Purification and characterization of a novel thermostable α-L- arabinofuranosidase from a color-variant strain of Aureobasidium pullulans

Abstract

A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced α-L-arabinofuranosidase (α-L-AFase) when grown in liquid culture on oat spelt xylan. An extracellular α-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°C, displayed optimal activity at 75°C and pH 4.0 to 4.5, and had a specific activity of 21.48 μmol · min-1 · mg-1 of protein against p- nitrophenyl-α-L-arabinofuranoside (pNPαAF). The purified α-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 pNPαAF, arabinan, and debranched arabinan at 75°C and pH 4.5 were 0.26 mM, 2.14 mg/ml, and 3.25 mg/ml, respectively. The α-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).