Purification and characterization of alpha-L-arabinofuranosidase from Bacillus stearothermophilus T-6.
Bacillus stearothermophilus T-6 produced an alpha-L-arabinofuranosidase when grown in the presence of L-arabinose, sugar beet arabinan, or oat spelt xylan. At the end of a fermentation, about 40% of the activity was extracellular, and enzyme activity in the cell-free supernatant could reach 25 U/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 20000, and the enzyme was purified eightfold by anion-exchange and hydrophobic interaction chromatographies. The molecular weight of T-6 alpha-L-arabinofuranosidase was 256,000, and it consisted of four identical subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The native enzyme had a pI of 6.5 and was most active at 70 degrees C and at pH 5.5 to 6.0. Its thermostability at pH 7.0 was characterized by half-lives of 53, 15, and 1 h at 60, 65, and 70 degrees C, respectively. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave a Vmax, a Km, and an activation energy of 749 U/mg, 0.42 mM, and 16.6 kcal/mol, (ca. 69.5 kJ/mol), respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by 1 mM Hg2+. T-6 alpha-L-arabinofuranosidase released L-arabinose from arabinan and had low activity on oat spelt xylan. The enzyme acted cooperatively with T-6 xylanase in hydrolyzing oat spelt xylan, and L-arabinose, xylose, and xylobiose were detected as the end reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)
Gilead
S
S
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa.
Shoham
Y
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D005285:Fermentation; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment
D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase