Purification and characterization of a novel thermostable alpha-L-arabinofuranosidase from a color-variant strain of Aureobasidium pullulans.
A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced alpha-L-arabinofuranosidase (alpha-L-AFase) when grown in liquid culture on oat spelt xylan. An extracellular alpha-L-AFase was purified 215-fold to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE Bio-Gel A agarose column chromatography, gel filtration on a Bio-Gel A-0.5m column, arabinan-Sepharose 6B affinity chromatography, and SP-Sephadex C-50 column chromatography. The purified enzyme had a native molecular weight of 210,000 and was composed of two equal subunits. It had a half-life of 8 h at 75 degrees C, displayed optimal activity at 75 degrees C and pH 4.0 to 4.5, and had a specific activity of 21.48 mumol min-1. mg-1 of protein against p-nitrophenyl-alpha-L-arabinofuranoside (pNP alpha AF). The purified alpha-L-AFase readily hydrolyzed arabinan and debranched arabinan and released arabinose from arabinoxylans but was inactive against arabinogalactan. The K(m) values of the enzyme for the hydrolysis of pNP alpha AF, arabinan, and debranched arabinan at 75 degrees C and pH 4.5 were 0.26 mM, 2.14 mg/ml, and 3.25 mg/ml, respectively. The alpha-L-AFase activity was not inhibited at all by L-arabinose (1.2 M). The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM).
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Fermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Peoria, Illinois 61604, USA. sahabc@mail.ncaur.usda.gov
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D000645:Ammonium Sulfate / Q000494:pharmacology; D001089:Arabinose / Q000031:analogs & derivatives / Q000378:metabolism; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003470:Culture Media / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005685:Galactans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D003470:Culture Media; D005685:Galactans; D011134:Polysaccharides; D014990:Xylans; C030080:araban; D020245:p-Chloromercuribenzoic Acid; C017567:4-nitrophenyl-alpha-L-arabinofuranoside; C085118:arabinoxylan; D004492:Edetic Acid; D001089:Arabinose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase; C005653:arabinogalactan; D000645:Ammonium Sulfate; D004229:Dithiothreitol