title: Properties of recombinant trehalose synthase from Deinococcus radiodurans expressed in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Food Chemistry, Technology and Biotechnology, Faculty of Chemistry, Gdansk University of Technology, Poland. pawel.filipkowski@pg.gda.pl
authors.forename: Paweł
authors.initials: P
authors.lastname: Filipkowski
authors.forename: Olga
authors.initials: O
authors.lastname: Pietrow
authors.forename: Anna
authors.initials: A
authors.lastname: Panek
authors.forename: Józef
authors.initials: J
authors.lastname: Synowiecki
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D003470:Culture Media / Q000378:metabolism; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013997:Time Factors; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D008320:Maltose; D005964:Glucosyltransferases; C099692:trehalose synthase