title: [Thermostability of alkaline phosphatase of Meithermus ruber bacteria: cloning of the gene, expression in Escherichia coli cells and biochemical characterization of the recombinant protein].
abstract: The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60-65 degrees C and pH 11.0 and had K(m) = 0.055 mM as estimated with p-nitrophenyl phosphate (pNPP). The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60 degrees C and being completely inactivated in 2 h at 80 degrees C. In substrate specificity assays, the highest enzymic activity was observed with pNPP and dATP. Vanadate, inorganic phosphate, and SDS inhibited M. ruber alkaline phosphatase, while thiol-reducing agents had virtually no effect. The enzymic activity strongly depended on exogenous Mg2+ and declined in the presence of EDTA.
authors.affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182 Russia. yurchenko@mail.ru
authors.forename: Iu V
authors.initials: IuV
authors.lastname: Iurchenko
authors.forename: I S
authors.initials: IS
authors.lastname: Khromov
authors.forename: A V
authors.initials: AV
authors.lastname: Budilov
authors.forename: S M
authors.initials: SM
authors.lastname: Deev
authors.forename: A Iu
authors.initials: AIu
authors.lastname: Sobolev
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D034301:Deinococcus / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase