title: Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase.
abstract: A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4(G)-β-D-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.
authors.affiliation: Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka, Kanagawa, 237-0061, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Ohta
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hatada
authors.forename: Yuko
authors.initials: Y
authors.lastname: Hidaka
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shimane
authors.forename: Keiko
authors.initials: K
authors.lastname: Usui
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ito
authors.forename: Koki
authors.initials: K
authors.lastname: Fujita
authors.forename: Gaku
authors.initials: G
authors.lastname: Yokoi
authors.forename: Marina
authors.initials: M
authors.lastname: Mori
authors.forename: Shona
authors.initials: S
authors.lastname: Sato
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Miyazaki
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishikawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Tonozuka
mesh_terms: D000192:Actinomycetales / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering; D043324:beta-Fructofuranosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D043324:beta-Fructofuranosidase