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A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.

The present study reports the successful and effective degradation of the persistent herbicide dymron catalyzed by the oxidative enzyme laccase in the presence of a reaction mediator (a laccase/mediator system). Using 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the mediator, over 90% of dymron was degraded within 24 h, while the half-life of dymron is 50 days in soil. The results suggested that oxidation of dymron resulted in the production of decomposed compounds with a single aromatic ring. We also found that edible surfactants and a dishwashing detergent were useful to solubilize dymron in an aqueous solution and did not inhibit the oxidative degradation. Degradation proceeded at acidic pH and in a broad range of temperatures (303-353 K). The use of natural mediators also allowed the oxidative degradation of dymron to some extent. In conclusion, we propose the possible use of a laccase/mediator system for the treatment of soils and drainwater contaminated with herbicides.