Regeneration of NADPH by cactus chloroplasts: Coupling reaction with P450 monooxygenase

A stable photoreaction system was developed using cactus chloroplasts coupled with the fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase expressed in yeast microsomes. The fused enzyme catalysed o-deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin using molecular oxygen and NADPH produced by photosystems I and II. Cactus chloroplast were found to constitute a good material for use as a NADPH-regenerating system in bioreactors. For NADPH formation, cactus chloroplasts showed twice as much activity as those of spinach when the same chlorophyll concentration was used at 30 degrees C, as well as being more thermostable. The optimal temperature for NADPH formation with cactus chloroplasts was 42.5 degrees C, whereas for spinach it ranged from 20 to 30 degrees C. Cactus chloroplasts can thus be applied in bioreactor NADPH-regenerating systems using oxido-reductive enzymes like P450s.