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ARS Home » Research » Publications at this Location » Publication #80929
Title: PURIFICATION AND CHARACTERIZATION OF A NOVEL THERMOSTABLE ALPHA-L-ARABINOFURANOSIDASE FROM A COLOR VARIANT STRAIN OF AUREOBASIDIUM PULLULANS

Author
item SAHA, BADAL
item BOTHAST, RODNEY

Submitted to: Journal of Applied & Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/29/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Various agricultural residues such as corn fiber, corn cobs and wheat straw contain 20-35% hemicellulose. The structure of hemicellulose is complex and contains side chains. These side chains restrict enzymatic hydrolysis. We undertook this research to develop a suitable enzyme that hydrolyzes these side chains for the subsequent production of fuel ethanol and other value-added chemicals. We found that a yeast-like fungus produced a highl thermostable enzyme that has the ability to readily hydrolyze hemicellulose and release simple sugars. The enzyme has been purified and characterized. To our knowledge, this is the first such enzyme reported to have such a high thermoactivity. The enzyme has potential for use in the production of fermentable sugars from various hemicellulosic agricultural residues such as corn fiber and corn cob and to improve animal feed digestibility by hydrolyzing a major component of animal feed.

Technical Abstract: 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 232-fold to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE Bio-Gel A agarose column chromatography, gel filtration on Bio-Gel A-0.5 m, arabinan-Sepharose 6B affinity chromatography and SP-Sephadex C-5 column chromatography. The purified enzyme had a native molecular weight of 190,000 and was composed of two equal subunits. It had a half-life of 8 h at 75 deg C and displayed optimal activity at 75 deg C and pH 4.0-4.5, and had a specific activity of 21.48 umol. min**-1. mg**-1 protein against p-nitrophenyl-alpha-L-arabinofuranoside (pNPalphaAF). The purified alpha-L-AFase readily hydrolyzed arabinan and debranched arabinan and released arabinose from arabinoxylans but was inactive against arabinogalactan. The Km values of the enzyme for the hydrolysis of pNPalphaAF, arabinan and debranched arabinan at 75 deg 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 metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), ethylenediaminetetraacetate (10 mM) or dithiothreitol (10 mM). The enzyme has potential for use in the production of fermentable sugars from various hemicellulosic agricultural residues such as corn fiber and corn cob and to improve animal feed digestibility by hydrolyzing arabinoxylans, a major component of animal feed.