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

1 Citation (Scopus)

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, KM = 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.

Original languageEnglish
Pages (from-to)841-848
Number of pages8
JournalMolecular Biology
Volume37
Issue number6
DOIs
Publication statusPublished - Nov 2003
Externally publishedYes

Fingerprint

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

Keywords

  • Alkaline phosphatase
  • Gene cloning
  • Gene expression
  • Meiothermus ruber
  • Thermostable enzyme

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics

Cite this

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: Molecular Biology, Vol. 37, No. 6, 11.2003, p. 841-848.

Research output: Contribution to journalArticle

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