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Galactose Oxidase

Official Full Name
Galactose Oxidase
Galactose oxidase is an extracellular copper-containing enzyme, secreted by the deuteromycete fungus Dactylium dendroides. It catalyzes the oxidation of a range of primary alcohols, including D-galactose, to the corresponding aldehyde, with reduction of oxygen to hydrogen peroxide.
EC; D-galactose oxidase; β-galactose oxidase; 9028-79-9; Galactose Oxidase

Product Name
EC No.
CAS No.9028-79-9
EC No.
CAS No.9028-79-9
SourceDactylium dendr...
CAS No.9028-79-9
SourceDactylium dendr...
Related Services
Related Protocols
galactose_oxidase -Enzymatic Assay Protocol
Related Reading

Galactose oxidase (GO, GAO, EC is a mononuclear copper enzyme that catalyzes the two-electron oxidation of a large number of primary alcohols to their corresponding aldehydes, coupled with the reduction of dioxygen to hydrogen peroxide. The protein is a single polypeptide with molecular weight of 65-68 kDa. GO can exist in three distinct oxidation states: the highest state with Cu(II) and tyrosyl radical, the intermediate state with Cu(II) and tyrosine, and the lowest state with Cu(I) and tyrosine. The utilization of GAO is feasible in biosensors and other analytical techniques detecting galactose and galactose-containing saccharides, such as lactose. For applications in chemical synthesis, various new mono-, oligo- and polysaccharide derivatives have been prepared by the GAO-catalyzed oxidation.


Galactose Oxidase

The structure of galactose oxidase have been extensively studied. Galactose oxidase contains 639 amino acids. It has three domains with a single copper (II) ion located in a site near the surface of the β-propeller domain, close to the pseudo seven-fold axis. Domain 1 (residues 1-155) is a β-sandwich consisting of eight antiparallel β-strands. It contains a possible binding site for Na+ or Ca2+, which may serve structural roles in the protein. Another feature of Domain 1 is the presence of a carbohydrate binding site that direct the enzyme to bind to extracellular carbohydrates. Domain 2 (residues 156-552) contains the copper binding site. The β-strands in Domain 2 are organized as a seven-fold propeller, and each of the seven structural units is a subdomain consisting of four antiparallel β-strands. Domain 3 (residues 553-639) consists of seven anti-parallel β-strands and forms a “cap” over Domain 2. One histidine (His581) of Domain 3 serves as the ligand for copper, contributing to the metal-containing active site of the enzyme.

The crystal form grown in acetate buffer at pH 4.5 reveals the copper site to be square-pyramidal, with Tyr272, His496, His581 and an acetate ion as equatorial ligands and Tyr495 as the axial ligand. An unusual thioether bond exists between one of the C carbons of the Tyr272 ring and the Sg of Cys228 in the active site. An extended aromatic region is formed by the co-planar side chains of Tyr272 and Cys228 stacking with the indole ring of Trp290. In crystals grown at pH 4.5 in acetate then transferred to buffer at pH 7.0 lacking acetate, the structure of the copper site is essentially unchanged except for replacement of the acetate ion by water.

Mechanism of Action

The enzyme catalyses a two-electron redox reaction according to the general scheme: RCH2OH+O2→RCHO+H2O2. The catalytic mechanism of galactose oxidase consists of four major stages. In the first stage, the substrate is oxidized by the double redox center. The hydroxyl group is deprotonated by Tyr495 after the solvent coordination site is occupied by the hydroxyl group of substrate alcohol, followed by the release of Tyr495. This step makes the alcohol more prone to oxidation. The proton on the carbon used for the attached carbon is then transferred to Tyr272 in combination with the oxidation of the substrate. One electron goes to the copper(II) center, which is then reduced to copper(I), the other electron goes to the radical ligand. Meanwhile, Tyr272 radical is also reduced. The result of the first stage is the removal of two electrons and the removal two hydrogen atoms from the substrate. The second stage is the release of oxidized substrate and the coordination of dioxygen at the substrate coordination site. The third stage is the rapid reduction of dioxygen by copper(I) to form superoxide. The fourth stage is the deprotonation of Tyr496 by hydroperoxide, which is released as H2O2. Subsequent axial coordination of Tyr496 and equatorial coordination of new substrate molecule to the copper center completes the turnover of the enzyme.

Galactose OxidaseFigure 2. Catalytic mechanism of galactose oxidase.

It is clear that galacto aldehydes are the main products of GAO-catalyzed oxidation. However, several side products can also form. For example, following 72 hours oxidation of methyl α-D-galactopyranoside by GAO, 27% of products could not be identified. In other studies, some of the side products have been identified and characterized, such as an α,β-unsaturated aldehyde. In addition to the formation of GAO-generated side products, the high reactivity of main aldehyde products leads to the formation of hydrates and hemiacetals by reversible reactions. This phenomenon must be taken into account especially when quantifying the degree of oxidation.


Various immobilization techniques have been used in biosensor studies using GAO. For example, GAO was immobilized within a laponite clay film coated on a Pt electrode surface to construct a multipurpose amperometric sensor. With di-isothiocyanate stilbene as a bridging compound, GAO has also been attached onto monolayers of aminated thiols on Au or Ag surfaces, whereby the stilbene creates a crosslink between the amino groups of both GAO and the thiol coatings.

Galactose is not an essential nutrient for humans but it can be utilized as energy source or in the biosynthesis of some biomolecules, such as glycolipids and glycoproteins. Accordingly, a deficiency of the enzymes participating in the metabolism of galactose can lead to health problems, such as hepatosplenomegaly, bleeding disorders, Escherichia coli sepsis, and cataract. Thus galactose biosensors especially for serum samples have been developed. GAO-based biosensors have also facilitated investigations of protein glycosylation. Immobilized GAO oxidized and thus tagged galactose-terminated glycans, which could then be reduced by NaBD4 and analyzed with LC-MS or labelled with aminooxy biotin and analyzed with MS/MS. A similar approach has been used in chemiluminescent imaging of glycan expression.

Environmental applications of GAO include O2 removal, which is required for on-site monitoring of nitrate ions in water or soil by electrochemical analysis, as nitrate is consumed by nitrate reducing bacteria in anaerobic conditions. The addition of GAO, glucose oxidase, or pyranose 2-oxidase with the suitable substrates and catalase removed O2 completely from the samples in 1h.


  1. Parikka K, Master E, Tenkanen M. Oxidation with galactose oxidase: Multifunctional enzymatic catalysis [J]. Journal of Molecular Catalysis B Enzymatic, 2015, 120:47-59.
  2. Reynolds M P, Baron A J, Wilmot C M, et al. Structure and mechanism of galactose oxidase: catalytic role of tyrosine 495 [J]. Jbic Journal of Biological Inorganic Chemistry, 1997, 2(3):327-335.

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