Derivatization of Proteinase K with Heavy Atoms Enhances Its Thermal Stability

Abstract

Proteinase K is widely used in molecular biology for its broad substrate specificity, wide pH stability, and high hydrolysis activity. Aminolysis by proteinase K is also attractive for chemoenzymatic peptide synthesis. However, the relatively low thermal stability of the native protease limits the hydrolysis and aminolysis reactions in industrial processes. Here, we enhanced the thermal stability of proteinase K using a heavy-atom derivatization technique. The denaturation temperature of proteinase K derivatized with praseodymium (Pr) ions was 16.2 and 5.9 °C higher than those of metal-free and Ca-binding proteinase K, respectively. Isothermal titration calorimetry (ITC) measurements demonstrated that Pr-ion binding to proteinase K showed endothermic peaks, whereas Ca-ion binding showed exothermic peaks, indicating that the binding mode of Pr ions was different from that of Ca ions, even though the crystal structures of proteinase K with Pr and Ca ions were identical. Hydrolytic activity of Pr-derivatized proteinase K showed that the hydrolytic activity was 46-fold higher at 70 °C using synthetic nitroanilide substrate and 9- and 76-fold higher at 70 and 80 °C using fluorescein isothiocyanate-labeled casein, respectively, in comparison with the native proteinase K. Furthermore, based on the yield of chemoenzymatic peptide syntheses, the aminolysis activity of Pr-derivatized proteinase K was 3.5- and 9.5-fold higher than that of the native proteinase K at 50 and 60 °C, respectively. The present results demonstrate that stabilization of the protease with heavy atoms can broaden its potential as an efficient biocatalyst not only for hydrolysis but also for aminolysis of peptides, polypeptides, and proteins.