The Molecular Encapsulation Of Horseradish Peroxidase And Associated Application In The Elimination Of Environmental Hormone

Environmental hormones are a class of chemical substances that interferes with organism's endocrine by external factors,and it has the characteristics of large toxicity,stable existence and difficult to degrade.It is urgent mission to find a green,environmentally friendly and efficient way to remove environmental hormones.Horseradish peroxidase(HRP)can efficiently catalyze the removal of environmental estrogens by hydrogen peroxide.However,the poor stability of HRP,low substrate tolerance and narrow range of p H adaptation limit the application of HRP in the removal of environmental estrogens.The molecular encapsulation method is used to react with the modifier through the group on the surface of protein,and then encapsulates a single protein molecule by polymerization method or adsorption method,so that the activity and stability of protein are improved.In this paper,the chemical modification of horseradish peroxidase was carried out by molecular encapsulation method,and the free radical copolymerization was initiated to prepare the nano-gel containing HRP,and the effect of nano-gel enzyme on the removal of environmental estrogens was discussed.Finally,the effect of EDC/Sulfo-NHS method on the stability of nanogel enzyme was studied.As the first of this study,the surface of horseradish peroxidase was modified by molecular modification to introduce the double-bond copolymer.The zwitterionic monomer was copolymerized on the surface of protease by in situ free radical polymerization to form nanogel enzyme.The nanoparticle size distribution is 20-100 nm,and the Zeta potential is-1.8m V.After molecular encapsulation,the thermal stability was also enhanced obviously.At 50?,the water bath for 180 minutes,its activity still remains 48%,while the activity of HRP was only 8%.According to the Michaelis constant measurement,the molecular encapsulation method does not substantially change the affinity of horseradish peroxidase to the substrate.Next,this study tried to use nanogel enzyme to remove environmental estrogens.Phenol,bisphenol A,4-chlorophenol,2,4-dichlorophenol and 4-methoxyphenol were selected as removal objects.In the study of the effect of H2O2 on the catalytic oxidation of phenol by nano-gel enzyme,the optimum concentration of H2O2 was 1m M.In the study of the effect of p H on the catalytic oxidation of phenol by nanogel enzyme,there was a good catalytic activity between p H 3.0 and 10.0,and the optimum p H was 9.6.In the study of the nano-gel enzyme catalyzed oxidation of phenolic environmental hormones,compared with horseradish peroxidase,the removal rate of phenol of nanogel enzyme was increased by 61.9%,and the removal rate of bisphenol was increased by 33.2%,the removal rate of 4-chlorophenol was increased by 29.8%,the removal rate of 2,4-dichlorophenol was increased by 10.2%,and the removal rate of 4-methoxyphenol was increased by 60.2%.The effect of horseradish peroxidase on the removal of different phenols is also different,which was related to its catalytic mechanism and oxidation products,but on the whole,the molecular embedding method improves the removal rate of horseradish peroxidase to phenolic compounds.There are only 7 amino groups on the surface of HRP,but 29 carboxyl groups.This project also attempts to use the carboxyl group of HRP to carry on the double bond graft reaction.The carboxyl group of HRP was activated by EDC/Sulfo-NHS,and then reacted with the double bond molecules containing amino groups(APMA and AA)to obtain the macromonomer of HRP.It was found that when the ratio of APMA to HRP was 10:1 the thermal stability of nanogel enzyme was 17.5 times higher than that of horseradish peroxidase,and the nanoparticle size distribution was 100 nm.In the study of the molecular encapsulation of horseradish peroxidase in different buffer systems,the two-step MES buffer system was more easily obtained with a high thermal stability nanogel enzyme than the PBS buffer system.indicating that APMA was more easily grafted on the horseradish peroxidase surface in the MES buffer solution.On the surface of biological mass spectrometry analysis,HRP-APMA grafted more double bonds than HRP-NAS and HRP-AA,indicating that it is feasible to use carboxyl groups to graft double bonds.Finally,HRP-APMA-gel was applied to the degradation experiment of indigo carmine,and its degradation rate was increased by 32.5% compared with horseradish peroxidase.The results indicate that the molecular encapsulation of horseradish peroxidase has strong catalytic degradation ability in dye wastewater treatment.