Synthesis And Application Of Novel Nano-Fe₃O₄ Composite Carrier

Penicillin acylase is capable of producing the 6-APA which serves as an intermediate for synthesis of semi-synthetic β-lactam antibiotics by catalytic hydrolysis of penicillin G potassium salt.It is capable of producing newβ-lactam antibiotics by catalyzing 6-APA and 7-ADCA.It’s instability to things such as heat,organic solvents,acids,and bases prove to be a major factor affecting the application of free PGA.In addition,free PGA has the drawback of being difficult to separate from the product.The above problems have become a major bottleneck in the industrial production of β-lactam antibiotics,and it is crucial that steps are taken to slow down and fix this matter effectively.Immobilization methods have been shown could increase the stability and recyclability of PGA effectively,but the enzyme activity after immobilization has decreased.In addition,due to the limitation of the structure of the immobilized carrier,there is a trade-off effect between the loading rate and the response rate of the immobilized PGA.Therefore,it is very necessary to design and synthesize a carrier which can improve improves factors such as enzyme activity,load rate,and response rate of the immobilized PGA effectively,but also easy to recovery.Research have shown that the composite carrier exhibits higher catalytic activity due to the advantages of combining the inorganic carrier with the organic carrier,at the same time,the recovery rate and operational stability of the immobilized PGA have been improved significantly.However,the inherent contradiction between carrier loading and response rate hasn’t been solved.Therefore,this paper combines the existing research progress,and the novel composite carrier was synthesized by using a reversible addition fragmentation chain transfer polymerization method(RAFT),by combining the thermo-sensitive block copolymer with the magnetic Fe3O4 nanoparticles,and the immobilization of PGA was carried out.The results show that the novel composite carrier has the advantages of properities in the process of immobilizing PGA,such as large load,fast response rate,high enzyme activity,easy recovery and good reusability.The main research contents are summarized as follows:(1)Fe3O4 nanoparticles with a particle size of about 10 nm were prepared by a reverse microemulsion method.Then the Gr was used as the index,and the surface of magnetic nanoparticles were modified by the silane coupling agent KH-570.After pre-processing the innovation of the system,the influence of operation conditions,such as the dosage of KH-570,water-to-alcohol ratio,temperature,pH,and time on the graft ratio of KH-570 were investigated and optimized systemically by silicon molybdenum chromogenic to obtain the optimum conditions:CKH-570=16.0000 g/L,Vwater/ethanol=5%,T=25℃,t=2.5 h,pH=6.Under the optimum conditions,the graft ratio of KH-570 on the surface of magnetic nanoparticles reached 178.15%,and the FTIR、TEM、XRD、VSM and ICP were used to characterize nanoparticles before and after modification.(2)The G was used as the index,1,4-dioxane as solvent,the RAFT reagent study of modified Fe3O4 nanoparticles was performed with RAFT reagent.Then the optimal conditions were obtained:T=65℃,t=14 h,n(KH-570):n(CPDTC)=1:2.Under the optimum conditions,the G of RAFT agent on the surface of the modified Fe3O4 nanoparticles reached 79.34%,which laid the foundation for the preparation of a novel magnetic and thermo-sensitive composite carrier successfully.Finally,and the FTIR、TEM、XRD、and VSM were used to characterize nanoparticles before and after modification by RAFT.(3)A magnetic nano-Fe3O4 composite thermo-sensitivity polymer PDEA with a certain molecular weight and a reactive end group was prepared by grafting monomer onto the surface of the modified nanoparticles by RAFT polymerization.Next graft the monomer HEMA to construct a carrier microenvironment suitable for PGA to exert catalytic activity,then adjust the addition amount of immobilized target monomer(MMA,GMA).Finally,a series of composite carriers with magnetic and thermo sensitivity were synthesized.Finally,and the FTIR、TEM、XRD、and VSM were used to characterize composite carriers.(4)The PGA was immobilized by the composite carriers,and the effects of the immobilized target monomer ratio and immobilization process conditions on EA,EAR and ELC were investigated.The results show that when t=28 h,C(%)=2.0%,T=28℃,pH=8,EA=32927 U/g,EAR=93.48%,ELC=136.42mg/g.The recovery rate and residual enzymatic activity of immobilized PGA were 96.22%and 85.0%after repeated use for 12 times.