Introduction
Galactose oxidase (GalOx1; d-galactose:oxygen 6-oxidoreductase, EC 1.1.3.9) is a monomeric 68-kDa enzyme that contains a single copper ion [1] and an amino acid-derived cofactor [2] and [3], formed by cross-linking of a Cys and a Tyr residue in the direct vicinity of the copper [4], [5] and [6]. The thioether bond of the Tyr-Cys cross-link is post-translationally generated [4] and [7] and has been shown to affect the stability, the reduction potential [8] and the catalytic efficiency of the enzyme [9] and [10]. It has been classified as a member of the carbohydrate active-enzyme family AA5, subfamily 2 [11]. GalOx catalyzes the two-electron oxidation [3] and [12] of the C6-hydroxyl group of nonreducing d-galactose residues [13] as well as a range of primary alcohols to the corresponding aldehydes with concomitant reduction of oxygen to hydrogen peroxide [14], [15], [16] and [17]. During catalysis both the metal ion and the cysteine-modified tyrosine group undergo 1-electron redox interconversions [18]. Despite a wide substrate specificity, GalOx is strictly regioselective and no secondary alcohols are oxidized [19]. However, the enzyme accepts a wide variety of primary alcohols such as benzyl alcohol [20], and glycerol [21] as reducing substrates. GalOx displays remarkable stereospecificity in its reaction with sugars [22], being highly sensitive for the orientation of the C4-OH group, and hence it shows activity with galactose but not with glucose. Because of this specificity, various analytical techniques are based on GalOx, such as the determination of lactose in milk and dairy products [23] or the histochemical examination of mucus-secreting cells [24]. Furthermore, GalOx has been used in biosensors for the measurement of galactose and its derivatives in biological fluids [25], to label galactose residues in glycoconjugates [26], and for the induction of interferon in human lymphocyte culture [27] and [28]. GalOx is viewed as a competitive and cost-effective catalyst compared to chemical conversion for the manufacturing of fine chemicals for pharmaceutical purposes or in food industry, for example GalOx was used for conversion of sugars like d-galactose to food-grade cross-linking agents [29], [30], [31] and [32]. Another important application for GalOx is the modification of cell surface carbohydrates and has been used in cell labeling studies and histochemical staining [19]. GalOx is interesting for the use in industrial processes such as derivatization of guar gum and related polymers as well [33] and [34].
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