Alcohol Oxidase

Description

Alcohol oxidase (AOX, EC 1.1.3.13) is a flavoenzyme, an oxidoreductase that catalyzes the oxidation of alcohol to the corresponding carbonyl compound and simultaneously releases hydrogen peroxide.
Alcohol oxidase catalyzes the oxidation of short-chain, primary, aliphatic alcohols to the respective aldehydes.
RCH2OH + O2 -->RCHO + H2O2
The enzyme has the highest affinity for methanol with the affinity decreasing with increasing chain length of the alkyl (R) group.
Alcohol oxidase plays a major role in the metabolism of methanol resulting in the formation of formaldehyde and has been detected in several genera of yeasts, such as Candida, Pichia, and Hansenula, that utilize methanol as a sole carbon and energy source.
Primarily localized in the peroxisome, alcohol oxidase has also been found in the cytoplasm. Monomers are synthesized in the cytosol and assembled into octomers in the peroxisome. Octomerization is thought to be chaparone mediated. Alcohol oxidase is of interest for the study of protein translocation into peroxisomes.

Chemical Properties

Specificity: Alcohol Oxidase is specific for short-chain, linear aliphatic alcohols and oxidizes methanol and ethanol. The following are oxidized at decreasing velocity rate: methanol = ethanol, npropanol, n-butanol. It also catalyzes the oxidation of allyl and propargyl alcohol, methyl and ethyl mercaptan and fromaldehyde at slower rates. Branched chain alcohols, C2-aldehydes or higher, ketones and organic acids are not substrates. However, some of these reagents can give false positive responses due to trace contamination with alcohol. Oxygen is the only known hydrogen acceptor. At air saturation, the apparent KM for alcohol oxidase is 0.7 mM using methanol and 9 mM using ethanol as the substrate. In oxygen saturated solutions the KM values are higher. The turnover number is 20 000 sec-1 .
The optimum temperature range is 40 – 45oC. The pH optimum is 7.2 with a working range of 5.5 – 9.5.
Inactivation: freezing does not inactivate the enzyme. Compared to other oxidases, it is relatively resistant to p-chloromercuribenzoate, heavy metals and hydrogen peroxide. Hydrogen peroxide does not inactivate the enzyme at concentrations many times higher than those reported to inactivate previously-described yeast alcohol oxidases (Hansenula, Candida).
Solubility of the enzyme is inversely related to temperature; refrigeration will often reverse cloudiness in solutions where precipitation is occurring. The enzyme is freely soluble in greater than 0.1 M Phosphate buffer above pH 7.0. The enzyme reversibly precipitates at below 0.05 M Phosphate and below pH 7.0. These boundaries can be extended under certain pH, temperature and ionic conditions. Alcohol Oxidase has a molecular weight of approximately 630 000 and is composed of eight subunits, each having one molecule of bound azide. Inhibitors include azide, Cu++, Ag+ , Hg++ , p-chloromercuribenzoate, hydroxylamine and NaF.

Uses

Alcohol Oxidase may be used to study protein translocation into peroxisomes. Product A2404 is from Pichia pastoris. It has been used for the bacterial expression and immunological verification of Hv-p68 cDNA clones .

Uses

Alcohol oxidase is used to catalyze the oxidation of short-chain, primary, aliphatic alcohols to their respective aldehydes . It may be used to study methanol metabolism is yeasts, such as Candida, Pichia, and Hansenula. It is useful to study protein translocation into peroxisomes. Product A6941 is from Candida boidinii and is provided as a lyophilized powder.

Application

Alcohol Oxidase from Hansenula sp. has been used:
in the immobilization on amperometric biosensor by glutaraldehyde cross linking
as a component in pre-mixed hydrogels for the electrode preparation in the biosensors
in the modification of screen printed Co-phthalocyanine/ Carbon electrode for alcohol biosensor preparation

Reactions

In enzymology, an alcohol oxidase (EC 1.1.3.13) is an enzyme that catalyzes the chemical reaction a primary alcohol + O2 ? an aldehyde + H2O2. Thus, the two substrates of this enzyme are primary alcohol and O2, whereas its two products are aldehyde and H2O2. This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with oxygen as acceptor.

General Description

Alcohol oxidase (AOX) is a homo-octamer, which has a molecular weight of 600-kDa. It has eight flavin adenine dinucleotide (FAD) cofactors.It belongs to the glucose-methanol-choline (GMC) oxidoreductase family.

Biochem/physiol Actions

Alcohol oxidase has the highest affinity for methanol. The affinity decreases with increasing chain length of the alkyl (R) group. Alcohol oxidase is primarily localized in the peroxisome but is also found in the cytoplasm .