Preperation Of Magnetic Immobilized Laccase And Its Application

Magnetic polymer microspheres developed recently are a kind of novel functional magnetic polymer materials ,whose application in bio-medicine,cytology,bio-engineering ,et al,has just attracted widespread interest .Laccase(EC1. 10. 3. 2), belonged to a family of muti-copper oxidases has important applied value in areas such as paper industry, bioremediation of toxic substances, biodetection and food industry. Immobilization technology of enzyme is an effective measure to achieve using repeatly of enzyme and improve stability. In addition, enzyme immobilized by magnetic polymer microspheres is easer to be separate and reclaim than other carriers. So immobilization of enzyme by magnetic polymer microspheres was studied in this article. The results of optimized conditions of immobilization of laccase,differences between free laccase and immobilized laccase, application of immobilized laccase in removal of phenols from apple juice and decoloration of dyes had be showed as follows:1. The laccase produced mutated Trametes gallic Fr{SAH-12) was purified to electrophoretic homogeneity by the step of sulfate precipitation and Sephadex G-75 chromatography.Purific-action of about 2.94 fold was achieved with activity recovery yield of 73.6%.After purification, its specific activity was 48.8 U/mg.2. Magnetic immobilized laccase has been prepared by immobilizing laccase onto magnetic polystyrene microspheres with glutaraldehyde.The results indicated that the optimized conditions of immobilization of laccase could be specified as:pH3.6,0.1ml magnetic polystyrene microspheres (20mg/ml) was activated by 5% glutaraldehyde for 6h,then added 0.3 ml laccase(0.3mg/ml) to immobilize for 8h.In this way, 888.8U/g carrier of catalytic activity, 17.6 U/mg of specific activity, 36% of recovery yield of immobilized laccase was available. Compared with the free enzyme, the affinity of immobilized laccase increasedtoward substrate and its stability was considerably improved. The measured Km values in magnetic immobilized enzyme were lower than free enzyme. The optimum temperature of magnetic immobilized enzyme was 60°C as same as free enzyme. Magnetic immobilized enzyme was 0.4 unit lower than free enzyme. Magnetic immobilized enzyme has excellent thermal stability, working stability and working stability. Magnetic immobilized enzyme could be enhanced by Cu2+ and inhibited by Fe2\ Hg2+, Cr3+. The influence of magnetic immobilized enzyme by EDTA and DMSO was lower than free enzyme.3. The application of magnetic immobilized laccases in the removal of polyphenol of apple juice was studied in this article. Experiments showed that The polyphenol of apple juice dealed with magnetic immobilized laccase in the optimized temperature 40 °C for 2h decreased 20%,while enzyme activity did not vary which indicated immobilized laccase was more stable than free enzyme. Therefore magnetic immobilized laccase had potential application in production of apple juice.4. Decolorization of five dyes containing Malachite Green, Crystal Violet, Methyl Orange, Cresol Red, Mthylene Blue by magnetic immobilized laccase was studied in this article. The result showed four dyes except Mthylene Blue were decolorized to different extents by magnetic immobilized laccase alone,althoug decolorization was much more efficient and rapid with small molecular compound ABTS(2,2,-azinobis-(3-ethyl benzthiazoline-6-sulphonate)),whereas Mthylene Blue could be decolorized only in the presence of ABTS. The decolorization efficiency of Malachite Green and Crystal Violet was the highest. Color removal of Malachite Green and Crystal Violet reached 93.3% and 90.6% after 24h reaction and all could reach 100% after lh and 12h in addition of ABTS, while Methyl Orange and Cresol Red could reach 85.3% and 66.7% after 24h. Mthylene Blue could not be decolorized by magnetic immobilized laccase alone, but the decolorization efficiency also could reach 40%...