Refolding of a denatured α-chymotrypsin and its smart bioconjugate by three-phase partitioning

-Chymotrypsin inactivated with 8 M urea and 100 mM dithiothreitol could be completely reactivated by subjecting it to three-phase partitioning (TPP). TPP consisted of adding 30% w/v ammonium sulfate and t-butanol (volume equivalent to aqueous solution of denatured -chymotrypsin) at 25°C. The activated -chymotrypsin was recovered as an interfacial precipitate between the upper organic and lower aqueous phase. It was found that this could be extended to a thermally inactivated smart bioconjugate of -chymotrypsin with Eudragit S-100 (a reversibly soluble-insoluble methmethacrylate). The thermally inactivated bioconjugate had to be further subjected to urea and dithiothreitol before refolding by three-phase partitioning. Ninety per cent of the activity of the bioconjugate could be recovered. The free enzyme and its bioconjugate which lost activity in the presence of 90% dioxane recovered 94 and 90% of their activities, respectively, by employing TPP. The refolded free enzyme and its bioconjugate were evaluated in terms of V _max/K _m and their fluorescence emission spectra.