Purification and Characterization of an Intracellular Chymotrypsin-Like Serine Protease from Thermoplasma volcanium
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An intracellular serine protease produced by <I>Thermoplasma (Tp.) volcanium</I> 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 <I>Tp. volcanium</I> protease reaction performed using <I>N</I>-succinyl-<small>L</small>-phenylalanine-<I>p</I>-nitroanilide as substrate revealed a <I>K</I><SUB>m</SUB> value of 2.2 m<small>M</small> and a <I>V</I><SUB>max</SUB> value of 0.045 μmol<SUP>−1</SUP> ml<SUP>−1</SUP> min<SUP>−1</SUP>. Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca<SUP>2+</SUP> and Mg<SUP>2+</SUP> at 2–12 m<small>M</small> 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.
- Agricultural and Biological Chemistry
Agricultural and Biological Chemistry 70(1), 126-134, 2006-01-23
Japan Society for Bioscience, Biotechnology, and Agrochemistry