title: Biochemical characterization of two recombinant ferredoxin reductases from Alcanivorax borkumensis SK2.
abstract: Alcanivorax borkumensis strain SK2 is a cosmopolitan oil-degrading oligotrophic marine γ-proteobacterium that exclusively uses petroleum hydrocarbons as sources of carbon and energy. Its ubiquity and unusual physiology suggest its global importance in the removal of hydrocarbons from polluted marine systems. The genome of A. borkumensis SK2 was recently sequenced. Two ferredoxin-nicotinamide adenine dinucleotide phosphate (NADPH) reductase genes (ABO_0145 and ABO_0203) have been annotated for this bacterium. In the present study, the expression, purification, and kinetic properties of these two genes were explored by constructing the prokaryotic expression vectors (pET21a) for the first time. Isopropyl β-D-thiogalactoside (0.5 mM) was used for induction of exponentially growing cells (30 °C, overnight). Most of the proteins were expressed in inclusion body. Partial purification of recombinant enzymes was performed by ion-exchange chromatography on a DEAE-sepharose column using only one linear gradient of sodium chloride ranging between 0 and 500 mM. The recombinant enzymes displayed reductase activity, which was optimal at pH 6.0 and 45 °C. Ferredoxin-NADPH reductases exhibited several outstanding properties that made them excellent model proteins to address broad biological questions. This study serves as the basis for further investigations of the biotechnological potential of these enzymes.
authors.affiliation: Department of Biochemistry, Institute of Biochemistry and Biophysics-IBB, University of Tehran, Tehran, Iran.
authors.forename: Afsaneh
authors.initials: A
authors.lastname: Teimoori
authors.forename: Shahin
authors.initials: S
authors.lastname: Ahmadian
authors.forename: Armin
authors.initials: A
authors.lastname: Madadkar-Sobhani
mesh_terms: D053521:Alcanivoraceae / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005818:Genetic Engineering; D007700:Kinetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D005287:Ferredoxin-NADP Reductase