Characterization of a family 54 α-L-arabinofuranosidase from Aureobasidium pullulans
Date
2008
Authors
De Wet B.J.M.
Matthew M.K.A.
Storbeck K.-H.
Van Zyl W.H.
Prior B.A.
Journal Title
Journal ISSN
Volume Title
Publisher
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. © 2007 Springer-Verlag.
Description
Keywords
Amino acids, Carbohydrates, DNA sequences, Enzyme activity, Genes, Proteins, Aureobasidium pullulans, Polymerase chain reaction, Plants (botany), alpha arabinofuranosidase, amino acid, arabinose, arabinoxylan, carbohydrate, fungal protein, genomic DNA, xylan endo 1,3 beta xylosidase, amino acid, carbohydrate, cereal, DNA, electrokinesis, enzyme activity, gene expression, inhibitor, modeling, polymerase chain reaction, protein, yeast, article, Aureobasidium pullulans, catalysis, crystal structure, enzyme purification, gel filtration, maize, nonhuman, oat, open reading frame, polyacrylamide gel electrophoresis, polymerase chain reaction, temperature, Arabinose, Ascomycota, Aspergillus, Binding Sites, Catalytic Domain, Chromatography, Gel, Cloning, Molecular, Computational Biology, DNA, Fungal, Electrophoresis, Polyacrylamide Gel, Enzyme Inhibitors, Enzyme Stability, Galactans, Gene Expression, Glycoside Hydrolases, Hydrogen-Ion Concentration, Kinetics, Molecular Weight, Open Reading Frames, Polymerase Chain Reaction, Protein Structure, Tertiary, Recombinant Proteins, Saccharomyces cerevisiae, Sequence Analysis, DNA, Substrate Specificity, Temperature, Xylans, Aspergillus kawachii, Aureobasidium pullulans, Larix, Saccharomyces cerevisiae, Triticum aestivum, Triticum aestivum subsp. spelta, Zea mays
Citation
Applied Microbiology and Biotechnology
77
5
77
5