title: An improved procedure for the purification of catalytically active alkane hydroxylase from Pseudomonas putida GPo1.
abstract: Bacterial alkane hydroxylases are of high interest for bioremediation applications as they allow some bacteria to grow in oil-contaminated environments. Furthermore, they have tremendous biotechnological potential as they catalyse the stereo- and regio-specific hydroxylation of chemically inert alkanes, which can then be used in the synthesis of pharmaceuticals and other high-cost chemicals. Despite their potential, progress on the detailed characterization of these systems has so far been slow mainly due to the lack of a robust procedure to purify its membrane protein component, monooxygenase AlkB, in a stable and active form. This study reports a new method for isolating milligramme amounts of recombinant Pseudomonas putida GPo1 AlkB in a folded, catalytically active form to purity levels above 90%. AlkB solubilised and purified in the detergent lauryldimethylamine oxide was demonstrated to be active in catalysing the epoxidation reaction of 1-octene with an estimated K (m) value of 0.2 mM.
authors.affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK. meng.xie@mail.mcgill.ca
authors.forename: Meng
authors.initials: M
authors.lastname: Xie
authors.forename: Hernan
authors.initials: H
authors.lastname: Alonso
authors.forename: Anna
authors.initials: A
authors.lastname: Roujeinikova
mesh_terms: D000473:Alkanes / Q000378:metabolism*; D000475:Alkenes / Q000378:metabolism*; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A / Q000235:genetics / Q000378:metabolism*; D004123:Dimethylamines / Q000737:chemistry; D004926:Escherichia coli; D006900:Hydroxylation; D007700:Kinetics; D010578:Petroleum / Q000378:metabolism*; D010957:Plasmids; D017510:Protein Folding; D016958:Pseudomonas putida / Q000737:chemistry / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism; D014169:Transformation, Bacterial
chemical_list: D000473:Alkanes; D000475:Alkenes; D004123:Dimethylamines; D010578:Petroleum; D011994:Recombinant Proteins; C014518:dodecyldimethylamine oxide; C037690:1-octene; D042926:Cytochrome P-450 CYP4A