Molecular characterization, catalytic, kinetic and thermodynamic properties of protease produced by a mutant of Bacillus cereus-S6-3.

The proteolytic strain Bacillus cereus-S6-3 was subjected to mutagenic treatments viz. UV irradiations and methyl methane sulfonate (MMS). The obtained mutant strain, B. cereus-S6-3/UM90 showed 1.34 fold over the parent strain. Molecular characterization of proteases from the parent (PP/S6-3) and mutant (PM/UM90) strains indicated that they were consisted of two domains and binds a zinc ion and 4 calcium ions in the active site. Amino acid sequence alignment of PM/UM90 protease showed 19 amino acid residues were substituted compared to that of the wild-type enzyme. However, both proteases contained equal number of aromatic and hydrophobic amino acids. Protease from PM/UM90 showed an effective improvement in thermal properties in terms of reaction temperature, t, the values of k, activation energy (E), and decimal reduction time (D) within the temperature range from 60 to 80 °C. In addition, the kinetic and thermodynamic parameters for substrate hydrolysis (i.e., K, V, ΔH*, ΔG*, ΔS*, k, V/K, k/K, ΔG* and ΔG*) showed a significant improvement of the catalytic efficiency for PM/UM90 protease. Furthermore, the correlation between thermodynamic properties and the patterns of amino acid substitution of wild-type enzyme to has been discussed.