Purification and characterization of an extracellular alpha-L-arabinosidase from a novel isolate Bacillus pumilus ARA and its over-expression in Escherichia coli |
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Authors: | Pei Jianjun Shao Weilan |
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Institution: | (1) Jiangsu Key Laboratory for Biodiversity and Bio-resources, Nanjing Normal University, Nanjing, Jiangsu, 210046, People’s Republic of China |
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Abstract: | The α-l-arabinosidase, AraB, was induced when Bacillus pumilus ARA was grown at 50°C in a minimal medium containing xylan. A 56-kDa protein with α-l-arabinosidase activity was purified from culture supernatant to gel electrophoretic homogeneity. The optimal activity was
at pH 6.4 and 60°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°C. The enzyme released arabinose from oat spelt xylan. Kinetic experiments at 60°C with p-nitrophenyl α-l-arabinofuranoside as substrate gave a K
m, and V
max of 1.05 mM and 240 U per mg of protein. The NH2-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. |
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