Purification and Characterization of Membrane-Bound Quinoprotein Alcohol Dehydrogenase from a Native Strain of Acetbacter

A Pyrroloquinoline quinone alcohol dehydrogenase of a native strain of Acetobacter sp. 15 has been purified and characterized in order to its biotechnological and industrial application. Enzyme assay method is done with the potassium ferricyanide (as an electron acceptor), McIlvaine buffer 0.1 M (pH 4), Triton X-100 10%, ethyl alcohol 1 M and enzyme solution. In the presence of SDS the Enzyme was dissociated into submits with four molecular weight: 13.7, 14, 23.3 and 40 kD. At pH 6 of phosphate buffer 0.01 M, the enzyme has K sub(m) 1.75 mM for ethanol as a substrate. In this study, substrate specificity, optimum pH and effect of Ethylenediamine tetra acetic acid (EDTA), metal ions on activity of ADH have been investigated. The data have shown that ethanol (100 mM) is the best substrate for the enzyme. The optimum pH of the enzyme activity was 4.0 and the enzyme was stable in pH 6-7.5. EDTA completely inhibited ADH activity via its binding to Ca super(2+). Metal ions such as Ca super(2+) (1.0 mM) increased about 2-fold ADH activity whereas Fe super(2+), Mg super(2+) and Zn super(2+) inhibited the enzyme activity. It seems that the effect of Ca super(2+) was a result of a functional role for Ca super(2+) in the enzyme, similar to what has been observed with quinoproteins glucose and methanol dehydrogenases.