Study On The Production Of Trehalose Synthase By Immobilized Enzyme

Trehalose exists in nature widely, including lower plants, alga, bacteria, fungi, yeasts, insects, and invertebrates. In most cases trehalose is the major sugar in the blood of insects. Trehalose is an specific protectant, which seems to protect all biological molecules. It can protect biological membranes, proteins, liposomes, and sensitive cytoderms etc. from damage due to drought, freezing and osmotic pressure changes. Because of its distinctive biological functions, trehalose may possibly have various industrial applications such as a preservative material for unstable food, cosmetics and medicines.On the basis of them my studies is carrying on in order to find one efficient way of actual industrious production-immobilized enzyme method. Concrete step include: 1. separate a kind of bacterium GX?0012 from soil and produce trehalose synthase enzyme and transform maltose to trehalose, following study find that trehalose synthase enzyme is a kind of enzyme in the cell. 2. substantive cultivate bacterium GX?0012 and enrich culture mycelium and the cells harvested were disrupted by ultrasonic, followed by leaching and centrifugation to obtain crude enzyme solution. 3. The crude enzyme was fractionated with ammonium sulfate and the active fractions were collected, dialyzed, concentrated and successively purified by column chromatography packing Sephadex G-100. The purity and molecular weight of the enzyme were determined by SDS polyacrylamide gel electrophoresis. 4. Coarse enzyme solution were immobilized by polysaccharide gel and produced immobilized enzyme. Activity analysis of immobilized enzyme were realized by trehalose content in reaction mixture of immobilized enzyme andmaltose.SDS polyacrylamide gel electrophoresis determined result showed that trehalose synthase enzyme appeared to be a dimmer. Their molecular weight was 55000 Da and 5000 Da respectively.The study result showed that optimal condition of producing immobilized enzyme: the concentration of aldehyde is 0.5%; the pH value of unite is 8.0; the temperature of unite is 15癈; the optimal time of unite is 12 hour; the ratio of quality for liquid stated enzyme and polysaccharide gel is 1:1; in the condition of these immobilized enzyme reaction activity is the highest and producing trehalose quantity is the most, the most content of trehalose can arrive to around 40%. Otherwise, the optimal reaction time is 18 hour for immobilized enzyme changing maltose to trehalose and we can acquire the highest content of trehalose. Otherwise by HPAEC,HPLC etc determined means, at last we can affirmed that immobilized trehalose synthase enzyme can transform maltose to D- (+) -trehalose and D- (+) -trehalose is the most (a , a )-trehalose in the nature.