Thermostable alkaline phosphatase of bacterium Meiothermus ruber: Gene cloning, expression in Escherichia coli, and biochemical characterization of the recombinant protein

Yu V. Yurchenko, I. S. Khromov, A. V. Budilov, S. M. Deyev, A. Yu Sobolev

Research output: Contribution to journalArticle

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 and pH 11.0 and had Km = 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°C and being completely inactivated in 2 h at 80°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.

Original languageEnglish
Pages (from-to)989-998
Number of pages10
JournalMolekulyarnaya Biologiya
Volume37
Issue number6
Publication statusPublished - 2003
Externally publishedYes

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Recombinant Proteins
Alkaline Phosphatase
Organism Cloning
Escherichia coli
Bacteria
Gene Expression
Enzyme Precursors
Vanadates
Reducing Agents
Enzymes
Protein Sorting Signals
Substrate Specificity
Sulfhydryl Compounds
Edetic Acid
Phosphates
Genes
nitrophenylphosphate

ASJC Scopus subject areas

  • Medicine(all)

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Thermostable alkaline phosphatase of bacterium Meiothermus ruber : Gene cloning, expression in Escherichia coli, and biochemical characterization of the recombinant protein. / Yurchenko, Yu V.; Khromov, I. S.; Budilov, A. V.; Deyev, S. M.; Sobolev, A. Yu.

In: Molekulyarnaya Biologiya, Vol. 37, No. 6, 2003, p. 989-998.

Research output: Contribution to journalArticle

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