Properties of recombinant trehalose synthase from Deinococcus radiodurans expressed in Escherichia coli.

Paweł Filipkowski, Olga Pietrow, Anna Panek, Józef Synowiecki

Department of Food Chemistry, Technology and Biotechnology, Faculty of Chemistry, Gdansk University of Technology, Poland. pawel.filipkowski@pg.gda.pl

Acta biochimica Polonica 2012

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A trehalose synthase gene from Deinococcus radiodurans (DSMZ 20539) containing 1659 bp reading frame encoding 552 amino acids was amplified using PCR. The gene was finally ligated into pET30Ek/LIC vector and expressed after isopropyl β-d-thiogalactopyranoside induction in Escherichia coli (DE3) Rosetta pLysS. The recombinant trehalose synthase (DraTreS) containing a His(6)-tag at the C-terminus was purified by metal affinity chromatography and characterized. The expressed enzyme is a homodimer with molecular mass of 126.9 kDa and exhibits the highest activity of 11.35 U/mg at pH 7.6 and at 30°C. DraTreS activity was almost unchanged after 2 h preincubation at 45°C and pH 7.6, and retained about 56% of maximal value after 8 h incubation at 50°C. The DraTreS was strongly inhibited by Cu(2+), Hg(2+), Zn(2+), Al(3+) and 10 mM Tris. The K(m) value of maltose conversion was 290.7 mM.

Citation

Paweł Filipkowski, Olga Pietrow, Anna Panek, Józef Synowiecki. Properties of recombinant trehalose synthase from Deinococcus radiodurans expressed in Escherichia coli. Acta biochimica Polonica. 2012;59(3):425-31