TY - JOUR
T1 - An improved procedure for the purification of catalytically active alkane hydroxylase from Pseudomonas putida GPo1
AU - Xie, Meng
AU - Alonso, Hernan
AU - Roujeinikova, Anna
PY - 2011/10
Y1 - 2011/10
N2 - 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 Km value of 0.2 mM.
AB - 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 Km value of 0.2 mM.
KW - biocatalysis
KW - alkane hydroxylase
KW - biomodification of hydrocarbons
KW - alkane biodegradation
UR - http://www.springerlink.com/content/b437l58n53964714/fulltext.pdf
U2 - 10.1007/s12010-011-9300-5
DO - 10.1007/s12010-011-9300-5
M3 - Article
VL - 165
SP - 823
EP - 831
JO - Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology
JF - Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology
SN - 0273-2289
IS - 3
ER -