Characterization of a family 54 alpha-L-arabinofuranosidase from Aureobasidium pullulans.

A glycosyl hydrolase family 54 (GH54) alpha-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 degrees C and between pH 3.5 and pH 4. The enzyme had a K (m) value for p-nitrophenyl-alpha-L-arabinofuranoside of 3.7 mM and a V (max) of 34.8 micromol 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 alpha-1,5-debranched arabinan. AbfA displayed low activity against alpha-1,5-L-arabino-oligosaccharides. The enzyme acted synergistically with endo-beta-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.