Screening, Expression And Immobilization Of Inulinase

The production of fructo-oligosaccharides with endoinulinase owns many advantages that other methods can not achieve. Aspergillus fumigatus CL1and Aspergillus niger CL2that showed higher endoinulinase activity was isolated from soil of orchard.The endoinulinase encoded by endoinulinase gene from A. fumigatus was firstly overexpressed in Pichia pastoris with a maximum endoinulinase activity of3856U/ml in the5-1fermentor and the biochemical characterization of recombinant endoinulinase was investigated. The results showed that the molecular weight of recombinant endoinulinase was estimated to be58kDa by SDS-PAGE. The optimum pH and temperature of this enzyme were6.0and559℃, respectively. Its activity was enhanced by Ca2+, Fe2+and Mn2+and strongly inhibited by Ag+, Cu2+, EDTA and SDS. The Km and Vmax values versus inulin were2.18mM and1593U/mg respectively. The major products hydrolyzed from both pure inulin and processed Helianthus tuberosus by the recombinant endoinulinase were fructo-oligosaccharides with DP of3,4and5.Several carriers including chitosan, chitosan-PEG, amino resin HA, epoxy resin EP and macroporous anion exchange resin D201were applied to endoinulinase immobilization. The immobilization with chitosan showed a better efficiency with activity recovery of39.8%. The optimum temperature of immobilized INUCLl enhanced5℃compared with free INUCL1, meanwhile, the thermal stability of immobilized INUCLl was improved greatly. After ten batches of continuous hydrolysis, the immobilized INUCL1maintained about80%of its original activity, which proved the immobilized INUCL1was stable.The exoinulinase gene from Paenibacillus polymyxa was expressed in Escherichia coli BL21and its biochemical characterization was studied. When inulin was used as substrate, the optimum pH and temperature of this enzyme were6.0and30℃, respectively. Its activity was enhanced by Na+, K+, Li+and Mn2+. The Km and Vmax values with inulin as the substrate were53mM and14.3U/mg respectively. When sucrose was used as substrate, the optimum pH and temperature of this enzyme were7.0and30℃. Its activity was enhanced just by Mn2+. The Km and Vmac values with inulin as the substrate were5.38mM and80.1U/mg respectively. Only fructose was detected when pure inulin was hydrolyzed by this enzyme.