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Thermostable Alkaline Phosphatase of Bacterium Meiothermus ruber: Gene Cloning, Expression in Escherichia coli, and Biochemical Characterization of the Recombinant Protein

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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°C, pH 11.0, K M = 0.055 mM with p-nitrophenyl phosphate. The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60°C and being completely inactivated in 2 h at 80°C. In substrate specificity assays, the highest activity was observed with p-nitrophenyl phosphate and dATP. Vanadate, inorganic phosphate, and SDS were inhibitory, while thiol-reducing agents had virtually no effect. The enzyme activity strongly depended on exogenous Mg2+ and declined in the presence of EDTA.

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Authors and Affiliations

  1. Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia

    Yu. V. Yurchenko, I. S. Khromov & A. Yu. Sobolev

  2. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia

    A. V. Budilov & S. M. Deyev

  3. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia

    S. M. Deyev

Authors
  1. Yu. V. Yurchenko
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  2. I. S. Khromov
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  3. A. V. Budilov
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  4. S. M. Deyev
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  5. A. Yu. Sobolev
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Yurchenko, Y.V., Khromov, I.S., Budilov, A.V. et al. Thermostable Alkaline Phosphatase of Bacterium Meiothermus ruber: Gene Cloning, Expression in Escherichia coli, and Biochemical Characterization of the Recombinant Protein. Molecular Biology 37, 841–848 (2003). https://doi.org/10.1023/B:MBIL.0000008352.79909.f2

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  • DOI: https://doi.org/10.1023/B:MBIL.0000008352.79909.f2

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