Purification and characterization of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium

• Published in: Bioscience, biotechnology, and biochemistry Vol. 70; no. 1; pp. 126 - 134
• Main Authors: Kocabiyik, S.(Middle East Technical Univ., Ankara (Turkey)), Ozdemir, I
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01-01-2006; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: AISLAMIENTO; Archaea; Biological and medical sciences; Calcium - chemistry; Calcium - pharmacology; Cations, Divalent - chemistry; Cations, Divalent - pharmacology; chymotrpsin-like protease; ENZYME PREPARATIONS; EXTRACTOS; EXTRACTS; EXTRAIT; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Hydrolysis - drug effects; ISOLATION; ISOLEMENT; Magnesium - chemistry; Magnesium - pharmacology; MICRO-ORGANISME THERMOPHILE; MICROORGANISMOS TERMOFILOS; Peptides - metabolism; PREPARACION ENZIMATICA; PREPARATION ENZYMATIQUE; PROTEASAS; PROTEASE; PROTEASES; PURIFICACION; PURIFICATION; SERINA; SERINE; Serine Endopeptidases - chemistry; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; serine protease; Serine Proteinase Inhibitors - pharmacology; Substrate Specificity; Temperature; thermophilic; THERMOPHILIC MICROORGANISMS; Thermoplasma - enzymology; Thermoplasma volcanium; thermophilic; Purification; Serine endopeptidases; Archaeobacteria; Enzyme; Thermoplasma volcanium; Archaea; Thermophily; serine protease; Characterization; Peptidases; Chymotrypsin; chymotrpsin-like protease; Bacteria; Hydrolases; Intracellular; Thermoplasma
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.70.126

An intracellular serine protease preduced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and alpha-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 deg 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-beta-nitroanilide as substrate revealed a Ksub(m) value of 2.2 mM and a Vsub(max) value of 0.045 micromolsup(-1) mlsup(-1) min1. Peptide hydrolyzing activity was enhanced by 2-fold in the presence of Casup(2+) and Mgsup(2+) t 2-12mM 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.