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Expression of Deinococcus geothermalis Trehalose Synthase Gene in Escherichia coli and Its Enzymatic Properties
Abstract
A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1,692 bp reading-frame encoding 564 amino acids was amplified using PCR. The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl alfa-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 40C, respectively. The activity of DgeoTreS was almost unchanged after 8 h preincubation at 40C and pH 7.6, and retained about 57 % of maximal value after 8 h of incubation at 55C. 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.
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- Category:
- Articles
- Type:
- artykuł w czasopiśmie wyróżnionym w JCR
- Published in:
-
AFRICAN JOURNAL OF BIOTECHNOLOGY
no. 11(67),
pages 13131 - 13139,
ISSN: 1684-5315 - Language:
- English
- Publication year:
- 2012
- Bibliographic description:
- Filipkowski P., Panek A., Felczykowska A., Pietrow-Tobiszewska O., Synowiecki J.: Expression of Deinococcus geothermalis Trehalose Synthase Gene in Escherichia coli and Its Enzymatic Properties// AFRICAN JOURNAL OF BIOTECHNOLOGY. -Vol. 11(67), (2012), s.13131-13139
- Verified by:
- Gdańsk University of Technology
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