Abstract
A glycosyl hydrolase family 54 (GH54) α-l-arabinofuranosidase gene (abfA) of Aureobasidium pullulans was amplified by polymerase chain reaction from genomic DNA and a 498-amino-acid open reading frame deduced from the DNA sequence. Modeling of the highly conserved A. pullulans AbfA protein sequence on the crystal structure of Aspergillus kawachii AkabfB showed that the catalytic amino acid arrangement and overall structure were highly similar including the N-terminal catalytic and C-terminal arabinose binding domains. The abfA gene was expressed in Saccharomyces cerevisiae, and the heterologous enzyme was purified. The protein was monomeric, migrating at 49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and eluting at 36 kDa upon gel filtration. AbfA showed maximal activity at 55°C and between pH 3.5 and pH 4. The enzyme had a K m value for p-nitrophenyl-α-l-arabinofuranoside of 3.7 mM and a V max of 34.8 μmol min−1 mg protein−1. Arabinose acted as a noncompetitive inhibitor with a K i of 38.4 mM. The enzyme released arabinose from maize fiber, oat spelt arabinoxylan, and wheat arabinoxylan, but not from larch wood arabinogalactan or α-1,5-debranched arabinan. AbfA displayed low activity against α-1,5-l-arabino-oligosaccharides. The enzyme acted synergistically with endo-β-1,4-xylanase in the breakdown of wheat arabinoxylan. Binding of AbfA to xylan from several sources confirmed the presence of a functional carbohydrate-binding module.
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Acknowledgment
We thank the National Research Foundation for financial support. The assistance of Marc Claeyssens is gratefully acknowledged with the protein modeling. Excellent technical assistance was provided by Annatjie Hugo and Yolanda Engelbrecht.
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Fig. S1
Computer model of the three-dimensional structure of AbfA. a Overlay of the AbfA model (blue) and the solved crystal structure of AkabfB (green). b Molecular surface of AbfA, with the surface colored blue for positive and red for negative according to the electrostatic potential. c Ribbon structure of AbfA, with the four disulfide bridges indicated in yellow. d Topological diagram of the catalytic domains active pocket. The side chains of the amino acid residues, Cys178, Cys179, Met197, Trp208, Asp221, Glu223, Asn224, Leu226, and Asp299, which are important for catalysis, are indicated (GIF 142 kb)
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de Wet, B.J.M., Matthew, M.K.A., Storbeck, KH. et al. Characterization of a family 54 α-l-arabinofuranosidase from Aureobasidium pullulans . Appl Microbiol Biotechnol 77, 975–983 (2008). https://doi.org/10.1007/s00253-007-1235-y
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DOI: https://doi.org/10.1007/s00253-007-1235-y