title: Cloning and expression of alkane hydroxylase-1 from Alcanivorax borkumensis in Escherichia coli.
abstract: Enzymes with hydroxylating activity on alkanes have potential application as biotransformation catalysts in chemical and pharmaceutical industry. Genome of Alcanivorax borkumensis, a marine bacterium with hydrocarbon dissimilation activity, contains at least two P450 monooxygenases and two nonheme monooxygenases, AlkB1 and AlkB2, respectively. Presumably, all these enzymes possess alkane hydroxylating activity. Both AlkB1 and AlkB2 are membrane proteins. Two accessory proteins, rubredoxin and rubredoxin reductase, supply the reducing equivalent from nicotinamide adenine dinucleotide phosphate reduced (NADPH to hydroxylases. Rubredoxin reductase catalyses the reduction of rubredoxin by oxidation of NADPH, and rubredoxin transfers the electrons to the alkane hydroxylase to complete the hydroxylation reaction. Here, we sought to investigate the expression of alkB1 gene in Escherichia coli. Therefore, we amplified alkB1 gene from A. borkumensis genome by polymerase chain reaction and cloned it in the expression vector pET26 upstream of His-tag sequence. Predisposed BL21 (DE3) cells were transformed by the recombinant vector. At last, expression of recombinant enzyme was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Regarding the potential ability of this enzyme in hydroxylation of long-chained alkanes, the application of it would be studied in petroleum downstream industries.
authors: Department of Genetics, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors: Simin Zadehgan
authors: SZ
authors: Eidani
authors: Mahvash Khodabandeh
authors: MK
authors: Shahraki
authors: Fatemeh
authors: F
authors: Gasemisakha
authors: Mehrdad
authors: M
authors: Hahsemi
authors: Bijan
authors: B
authors: Bambai
mesh_terms: D053521:Alcanivoraceae / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006639:Histidine; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D042926:Cytochrome P-450 CYP4A