Purification and characterization of a novel alpha-L-arabinofuranosidase from Pichia capsulata X91.
An intracellular alpha-L-arabinofuranosidase from Pichia capsulata X91 was purified and characterized. The enzyme was purified to homogeneity from a cell-free extract by ammonium sulfate treatment, Concanavalin A-Sepharose, ion-exchange chromatography with DEAE Bio-Gel A agarose, arabinose-Sepharose 6B affinity chromatography, and hydroxyapatite column chromatography. The apparent molecular mass of the enzyme was estimated to be 250 kDa by native-PAGE. The enzyme molecule was suggested to be a tetramer with a subunit molecular mass of 72 kDa by SDS-PAGE. The enzyme had an isoelectric point at 5.1, and was most active at pH 6.0 and at around 50 degrees C. The alpha-L-arabinofuranosidase was active at ethanol concentrations of wine. The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate. The enzyme hydrolyzed beet arabinan and arabinogalactan, and efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice. A considerable amount of monoterpenols was produced in the Muscat wine coupled with the enzyme addition.
Yanai
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Mercian Corporation, Wine & Spirits Research Institute, Fujisawa, Japan.
Sato
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D004795:Enzyme Stability; D005524:Food Technology; D005685:Galactans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity; D013696:Temperature; D014920:Wine
D005685:Galactans; D011134:Polysaccharides; C030080:araban; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase; C005653:arabinogalactan