Purification and biochemical characterization of an alpha-glucosidase from Xanthophyllomyces dendrorhous.
Xanthophyllomyces dendrorhous grown in different media shows amylolytic activity, consisting in an extracellular exo-acting enzyme able to hydrolysed alpha,1-4 glycosidic bonds from soluble starch, which also cleaves maltose and malto-oligosaccharides. The enzyme was purified, using basically a couple of chromatography process on DEAE-Sephacel. It is a glycoprotein with a molecular weight estimated to be 60.2 kDa based on its mobility in SDS-PAGE and 115 kDa based on gel filtration. N-linked carbohydrate accounts for 12% of the total mass. It exhibited optimum activity at pH 5.5 and 45 degrees C. Thermostability analysis indicated that it was stable to thermal treatment up to 50 degrees C; 50% of the activity was maintained after 3 h. The rate parameters measured for the hydrolysis of starch and various chain length malto-oligosaccharides shows high catalytic efficiency, calculated by the relationship V(cat)/K(m), for malto-oligosaccharides, such as maltotriose (873 mM(-1) min(-1)), or maltoheptose (698 mM(-1) min(-1)). The new enzyme hydrolysed soluble starch with nearly 3.5- and 1.4-fold lower efficiency than that for maltotriose and maltose, respectively. No activity was found on heterogeneous substrates, such as sucrose and aryl alpha-glucoside, or on isomalto-oligosaccharides. In accordance to substrate specificity profile, the new enzyme was classified as an alpha-glucosidase.
Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, Cantoblanco, 28049 Madrid, Spain.
Dolores
D
Marín
Dolores
D
Linde
María
M
Fernández Lobato
D001487:Basidiomycota / Q000201:enzymology* / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D008970:Molecular Weight; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013213:Starch / Q000378:metabolism; D000520:alpha-Glucosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D013213:Starch; D000520:alpha-Glucosidases