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

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.


MELLATI, AA; YUCEL, M; ALTINORS, N; Gündüz, Ufuk (1993-10-01)
The M2-type pyruvate kinase was purified from human meningioma by ammonium sulfate precipitation, followed by ion exchange and affinity chromatography. The specific activity of the purified enzyme was 33.4 U/mg with a yield of 6.5%.
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Izgu, F; Altinbay, D (Informa UK Limited, 2004-03-01)
K5-type yeast killer protein in the culture supernatant of Pichia anomala NCYC 434 cells was concentrated by ultrafiltration and purified to homogenity by ion-exchange chromatography with a POROS HQ/M column followed by gel filtration with a TSK G2000SW column. The protein migrated as a single band on discontinous gradient SDS-PAGE and had a molecular mass of 49000 Da. The pI value of the K5-type killer protein was measured at pH 3.7 by high voltage vertical gel electrofocusing. The result of an enzyme immu...
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Takac, S; Elmas, S; Çalık, Pınar; Ozdamar, TH (2000-06-01)
The separation from fermentation medium of extracellular serine alkaline protease (SAP) enzyme produced by Bacillus licheniformis was investigated using a crossflow ultrafiltration system. SAP was separated from the high molecular weight neutral protease (NP) and amylase (AMY) enzymes and from the low molecular weight organic acids and amino acids in a crossflow ultrafiltration system with 30000 Da and 10000 Da MWCO polysulfone membranes, respectively. The effects of transmembrane pressure (TMP), recirculat...
Synthesis and properties of a novel redox driven chemiluminescent material built on a terthienyl system
Atilgan, Nurdan; Algi, Fatih; Önal, Ahmet Muhtar; CİHANER, ATİLLA (Elsevier BV, 2009-07-18)
A novel redox driven chemiluminescent material built on a terthienyl system, namely 5,7-di-ethylenedioxythiophen-2-yl-2,3-dihydro-thieno[3,4-d]pyridazine-1,4-dione (ETE-Lum), which is soluble in both organic media and basic aqueous solution was synthesized and characterized. Furthermore, its polymer, PETE-Lum, which is one of the most rare examples of chemiluminescent polymeric materials bearing a pyridazine unit, was obtained successfully by electrochemical means. Both of the materials give chemiluminescen...
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A convenient and new method for the synthesis of 1,2,3,5-tetrasubstituted pyrrole derivatives starting from 1,3,-dicarbonyl compounds through acid catalyzed cyclization reaction is described. Alkylation of 1,3-dicarbonyl compound with propargyl bromide followed by one step cyclization with the introduction of primary amines in the presence of catalytic amount of triflouroacetic acid (TFA) affords the corresponding pyrrole derivatives in high yields. The investigations on the studies of developing a new meth...
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.