Characterization of pyranose dehydrogenase from Agaricus meleagris and its application in the C-2 specific conversion of D-galactose.
Pyranose dehydrogenase (PDH) of the mushroom Agaricus meleagris was purified from mycelial culture media to substantial homogeneity using ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric polypeptide with a molecular mass of 66,547Da as determined by matrix-assisted laser desorption/ionisation mass spectrometry containing approximately 7% carbohydrate and covalently bound flavin adenine dinucleotide. The enzyme exhibited a broad sugar substrate tolerance, oxidizing different aldopyranoses to the corresponding C-2 or C-2,3 (di)dehydro sugars. Preferred electron donors with the highest k(cat)/K(m) values were major sugar constituents of cellulose and hemicellulose, namely d-glucose, D-galactose, l-arabinose, D-xylose and cellobiose. This indicates a possible physiological role of the enzyme in lignocellulose breakdown. PDH showed no detectable activity with oxygen, and its reactivity towards electron acceptors was limited to various substituted benzoquinones and complexed metal ions, with the ferricenium ion and the benzoquinone imine 2,6-dichloroindophenole displaying the highest k(cat)/K(m). The enzyme catalyzed in up to 95% yields the regiospecific conversion of D-galactose to 2-dehydro-D-galactose, an intermediate in a possible biotechnologically interesting process for redox isomerization of D-galactose to the prebiotic sugar D-tagatose.
Sygmund
Christoph
C
Division of Food Biotechnology, Department of Food Sciences and Technology, BOKU - University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria.
Kittl
Roman
R
Volc
Jindrich
J
Halada
Petr
P
Kubátová
Elena
E
Haltrich
Dietmar
D
Peterbauer
Clemens K
CK
D000363:Agaricales / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature
D002237:Carbohydrate Dehydrogenases; D005947:Glucose; D005690:Galactose