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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 His6-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 Cu2+, Hg2+, Zn2+, Al3+ and 10 mM Tris. The Km value of maltose conversion was 290.7 mM.
High precision measurements of ultrasonic velocity in aqueous suspension of phosphatidylcholine liposomes in the presence of trehalose, sucrose and glucose have demonstrated its dependence on the concentration of the two disaccharides. This finding is interpreted in terms of replacement by the latter of water molecules bound to phosphate head groups on the liposome surface.
The present study investigated the effect of Ca2+, Mg2+, Zn2+, Mn2+, Co2+ and Cu2+ on protein and trehalose synthesis, fermentation activity and biomass yield in two strains of baker's yeast Saccharomyces cerevisiae cultured on synthetic Rose's medium. The experiments showed an increase in trehalose content (by 15-60%) and fermentation activity (by 20-50%) in yeast cultured on the medium supplemented with Mg2+, Zn2+ and Mn2+. Ion pairs, and especially Zn2+ and Mn2+, exerted positive synergistic action on fermentation activity and volume of trehalose synthesis. The addition of Mn2+ or Zn2+ (10-20 µM) to the medium reduced negative effects of the increased concentrations of Cu2+ and Co2+ on the cells of the tested yeast.
Two recombinant trehalose synthases from Deinococcus geothermalis (DSMZ 11300) were compared. A significant influence of the artificial polyhistidine tag was observed in protein constitution. The recombinant trehalose synthase from D. geothermalis with His6-tag has a higher Km value of 254 mM, in comparison with the wild-type trehalose synthase (Km 170 mM), and displayed a lower activity of maltose conversion when compared to the wild type. Moreover, differences in properties like temperature, pH, thermal- and pH-stability were observed. Presence of the histidine tag caused a decrease of thermal resistance in case of trehalose synthase with His6-tag. These data confirmed a suggestion that the introduction of the histidine domain produces in some seldom cases undesirable changes in the protein.
Introduction. The influence of infection with two species of entomopathogenic nematodes of Steinernematidae family on metabolism of glycogen and trehalose of the host was studied. Material and methods. Last instar larvae (L₇) of Galleria mellonella were experimentally infected with Steinernema affinis and S. feltiae. At 6, 12, 18 and 24 h after infection concentrations of trehalose and glycogen as well as activity of trehalase and α-amylase were determined. Results. The content of glycogen was lower in insects infected with S. feltiae than in the controls and animals infected with S. affinis. The content of trehalose was higher in insects from both infected groups than in the controls. Its concentration was slightly higher in larvae infected with S. affinis than in those infected with S. feltiae. The activity of α-amylase after infection with S. affinis was low. It was significantly higher in insects infected with S. feltiae. In animals of both infected groups, following a significant reduction at 6 h, the activity of trehalase remained at a similar level, higher than in the controls. In the paper the effects of infection with (i) different species of entomopathogenic nematodes and (ii) the importance of the developmental stage of the insect-host for changes in its metabolism of glycogen and trehalose were discussed.
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