Optimization of Protease Activity by Bacillus haynesii BK1H Using Response Surface Methodology

Abstract

Proteases are enzymes that catalyze the hydrolysis of peptide bonds into oligopeptides and amino acids. Previous bacterial studies reported that eight proteolytic isolates had been isolated from Bledug Kuwu mud, Central Java. One of these isolates is BK1H which has a proteolytic index of 2.960 making it possible to be studied further. In this study, optimization of the protease activity of BK1H was carried out using 3 factors namely temperature, pH, and divalent metal cations. This research was conducted through several stages, (1) Bacterial culture regeneration, (2) Protease fermentation using the SmF method, (3) Determination of the type of protease, (4) One Factor at a Time (OFAT), (5) Plackett-Burman Design (PBD), (6) Response Surface Methodology-Central Composite Design (RSM-CCD), and (7) Confirmation of RSM-CCD optimization results on experimental results. OFAT experiment results showed the lowest activity and the highest activity was obtained at 37°C and 57°C; pH 6 and 8; and CaCl2 0.5 and 1.0 mM. The significance test of the 2 factors namely temperature and pH has a p value <0.05, which indicates that these factors have a significant effect on protease activity. The optimum factor value in the RSM-CCD has a temperature of 63.10°C and pH 10.82, with the predicted activity of 149.910 U/mL, while the experimental results have activity of 157.630 ± 16.240 U/mL.