Expression of Deinococcus geothermalis trehalose synthase gene in Escherichia coli and its enzymatic properties

  • Pawel Filipkowski
  • Anna Panek
  • Felczykowska Agnieszka
  • Olga Pietrow
  • Józef Synowiecki
Keywords: Trehalose synthase, Deinococcus geothermalis, transglucosylation, gene expression, Escherichia coli

Abstract

A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in Escherichia coli BL21(DE3)pLysS. The recombinant trehalose synthase (DgeoTreS) containing a His6 tag at the C-terminus was purified by metal affinity chromatography and characterized. The expressed enzyme is a homodimer with deduced molecular mass of 64.69 kDa for each subunit, and exhibits the highest activity at pH and temperature of 7.6 and 40°C, respectively. The activity of DgeoTreS was almost unchanged after 8 h preincubation at 40°C and pH 7.6, and retained about 57% of maximal value after 8 h of incubation at 55°C. The DgeoTreS was highly inhibited by Cu2+, Hg2+ and 10 mM Tris as well as by EDTA when its concentration exceeded 1 mM, but slightly activated by 1 mM dithiotreitol. The Km and kcat values of maltose conversion were 254 mM and 31.86 s-1, respectively.

Keywords: Trehalose synthase, Deinococcus geothermalis, transglucosylation, gene expression, Escherichia coli

Published
2016-01-22
Section
Articles

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eISSN: 1684-5315