Cloning and characterization of a rhamnose isomerase from Bacillus halodurans.
Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an L-rhamnose isomerase (L-RhI). The identified L-RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k(cat) of 8,971 min⁻¹ and a k(cat)/K(m) of 17 min⁻¹mM⁻¹ for L-rhamnose. Although L-RhIs have been characterized from several other sources, B. halodurans L-RhI is distinguished from other L-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans L-RhI a good choice for industrial applications. This work describes one of the most thermostable L-RhI characterized thus far.
Prabhu
Ponnandy
P
Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea.
Doan
Thanh Thi Ngoc
TT
Jeya
Marimuthu
M
Kang
Lin-Woo
LW
Lee
Jung-Kul
JK
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D016366:Open Reading Frames; D055503:Protein Multimerization; D012210:Rhamnose / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013696:Temperature
D019747:Aldose-Ketose Isomerases; C062878:L-rhamnose isomerase; D012210:Rhamnose