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

Результат исследований: Материалы для журналаСтатья

Аннотация

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.

Язык оригиналаАнглийский
Страницы (с-по)989-998
Число страниц10
ЖурналMolekulyarnaya Biologiya
Том37
Номер выпуска6
СостояниеОпубликовано - 2003
Опубликовано для внешнего пользованияДа

ASJC Scopus subject areas

  • Medicine(all)

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