Characterization of a glycoside hydrolase family-51 α-L-arabinofuranosidase gene from Aureobasidium pullulans ATCC 20524 and its encoded product(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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The genomic DNA and cDNA encoding α-L-arabinofuranosidase were cloned from the dimorphic fungus Aureobasidium pullulans ATCC 20524 and sequenced. The open reading frame (2097 bp) of the α-L-arabinofuranosidase gene abfB was interrupted by five introns of 49, 49, 50, 65, and 49 bp. The gene encoded a presumed signal peptide of 17 residues and a mature protein of 682 residues with a calculated M_r of 74,230 Da and a theoretical isoelectric point of 4.95. Glu-362 and Glu-440 residues are likely involved in catalytic reactions as an acid/base and a nucleophile, respectively. The protein possessed 15 potential N-glycosylation sites. The deduced amino acid sequence of the abfB gene product was 58% identical to the Penicillium purpurogenum ABF 2, which belongs to the glycoside hydrolase family-51 α-L-arabinofuranosidase. The abfB cDNA was functionally expressed in the yeast Pichia pastoris. The recombinant enzyme, AbfB, was purified from the culture filtrate, and it appeared as a single band on SDS-PAGE with an apparent Mr of 110 kDa. AbfB showed α-L-arabinofuranosidase activity of 56.6 U/mg of protein toward p-nitrophenyl (pNP) α-L-arabinofuranoside at optimal pH 4.5 and 75℃. The enzyme exhibited apparent K_m and V_<max> values of 6.27 mM and 78.1 μmol/mg/min, respectively, for pNP α-L-arabinofuranoside. The enzyme was highly active on rye arabinoxylan as well as pNP α-L-arabinofuranoside, but it showed weak activity toward α-(1→5)-L-arabinobiose, α-(1→5)-L-arabinotriose, branched L-arabinan, linear α-(1→5)-L-arabinan, and arabinogalactan.

Characterization of a glycoside hydrolase family-51 α-L-arabinofuranosidase gene from Aureobasidium pullulans ATCC 20524 and its encoded product(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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