An efficient method for the immobilization of inulinase using new types of polymers containing epoxy groups

New glycidyl methacrylate copolymers containing different numbers of epoxy groups were synthesized and used to develop effective procedures for inulinase immobilization. The beneficial characteristics of the carriers included a high degree of crosslinking, stability at ambient temperature, an appropriate surface, and the presence of reactive epoxy groups. Some factors affecting the efficiency of immobilization of crude inulinase, including the kind and amount of carrier, the number of epoxy groups, as well as buffer pH and buffer concentration were examined. The yield of immobilization of this enzyme on the investigated type of microspheres was higher than on the commercial carrier, Eupergit(®) C. After immobilization, the optimum temperature for inulinase activity shifted from 55 to 45 °C, whereas the optimum pH = 5 remained unchanged. The basic parameters of inulin hydrolysis were examined, and the possibility of applying the obtained biocatalyst in continuous conditions was tested. Inulin at a concentration of 0.5 % (w/v) was almost completely hydrolyzed to fructose (in a yield of 98 %) at a flow rate of 0.1 mL/min. A tenfold increase in the speed of flow resulted in an increase in the yield of oligosaccharides (DP2-DP6) up to ~41 % in the overall hydrolysate, as analysed by HPLC-RID and LC-ESI/MS. These results indicate that two forms of inulinase, an exo- and an endo-acting enzyme, were immobilized on our carrier. The enzyme showed good operational stability in a packed column over 28 days. There were no significant decreases in the efficiency of continuous hydrolysis during this time (about 17.4 % in comparison to its initial value).