The influence of copper on alkaline protease stability toward autolysis and thermal inactivation

Abstract

Alkaline proteases are one of the most important group of enzymes that are indispensable in a number of different industrial sectors. In this work, the effect of copper ions (Cu2+) was investigated for improving the thermostability and hydrolytic performance of Bacillus clausii GMBE 42 alkaline protease at different temperatures (45-65 degrees C). Maximal residual activity was observed in the presence of 5 mM CuCl2. The enzyme was thermoinactivated according to first-order kinetics. A stabilization effect caused by copper ions was the result of a decrease in both autolysis and thermoinactivation rates. Thermodynamic analysis of the thermoinactivation process showed that Ea,i, ?Gi, and ?Hi values of the enzyme were higher in the presence of copper ions, but there was no measurable change in ?Si values. These results show the thermostabilizing potential of copper ions on the enzyme. Lower Km values and higher kcat and kcat/Km values were obtained in the presence of copper ions, which is an indication of the nonessential activation of the enzyme by copper ions. Thermodynamic analysis of casein hydrolysis showed that in presence of copper ions Ea, ?G?, ?H?, ?GE-S?, and ?GE-T? values of enzyme were lower, but there was no change in ?S? values. This is so far the first study that investigates the effect of cations on the basic catalytic and thermodynamic properties of an alkaline serine protease, which may be used to remove protein wastes from various industries such as food and leather processing.