title: The use of immobilized cytochrome P4502C9 in PMMA-based plug flow bioreactors for the production of drug metabolites.
abstract: Cytochrome P450 enzymes play a key role in the metabolism of pharmaceutical agents. To determine metabolite toxicity, it is necessary to obtain P450 metabolites from various pharmaceutical agents. Here, we describe a bioreactor that is made by immobilizing cytochrome P450 2C9 (CYP2C9) to a poly(methyl methacrylate) surface and, as an alternative to traditional chemical synthesis, can be used to biosynthesize P450 metabolites in a plug flow bioreactor. As part of the development of the CYP2C9 bioreactor, we have studied two different methods of attachment: (1) coupling via the N-terminus using N-hydroxysulfosuccinimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and (2) using the Ni(II) chelator 1-acetato-4-benzyl-triazacyclononane to coordinate the enzyme to the surface using a C-terminal histidine tag. Additionally, the propensity for metabolite production of the CYP2C9 proof-of-concept bioreactors as a function of enzyme attachment conditions (e.g., time and enzyme concentration) was examined. Our results show that the immobilization of CYP2C9 enzymes to a PMMA surface represents a viable and alternative approach to the preparation of CYP2C9 metabolites for toxicity testing. Furthermore, the basic approach can be adapted to any cytochrome P450 enzyme and in a high-throughput, automated process.
authors.affiliation: Basic Pharmaceutical Sciences, West Virginia University, 1 Medical Center Drive, P.O. Box 9530, Morgantown, WV, 26506, USA.
authors.forename: Lance A
authors.initials: LA
authors.lastname: Wollenberg
authors.forename: Jarod L
authors.initials: JL
authors.lastname: Kabulski
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Powell
authors.forename: Jifeng
authors.initials: J
authors.lastname: Chen
authors.forename: Darcy R
authors.initials: DR
authors.lastname: Flora
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Tracy
authors.forename: Peter M
authors.initials: PM
authors.lastname: Gannett
mesh_terms: D019149:Bioreactors; D065729:Cytochrome P-450 CYP2C9 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D008658:Inactivation, Metabolic / Q000235:genetics; D019904:Polymethyl Methacrylate / Q000737:chemistry / Q000378:metabolism
chemical_list: D055767:Immobilized Proteins; D019904:Polymethyl Methacrylate; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9