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.

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 30oC. DraTreS activity was almost unchanged after 2 h preincubation at 45oC and pH 7.6, and retained about 56% of maximal value after 8 h incubation at 50oC. 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.

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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 His 6 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 Cu 2+ , Hg 2+ and 10 mM Tris as well as by EDTA when its concentration exceeded 1 mM, but slightly activated by 1 mM dithiotreitol. The K m and k cat values of maltose conversion were 254 mM and 31.86 s-1 , respectively.

Magnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3-aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si-O-Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support using glutaraldehyde as a coupling agent. Obtained preparations had specific activity of 148 U/g of the support when measured at 558C using maltose as substrate. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K m value and a lower energy of activation. The immobilization was almost completely terminated after 30 min of the reaction at 308C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10 mM. The immobilization did not influence considerably on optimum pH and temperature of substrate hydrolysis catalyzed by investigated enzyme (558C, pH 4.5). Moreover, immobilized glucoamylase was easily separated from the reaction medium by an external magnetic field and retained about 60% of initial activity after nine repeated cycles of enzyme reaction followed by magnetic separation.