Fabrication of single carbonic anhydrase nanogel against denaturation and aggregation at high temperature

A two-step procedure to encapsulate a single bovine carbonic anhydrase (BCA) molecule into a spherical nanogel was proposed. BCA was reacted first with N-acryloxysuccinimide to introduce surface vinyl groups, followed by in-situ aqueous polymerization. Characterization of the nanogel by dynamic light scattering, transmission electron microscopy, and atomic force microscopy confirmed that each nanogel contained a single BCA molecule. The encapsulated BCA maintained 70% of the activity of its free counterpart, but exhibited an increase in the molten temperature from 64 to 81 degrees C demonstrated by differential scanning calorimetry and an extension of the half-life from less than 3 to over 90 min at 75 degrees C. Circular dichroism spectroscopy indicated that the encapsulation and the multi-point covalent linkage between BCA and the polymer shell strengthened the secondary structure and thus inhibited the aggregation at high temperature. The uniform BCA nanogel with enhanced structural stability against denaturation and aggregation expands the applications of BCA catalysis, particularly those carried out at high temperatures.