A thermostable alpha-arabinofuranosidase from xylanolytic Bacillus pumilus: purification and characterisation.
Bacillus pumilus PS213 secretes an alpha-L-arabinofuranosidase (AF) when grown in the presence of arabinogalactan or oat meal. The enzyme has been purified to homogeneity and characterised. Its molecular mass, as determined by gel filtration, is 220 kDa, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of approximately 60 kDa. According to the result of the mass spectrometry analysis showing a molecular mass of 56 kDa, the enzyme should be a homotetramer. The isoelectric point was found to be 5.2, the enzyme activity was optimal at 55 degrees C and pH 7.0. The enzyme retained 80% of its activity after 2 h at 65 degrees C and lost 50% of activity at 75 degrees C after 135 min. The Michaelis constant K(m) and V(max) for p-nitrophenylarabinofuranoside at 37 degrees C were 1.7 mM and 52.9 U mg(-1), respectively. N-terminal sequence analysis and internal peptide fragments showed homology with glycosyl hydrolases of family 51.
Degrassi
Giuliano
G
Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Area Science Park, Pardiciano 99, I-34012 Trieste, Italy. degrassi@icgeb.org
Vindigni
Alessandro
A
Venturi
Vittorio
V
D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology; D002244:Carbon / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011786:Quality Control; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature; D014990:Xylans / Q000737:chemistry / Q000378:metabolism
D014990:Xylans; D002244:Carbon; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase