1-(N,N`-DIMETHYLAMINE)-4-(4-MORPHOLINE)BENZENE as electron transfer mediator in oxidoreductases‘ catalysed reactions

Abstract

This paper focuses on the investigation of the reactivity of mediator 1-(N,N`-dimethylamine)-4-(4-morpholine)benzene (AMB) with fungal laccases, PQQ-dependent dehydrogenases and NADH by spectrophotometric and electrochemic methods. The redox potential (E) of the mediator AMB was determined from cyclic voltamperograms at variuos potential scan rate and was equal to 157±1 mV vs SCE at buffer solution pH 7.0. NADH and AMB+. reactivity was investigated by registering the increase of catalytic current of AMB oxidation. Bimolecular constant of NADH and AMB+. reactivity is equal to (7.4±0.1)×102 M-1s-1. AMB oxidation kinetics was investigated spectrofotometically by registering the increase of AMB oxidation product – AMB radical cation absorbtion at 604 nm of wavelength. The bimolecular AMB and laccase reactivity constant kox was calculated as ratio of kcat and KM. The values of kox for various laccases are in the interval from (9.7±1.6)×103 M−1s−1 (Didymocrea sp. laccase with AMB at pH 7.0) to (5.1±0.9)×105 M−1s−1 (Ulocladium botrytis laccase with AMB at pH 7.0). The reactivity of the oxidized form of AMB (AMB+.) with PQQ-dependent dehydrogenases was investigated by monitoring the synergetic substrate ferricyanide reduction rate dependence on AMB concentration. The values of bimolecular reactivity constants kred are (1.7±0.2)×105 M-1s-1 for AMB+. reactivity with PQQ-dependent alcohol dehydrogenase from Pseudomonas putida (ADH IIG) at pH 7.0 and (1.9±0.3)×106 M-1s-1 for reactivity of PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus with AMB+. at pH 7.0 .