Functionalization of the cytochrome P450cam monooxygenase system in the cell-like aqueous compartments of water-in-oil emulsions.
The functionalization of the cytochrome P450cam monooxygenase system, which requires electron transfer among three different proteins, was investigated in the micro-scale aqueous compartments of stable water-in-oil (W/O) emulsions formed with the nonionic surfactant tetraethylene glycol dodecyl ether. Neither an organic-aqueous biphasic system nor a non-emulsified organic-aqueous solution containing the same amount of surfactant showed substantial hydroxylation of camphor, a natural substrate of P450cam, whereas substantial monooxygenation activity was detected when stable aqueous compartments were provided by the formation of W/O emulsions. Since the camphor hydroxylation in W/O emulsions was modest, we explored the integration of an enzymatic NADH regeneration system in order to effectively provide a reducing equivalent. Two different dehydrogenases, bacterial glycerol dehydrogenase (GLD) and yeast alcohol dehydrogenase (ADH), were selected, and each of these was coupled with the P450cam catalytic cycle in W/O emulsions. As a result, the camphor hydroxylation rate was successfully improved by approximately 5-fold when GLD was employed under optimized conditions. These results reveal the potential utility of the micro-scale cell-like aqueous compartments of W/O emulsions for multicomponent enzymatic reactions especially for substrates with low aqueous solubility.
Michizoe
Junji
J
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Ichinose
Hirofumi
H
Kamiya
Noriho
N
Maruyama
Tatsuo
T
Goto
Masahiro
M
D032701:Biomimetics / Q000379:methods*; D002468:Cell Physiological Phenomena; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004655:Emulsions / Q000737:chemistry; D004789:Enzyme Activation; D009821:Oils / Q000737:chemistry*; D014867:Water / Q000737:chemistry*
D004655:Emulsions; D009821:Oils; D014867:Water; D003577:Cytochrome P-450 Enzyme System