Activity of immobilised rat hepatic microsomal CYP2E1 using alumina membrane as a support.
Porous alumina membranes are attractive materials for the construction of biosensors and also have utility for the production of immobilised enzyme bioreactors. Microsomes from rat liver were adsorbed onto alumina membrane activated by silane. Microsomal membranes were pumped through the channels where they became immobilised by binding to amine groups on the surface of the alumina membrane. In an effort to gain a quantitative understanding of the effects of microsomal film growth on enzyme activity, we compared the para-nitrophenol (pNP) hydroxylase activity of the microsomes by varying the amount of microsomes fixed in alumina microchannels. The alumina membrane was placed in a fluidic device at a fast flow that afforded short residence time (seconds) to obtain transformation of pNP to 4-nitrocatechol (pNC), which was detected by LC-MS/MS. This enabled the use of this bioreactor where CYP2E1 activity is low and tissue sources are limiting. The microsomes, successfully immobilised on the alumina membranes, were used to produce stable biocatalytic reactors that can be used repeatedly over a period of 2 months.
Tanvir
Shazia
S
Laboratoire de Génie Enzymatique et Cellulaire, UMR 6022 CNRS, Université de Technologie de Compiègne, BP 20529-60205 Compiègne Cedex, France. tanvirsh@utc.fr
Morandat
Sandrine
S
Frederic
Nadaud
N
Adenier
Hervé
H
Pulvin
Sylviane
S
D000327:Adsorption; D000537:Aluminum Oxide / Q000737:chemistry*; D000818:Animals; D019149:Bioreactors; D002396:Catechols / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006046:Gold; D006900:Hydroxylation; D008297:Male; D008567:Membranes, Artificial; D008862:Microsomes, Liver / Q000201:enzymology* / Q000648:ultrastructure; D009596:Nitrophenols / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D002396:Catechols; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D009596:Nitrophenols; D006046:Gold; D019392:Cytochrome P-450 CYP2E1; D000537:Aluminum Oxide; C001833:4-nitrocatechol; C024836:4-nitrophenol