Zwitterionic poly(carboxybetaine) microgels for enzyme (chymotrypsin) covalent immobilization with extended stability and activity

There is emerging evidence that biocompatible zwitterionic materials can prevent nonspecific interactions within protein systems and increase protein stability. Here, a zwitterionic microgel was synthesized from poly (carboxybetaine methyl methacrylate) (pCB) using an inverse emulsion, free radical polymerization reaction technique. The microgel was loaded with a model enzyme, α‐chymotrypsin (ChT), using a post‐fabrication loading technique. A reaction scheme was developed and studied for covalent immobilization of ChT within the microgel. Confocal laser microscopy studies showed that immobilized ChT (i‐ChT) was distributed within the hydrogel. The enzyme‐immobilized microgels showed excellent reusability (72% of its initial activity after 10 uses) and could undergo several freezing/drying/rehydration cycles while retaining enzymatic activity. The i‐ChT activity, half‐life, and conformational stability were studied at varying pH and temperatures with results compared to free ChT in buffer. ChT immobilized within pCB hydrogel showed increased enzymatic stability as observed by a 13°C increase in the temperature at which i‐ChT loses activity compared to free ChT. Furthermore, enzyme half‐life increased up to seven‐fold for the pCB immobilized ChT, and the increased stability resulted in higher activity at elevated pH. The i‐ChT was most active at pH of 8.5 and was partially active up to the pH of 10.2.