Purification and Characterization of an Intracellular Chymotrypsin-Like Serine Protease from Thermoplasma volcanium

Abstract

An intracellular serine protease produced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and α-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 °C. The purifed enzyme hydrolyzed synthetic peptides preferentially at the carboxy terminus of phenylalanine or leucine and was almost completely inhibited by PMSF, TPCK, and chymostatin, similarly to a chymotrypsin-like serine protease. Kinetic analysis of the Tp. volcanium protease reaction performed using N-succinyl-L-phenylalanine-p-nitroanilide as substrate revealed a K_m value of 2.2 mM and a V_max value of 0.045 μmol⁻¹ ml⁻¹ min⁻¹. Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca²⁺ and Mg²⁺ at 2–12 mM concentration. The serine protease is a monomer with a molecular weight of 42 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and zymogram activity staining.