Production Of Fructooligosaccharides With Immobilized Fructosyltransferase On Anion-exchange Resin

Fructosyltransferase was immobilized with porous anion-exchange resin to produce fructooligosaccharides (FOS). The preparation of free enzyme, selection of carrier, optimization of immobilization, properties of immobilized enzyme and continuous production of FOS were mainly investigated in this thesis.Introcellular fructosyltransferase was extracted from the cells of Aspergillus niger with ultrasonic treatment, and the ratio of extraction was up to about 50%. D201 anion-exchange resin was selected as a carrier to immobilize fructosyltransferase through absorption and crosslinking. The microstructure of immobilized enzyme was observed by SEM.400 U enzyme per g wet resin was immobilized for 8 h at 30℃ and pH 5.0～5.5, then crosslinked with 0.01～0.05% (final concentration) glutaraldehyde for 8 h at 4℃. The highest enzyme activity retained 30.2%.The optimum temperature and pH of the immobilized fructosyltransferase were 50℃ and 5.5, respectively, whereas for free enzyme, were 50℃ and 5.0. Thermal and pH stability were both improved by immobilization comparing with the free enzyme. The Km value was 0.52 M (for sucrose), which was 2 times higher than that (0.29 M) of the free enzyme. This indicated that the affinity between enzyme and substrate decreased significantly.The highest efficiency of production of FOS (50.34%, based on total sugar), using a column packed with immobilized fructosyltransferase, was obtained with 50% (w/w) sucrose at a flow rate of 0.48 ml/min at 50℃. The volumetric productivity in the reactor was 222.91 g FOS /l /h. Influenced by the inhibition from glucose and the invertase from Aspergillus niger, the concentration of FOS in the reaction mixture was relatively low (about 50%).After continuous operation of the column packed with immobilized enzyme over 13 days (more than 8 h per day), 87% of initial enzyme activity was kept. The half-life of the immobilized enzyme at 50℃ was estimated to be 21 days. Mainly because of the higher concentration of substrate (50～60%) and operation temperature(50℃), no microbial contamination was found during production of FOS (about one month). In addition, the mechanical strength of the immobilized enzyme was unchanged.