Development of a laccase/syringaldazine system for NAD(P)H oxidation

Abstract

The kinetics of laccase-catalyzed NAD(P)H oxidation in the presence of syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine) was investigated spectrophotometrically and by measuring theconsumption of oxygen. Laccases from Didymocrea sp. (DsL) and Trichaptum abietinum (TaL) were used.These enzymes showed different pH profiles towards syringaldazine. DsL showed a maximum activityat pH 8.0 that is unusual for fungal laccases. The maximal activity of TaL was observed at pH 5.5. Akinetic mechanism for the laccase-catalyzed syringaldazine oxidation and disproportionation of reactionproducts as well as their reaction with NAD(P)H was suggested. Since TaL was slightly more reactive withsyringaldazine than DsL, the calculated constants of enzymatic reaction were different for the investigatedlaccases. The constant of NAD(P)H oxidation by oxidized syringaldazine (tetramethoxy azobismethylenequinine, TMAMQ) is 1.0 × 104M−1s−1, which was sufficient for effective NAD(P)H oxidation. The produc-tion of an enzymatically active NAD+was proved by coupling the laccase/mediator system with alcoholoxidation catalyzed by yeast alcohol dehydrogenase.