64] Primary and secondary alcohol dehydrogenases from Gluconobacter

Abstract

This chapter describes the purification and the properties of two soluble nicotinamide adenine dinucleotide (NAD)-linked primary and secondary alcohol dehydrogenases and two particulate primary and secondary alcohol dehydrogenases from Gluconobacter oxydans (suboxydans), strain SU (NCIB 9108). In the case of soluble NAD-linked primary and secondary alcohol dehydrogenases, the assay is based on the rate of reduction of NAD at 340 mμ in the presence of an alcohol. In the course of assay of two particulate primary and secondary alcohol dehydrogenases, the particles contain the complete electron transport chain. The oxidation of alcohols and glycols by the particulate enzymes can thus be followed by measuring the rate of oxygen uptake in the Warburg respirometer. Once solubilized, the electron transport to oxygen is broken. Therefore the oxidation of primary and secondary alcohols is routinely determined spectrophotometrically by the reduction of 2,6-diehlorophenolindophenol. In spite of the disadvantages inherent to the use of this electron acceptor, the assay was found to be reproducible for routine determinations in following the purification of the enzymes.