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.
authors.affiliation: Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan. k.ohta@cc.miyazaki-u.ac.jp
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Ohta
authors.forename: Shinya
authors.initials: S
authors.lastname: Fujii
authors.forename: Chihiro
authors.initials: C
authors.lastname: Higashida
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005685:Galactans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007438:Introns / Q000235:genetics; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology / Q000235:genetics; D010802:Phylogeny; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D005685:Galactans; D011134:Polysaccharides; D011994:Recombinant Proteins; D014990:Xylans; C030080:araban; C085118:arabinoxylan; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase; C005653:arabinogalactan
keywords: Aureobasidium pullulans; DIG; GH; Gene expression; Glycoside hydrolase family-51; ORF; Pichia pastoris; digoxigenin; glycoside hydrolase; nt; nucleotide(s); open reading frame; p-nitrophenyl; pNP; α-l-Arabinofuranosidase