Effects of encapsulation in sol-gel silica glass on esterase activity, conformational stability, and unfolding of bovine carbonic anhydrase II

Bovine carbonic anhydrase II retains its overall conformation when encapsulated in silica monoliths by the sol-gel method. Upon gradual heating the enzyme in solution precipitates at 64 degrees C, whereas the encapsulated enzyme does not; it unfolds, with the nominal melting temperature of 51 +/- 3 degrees C. Even at 74.0 degrees C, the encapsulated enzyme is only similar to 77% unfolded, but it does not refold upon cooling. Upon treatment with guanidinium chloride, the degree of enzyme unfolding is 100% in solution but only similar to 83% within the silica matrix. Again, the enzyme does not refold upon removal of the denaturing agent from the glass. The glass matrix constrains the motions of the encapsulated protein molecules and prevents both their full unfolding and refolding. The former may be taken for "stabilization", the later for "destabilization" of the native conformation. Evidently, the effect of the glass on the encapsulated protein cannot be described in these general terms. The encapsulated enzyme obeys the Michaelis-Menten kinetic law as it catalyzes hydrolysis of p-nitrophenyl acetate. The apparent Michaelis constant (K'(M)) is practically the same in the glass and in solution, but the apparent turnover number (k'(cat)) and specific activity for the encapsulated enzyme are only 1-2% of these values for the enzyme in solution. Because the substrate diffuses slowly into silica pores, most of the catalysis is due to the enzyme embedded near the surfaces of the glass monolith. Effect of encapsulation on structure and activity of proteins should be studied in quantitative detail before protein-doped glasses can be used as reliable biosensors, heterogeneous catalysts, and composite biomaterials.