SPECTROSCOPIC AND KINETIC-STUDIES OF LIPASES SOLUBILIZED IN REVERSE MICELLES

The conformation and activity of three different lipases have been stu died in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosucci nate (AOT) in isooctane. In the case of human pancreatic lipase, the c onformation of the polypeptide chain-as judged from far-UV circular di chroism measurements-is only slightly altered after the enzyme is tran sferred from a bulk aqueous solution into the microenvironment of reve rse micelles. Significant spectral changes in the near-UV circular dic hroism and fluorescence spectrum indicate, however, that the solvation of aromatic amino acid side chains is considerably different in rever se micelles. Conversely, the circular dichroism spectra of the lipases from Candida rugosa and Pseudomonas sp. are considerably different in reverse micelles, compared with the spectra in aqueous solution, indi cating that both enzymes loose the native structure at the water/AOT/o il interface. Bound substrate and/or product can prevent this denatura tion. While Pseudomonas sp. and human pancreatic lipase are inhibited by tetrahydrolipstatin (THL), the lipase from Candida rugosa is not. T hese data, together with additional activity and inhibition data, indi cate that the micellar microenvironment accentuates the difference bet ween the different enzymes in terms of the relation structure/activity .