Novel Zwitterionic Polymers For Enzyme Immobilization And Their Interactions With Enzymes

Enzyme is a kind of biocatalyst with high efficiency and specificity,but industrial applications of natural enzymes are often hampered by their drawbacks including instability and difficulty in recycled use.Enzyme immobilization is one of the most effective methods to improve the stability and reusability of enzyme,and the structures and surface properties of support materials affect the performance of immobilized enzymes.Therefore,in this work,two novel zwitterionic polymers were used to modify the immobilized carriers to improve the performance of immobilized enzyme,and then the effect of a water-soluble zwitterionic polymer on various enzyme activities and structures was studied.Firstly,two novel zwitterionic polymer-grafted silica nanoparticles(SNPs-p OD and SNPs-pID)were fabricated and used as supports to covalently immobilize Candida rugosa lipase(CRL).The polymers,p OD with cetane side chains and pID(water-soluble)with short alkyl side chains,were the products of the reaction of poly(maleic anhydride-alt-1-octadecene)or poly(isobutylene-alt-maleic anhydride)and N,N-dimethylethylenediamine,respectively.The immobilized enzyme(SNPs-p OD-CRL)exhibited a specific activity 2.36 times higher than the free enzyme,which was higher than SNPs-pID-CRL(1.21 times).After heat teatment of 3 h at 50^oC,SNPs-pID-CRL still retained 73%of its original activity,while the residual activities of SNPs-p OD-CRL and free CRL decreased to 31%and 11%,respectively.In addition,the two immobilized enzymes displayed improved p H tolerance and storage stability,as well as good reusability.In order to expand the application of pID,two CRL-pID conjugates with different grafting densities were prepared by directly bonding pID with CRL via the reaction between cyclic anhydride and amino group.In comparison with native CRL,both modified enzymes exhibited an activity 2.2 times higher than the free enzyme,and showed an increase in the maximum reaction rate(V_max)and a decrease in the Michaelis constant(K_m).Moreover,the thermostability and p H tolerance of the modified enzymes were significantly improved.Spectroscopic studies showed the modification of pID results in a blue-shift of the maximum fluorescence emission peak and the increases in?-helix and?-fold contents of the enzyme,indicating the change in the microenvironment and conformation of the enzyme.In order to reveal the mechanism of the effect of pID on enzyme activity and structure,the interactions between pID and CRL,lysozyme(Lys),catalase(CAT)and laccase(Lac)were systematically studied.The results showed that pID reduced Lys activity at high concentrations,but it increased the activity of the other three enzymes,especially CRL.Besides,pID was not conducive to the stability of Lys and CAT,but could significantly enhance the thermal stability of CRL and Lac.Fluorescence spectroscopic analysis showed that pID effectively quenched the intrinsic fluorescence of CRL,Lys and CAT through static quenching mechanism.pID mainly bound to enzymes through hydrophobic and electrostatic interactions,and there was a single binding site of pID with the enzyme in order of binding capacity CRL>Lys>CAT.The results of synchronous fluorescence spectrum indicated that under the interaction with pID,only the microenvironment structure of tryptophan residues in CRL and Lys were altered,while the microenvironments of tyrosine and tryptophan in CAT molecule changed.These results suggest that pID has different interaction mechanisms with different enzymes and it is not a universal protein stabilizer and that its efficacy depends on the protein structure.