Study On Enzymatic Properties Of Trehalose Synthase And Condition Of Enzyme Immobilization

Trehalose is a secure natural saccharide, It is non-reducing and stabilized to heat and acid. Because trehalose exhibit protective action against damage of biologic molecule, it could be widely used in molecular biology , pharmaceuticals, foodstuff, cosmetic and agriculture etc.. Trehalose's uses are restricted due to the difficulty in producing it. This study mainly discussed the synthesis of trehalose by trehalose synthase. The main research contents include the screen of Pseudomonas putiada P06 which producing trehalose synthase, the purification of trehalose synthase enzyme, immobilized enzyme and the properties of trehalose synthase.Based on Pseudomonas putiada P06, the mutant strain P06-3-5 which was treated with ultraviolet and NTG inducement was screened and got. The ability of the mutant to produce trehalose synthase increased 1.25 times compared with P06. P06-3-5 had a good genetic stability after 10 generations by transfer of culture experiment.Trehalose synthase is a kind of intercellular enzyme, the cell of P06-3-5 was disrupted to release the enzyme. The crude enzyme was purified by suing gel filtration chromatography of Sephadex G75 and SephadexG200. The enzyme appeared to be a dimmer since determined by SDS Polyacrylamide gel electrophoresis (SDS-PAGE). Their molecular weight were 55000Da and 50000Da respectively. And then the characteristic of the enzyme was worked over.The effects of four carriers for enzyme immobilization were firstly researched, and the condition of enzyme immobilization which had a good enzyme activity and stability was optimized. The concentration of aldehyde was 0.5%, the ratio of quality for liquid stated enzyme and polysaccharide gel was 1:1, the pH value of unite was 8.0, the temperature of unite was 15℃, the optimal time of unite was 12 hours.The trehalose synthase was immobilized on bacterial cellusose by adsorption and crosslinking. The optimum condition for immobilization was investigated, and stated as 20μL trehalose synthase was adsorbed with 1g dry bacterial cellusose at pH7.0 and 15℃for 20h, followed by being treated with 6% glutaraldehyde at 15℃for 20h. Some properties of the immobilized enzyme were compared with those of the nature enzyme. The optimum pH of the immobilized enzyme was 7.4, which was 0.4 higher than that of nature enzyme.Besides, its optimum reaction temperature was 45℃, which was 5℃higher than that of the nature enzyme. The stabilities of trehalose synthase on enduring pH changing ,heat treatment and multiple utilization were obviously improved after immobilization.The reaction conditions for production of trehalose by trehalose synthase were detailed studied. The effects of the maltose concentration, incubation time and temperature, reaction rotate speed were studied above all. The maltose concentration had very little influence on the conversion ratio. At the beginning of reaction,the longer the time and the higher the temperature,the more trehalose. But in the end , the conversion ratio was less. The optimal reaction time was 18 hour and reaction temperature was 40℃for immobilized enzyme changing maltose to trehalose and the highest content of trehalose was acquired.