Oil pollution is an environmental problem of increasing importance. Alcanivorax borkumensis, with a high potential for biotechnological applications, is a key marine hydrocarbonoclastic bacterium and plays a critical role in the bioremediation of oil-polluted marine systems. In oil degrading bacteria, the first step of alkane degradation is catalyzed by a monooxygenase. The reducing electrons are tunneled from NAD(P)H via rubredoxin, one of the most primitive metalloproteins, to the hydroxylase. Rubredoxin reductase is a flavoprotein catalyzing the reduction of rubredoxin. There are two rubredoxin genes, alkG and rubA, in A. borkumensis genome. In this work, the genes encoding rubredoxin reductase (ABO_0162, rubB) and AlkG(ABO_2708, alkG) were cloned and functionally overexpressed in E. coli. Our results demonstrate that RubB could reduce AlkG, therefore compensating for the absence of AlkT, also a rubredoxin reductase, missing in A. borkumensis SK2 genome. These results will increase our knowledge concerning biological alkane degradation and will lead us to design more efficient biotransformation and bioremediation systems.

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.