title: Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium.
abstract: The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50 degrees C, lower than that of E. coli APase by 30 degrees C. The specific activity of SIB1 APase at 50 degrees C was 3.1 fold higher than that of E. coli APase at 80 degrees C. SIB1 APase lost activity with a half-life of 3.9 min at 70 degrees C, whereas E. coli APase lost activity with a half-life of >6 h even at 80 degrees C. Thus SIB1 APase is well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Mizutani
authors.forename: Tadao
authors.initials: T
authors.lastname: Tsuji
authors.forename: Naoto
authors.initials: N
authors.lastname: Ohtani
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Haruki
authors.forename: Masaaki
authors.initials: M
authors.lastname: Morikawa
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008670:Metals; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000201:enzymology*; D013696:Temperature
chemical_list: D008670:Metals; D000469:Alkaline Phosphatase