Purification Scheme and Biochemical Profile of Metallo-alkaline Protease Produced by B.t. Dendrolimus IP 4A/4B

Abstract

Objective: Purify and classify alkaline protease (AP), produced by Bacillus thuringiensis (B.t.) dendrolimus 4A/4B. Methods: Inoculum preparation, production media, enzymes assays, protein determination, ammonium sulfate precipitation, acetone fractionation, Sephadex Gel chromatography filtration. Results: The purification scheme, (a) ammonium sulfate fractionation, the most active fraction with 1.93 purification fold was at 60-75% saturation; (b) acetone precipitation, where the most active fraction was at 30-45% with purification fold of about 3.92 times; (c) Column chromatography on G-100 Sephadex with final purification folds 5.36 times of the crude enzyme with final yield recovery 1.9%. Purified AP showed stability against H2O2 1%, 5% and 10% (v/v), and retained 111%, 101% and 66% of its activity, respectively. AP retained 82%, 87% and 101% of its activity with 10, 5 and 1mM PMSF, respectively. Enzyme activity was completely inhibited by 10mM EDTA, while the enzyme retained about 125% of its activity with 10mM Mercapto-ethanol. CaCl2 and MgCl2 (10mM) showed activation effect, increasing the activity to 136% and 147% compared to the control, respectively. The optimum pH are 8.0 and 10.0. AP activity was increased through 30ºC to 50ºC. At 60˚C, the enzyme retained ~48% of its original activity. AP concentration 0.014mg protein/mL has maximum activity (6.750U/mL/min). The highest maximum velocity (Vmax) was 31.821μg tyrosine/mL/min. Km value 0.833%, w/v casein concentration. Conclusion: Thus, this inhibition profile suggested that the AP produced belongs to the class of metalo-proteases.