The Hydrolysis Of Inulin With Immobilized Inulinase On Cotton Cloth

Inulinase from Aspergillus ficuum SK004 was immobilized on cotton cloth activated with p-toluenesulfonyl chloride (tosyl chloride). The best carrier was cotton cloth with soft nap selected from fifteen kinds of carriers. The analysis of fibril micrographs by SEM proved that cotton cloth with soft nap was a suitable material for inulinase immobilization. The activity of immobilized inulinase in 0.2 mol/L acetic buffer (pH 4.5) was stable for 8 weeks at 4℃. The enzyme remained in the supernatant after immobilization can be reused for preparation of immobilized inulinase after dialysis and ultrafiltration. The reutilization capacity of fiber used for the preparation of the immobilized inulinase was assessed.The best conditions was obtained through the single factor and orthogonal experiments. Cotton cloth was soaked in 0.5 mol/L NaOH solution for 3 h. Dry pyridine was then added to the cloth with the optimal amount of 10mL to each gram of dry cotton for 3 h. The ratio of tosyl (g) /pyridine (mL) was 1g:1mL, tosylation time was 3 h. 80 U enzyme per g dry cotton cloth was immobilized for 12 h at 25℃. The optimal coupling pH was found to be 4.5 and ionic strength was 0.2 mol/L. The highest enzyme activity yield achieved 83.60%.The crude inulinase preparation was partially purified by active carbon, ammonium sulfate fractionation and dialysis. And the purification multiple was 5.67, enzyme activity yield was 83.73%. The Km was 167.14 mg/mL(for inuline). After immobilization, Km raise both for inuline and sucrose, I/S ratio slightly droped.The optimum temperature and pH for inulin hydrolysis with the immobilized inulinase were 60℃and 5.0, respectively, whereas for free enzyme, were 60℃and 4.5. Thermal stability and pH stability had no significant change. Ca2+, Na+ and Zn2+ activated enzyme activity relatively. EDTA turned from original inhibitor into activator.Continuous production of fructose from inulin was investigated using the immobilized inulinase in a packed-bed column reactor. At the space velocity of 0.2mL/min, 5%(w/v)inuline solution was completely hydrolyzed and the volumetric productivity was 43 g·L-1·h-1 at 50℃; the DH of 10%(w/v) inuline solution was 70% and the volumetric productivity was 53 g·L-1·h-1. The operational half-life of the immobilized inulinase was 19 d at 50℃and 32 d at 40℃. In batch reactor the extent of 10% inuline hydrolysis attained 96.94% in 4 h and the productivity was 242.36 mg·L-1·h-1 when the ratio of substrate and immobilized inulinase was 100ml∶1g.