title: Characterization of a thermophilic L-rhamnose isomerase from Caldicellulosiruptor saccharolyticus ATCC 43494.
abstract: L-Rhamnose isomerase (EC 5.3.1.14, l-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-rhi gene encoding L-RhI was PCR-cloned from Caldicellulosiruptor saccharolyticus ATCC 43494 and then expressed in Escherichia coli. A high yield of active L-RhI, 3010 U/g of wet cells, was obtained after 20 °C induction for 20 h. The enzyme was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 90 °C. The enzyme was stable at pH values ranging from 4 to 11 and retained >90% activity after a 6 h incubation at 80 °C and pH 7-8. Compared with other previously characterized L-RhIs, the L-RhI from C. saccharolyticus ATCC 43494 has a good thermostability, the widest pH-stable range, and the highest catalytic efficiencies (k(cat)/K(M)) against L-rhamnose, L-lyxose, L-mannose, D-allose, and D-ribose, suggesting that this enzyme has the potential to be applied in rare sugar production.
authors.affiliation: Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan.
authors.forename: Chia-Jui
authors.initials: CJ
authors.lastname: Lin
authors.forename: Wen-Chi
authors.initials: WC
authors.lastname: Tseng
authors.forename: Tsuei-Yun
authors.initials: TY
authors.lastname: Fang
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009005:Monosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D009005:Monosaccharides; D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C062878:L-rhamnose isomerase