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

摘要

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 the consumption 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 activity at pH 8.0 that is unusual for fungal laccases. The maximal activity of TaL was observed at pH 5.5. A kinetic mechanism for the laccase-catalyzed syringaldazine oxidation and disproportionation of reaction products as well as their reaction with NAD( P)H was suggested. Since TaL was slightly more reactive with syringaldazine than DsL, the calculated constants of enzymatic reaction were different for the investigated laccases. The constant of NAD(P)H oxidation by oxidized syringaldazine (tetramethoxy azobismethylene quinine, TMAMQ) is 1.0 × 10~4 M~(-1) s~(-1), which was sufficient for effective NAD(P)H oxidation. The production of an enzymatically active NAD~+ was proved by coupling the laccase/mediator system with alcohol oxidation catalyzed by yeast alcohol dehydrogenase.