Cloning and characterization of a novel l-arabinose isomerase from Bacillus licheniformis

• Published in: Applied microbiology and biotechnology 2008-11, Vol.81 (2), p.283-290
• Main Authors: Prabhu, Ponnandy, Kumar Tiwari, Manish, Jeya, Marimuthu, Gunasekaran, Paramasamy, Kim, In-Won, Lee, Jung-Kul
• Format: Article
• Language: English
• Subjects: Aldose-Ketose Isomerases - chemistry; Aldose-Ketose Isomerases - genetics; Aldose-Ketose Isomerases - metabolism; Amino acids; Arabinose - metabolism; Bacillus - enzymology; Bacillus - genetics; Bacillus licheniformis; Biological and medical sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Characterization; Chromatography; Chromatography, Gel; Cloning; Cloning, Molecular; Cobalt; Cobalt - pharmacology; Coenzymes - pharmacology; Dimerization; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; Electrophoresis, Polyacrylamide Gel; Enzymatic activity; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genomes; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; l-Arabinose isomerase; Life Sciences; Manganese - pharmacology; Microbial Genetics and Genomics; Microbiology; Molecular Weight; Open Reading Frames; Proteins; Rare sugar; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Studies; substrate specificity; Temperature
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-008-1652-6

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 ~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 Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°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.