title: Characterization of a thermophilic L-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1.
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 Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active L-RhI, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. L-RhI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 75 °C. The enzyme was stable at pH values ranging from 5 to 9, and the activity was fully retained after a 2 h incubation at 40-70 °C. L-RhI from T. saccharolyticum NTOU1 is the most thermostable L-RhI to date, and it has a high specific activity (163 U/mg) and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used 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: Tien-Hsiang
authors.initials: TH
authors.lastname: Lin
authors.forename: Shiu-Mei
authors.initials: SM
authors.lastname: Liu
authors.forename: Wen-Shyong
authors.initials: WS
authors.lastname: Tzou
authors.forename: Tsuei-Yun
authors.initials: TY
authors.lastname: Fang
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005632:Fructose / Q000096:biosynthesis; D015870:Gene Expression; D005947:Glucose / Q000096:biosynthesis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D046950:Thermoanaerobacterium / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C003243:psicose; D005632:Fructose; C002055:allose; D019747:Aldose-Ketose Isomerases; C062878:L-rhamnose isomerase; D005947:Glucose