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

Abstract

An intracellular serine protease produced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and -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 mol À1 ml À1 min À1 . Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca 2þ and Mg 2þ 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.

Cloning and overexpression of a thermostable signal peptide peptidase (SppA) from Thermoplasma volcanium GSS1 in E. coli

“…5B). This result indicates that Tpv SppA, like some other mi- crobial SP, possibly requires Ca 2+ ion for activity [13,22,23]. Bajorath et al [24] showed that Ca 2+ plays an important role in regulating and maintaining the enzymatically active conformation of the proteinase K, by triggering a concerted sets of movements when it occupies the tight binding site, thus affecting the geometry of the substrate binding site and catalytic triad.…”

“…Peptide hydrolyzing activities were assayed by continuous monitoring of the release of p-nitroaniline (pNA) from oligopeptidyl-p-nitroanalide substrates, Ala-Ala-Phe-pNA and N-Suc-Ala-Ala-Pro-Phe-pNA at 410 nm in a thermostatically controlled spectrophotometer (Schimadzu UV-1601A Spectrophotometer, Kyoto, Japan) as described before [13]. The reaction mixture (total volume 820 µL) contained 720 µL assay buffer (50 mM Tris HCl, pH 7.5, 5 mM CaCl 2 ), 50 µL enzyme solution and 600 µM substrate.…”

“…Owing to this unique feature, there has been an increasing interest in the archaeal SP for a variety of applications in industry and biotechnology, as well as in basic enzymology and molecular biology research. To date, SP from several thermophilic and hyperthermophilic archaea including the genera Pyrococcus (pyrolysin and PfpI) [6,7], Desulforococcus (archaelysin) [8], Sulfolobus [9,10], Thermococcus [11,12] and Thermoplasma [13] have been identified. Recent analyses of complete genome sequences of prokaryotes, including archaea revealed even more expansive ORFs with common or novel functional domains of SP [14,15].…”

Protein Purification by Affinity Chromatography

“…A support containing trypsin immobilized on agarose was used to purify trypsin inhibitor from liver of Oreochromis niloticus (Leite et al, 2011). Chromatography on -casein-Agarose was useful for purification of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium (Kocabiyik & Ozdemir, 2006).…”

Biotechnology in Turkey: An overview

“…A line of Kocabıyık's research activities involves structure-function analysis of antibiotic resistance enzyme and thermophilic enzymes to reveal molecular mechanisms responsible for the catalysis, substrate specificity and enzyme stability. She is using a number of complementary approaches that entail cloning of appropriate genes, purification of target enzymes, rigorous characterization of their catalytic functions and probing structure-function relationships by molecular biological methods, such as site-specific mutagenesis [114][115][116][117][118]. Gülay Özcengiz has also been researching in molecular genetics [119][120][121].…”