Colorimetric alcohol assays with alcohol oxidase

Abstract A method is described for manufacturing crude alcohol oxidase (EC 1.1.3.13) preparations which are suitable for application in colorimetric alcohol assays. The procedure involves a one-step removal of catalase activity from a partially purified preparation of alcohol oxidase from the yeast Hansenula polymorpha via dialysis against 3-amino-1,2,4-triazole and hydrogen peroxide. Thus, the irreversible inactivation of more than 90% of the catalase present was achieved, which is prerequisite for the use of alcohol oxidase preparations in colorimetric alcohol assays via peroxidase-mediated oxidation of dyes. This type of assay was shown to be rapid, accurate and sensitive. The influence of the relative concentrations of the various assay constituents such as alcohol oxidase, catalase and peroxidase is discussed. It is concluded that this colorimetric alcohol assay is particularly suitable for the determination of ethanol in fermentation broths, both in qualitative and in quantitative tests.