title: Creation of a thermostable firefly luciferase with pH-insensitive luminescent color.
abstract: The thermal instability and pH-sensitive spectral property of firefly luciferase have hampered its use as a sensitive multicolor luminescent label or bioluminescent resonance energy transfer donor. With the intention of improving the thermostability of a previously found firefly Hotaria parvula luciferase mutant with minor pH-sensitive spectral change (V368A), further mutation (E356R) was introduced by taking a reportedly stabilized mutant of Photinus pyralis luciferase into account. The double mutant E356R/V368A showed significantly improved thermostability because > 90% activity remained after incubation for 1 h at 45 degrees C, with its specific activity being maintained. Unlike the wild type or V368A, E356R/V368A showed no change in the emission maximum of 568 nm even at pH 6.3, also implying a mutual relationship between thermostability and the proportion of yellow-green luminescent peak under acidic condition.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Tokyo, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kitayama
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Yoshizaki
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohmiya
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D000818:Animals; D001483:Base Sequence; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D004247:DNA / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D049449:Luminescence; D016297:Mutagenesis, Site-Directed; D018462:Photobiology
chemical_list: D004247:DNA; D008156:Luciferases