Cloning and characterization of a novel L-arabinose isomerase from Bacillus licheniformis.
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.
Prabhu
Ponnandy
P
Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul, Korea.
Tiwari
Manish Kumar
MK
Jeya
Marimuthu
M
Gunasekaran
Paramasamy
P
Kim
In-Won
IW
Lee
Jung-Kul
JK
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001089:Arabinose / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology; D003067:Coenzymes / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
D003067:Coenzymes; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D003035:Cobalt; D008345:Manganese; D001089:Arabinose; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase