Kinetic and spectroscopic behaviour of a lipase-microgel derivative in aqueous and micellar media

The kinetic and spectroscopic behaviour of the native and immobilized isolipase B from C. rugosa were compared in water and in micelles. A negative interaction of the lipase with the microgel matrix reduced the catalytic efficiency by 34 times and produced a blue shift in the protein emission spectrum in water. However, the immobilization caused the opposite variation in its kinetic and spectrofluorimetric behaviour in the micellar medium. The crosslinked polymer (hydrodynamic radius 400 +/- 20 Angstrom) modified the micellar aggregate. It increased the water activity of poorly hydrated micelles (small W-0 = [H2O]/[surfactant] values). The emission fluorescence of 2-naphtol, solubilized through the tensioactive molecules in the micellar interface, varied according to the increase of the micellar size. This microgel increased the polarity of the micellar aqueous phase, as shown in the red shifted spectrum of 1-naphthol-6,8-disulphonate. The protein was sensitive to this change of polarity and its fluorescence emission was also red shifted in the presence of the microgel. These changes on the micellar droplets and the subsequent modification on the Trp environment of the immobilized protein affected the k(cat) and K-m,K-app values. The polymer decreased the stability of lipase B in water. This negative enzyme-microgel interaction was favoured in the micelles of the smallest W-0 values. A model to explain the activity and stability of the enzyme-microgel system in micelles has been proposed here. This model considers the structural variations in the micelle because of the microgel (A(w), droplet size). (C) 1998 Elsevier Science B.V.