Guanidine Hydrochloride-Induced Denaturation of Pseudomonas

The guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase (PCX,) was studied at pH 7 by monitoring the changes in the fluorescence and circular dichroism of the enzyme. The denaturation was irreversible as a whole, and the addition of Ca2+ ions decreased the velocity of the denaturation. The denaturation process was well explained consistently by a two-step mechanism, as follows: Ca2+ N-Ca24 D,-»DF where N is the native state of PCL, D, an intermediate denatured-state which can be refolded into the native state, and DF the final denatured-state that can not be renatured. Ethanol (10%) increased the denaturation velocity by decreasing the refolding step, D, + Ca2+-»N-Ca2+, which would be caused by the stabilization of D, by ethanol. Key words: denaturation, guanidine hydrochloride, lipase from Pseudomonas cepacia, unfolding. Lipase [EC 3.1.1.3] catalyzes the hydrolysis of triglycer-ides to produce free fatty acids and glycerols. This enzym