Immobilization of a mediator onto carbon cloth electrode and employment of the modified electrode to an electroenzymatic bioreactor.

5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) was selected as an electron transfer mediator and was covalently immobilized onto high porosity carbon cloth to employ as a working electrode in an electrochemical NAD(+)-regeneration process, which was coupled to an enzymatic reaction. The voltammetric behavior of DTNB attached to carbon cloth resembled that of DTNB in buffered aqueous solution, and the electrocatalytic anodic current grew continuously upon addition of NADH at different concentrations, indicating that DTNB is immobilized to carbon cloth effectively and the immobilized DTNB is active as a soluble one. The bioelectrocatalytic NAD+ regeneration was coupled to the conversion of L-glutamate into alpha-ketoglutarate by L-glutamate dehydrogenase within the same microreactor. The conversion at 3 mM monosodium glutamate was very rapid, up to 12 h, to result in 90%, and then slow up to 24 h, showing 94%, followed by slight decrease. Low conversion was shown when substrate concentration exceeding 4 mM was tested, suggesting that L-glutamate dehydrogenase is inhibited by alpha-ketoglutarate. However, our electrochemical NAD+ regeneration procedure looks advantageous over the enzymatic procedure using NADH oxidase, from the viewpoint of reaction time to completion.