Optimization Of Fermentation Conditions For The Production Of Dextranase By Chaetomium Gracile And Its Application

In the process of cane sugar production, Leuconostoc mesenteroides are fermentative in nature and result in the destruction of large amounts of sugar which produce dextran. Dextran which exist in the sugarcane juice is not only the main ingredients of acid flocculate in the white granulated sugar, but confuse photometric readings, making false increase the purity of sugar juice, influencing production management, and increasing the sugar liquid viscosity, impacting boiled sugar and purging operation. Hydrolysising dextran with dextranase is currently recognized as the most effective method in the sugarcane industry. The paper focused on fermenting dextranase with Chaetomium gracile which got approve from U.S. Food and Drug Administration (FDA) and GB2760-2011"Standards of Using Food Additives", separating dextranase and testing the application effect in sugarcane juice clarification and its optimal process technology conditions. The main contents and results were as follows:(1) Within the range of the composition, the best carbon source of Chaetomium gracile to produce dextranase was dextran crude anhydride, dextran concentration was25g/L; the best nitrogen source was yeast extract, yeast extract concentration was10g/L; K2HPO4and MgSO4has an important effect on enzyme production, the concentration of them were buth2.5g/L. The optimum condition of fermentation in orthogonal design were:fermentation time was92h, initial pH value was5.5, installs liquid volume was25mL/250mL flask, vaccination quantity was3%. Carrying out three parallel experiments under the best optimization conditions, enzyme activity was231.5U/mL.(2) The crude enzyme was purified by ammonia sulfate fractional precipitation and gel filtration chromatography on Sepharose6B. A electrophoresis pure dextranase was obtained after two-steep purification, whose molecular weight was70kDa. The specific activity was18478.1U/mg, purity multiple was7.7and its recovery rate was29.2%.(3) The characterization of dextranase was researched. The optimal pH of dextranase was5.5, with relative activity about80%at the pH rang from4.0to7.0. The optimal temperature was60℃, dextranase was stable when it was treated under60℃. Dextranase could be inhibited by Cu2+, Fe3+and Hg2+. The dextranase has a specificity to sequential α-1,6glucosidic bond. The Km and Vmax of dextranase to dextran2000kDa,500kDa and70kDa were3.2×10-3,5.8×10-3and7.1×10-3mg/mL,9.1,5.9and4.8mg/(mL-min) respectively.(4) The kinetic models were obtained. A simple model was proposed using the Logistic equation for growth, the Luedeking-Piret equation for dextranase and the Luedeking-Piret-like equation for dextran consumption. The cell growth modes was the dextranase biosynthesis model was: The model values fitted well with the experimental datas.(5) The same amount of "Non-concentrated"dextranase was added to the mix juice, clearjuice, syrup, the results showed that20%,13%and5%of dextran were removed respectively."Non-concentrated"dextranase was added to the mixed juice, according to the process sugar refineryeach, clear juice was prepared. Sedimentation velocity and filtration rate increased30%-40%, color and turbidity dropped60%,20%dextran was removed. The results showed that dextran makes the optical rotations and the purity of sugarcane juice dropped, but the sucrose of sugarcane juice analysised by High performance liquid chromatographic showed that they were the same.