Optimization of Parameters that Affect the Activity of the Alkaline Protease from Halotolerant Bacterium, Bacillus acquimaris VITP4, by the Application of Response Surface Methodology and Evaluation of the Storage Stability of the Enzyme

Background: It was previously shown that the activity of a serine protease from a moderately halotolerant Bacillus aquimaris VITP4 strain is active in a wide range of pH and temperatures and could be modulated by the presence of the divalent metal ions.  Objectives: In the present study, a quantitative analysis was done in order to explore the parameters that are contributing to the protease activity. Materials and Methods: Changes in the secondary structure of the enzyme was determined by circular dichroism analysis.  The conditions for the optimal activity was investigated by Response Surface Methodology. Stability of the enzyme was determined by thermal inactivation experiments.  Results: The initial one-factor-at-a-time experiments have indicated that the activity of the enzyme could be enhanced not only by the presence of low concentrations of NaCl but also by divalent metal ions, such as Ca2+, Mn2+ and Cu2+. A clear dependence of the activity to the secondary structure  of the enzyme could be established using circular dichroism spectroscopy. In the next level of optimization, four factors; viz. pH, temperature, concentration of Ca2+, and Mn2+ were used to optimize the conditions required for the maximal activity of the enzyme by Response Surface Methodology, and the data could be explained using quadratic model. Under optimal condition of 43°C,  pH 8.0, 8.2 mM Ca2+, and 4.3 mM Mn2+ a 1.5 times enhancement in the enzyme activity could be achieved. The storage stability of the enzyme under these selected conditions has indicated a non-linear relation between the conditions for the enzymatic activity as well as stability.  However, the condition for the maximal stability (267±18 min) has corresponded to that of the optimal conditions for the maximal activity. Conclusions: This study, for the first time, has explored the possibility of using statistical methods for identifying the optimal conditions for alkaline protease activity isolated from the halotolerant Bacillus aquimaris VITP4.