Effect of Temperature on Coenzymes Associated with Cytochrome P450

Abstract

Cytochrome P450 are heme-containing enzymes that have been deemed to be the most active group of monooxygenases reaction.   It is a self-sufficient protein whose activities rely on the various associated coenzymes. Expression of the catalytic module of cytochrome P450 gene (CYP102A2) containing pET-28a in Escherichia coli BL21 (DE3) across temperatures ranging from 19 ºC to 37 ºC. The solubility of the expressed recombinant proteins on SDS-PAGE showed that the recombinant protein is both secreted and membrane-bound protein across all the temperature treatments. The highest expressed recombinant protein of interest (P.O.I.) band observed in lane 15 was membrane-bound and was optimum at 28 ºC, while the most prominent expressed P.O.I. bands in lane 16 from the secreted proteins was optimum at 30 ºC. The estimated flavin adenine dinucleotide (F.A.D.) and flavin mononucleotide (FMN) showed optimum concentrations of 15 mM and 14 mM respectively at the optimum temperature of 25 ºC, while the estimated heme optimum concentration (2.4 mM) was recorded at the optimum temperature of 35 ºC. These observations showed that it is little or no connection between the quantity of the expressed recombinant cytochrome P450 and the estimated coenzymes and cofactors concentrations in response to the temperature parameter. The enzymatic activity of the recombinant P.O.I. at a selected temperature of 25 ºC and 35 ºC showed exponential slope within the period of 60 seconds at 550 nm wavelength of reaction; however, a wide range of absorbance ranging from 0.1 A to 0.7 A and 0. 689 A - 0.998 A was recorded, respectively. The increase in activity may also be attributed to the fact that higher temperature tends to support an increase in the rate of reactions, hence the rate of monoxygenation.