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Characterization of a glycoside hydrolase family-51 α-l-arabinofuranosidase gene from Aureobasidium pullulans ATCC 20524 and its encoded product

Paper ID Volume ID Publish Year Pages File Format Full-Text
20757 43190 2013 6 PDF Available
Title
Characterization of a glycoside hydrolase family-51 α-l-arabinofuranosidase gene from Aureobasidium pullulans ATCC 20524 and its encoded product
Abstract

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 Mr 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°C. The enzyme exhibited apparent Km and Vmax 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.

Keywords
α-l-Arabinofuranosidase; Aureobasidium pullulans; Glycoside hydrolase family-51; Gene expression; Pichia pastorisDIG, digoxigenin; GH, glycoside hydrolase; nt, nucleotide(s); ORF, open reading frame; pNP, p-nitrophenyl
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Characterization of a glycoside hydrolase family-51 α-l-arabinofuranosidase gene from Aureobasidium pullulans ATCC 20524 and its encoded product
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 116, Issue 3, September 2013, Pages 287–292
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us