Catalytic, kinetic and thermodynamic properties of stabilized Bacillus stearothermophilus alkaline protease

Bacillus stearothermophilus alkaline protease was conjugated to several oxidized polysaccharides of different chemical structure. The conjugates were evaluated for the kinetic and thermodynamic stability. The conjugated enzyme with oxidized pectin had the highest retained activity (79.5%) and the highest half-life (T1/2) at 50 °C and pH 9.0. Compared to the native protease, the conjugated preparation exhibited lower activation energy (Ea), lower deactivation constant rate (kd), higher T1/2, and higher D values (decimal reduction time) within the temperature range of 50-60 °C. The thermodynamic parameters for irreversible inactivation of native and conjugated protease indicated that conjugation significantly decreased entropy (ΔS*) and enthalpy (ΔH*) of deactivation. The calculated value of activation energy for thermal denaturation (Ead) for the conjugated enzyme was 20.4 KJ mole−1 higher over the native one. The results of thermodynamic analysis for substrate hydrolysis indicated that the enthalpy of activation (ΔH*) and free energy of activation (free energy of substrate binding) ΔG*E-S and (ΔG*), (free energy of transition state) ΔG*E-T values were lower for the modified protease. Similarly, there was significant improvement of kcat, kcat/Km values. The enzyme proved to be metalloprotease and significantly stimulated by Ca2+ and Mg2+ whereas Hg2+, Fe3+ Cu2+ and Zn2+ inhibited the enzyme activity. There was no pronounced effect on substrate specificity after conjugation.