Study On Preparation Of Magnetic Supports And Their Application For Immobilized Cellulase

In recent years, the enzyme immobilization has been shown to improve catalytic activity, stability with p H, tempreture and reusability which as a essential method. Cellulase is a mixture of enzymes that act synergistically to convert cellulose into bioethanol. However, the cost of cellulase is very high of the hydrolysis costs, and the hydrolysis efficiency is always undesirable. Therefor, cellulase immobilization has been suggested as a viable method, which providing a facile route to recycle and reuse enzyme. In this paper, a series of magnetic supports are prepared. The cellulase was immobilized on support by different methods, and the properties of immobilized celluase were researched.1. The carboxylation magnetic Fe3O4 nanoparticles were successfully prepared by co precipitation of Fe2+/Fe3+ with oleic acid as modifier and consequent oxidation of C=C into —COOH by KMn O4 solution, then, cellulase was directly bonded via electrostatic adsorption. The magnetic support and immobilization cellulase were characterized with X-ray diffraction(XRD), fourier transform infrared spectroscopy(FT-IR) and vibrating sample magnetometry(VSM). The effect of those factors such as different enzyme concentration, immobilization time and p H value on loading efficiency and specific activity of immobilization cellulase were investigated, and the properties of immobilized enzyme w ere studied. The results showed that the average grain size of magnetic support was 8.9 nm and saturated magnetic intensity of immobilized cellulase was 39.98 emu/g; the optimal immobilization conditions were enzyme concentration of 2.0 mg/m L, immobilization time of 6 h and p H of 4.8. The loading efficiency is 96.2% and specific activity was 1.22 IU/mg, respectively. The optimal tempreture and p H value condition were 4.2 at 50℃. Immobilized cellulase has a better heat resistance, p H stability than the free enzyme. The relative activity was 45.6% at temperature of 60℃ for 3 h incubation, and the relative activity of the immobilized cellulase was about 58.4% after 10 cycles.2. Magnetic Fe3O4/attapulgite composite were rapidly prepared using a chemical co-precipitation method, and then, chain chitosan molecules have self-assembled on the Fe3O4/attapulgite composite by electrostatic interaction. Cellulase was immobilized via glutaraldehyde cross-linking method. The synthesized magnetic composites and immobilized cellulase were characterized by FT-IR, XRD, TEM, and VSM. The results present that Fe3O4 nanoparticales has grown outside from attapulgite noodles surface.The chitosan could self-assembl and wrapp around the Fe3O4/attapulgite composite surface, and improve the dispersibility of magneitic nanocomposite. The saturated magnetic intensity of carrier and immobilized cellulase was 35.19 emu·g-1 and 30.47 emu·g-1, respectively. The loading efficiency was 76.72%, amount of celluase immobilized was 126.63 mg/g and activity of immobilization cellulase was 1.51 IU/mg. The optimal tempreture and p H value condition were 4.8 at 50℃. The immobilized cellulase has a wider range of tempreture and p H value condition s than free cellulase. The relative activity of immobilized cellulase was 32.4% for 3 h incubation at 60℃, and it has a impressive heat stability which was compared with free cellulase and the relative activity of the immobilized cellulase was about 23.3% after 10 cycles, saccharification yield maintained 25% after 10 cycles.3. A new strategy was developed for oriented f abrication cellulase/chitosan/ Fe3O4 composite. Different from traditional covalent methods, before immobilization, cellulase was mixed with cellulose firstly, which allows active-site of cellulase combined with cellulose tightly. And then, the chitosan coated Fe3O4 nanoparticles were activated by GDA so as to immobilize cellulase by chemical binding immediately. After hydrolyzing cellulose, the immobilized cellulase was prepared. The synthesized magnetic composites and immobilized cellulase were characterized by FT-IR, XRD, TEM, and VSM. The result shows that cellulose template has been removed successfully in the immobilized cellulase and the flexible hollow composite of immobilized enzyme structure was performed. the immobilized cellulase was supermagnetic which has high saturated magnetic intensity of 24.6 emu/g.The loading efficiency was 50.19%, the amount of celluase immobilized was 202.63 mg/g and activity of immobilization cellulase was 3.53 IU/mg. The immobilized celluase has higher stability compared with free enzyme over wider temperature and p H ranges. The relative activity of immobilized cellulase still remained at 76.10% after 9 h test at 60℃. The relative activity of immobilized cellulase was 45.6% after 17 cycles and saccharification yield maintained 50% which has much better reusability after recovered by magnet.