Silk fibroin-based hydrogels as a protective matrix for stabilization of enzymes against pH denaturation

A significant challenge remains to protect proteins (including enzymes) from inactivation against pH. This study investigated stabilization against pH denaturation of carbonic anhydrase (CA) in silk fibroin (SF)-based hydrogels. CA was immobilized into SF-based hydrogels by a Ru(II) mediated photochemical method, giving rise to ∼100% immobilization efficiency and >60% activity of the free enzyme at the optimum pH value of 8. At an unfavorable acidic pH value of 3, the immobilized CA achieved >20% of the initial activity and ∼8% in the second cycle even after 12 h storage, while the free enzyme lost its activity completely. Fourier transform infrared spectroscopy (FTIR) results suggested that the protective effect of the SF-based hydrogels on the pH stability of CA could be attributed to unique structural feature of the SF molecules, enabling strong intermolecular interactions between SF and CA, and thus increasing protein rigidity against pH denaturation. The same approach was applied to lysozyme and xylanase, and achieved protein stabilization of both enzymes at an unfavorable basic pH value of 9. The SF-based hydrogels demonstrated great potential as a promising matrix for protein immobilization against pH denaturation.