Preparation Of Hydrogel-immobilized Enzyme And Its Application In Dye Degradation

With the rapid development of the industry,the pollution of dye effluents discharged by the printing and dyeing industry pose serious destruction to the ecological environment.Enzyme-catalyzed degradation exhibits many advantages in treating dye effluents,such as high efficiency,high selectivity,and environmental friendliness.Laccase and horseradish peroxidase（HRP）show excellent dye removal performance,and their catalytic product is H₂O,which is in line with the concept of green chemistry.However,the inherent shortages of free enzymes,such as enzyme deactivation and unrenewable,limit the application of free enzymes in dye effluents treatment.Enzyme immobilization provides protection for the enzyme while retaining its original activity,and broadens the resistant to environmental changes,such as extreme pH and temperature.Enzyme immobilization strategies include adsorption method,embedding method and chemical method.The embedding is a relatively better immobilized enzyme method due to convenient operation,unchanged the chemical structure of enzyme and least impact on the enzyme activity.There are various carrier materials for immobilized enzymes.Among them,widely distributed and cheap natural polymers exhibit the best biocompatibility with enzymes.Metal-organic frameworks exhibit excellent thermal and chemical stability,which can effectively improve the tolerance of immobilized enzymes to harsh environments.Immobilized enzymes with hydrogels provides a good hydrophilic environment for the enzyme,which is beneficial to the expression of the enzyme activity.Enzymatic cross-linking is an efficient and environmentally friendly method for the preparation of hydrogels.The in-situ embedding,and immobilization of enzymes is completed during the formation of hydrogels.The prepared hydrogels pose immobilized enzyme properties.Alginic acid（SA）is an excellent natural polymer for preparing hydrogels and immobilizing enzyme due to its low price and non-toxicity.In this thesis,tyramine-modified alginate（SA-Tyr）was synthesized,and the hydrogel of in situ encapsulated enzyme was prepared by utilizing the catalytic properties of laccase and HRP on tyramine.The prepared hydrogels were used for dye degradation.The specific research as follows:（1）Two different laccase-catalyzed hydrogels with highly adsorbent graphene oxide（GO）or without GO were prepared by mixing the SA-Tyr solution with laccase.The network structure of the prepared hydrogels was perfected by the secondary cross-linking using Ca²⁺.Experimental results showed that the enzymatic gelation time shortened with increasing the concentration of laccase and SA-Tyr,and the gelation could be completed within 30 seconds.The gelation time increased with increasing the concentration of doped GO.The equilibrium swelling ratio of the double-crosslinked hydrogels is much lower than that of single crosslinked hydrogels.Rheological analysis showed that the gelation time shortened with increasing the concentration of SA-Tyr.The laccase-catalyzed crosslinking hydrogel exhibited excellent mechanical properties in the rheological frequency sweeping and compression test.Frequency sweeping showed that the storage modulus G’of the prepared gel remained stable in the range of1-100 Hz.The Young’s modulus of the double-crosslinked hydrogel with 2 wt%of SA-Tyr was 149.3 k Pa,and achieved a larger compression deformation.Free laccase and immobilized laccase hydrogels were used for the degradation of the anthraquinone dye Remazol Brilliant Blue R（RBBR）.The immobilized laccase maintained a good decolorization rate under extreme pH and temperature,while the free laccase was almost inactivated.Experimential results indicated that the best decolorization conditions of the laccase-immobilized hydrogels for RBBR:pH=5,react at 40℃for350 min,and the decolorization rate of dyes reached 81.3%.The decolorization rate of immobilized laccase hydrogels decreased only 13%after 9 cycles of use,and retained70.4%of the initial activity after 29 days of storage.（2）The metal-organic framework material ZIF-8 was used to embed HRP to obtain HRP@ZIF-8.HRP@ZIF-8 was dispersed in SA-Tyr solution,and the double crosslinked HRP@ZIF-8-embedded hydrogel（HRA@ZIF-8@SA-Tyr）with Ca²⁺ionic cross-linking and enzymatic cross-linking was prepared under the action of Ca²⁺and H₂O₂.The HRA@ZIF-8@SA-Tyr hydrogel microspheres were used for the degradation experiment of the azo dye Congo red.The optimal decolorization conditions were obtained:pH 5.0,and the reaction was carried out at 40℃for 150 min,and the maximum decolorization rate of Congo red reached to 75%.The immobilized enzyme showed better temperature and pH tolerance,and retained 65.6%of the decolorization rate after seven decolorization cycles.