Study On Immobilization Of Laccase Based On Ordered Mesoporous Silical Carrier And Its Performance

In recent years,the rapid development of industrial economy has caused serious water pollution problems.Phenolic pollutants in industrial wastewater are discharged into the surrounding environment,seriously endangering the ecological environment and human health.As a kind of natural polyphenol oxidase,laccase has excellent ability to catalyze the oxidation of refractory pollutants such as phenols.The final product is environmentally friendly.So laccase is widely used in environmental pollution control and other fields.However,free laccase is difficult to separate from the reaction system and is easily affected by the external environment,which limit its industrial application.Ordered mesoporous silica has become a hot carrier material for enzyme immobilization research due to its advantages of easy synthesis,high specific surface area,large and adjustable pore size.In this study,first,the degradation effect of free laccase on phenolic compounds（2,4-dichlorophenol,phenol,bisphenol A）was investigated.Then,the ordered mesoporous silica material COK-12 was directly synthesized by a sol-gel method using triblock copolymer P123 as a template agent and sodium silicate as an inexpensive silicon source.The microscopic morphology and material properties of the materials were characterized by SEM,TEM,XRD,FTIR and BET.Furthermore,ordered mesoporous silica COK-12 was used to immobilize laccase.At the same time,the immobilization conditions and enzymatic properties of immobilized laccase were discussed,and compared with free laccase.Finally,the influencing factors and removal effects of immobilized laccase on the removal of 2,4-dichlorophenol（2,4-DCP）,phenol and bisphenol A（BPA）pollutants were studied.The results show that:（1）Discussion on the catalytic degradation of phenolic pollutants by free laccase.Free laccase had excellent performance of degrading phenolic pollutants,under optimal conditions（2,4-DCP（100 mg/L）:pH=6.0,40℃,5.0 g/L enzyme;BPA（50mg/L）:pH=6.0,30℃,10.0 g/L enzyme;phenol（50 mg/L）:pH=5.0,35℃,12.5 g/L enzyme）.The degradation rates were 94.97%,47.52%and 42.56%,respectively.The degradation effect of laccase on 2,4-DCP was better than that of BPA and phenol.The characteristic peaks of each compound were obviously shifted by the full-wavelength spectral line.Pollutants were gradually being transformed into other substances.（2）Synthesis and characterization of ordered mesoporous silica COK-12material.The microscopic morphology of the material was observed by SEM image to be uniform spherical particles with a particle size of about 700 nm.It was seen from the TEM image that the material had no obvious crystal form.The pore size was about 10 nm,and the mesopores were neatly arranged.The characteristic diffraction peaks of mesoporous silica were obtained by XRD.Some diffraction peaks were missing,and the order was reduced.The groups in the material phase were obtained by FTIR spectral analysis.The specific surface area,average pore diameter and pore volume of the material were calculated by nitrogen adsorption and desorption tests to be 864.50 m~2/g,10.17 nm and 0.74 cm~3/g,respectively.The above characterization tests confirmed the successful synthesis of COK-12.（3）Optimization of laccase immobilization conditions and study of enzymatic properties.Under the optimal immobilization conditions（time:5 h;enzyme concentration:2.0 g/L;pH=4.0）,the enzyme activity of immobilized laccase COK-12-Lac was 21.50 U/g.The recovery rate of enzyme activity was as high as87.74%.Compared with free laccase,the adaptability of immobilized laccase to temperature and pH was widened.After immobilized laccase was stored for 10 days,the remaining enzyme activity could still reach 67.39%,while the free laccase was basically lost when stored for 7 days.The immobilized laccase still maintained high activity after 5 consecutive cycles of reaction.The remaining enzyme activity still reached 70.19%,which made immobilized laccase can be reused.The Michaelis-Menten constant Km of immobilized laccase(7.83×10^-5mol/L)was slightly higher than that of free laccase(6.32×10^-5mol/L).In summary,the immobilized laccase COK-12-Lac had good enzymatic properties.The pH stability,thermal stability,storage stability,and operational stability were significantly enhanced.In addition,other modified materials（COK-12@Cu O,COK-12-NH₂,COK-12-CS,COK-12@Fe₂O₃）were prepared in the experiment.The activity of laccase immobilized with these materials did not achieve the expected effect.（4）Study on the performance of immobilized laccase to remove phenolic pollutants.Under the optimal reaction conditions（2,4-DCP:pH=7.0,40℃,0.100 g immobilized enzyme;BPA:pH=5.0,35℃,0.100 g immobilized enzyme;phenol:pH=6.0,35℃,0.100 g immobilized enzyme）,the removal rates were 67.26%,38.03%and 37.42%,respectively.The removal of pollutants was the result of the combined effect of carrier adsorption and laccase degradation.Among them,the carrier adsorption rate was less than 10%,and enzymatic degradation played the main role.The removal rate of various pollutants by immobilized laccase decreased with the increase of the initial reaction substrate concentration.The high substrate concentration inhibited the enzymatic reaction.By introducing the ABTS mediator,the pollutant removal effect was effectively improved,and the removal rates were increased to 79.03%,46.29%and 48.37%,respectively.The introduction of divalent metal cations can promote or inhibit the removal of phenolic pollutants by immobilized laccase.Mg²⁺and Zn²⁺can promote the removal of 2,4-DCP.Mg²⁺can promote the removal of BPA.Zn²⁺and Fe²⁺can promote the removal of pheno.Co²⁺,Ni²⁺and Cu²⁺can inhibit the removal of 2,4-DCP,while Mg²⁺,Mn²⁺and Ni²⁺can inhibit the removal of phenol.The degradation paths of the three phenolic pollutants were complex and there were many intermediates.In conclusion,the immobilized laccase COK-12-Lac has a good effect on the removal of phenolic pollutants and can be recycled.