| Sucrose and starch are two major reproducible bio-resource on the earth and easy available around the world. To explore enzymatic methods to produce value-added products from sucrose and starch are the main purpose of this investigation.T~ehalose synthase catalyses maltose into trehalose. Trehalose exists in nature widely, including low foliage, algae, bacterium, fungus, yeast, insects, and invertebrates.In most cases trehalose is the major sugar in the blood of insects. b~eha1ose is an aspecific protectant, which can protect biological membranes, proteins , liposomes , and sensitive cytoderms etc. from damage due to drought , freezing and osmotic pressure changes. behalose may possibly have various industrial applications such as a preservative material for unstable food, cosmetics, and medicines. The trehalose synthase studied was obtained by cultivating a strain of microorganism designated as GX-0012. The resulting cell was separated, and disrupted to release the enzyme. The crude enzyme was purified by using gel filtration chromatography of Sephadex G100 and SephadexR G200, and anion-exchange chromatography with Pharmacia MonoTh~ Q packed column. The enzyme appeared to be a dimer since determined by SDS Polyacrylamide gel electrophoresis (SDS-PAGE). Thefr molecular weight were 55,0(M) Da and 50,000 Da respectively.Fuctosyltransferase (Ftase) converts sucrose effectively to fructoolingosaccharide (FOS). As a functional sweetener, FOS has been found in various fruits and vegetables in nature, and has two characteristic properties, nondigestibility and selective utilization by beneficial intestinal bacteria, which make them useful as low-calorie dietary fiber for relief of constipation, improvement of blood lipid composition, cholesterol reduction, and suppression of intestinal putrefactive substances. Because of 2 -these beneficial physiologic functions, FOS has attracted more and more attentions worldwide.The strain GX-0010 was cultivated for Ftase. After the same pretreatment as to obtained crude enzyme of trehalose synthase mentioned above, the resulting crude Ftase wse further purified by using weak anion-exchange chromatography of DEAE-Sephadex A50, and Resource~ Q, followed by gel filtration chromatography of SephadexR G75. The purity and molecular weight of the enzyme were also determined by SDS-PAGE. Molecular weight of the enzyme is 89,000 DL The enzymatic properties of immobilized FT were investigated and compared with those of the free enzyme. It was found that both free and immobilized FTase have the pH optimum of 5.5 and were stable over a pH range of 5.0 7.5. The optimal temperature and thermostability of the immobilized enzyme was improved over that of the free enzyme, resulting in a temperature range for activity of 4 -55C and a retention of 87.5% of its optimal activity when subjected to 55C for 60 miii, compared to the free enzyme which exhibited a 45-52(2 activity range and retention of 61.6% of its optimal activity. The activity of the free enzyme was significantly inhibited by certain heavy metal ions, e.g. 0.00001 molIL Hg~ and 0.0001 mol/L Ag~, but the immobilized enzyme was only partly affected under the same conditions. Both the free and immobilized enzyme were completely inhibited by 0.001 molIL Ti2~ . The Km of the immobilized FTase (386 mmol/L) was about 1.8-fold that of free enzyme (215 mmoIIL).Because these enzymes catalyse cheap and available material as substates into functional sacchrides, with further studies going on, both enzymes will be applied in more industrial fields. |
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