Journal article
Cloning and characterization of a novel L-arabinose isomerase from Bacillus licheniformis
Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul, Korea.1
Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an L-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da.
The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni-NTA chromatography. The molecular mass of the purified enzyme was estimated to be approximately 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer.
The enzyme required a divalent metal ion, either Mn(2+)or Co(2+), for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50 degrees C, respectively, with a k (cat) of 12,455 min(-1) and a k (cat)/K (m) of 34 min(-1) mM(-1) for L-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for L-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of L-ribulose.
This work describes one of the most catalytically efficient L-AIs characterized thus far.
Language: | English |
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Publisher: | Springer Berlin Heidelberg |
Year: | 2008 |
Pages: | 283-290 |
ISSN: | 14320614 and 01757598 |
Types: | Journal article |
DOI: | 10.1007/s00253-008-1652-6 |
Aldose-Ketose Isomerases Arabinose Bacillus Bacillus licheniformis Biotechnology Characterization Chemistry Chromatography, Gel Cloning, Molecular Cobalt Coenzymes DNA, Bacterial Dimerization Electrophoresis, Polyacrylamide Gel Enzyme Stability Escherichia coli Gene Expression Hydrogen-Ion Concentration Isoelectric Point Kinetics L-arabinose isomerase Manganese Microbial Genetics and Genomics Microbiology Molecular Weight Open Reading Frames Rare sugar Recombinant Proteins Sequence Analysis, DNA Substrate specificity Temperature l-Arabinose isomerase