Cloning And Expression Of Trehalose Synthase Gene For Trehalose Production

Trehalose has been found in a large number of prokaryotic and eukaryotic cells, including mycobacteria, streptomycetes, enteric bacteria, archaea, yeast, fungi, algae, plants, and animals. It is not only used as structural components and energy substance in vivo, but also can protect proteins, lipid membranes, and cells from desiccation, refrigeration, and other harsh environments. This disaccharide plays an important role in pharmaceuticals, foods, and cosmetics filed. Because of its wide utilization, various methods for synthesis of trehalose have been reported, but the large-scale production is still in demand. The aim of this study is to find new trehalose synthase, simplify the production process, and increase the trehalose production.In this thesis, the trehalose synthase genes of Pseudomonas stutzeri A1501 was cloned and constructed the recombinant expression plasmid on the carrier of pETDuet, and it was expressed in E. coli BL21(DE3), and get the high enzyme recombinant bacteria. This study can reduce the cost and has the great important in improving the trehalose yields and upgrading the industry level. The pure enzyme was obtained after purification by Ni column. The molecular weight of the enzyme is about 70 kDa, the activity increased from 5.4 U/mg to 77.3 U/mg after purified, the purification factor was 14.3. Recombinant trehalose synthase optimal pH was 8.5, the optimal reaction temperature is 35 ℃, and Zn2+ and Cu2+ inhibited on trehalose synthase enzyme activity strongly.The trehalose synthase was immobilized on TiO2 by adsorption. The optimum condition for immobilization was investigated, enzyme amount is 100 U/carrier (g), the temperature of 4℃ oscillation adsorption 4 h. Immobilization of trhealose synthase on TiO2 the optimum pH was 8 and the optimal reaction temperature was 35℃. The optimal initial maltose concentration was 200 g/L, after reaction 12 h, trhealose synthase concentration reached the top 95.5 g/L, and trehalose conversion rate was 53.7%.Permeability of cells can maintain the original state, and maintain a high enzyme activity. Permeation were used after the BL21(pETDuet-treS) cells expression. Therefore,1% of chloroform dosage and 20 min for permeabilization were chosen in the following experiments. Under the optimal permeabilization conditions, the catalytic reaction optimal is pH 8, the optimal reaction temperature is 45℃. The biocatalytic process produced trehalose at a high cell concentration (17.7 g dry cell). Therefore, the novel process established in this study could also be used as a promising route for the production of trehalose and conversion rate reached 62.7%, the concentration of trehalose up to 94.1 g/L in a relatively short time (4 h).