Studies On Formation Of Trehalose By Trehalose Synthase In Permeabilized Cells

Trehalose is a natural functional disaccharide. It is non-reducing and stabilized to heat and acid. It also can protect biologic molecule , such as DNA and protein. So trehalose could be widely used in foodstuff.. pharmaceuticals. cosmetic.. and agriculture. Trehalose抯 uses are restricted due to the difficulty in producing it. This study mainly discussed the synthesis of trehalose by a microbial enzyme, trehalose synthase. The best strain which producing trehalose synthase, Pseudomonas putida H76, was selected from eight strains. Its fermentation medium and condition were investigated. The formula of culture medium was optimized as: maltose 3%, glucose 3%, peptone 2%, yeast extraction 0.7%, MgSO4.7H20 0.2%. The optimum culture conditions were: tenperature 30C, pH7.2, capacity lOOm] per 500m1 triangular bottle and time 52hrs. The permeabilization of Pseudomonas putida cells in relation to trehalose synthase activity was studied using different organic solvents and detergents. The performance of these solvents was dependent on the incubation temperature, time, and the concentration of cells. Maximum enzyme activity was achieved with 2% to]uene and 0.5% OP?100, at 35t, 0.Shr. The expression of intracellular trehalose synthase activity was increased 117.8梖old with respect to untreated cells. After freeze梔rying, its activity increased 2.6梖old more. The properties of trehalose synthase in the permeabilized cells were investigated systematically. The enzyme exhibited an optimum temperature of 35 C, and an optimum pH of 7.0?.8. The enzyme was stable at 30?0C, PH 6.6?.8. The activity of trehalose synthase was increased by addition of Mg2~, K~ and Na~, but was inhibited by 2+ 2+ 2+ 2+ 2+ Zn , Mn , Cu , Ca Ba . The enzyme catalyzed the conversion of maltose into trehalose by intramolecular transglucosylation, and it can slightly hydrolyze maltose into glucose, but it was inactive on other saccharieds. Stability of the enzyme system remained practically unaltered during refrigeration for up to 1 month. -2- 2001 ~ 6 ~ The reaction condition for production of treha]ose by trehalose synthase were detailed studied. The effects of the maltose concentrations, incubation time and temperature were studied above all. The maltose concentration has little influence on the yield. At the beginning of reaction, the longer the time, and the higher the temperature, the more trehalose. The optium conditions of reaction were gained with orthogonal experiment design: temperature 3Q0 C, pH7.0, time 24hrs, the amount of enzyme 375Ulgmaltose. Master candidate: Xue lu Major: Food science Tutor: Vice Prof. MaYing...