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_1/2, the values of k_d, activation energy (E_a), 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_m, V_max, ΔH*, ΔG*, ΔS*, k_cat, V_max/K_m, k_cat/K_m, ΔG*_E-T and ΔG*_E-S) 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.

Keywords: Kinetic properties; Mutagenic treatments; Protease; Thermodynamic properties.