Immobilization and Application of Laccase for Bisphenol A Removal

Author:Pu Ming Yue

Supervisor:zou dong lei


Degree Year:2019





Mesoporous silica materials(MSU-F)is a kind of mesoporous silica particles with wormlike and highly ordered structure.Its special window channel structure makes crosslinked enzyme aggregates not to leak,and enzyme activity will be protected at the same time.Hydrogel is a functional polymer material which owns three-dimensional spatial network structure and strong hydrophilic property.It is neither soluble in water nor in general organic solvents.Entrapping the enzyme into hydrogel can greatly improve the activity of the enzyme and makes it possible to resist adverse environment.This article chosen two different kinds of laccase immobilization carrier,mesoporous silica and polyethylene glycol(PEG)hydrogels,to immobilize the laccase by adsorption/crosslinking and entrapment/crosslinking,respectively,optimizing the immobilization conditions according to the enzyme loading amount,activity recovery and etc,and utilized them for bisphenol A treatment,which was chosen as the typical endocrine disruptors;explored the influence factors on bisphenl A biotransformation behavior,and applied SER/Lac to the fluidized bed reaction system.By adding the mediator agents,the removal efficiency of bisphenol A was strengthened.We constructed a stabilized Lac in the mesoporous silica(SER/Lac)by adsorption followed by cross linking using the glutaraldehyde(GA)as cross linking agent,and when the mixture of the free laccase and mesoporous silicon foam(MSU-F)was shaking for 30 min in pH 3.5 buffer solution,a high efficiency of Lac immobilization was obtained with the enzyme loading amount of 30.3 wt%and the activity recovery of 12.1%,also the stability,including pH,temperature and storage stability were all improved.Kinetic parameters Km and Vmaxax were obtained from the Lineweaver-Burk plot using ABTS as substrate.The Km and Vmaxax values of SER/Lac were 31.96μM and 0.023μM/min,respectively.The Vmaxax of SER/Lac(0.023μM/min)was decreased by about 1.95 fold compared to free Lac(0.045μM/min).The prepared SER/Lac was utilized to biotransformate bisphenol A and much better removal efficiency was obtained compared with free laccase,and pH 5 was the most effective condition.The dosage of SER/Lac was proportional to the degradation efficiency of bisphenol A.But when the dosage was higher than 0.48 U/L,the degradation efficiency did not increase significantly,so the dosage of SER/Lac was set to 0.48U/L.The adsorption of bisphenol A by MSU-F itself can be neglected,so the removal of bisphenol A was mainly due to the laccase catalytic degradation.Biotransformation pathway and product analysis results showed that the acid condition was helpful for oligomer flocculation,and the bisphenol A itself may be participated in the formation of oligomer.According to the GC/MS analysis,the main ingredient in the supernatant was p-isopropenyl phenol,and the bisphenol A biotransformated pathway by laccase was proposed according to the HPLC/MS.Encapsulating enzyme into biocompatible materials is a widely used strategy for enzyme immobilization.In this study laccase entrapped in PEG hydrogel particles(Lac/particles)were synthesized via emulsion method assisted by photopolymerization and utilized them for treating bisphenol A based on their dual functions of adsorption and enzymatic catalysis.By optimizing the oil/water ratio,percentage of PEG precursor,and the ratio of polyethylene glycol diacrylate(PEGDA)and polyethylene glycol methacrylate(PEGMA),the Lac/particles with 100%conversion rate were obtained,and the resulting Lac/particles’surface was rough,and the particle size was focus on 137-535μm.High Lac/particles yield(100%)and entrapment efficiency(100%)were obtained,and the activity recovery was 18.9%.The Km and Vmaxax values of Lac/particles were 1649.35μM and 0.8342μM/min,respectively.As compared to free Lac,the Lac/particles showed excellent stability that can maintain nearly 100%of initial activity after 24 h shaking in various pHs of3-7,and the residual activity was 95.46%after storage for 23 days in 4℃.When the bisphenol A was treated with Lac/particles,it can be absorbed into Lac/particles quickly,and then be further enzymatically biotransformed even without continuous shaking,highlighting the synergistic effects of adsorption and enzymatic catalytic on bisphenol A removal,enabling the reusability of Lac/particles because of liberating the active sites after enzymatic oxidation.Similarly,because of its excellent adsorption capacity,the continuous stirring process was avoided and thus the power cost was saved.At the same time,the enzyme degradation products were trapped in the hydrogel sphere,so the second pollution was avoided.More importantly,the degradation of bisphenol A by Lac can release the adsorption sites on the hydrogel microspheres,thereby improving the reusability of the gel microspheres.However,because the products of enzymatic degradation were trapped in the hydrogel,its presence would occupy the site,thus affecting the reusability.The efficiency of bisphenol A biotransformation can be obviously improved by adding certain concentration of mediator.When the concentration of mediator was 0.1mM,the degradation rate of bisphenol A was increased by all the four mediators,including ABTS,HBT,AS and SA.The free radicals produced did not decrease the activity of Lac/particles so that the excellent stability was maintained.When natural meditor,AS and SA were mixed together to treat bisphenol A with Lac/particles,they played the role of coordination function and much better efficiency was obtained compared with single mediator.HBT was the weakest mediator in the four mediators for the promotion of bisphenol A biotransformation.The addition of copper ions can promote the activity of enzymes,but the presence of copper ions inhibits the catalytic of enzymes in the treatment of bisphenol A.When applying SER/Lac for continuous biotransformation of bisphenol A in a circulating fluidized bed reactor(FBR),fully contacted between SER/Lac and substrate could be achieved at flow rate of 7.2 mL/min,and the degradation efficiency of bisphenol A was 81%after 6 h.By increasing the concentration of SER/Lac in the reaction system,the fastest degradation efficiency of bisphenol A was also within 6 h.Compared with batch experiments,the required enzyme activity in the fluidized bed was much lower(2.1 U/(mg BPA·h))than in batch experiments(14.3 U/(mgBPA·h)).When the fluidized bed reactor was utilized for treating other phenol-contained substrates,such as E2 or phenol,the ideal results were also obtained,showing that using SER/Lac in the fluidized bed reactor for the treatment of endocrine disruptor wastewater was feasible.