title: Production, purification, and characterization of metalloprotease from Candida kefyr 41 PSB.
abstract: A thermostable metalloprotease, produced from an environmental strain of Candida kefyr 41 PSB, was purified 16 fold with a 60% yield by cold ethanol precipitation and affinity chromatography (bentonite-acrylamide-cysteine microcomposite). The purified enzyme appeared as a single protein band at 43kDa. Its optimum pH and temperature points were found to be 7.0 and 105°C, respectively. Kmand Vmaxvalues of the enzyme were determined to be 3.5mg/mL and 4.4μmolmL-1min-1, 1.65mg/mL and 6.1μmolmL-1min-1, using casein and gelatine as the substrates, respectively. The activity was inhibited by using ethylenediamine tetraacetic acid (EDTA), indicating that the enzyme was a metalloprotease. Stability of the enzyme was investigated by using thermodynamic and kinetic parameters. The thermal inactivation profile of the enzyme conformed to the first order kinetics. The half life of the enzyme at 95, 105, 115, 125 and 135°C was 1310, 610, 220, 150, and 86min, respectively.
authors.affiliation: Cumhuriyet University Faculty of Engineering, Department of Bioengineering, 58140 Sivas, Turkey.
authors.forename: Sevgi
authors.initials: S
authors.lastname: Yavuz
authors.affiliation: Inönü University Faculty of Science, Department of Molecular Biology and Genetics, 44280 Malatya, Turkey.
authors.forename: Samet
authors.initials: S
authors.lastname: Kocabay
authors.affiliation: Cumhuriyet University Faculty of Science, Department of Molecular Biology and Genetics, 58140 Sivas, Turkey.
authors.forename: Serap
authors.initials: S
authors.lastname: Çetinkaya
authors.affiliation: Cumhuriyet University Faculty of Science, Department of Molecular Biology and Genetics, 58140 Sivas, Turkey. Electronic address: bakkaya@cumhuriyet.edu.tr.
authors.forename: Birnur
authors.initials: B
authors.lastname: Akkaya
authors.affiliation: Cumhuriyet University, Vocational School of Health Services, 58140 Sivas, Turkey.
authors.forename: Recep
authors.initials: R
authors.lastname: Akkaya
authors.affiliation: Cumhuriyet University Faculty of Science, Department of Molecular Biology and Genetics, 58140 Sivas, Turkey.
authors.forename: Ali Fazil
authors.initials: AF
authors.lastname: Yenidunya
authors.affiliation: Cumhuriyet University, Faculty of Medicine, Department of Microbiology, 58140 Sivas, Turkey.
authors.forename: Mustafa Zahir
authors.initials: MZ
authors.lastname: Bakıcı
mesh_terms: D002175:Candida / Q000201:enzymology*; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D045726:Metalloproteases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D059748:Proteolysis; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D012997:Solvents; D008345:Manganese; D045726:Metalloproteases; D015032:Zinc
keywords: Candida kefyr; Metalloprotease; Organic solvent stable; Thermostable; co-Catalytic active