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) produ ced alpha-L-arabinofuranosidase (alpha-L-AFase) when grown in liquid c ulture on oat spelt xylan. An extracellular alpha-L-AFase nas purified 215-fold to homogeneity front the culture supernatant by ammonium sul fate treatment, DEAE Bio-Gel A agarose column chromatography, gel filt ration on a Bio-Gel A-0.5m column, arabinan-Sepharose 6B affinity chro matography, and SP-Sephadex C-50 column chromatography. The purified e nzyme 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 mu mol . min(-1) . mg(-1) of protein against p-nitr ophenyl-alpha-L-arabinofuranoside (pNP alpha AF). The purified alpha-L -AFase readily hydrolyzed arabinan acid debranched arabinan and releas ed arabinose from arabinoxylans but was inactive against arabinogalact an. The K-m values of the enzyme for the hydrolysis of pNP alpha AF, a rabinan, 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 activi ty 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 b y p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (1 0 mM).