Purification and characterization of an extracellular alpha-L-arabinosidase from a novel isolate Bacillus pumilus ARA and its over-expression in Escherichia coli.
The alpha-L-arabinosidase, AraB, was induced when Bacillus pumilus ARA was grown at 50 degrees C in a minimal medium containing xylan. A 56-kDa protein with alpha-L-arabinosidase activity was purified from culture supernatant to gel electrophoretic homogeneity. The optimal activity was at pH 6.4 and 60 degrees C over a 10-min assay. The purified enzyme was stable over a pH range of 5.2-7.6 and had a 1-h half life at 70 degrees C. The enzyme released arabinose from oat spelt xylan. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as substrate gave a K (m), and V (max) of 1.05 mM and 240 U per mg of protein. The NH(2)-terminal amino acid sequence of the enzyme was determined, and its gene araB was subsequently cloned, sequenced, and over-expressed in Escherichia coli. The open reading frame of araB consists of a 1,479-bp fragment encoding a protein of 472 amino acids, which belonged to family 51 of the glycoside hydrolases with an identity of 67% to the protein encoded by abfB of Bacillus subtilis 168.
Pei
Jianjun
J
Jiangsu Key Laboratory for Biodiversity and Bio-resources, Nanjing Normal University, Nanjing, Jiangsu, 210046, People's Republic of China.
Shao
Weilan
W
D001089:Arabinose / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014990:Xylans / Q000378:metabolism*
D004269:DNA, Bacterial; D014990:Xylans; D001089:Arabinose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase