Purification and Characterization of Membrane-bound Aldehyde Dehydrogenase fromGluconobacter suboxydans

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Abstract
Membrane-bound aldehyde dehydrogenase was purified to almost homogeneous state from membrane fraction of Gluconobacter suboxydans IFO 12528 by a procedure involving solubilization of the enzyme by Triton X-100 and subsequent fractionation on DEAE-cellulose and CM-cellulose. A c-type cytochrome was tightly bound to the dehydrogenase protein and existed as an enzyme-cytochrome complex. Molecular weight of the enzyme was determined to be about 140,000, and SDS gel electrophoresis showed the presence of two subunits having a molecular weight of 86,000 and 55,000. Aliphatic aldehydes except formaldehyde were oxidized very rapidly in the presence of dyes, such as 2, 6-dichlorophenolindophenol, phenazine methosulfate or ferricyanide, but NAD, NADP or oxygen were not available as an electron acceptor. Optimum pH of aldehyde oxidation was observed at 4.0. The enzyme was stable at pH 5–6 and stability of the enzyme was much enhanced by the coexistence of sucrose and benzaldehyde. An apparent Michaelis constant for acetaldehyde was observed to be 0.4 mm with membrane particle and 3.3 mm with the solubilized enzyme. Evidence that the membrane-bound aldehyde dehydrogenase could be available for microdetermination of acetaldehyde was also presented. A trace amount of acetaldehyde was satisfactorily assayed.