The Study On The Expression Stability Of Recombinant Dextransucrase Genetic Engineering Strain And The Enzymatic Preparation Of Crystalline Fructose

Dextransucrase (EC.2.4.1.5) is a kind of glucosyltransferases which catalyzes the synthesis of dextran with D-glucosyl moieties from sucrose. The enzyme has been utilized in several fields, such as pharmaceutical, food, chemical industry and the environmental protection lately. Because of the characters of wild-type strains, the purification for this enzyme was turned out to be difficult, and this difficulty also obstructs the extractions of production as well as the studies on dextransucrase itself. So the recombinant genetic strain (BL21(DE3)/pET28-dexYG) was structured through molecular biological technique. Although the trouble was solved preferably, some questions including the stability of expression have appeared.In this study, the stability of expression was explored in detail according batches and resistance pressure research. Kanamycin screening was an effective and simple method and it is suggest that resistance screening should be established in every 6 or 7 batches. The values grow with diluted multiples until 8 times using the former means to measure the activity of dextransucrase. Those conclusions make contributions to an improvement. Dextransucrase was so sensitive to temperature that half of the original activity was lost in 30℃ for 3 hours. Appropriate dilution helps to gain the stability. Then enzymatic reaction characteristics were referenced on a base of the optimum conditions which were concluded by the former researchers. Several factors such as the concentration of substrate, the dosage of enzyme, the time of catalysis and the velocity of stirring were taken into account. The economical system was finally established:25～30℃, pH=5.4,125g/L for sucrose,2U/mL for enzymatic dosage, 150r/min for stirring speed,32h for reaction time. Competitive inhibition caused by the side reaction including the transglycosylation of fructose inhibited the synthesis of dextrans. And the high viscosity also obstructed contacts between enzymes and substrates during the later period.Through single-factor and orthogonality tests, the crystallization conditions of fructose in aqueous-alcohol solution system were studied. The results showed that a purity more than 90%was necessary to the whole crystallization system which containing water and alcohol with the ratio of 1:4. The optimum crystallization conditions were as following:85 Brix° for brix degree,10% of soluble solids amount for seed dosage,6 h for ripening time. The maximum crystallization yield was about 62.37%.Dextran was synthesized by dextransucrases. Fructose was released as byproduct in the catalytic liquid and the solution which is fit for cooling crystallization was obtained after rough purification and cation exchange chromatography. Finally, high efficiency liquid chromatography, infrared spectroscopy, X-ray powder diffraction, elementary analysis as well as scanning electron microscope were carried out to detect the crystallized products.This research has significances for the synthesis of dextran which was treated as the core in glucans industries and the retrieve of fructose from waste liquor as a kind of by-products.