Purification and characterisation of D-glucose oxidase from white-rot fungus Pleurotus ostreatus.
D-Glucose oxidase was purified 27.5-fold to apparent homogeneity with an overall yield of 23.8%, from Pleurotus ostreatus, through a purification procedure of ammonium sulphate precipitation, gel-permeation, anion-exchange and hydrophobic-interaction chromatography. The molecular mass determined by gel filtration was found to be 290 kDa. SDS/PAGE revealed that the enzyme consists of four subunits with a molecular mass of 70 kDa. The absorption spectra of the enzyme exhibit maxima at 280, 360 and 460 nm. The enzyme shows a fluorescence spectrum with an excitation maximum at 470 nm and an emission maximum at 530 nm. These results indicate that the prosthetic group of the enzyme is flavin and that the enzyme contains 4 mol flavin/mol enzyme. The enzyme is optimally active at 50 degrees C and at pH 5.5-6.0. It exhibits broad affinity for various sugars and specificity for D-glucose with Km value of 1.34 mM. 2,6-Dichloroindophenol, Wurster's blue, and 4-benzoquinone can function as electron acceptors but phenazine methosulphate cannot function as an electron acceptor. The enzyme is inhibited completely by mercuric chloride and partially by silver sulphate, sodium azide 8-hydroxyquinoline.
Shin
K S
KS
Department of Microbiology, College of Sciences, Taejon University, South Korea.
Youn
H D
HD
Han
Y H
YH
Kang
S O
SO
Hah
Y C
YC
D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005949:Glucose Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011124:Polyporaceae / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D005949:Glucose Oxidase