Novel Zwitterionic Polymers For Enzyme Immobilizations And Modifications

As a biocatalysit,enzyme has been widely used in medicine,food and fine chemical engineering.However,the main obstacle to the application of enzymes is that its spatial structure is susceptible to various environmental factors.Immobilization and modification are the most effective ways to improve enzyme performance.Thus,it is of great significance to develop new and effective immobilized materials and polymers to improve stabilities and efficiency of enzymes.Studies have shown that zwitterionic polymers could improve protein stability and maintain biological activity.In this paper,we first desiagned a new zwitterionic polymer carrier,silica nanoparticle-poly(glycidyl methecrylate)(S-p GMA)modified with cysteine(Cys),which was introduced zwitterionic groups,and named S-p GMA-Cys-L/H.Candida rugosa lipase(CRL)was fixed to S-p GMA(CRL-1)and S-p GMA-Cys-L(CRL-2)via reactions with epoxy groups,and S-p GMA-Cys-L(CRL-3)and S-p GMA-Cys-H(CRL-4)via reactions with carboxyl groups.The results revealed that the immobilization resulted in significantly higher enzymatic reaction efficiency and enzyme-substrate affinity than the free enzyme.More importantly,CRL-2,-3,-4,presented superior thermal stability and p H tolerance than those of CRL-1,which demonstrated that a hydrophobic segment-containing zwitterionic copolymer functionalized support can simultaneously improve lipase activity and stability.On the basis,zwitterionic monomer(sulfobetaine methacrylate,SBMA)and GMA were covlently grafted onto the surface of silica nanoperticles to obtain three carriers with different molar radios for CRL immobilization,and denoted as p(G100-S0)-CRL,p(G50-S50)-CRL and p(G10-S90)-CRL.The enzyme-substrate affinity after immobilization was higher than free one,while the activity varied with the grafted copolymer composition.In comparison to p(G100-S0)-CRL,p(G50-S50)-CRL and p(G10-S90)-CRL presented remarkably enhanced thermal stability and p H tolerance.These results further demonstrated that a hydrophobic segment-containing zwitterionic copolymer functionalized support has a significant effect on improving lipase activity and stability.In order to study the change of protein structure after the enzyme and zwitterionic polymer were combined in the immobilized enzyme,a study was made on the use of zwitterion and hydrophobic monomer mixed modified enzyme.A new enzyme-polymer conjugates synthesized by grafting polymerization onto CRL with zwitterionic carboxyl betaine methacrylate(CBMA),hydrophobic tert-butyl methacrylate(TBMA)and their equimolar mixture,which respectively created CRL-p CBMA,CRL-p TBMA,and CRL-p(T50-C50).All the enzyme conjugates presented improved catalytic activity,thermostability and enzyme-substrate affinity.The zwitterionic/hydrophobic balance is of vital importance to enhance the enzymatic performance.Spectroscopic characterizations revealed that the enhanced catalytic activity and stability of CRL-polymer conjugates were closely related to the changes in protein structure.The research has designed and synthesized a series of hydrophobic segment-containing zwitterionic copolymer functional materials,which were greatly improved the performance of enzymes.The findings provided new insights into the development of enzyme immobilization and modification.