Galactose oxidase (GalOx) is a secretory fungal enzyme with a broad substrate spectrum. The enzyme catalyzes the oxidation of primary alcohols at the C6-position to the corresponding aldehydes with concomitant reduction of molecular oxygen to hydrogen peroxide. Therefore, a two-electron redox chemistry is provided by a mononuclear copper ion active site and a tyrosine radical serving as the second redox cofactor. The copper is coordinated by four amino acid side chains: two tyrosines (Tyr272 and Tyr495) and two histidines (His496 and His581). Furthermore, Tyr272 is covalently linked at C to the sulphur atom of Cys228 under formation of a thioether bond. The genes encoding for GalOx from Fusarium oxysporum and F. sambucinum were successfully heterologously expressed both in Escherichia coli, and the F. oxysporum gene in Pichia pastoris as well. The corresponding enzymes were purified based on the His-tag, which is provided by the expression vectors, by IMAC followed by a polishing step of size-exclusion chromatography. Furthermore, the biochemical properties of the two galactose oxidases produced by E. coli were investigated. Amongst others, the pH optima and the effect of temperature (temperature optimum, thermal stability, differential scanning calorimetry) on GalOx activity were determined. Steady-state kinetic constants were measured for different electron donor substrates including D-galactose, 1-methyl--D-galactopyranoside and D-melibiose. GalOx is highly specific for molecular oxygen as an electron acceptor and no alternative acceptor could be found. The effect of various compounds (e. g. monovalent and divalent cations, nonionic detergents, EDTA, azide and cyanide) on GalOx activity was determined. The formation of the unique thioether bond in both expression systems was confirmed directly for the first time using mass spectrometry.