ABSTRACT
Milk clotting enzyme (MCE) produced by Bacillus subtilis KU710517 was conjugated to several activated polysaccharides. Among all the conjugates, the enzyme conjugated with polyethylene glycol (PEG) exhibited the highest retained activity (551U/mg protein) with a recovered activity of 95.3%. The activation energy of PEG-conjugated enzyme was calculated as 24.56kJ·mol-1which was lower than that of the native one (29.27kJ·mol-1) however, the temperature quotient (Q10) was about 1.08 for the two forms of the enzyme. The calculated half-life times of PEG-conjugated enzyme at 55 and 60°C were 317.78 and 128.6min respectively, whereas at the same temperatures the native enzyme had lower half-life times (53 and 19.6min respectively). The data of thermodynamic analysis for substrate catalysis including the specificity constant (Vmax/Km), turnover number (kcat), catalytic efficiency (kcat/Km), enthalpy of activation (ΔH*), free energy of activation (ΔG*), free energy for transition state formation ΔG*E-T and free energy of substrate binding ΔG*E-S were determined for both native and PEG-conjugated enzyme. In addition, the thermodynamic parameters for irreversible inactivation (ΔH, ΔG, ΔS) were evaluated. The calculated results indicated that the catalytic properties after the PEG-conjugation were significantly improved.
