β-sheet to α-helix conversion and thermal stability of β-Galactosidase encapsulated in a nanoporous silica gel

Abstract The effect on protein conformation and thermal stability was studied for β-Galactosidase (β-Gal) encapsulated in the nanopores of a silicate matrix (E β-Gal ). Circular dichroism spectra showed that, compared with the enzyme in buffer (S β-Gal ), E β-Gal exhibited a higher content of α-helix structure. Heating E β-Gal up to 75 °C caused a decrease in the content of β-sheet structure and additional augments on E β-Gal components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with S β-Gal . Steady state fluorescence spectroscopy analysis evidenced an E β-Gal structure less compact and more accessible to solvent and also less stable against temperature increase. While for S β-Gal the denaturation midpoint (Tm) was 59 °C, for E β-Gal it was 48 °C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65 °C and E β-Gal did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changes on the tertiary/cuaternary structure of E β-Gal leading to the loss of thermal stability and enzymatic activity.