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

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2006-01-01

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Kocabıyık, Semra

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An intracellular serine protease produced by Thermoplasma (Tp.) voleanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and et-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 degrees 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. value of 2.2 mm and a V-max value of 0.045 mu mol(-1) m1(-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.

Subject Keywords

Thermoplasma volcanium, Serine protease, Chymotrpsin-like protease, Archaea, Ther-mophilic

URI

https://hdl.handle.net/11511/38080

Journal

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY

DOI

https://doi.org/10.1271/bbb.70.126

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Department of Biology, Article

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Citation Formats

S. Kocabıyık, “Purification and characterization of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium,” BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, pp. 126–134, 2006, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/38080.