Preparation Of Morphology-controllable Carrier Materials And Study On The Catalytic Degradation Performance Of Immobilized Cellulase/Horseradish Peroxidase

Enzyme,a natural biocatalyst,has important application value in the degradation of bisphenol A,an environmental pollutant,and the conversion of cellulose into bioenergy.However,natural enzymes are sensitive to the external environment and can not be recycled.The high cost of application has become a key problem limiting their large-scale application in industry.Enzyme immobilization is one of the most commonly used techniques to improve enzyme performance.At present,the immobilized carrier has developed from a simple enzyme loading carrier material to a multifunctional carrier with multiple functions,which can participate in the catalytic reaction and build a synergistic/cascade catalytic system with enzymes,and the design and synthesis of immobilized material become the key control point to realize the stable circulation production of immobilized enzyme.Aiming at the catalytic degradation technology of cellulose and bisphenol A,we prepared functional carrier materials with controllable morphology for immobilizing cellulase and horseradish peroxidase,further studied their catalytic degradation performance,and discussed the application of immobilized enzyme in organic pollutant degradation and biomass energy production in this paper.The main research work is as follows:(1)Magnetic carbon spheres Fe₃O₄@C with core-shell structure were prepared by hydrothermal method.Based on the two-step method of adsorption and embedding,cellulase was immobilized.Firstly,the cellulase was adsorbed on the substrate by electrostatic interaction Fe₃O₄@C.Then,cellulase was embedded by in-situ deposition of silica to prepare microspheres with a diameter of about 200 nm.The thin layer of silica can enhance the physical adsorption of cellulase,reduce the mass transfer resistance effectively,and avoid the significant reduction of catalytic efficiency after the immobilization of cellulase.Through the complementary advantages of adsorption method and embedding method,the thermal stability,reusability and storage stability of cellulase were significantly improved.After incubation at 80?for 30 min,the relative enzyme activity of immobilized cellulase was still higher than 80%.Compared with the first cycle,78%of the relative enzyme activity was still retained after 9 cycles,while the retention rate of enzyme activity was 76%after 4 weeks storage at 4?.(2)Two kinds of Bi₂WO₆carriers with different morphologies were prepared by hydrothermal method and used for the immobilization of horseradish peroxidase and cellulase,respectively.First,Bi₂WO₆hollow microspheres were prepared and HRP was immobilized on the inner and outer surfaces of hollow microspheres.A special structure of artificial photocatalysis coupling cascade catalytic system was designed and synthesized,which was successfully used for the efficient degradation of phenolic organic pollutants in water.Active species capture experiments and ESR analysis showed that h⁺,·OH and·O₂^-played important roles in the degradation of BPA.Photogenerated electrons on Bi₂WO₆surface and generated H₂O₂could participate in the redox reaction of HRP and induce the synergistic effect of photocatalysis.Secondly,three-dimensional red blood cell like Bi₂WO₆was prepared and cellulase was immobilized on the surface of nanospheres.The stability of immobilized cellulase/Bi₂WO₆was significantly improved,and nearly 70%of its activity was retained after 8 cycles.The retention rate of enzyme activity was 77%at 4?for 18days.(3)Two kinds of two-dimensional modified CN based nanosheets,ACN and Ph CN,were prepared by thermal polycondensation of urea and 4-aminopyridine,while the latter was prepared by thermal polycondensation of urea and4-phenyl-3-thiosemicarbazide.First,the mesoporous ACN carrier material was prepared.The band gap of ACN was about 2.7 e V.It had photocatalytic performance,chemical inertness,thermal stability and biocompatibility.HRP was immobilized on ACN surface to construct a photocatalysis system.The catalyst had good cycle stability.The degradation rate of bisphenol A was as high as 94%,and the degradation rate was 2.53 and 25.24 times of ACN and CN.Secondly,mesoporous Ph CN nanosheets with smooth surface were prepared.The cellulase/Ph CN has excellent thermal stability,p H stability,storage stability,cycle stability and better substrate affinity compared with Fe₃O₄@C@cellulase-Si O₂and cellulase/Bi₂WO_6,.(4)CNT/CN,a two-dimensional carbon nanotube doped g-C₃N₄carrier material,was prepared and used for immobilization of horseradish peroxidase(HRP),and a photo enzyme coupled cascade catalytic system was successfully constructed.By loading CNT onto the surface of CN,the CNT/CN composite can significantly improve the charge separation efficiency and enhance the catalytic degradation activity;HRP/CNT/CN prepared by immobilizing HRP on the surface of CNT/CN showed higher catalytic degradation performance.When the loading of CNT and HRP were 1.5%and 10%,respectively,the HRP-10/CNT-1.5/CN had the highest catalytic degradation performance and could degrade BPA completely.The thermal stability and p H stability of HRP/CNT/CN were significantly higher than those of free enzyme.The retention rate of HRP/CNT/CN enzyme activity was close to 80%after 4 cycles,showing good cycle stability.The free radical capture experiments of HRP/CNT/CN composites were carried out by using ammonium bicarbonate as H⁺capture agent,ascorbic acid as·O₂^-capture agent,isopropanol as·OH capture agent and catalase as H₂O₂capture agent.In the process of BPA degradation,the active species were h⁺and·O^-₂.