半乳糖氧化酶 来源于树状指孢霉

半乳糖氧化酶可作为一种分析工具，用于特异性测定血浆、植物提取物和磷脂中的 D-半乳糖。它可用于表征几种聚合物中的末端 D-半乳糖苷单元。它也可用于测定乳糖含量。 来自树状指孢霉的半乳糖氧化酶已用作阿拉伯半乳聚糖的半乳糖氧化酶处理的组分。它也已用于与过氧化物酶共固定化，以制备用于半乳糖检测的生物传感器。 Galactose oxidase oxidizes galactose and some galactose derivatives in both free and polymeric forms. Oxidation occurs at the C6 position. The enzyme has a molecular weight of 68 ± 3 kDa, and the optimum pH is 7.0. Useful in the determination of lactose. Application Galactose Oxidase from Dactylium dendroides has been used as a component for galactose oxidase treatment of arabinogalactan. It has also been used to co-immobilise with peroxidase for the preparation of a biosensor for galactose detection. Galactose oxidase may be used as an analytical tool for the specific determination of D-galactose in blood plasma, plant extracts, and phospholipids. It could be used for the characterization of terminal D-galactoside units in several polymers. It may also be useful in the determination of lactose. 1、Specificity ：GAO has a wide substrate specificity, but remarkable stereospecificity, only oxidizing D-isomers of substrates (McPherson et al. 1992). GAO will oxidize galactose and some galactose derivatives in both free and polymeric form. Oxidation occurs at the C6 position. 2、Composition：GAO contains one Cu(II) atom yet catalyzes a two-electron transfer reaction (McPherson et al. 1992). The copper is bound by two tyrosines, and two histidines (Tyr272, Tyr495, His496, and His581). In a novel post-translational covalent modification, Tyr272 is linked by a thioether bond to cysteine (Cys228), suggesting the involvement of a tyrosine radical in the catalytic mechanism. Stabilization of the radical occurs because Tyr272 of the thioether bond is liganded to the copper, creating a stacking interaction with Trp290 (Whittaker et al. 1989, Ito et al. 1991, and Whittaker et al. 2005). The structure of the enzyme has revealed extensive beta-sheet secondary structure, consistent with the high stability of the enzyme (Kosman et al. 1974). Most extracellular proteins of eukaryotes are modified by glycosylation during passage through the ER and golgi, leading to greater glycosylation of extracellular than intracellular forms of a protein. Unusually, the intracellular form of GAO is more highly glycosylated (9% carbohydrate) and exhibits greater stability than the extracellular form (2% carbohydrate) (Medonca and Zancan 1988). Additionally, most proteins are modified by O- and/or N-glycosylation while GAO is only modified only by O-glycosylation (Kornfield and Kornfield 1985, and McPherson et al. 1992) 3、Molecular Characteristics：The gaoA gene contains a long open reading frame from +324 to +2507, including the mature protein-coding sequence (+521 to +2507). It also contains a long untranslated upstream region and a putative pro-sequence with a monobasic cleavage site (McPherson et al. 1992). 4、Characteristics of Galactose Oxidase： Protein Accession Number：P0CS93； Isoelectric point：7.75 (Theoretical) CATH Classification：Three domains: Class: Mainly Beta Architecture: Sandwich, 7 Propellor Topology: Jelly Rolls, Methylamind Dehydrogenase; Chain H, Immunoglobulin-like Molecular Weight 68.5 kDa (calculated from translated DNA sequence and SDS-polyacrylamide gel electrophoresis, McPherson et al. 1992) 68.0 ± 3.0 kDa (determined from physical measurements, Cooper et al. 1959) Optimal pH：7.0 (Cooper et al. 1959) Extinction Coefficient： 122,480 cm-1M-1 (Theoretical) E1%, 280 = 17.87 (Theoretical) Inhibitors Cyanide Diethyldithiocarbamate Azide Hydroxylamine EDTA Applications Quantitative determination of galactose in blood and other biological fluids (Frings and Pardue 1964, Hankin 1966, and Roth et al. 1965) Locating galactose histochemically (Roberts and Gupta 1965) Detecting and distinguishing glycoproteins (Itaya et al. 1975) 5、Galactose Oxidase Assay：Method The reaction velocity is measured in a peroxidase/o-tolidine coupled system as an increase in A425 resulting from the oxidation of galactose. One unit results in a change in A425 of 1.0 per minute at 25°C and pH 6.0 under the defined conditions. Reagents 0.1 M Potassium phosphate buffer, pH 6.0 0.5% o-tolidine. Note: o-tolidine has been reported to be carcinogenic. Handle with care. Peroxidase. Dissolve Worthington peroxidase (Code: HPOD) at a concentration of approximately 60 u/ml in reagent grade water. 10% galactose. Allow to come to equilibrium of mutarotation by allowing to stand overnight. Enzyme Dissolve at a concentration of 1 mg/ml in reagent grade water. Dilute further for assay to a concentration of 0.2 - 0.5 units/ml. Procedure Adjust spectrophotometer to 425 nm and 25°C. Prepare tolidine-buffer mixture by adding 0.1 ml tolidine to 12 ml 0.1 M potassium phosphate buffer pH 6.0. Pipette into each cuvette as follows: Tolidine-buffer solution 1.7 ml 10% Galactose 1.5 ml Peroxidase 0.1 ml Incubate in spectrophotometer at 25°C for 3 - 4 mintues to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml of appropriately diluted enzyme and record increase in A425/min. from initial linear portion of the curve.

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