Synthesis Of Polyethyleneimine-grafted Silica Nanoparticles And Its Application In Immobilized Enzyme Field

Biocatalysis technology is considered to be the best strategy to solve the problem of environmental pollution,resource and energy shortage,it also provides a novel idea for capturing and absorbing CO₂ in the environment efficiently.However,the stability of enzymes is very poor and they are difficult to reuse,which greatly limit the application of biocatalysts.In order to enhance the stability of enzymes and realize cyclic utilization,a novel Nano-scale carrier was constructed and applied to the immobilization of Carbonic Anhydrase?CA?and Phosphoenolpyruvate carboxylase?PEPcase?.Silica nanoparticles?SNPs?were covalently grafted with Polyethyleneimine?PEI?to prepare a series of Nano-scale carriers,which were named as SNPs-PEI,and BSA was used as a model protein to investigate the adsorption ability of SNPs-PEI.Compared to the microsphere PGMA-PEI,SNPs-PEI could adsorb protein more efficiently,and the maximum adsorption capacity of BSA was 260mg/g,which almost is 10 times that of the PGMA-PEI.The present results demonstrate that SNPs-PEI can be used for immobilization of enzymes.Next,the SNPs-PEI was used to immobilize CA by electrostatic interaction,and the activity and stability of the immobilized enzyme?SNPs-PEI@CA?were tested.The activity of SNPs-PEI@CA was up to 76%of the free enzyme,and its pH,temperature and storage stability were significantly enhanced.Especially,the SNPs-PEI@CA was shown that about 66.7%of the initial activity could be retained after 8 cycles of uses.Finally,the SNPs-PEI@CA was investigated for biocatalytic hydration of CO₂ and its precipitation as CaCO₃.The results show that the hydration efficiency of immobilized enzyme?60.7%?is very close to that of free enzyme?66.5%?because the adsorption and transformation of CO₂ by PEI enhanced the capability of immobilized enzyme to hydrate CO₂.Similarly,the SNPs-PEI was used as carrier to immobilize PEPcase.What's more,the activity,stability of the immobilized enzyme?SNPs-PEI@PEPcase?and the effects of substrate concentration on the activity of enzyme were investigated.The activity of SNPs-PEI@PEPcase was 4 times as much as that of free enzyme,and the stability was enhanced significantly.After 8 cycles,the immobilized enzyme still could maintain35.7%of the initial activity.Finally,the adsorption and transformation of CO₂ by PEI was verified by using CO₂ as the direct substrate.The result show that PEI can simulate the catalytic process of CA and convert CO₂ to HCO₃^-,which could be used as substrate of PEPcase.In conclusion,the research results provided a reference for the immobilization of CA and PEPcase and provides a novel idea for capturing and absorbing CO₂.What's more,the results could lay a foundation for the further study of the CA/PEPcase multienzymatic system.