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Appl Environ Microbiol, January 1998, p. 216-220, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Purification and Characterization of a Novel Thermostable -L-Arabinofuranosidase from a Color-Variant Strain of Aureobasidium pullulans

Badal C. Saha^* and Rodney J. Bothast

Fermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604

Received 5 August 1997/Accepted 29 October 1997

A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced -L-arabinofuranosidase (-L-AFase) when grown in liquid culture on oat spelt xylan. An extracellular -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 · min¹ · mg¹ of protein against p-nitrophenyl--L-arabinofuranoside (pNPAF). The purified -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 pNPAF, 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 -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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^* Corresponding author. Mailing address: USDA-ARS-NCAUR-FBR, 1815 N. University St., Peoria, IL 61604. Phone: (309) 681-6276. Fax: (309) 681-6686. E-mail: sahabc{at}mail.ncaur.usda.gov.

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