterization

Abstract

Objective: The study aimed to classify the alkaline protease produced by Bacillus thuringiensis aizawai HD283. Methods: The experimental methodology encompassed the following steps: preparation of the inoculum, formulation of production media, enzyme activity assays, protein quantification. Results: After ammonium sulfate fractionation, acetone precipitation, and G-100 Sephadex column chromatography purification steps, of Bacillus thuringiensis aizawai HD283 partial purified alkaline protease. The purified alkaline protease exhibited a significant activation response to H₂O₂ at concentrations of 1%, 5%, and 10% (v/v), retaining 412%, 342%, and 286% of its activity, respectively. The enzyme activity demonstrated notable increases of 224%, 226%, and 300% in the presence of 10 mM, 5 mM, and 1 mM PMSF, respectively. Alkaline protease activity was inhibited by 10 mM EDTA, AgNO3 and HgCl2 with 44, 60 and 67 % of control respectively, the enzyme's activity increased to approximately 572% in the presence of 10 mM mercaptoethanol. It also increased to 127%, 268%, 292%, and 703% compared to the control group with the addition of calcium chloride, choline chloride,
magnesium chloride, and manganese chloride (at 10 mM concentrations, respectively). Acetone at 50% concentration activated the enzyme by 177%. The optimal pH was found to be 8.0 and 10.0, and the enzyme's activity increased with temperature from 30°C to 70°C. The enzyme retained approximately 74% of its original activity at 60°C. At a protein concentration of 0.017 mg/mL, the enzyme's maximum activity was approximately about, 16.640 units/mL/min. The highest maximum velocity (Vmax) of the enzyme was achieved at 30.116 micrograms of tyrosine/ml/min, Km value of casein concentration was 5.448%. Conclusion: Based on the chemical classification scheme and the catalytic mechanism observed, the alkaline protease produced in this study was identified as belonging to the class of Thermophilic- Metallo-Alkaline Proteases.