Immobilization of P450 monooxygenase and chloroplast for use in light-driven bioreactors

P450 monooxygenases exhibit great potential for application to bioreactors for the decomposition of various hydrophobic chemicals including pollutant compounds. P450-containing microsomes were immobilized in spinach chloroplasts for use in light-driven bioreactors. We tested three methods (entrapment, adsorption and cross-linking) to immobilize chloroplasts and yeast microsomes containing a genetically engineered fusion enzyme between rat P450 1A1 and yeast P450 reductase. Entrapment in agarose gave the best activity for the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin under illumination of 6200 lx. We then tested three light-driven bioreactors (two column-type and one batch-type reactors developed) using the immobilized gels. A two-phase column-type reactor with separately immobilized microsomes and chloroplasts showed a higher conversion rate than a reactor with co-immobilization of both components. The reactor showed a turnover rate of 6.32 mol product/mol P450/min after a 40-min run, and 2.49 after a 180-min run. These turnover rates are higher than the values reported by others using immobilized microsomal P450s.