A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase

The thermal stability of the lipase from Chromobacterium viscosum was asses sed by deactivation (loss of activity), fluorescence, circular dichroism (C D) and static light scattering (SLS) measurements. Lipase fluorescence emis sion is dominated by the tryptophyl contribution. An increase in the tyrosy l contribution from 2 to 16% was only observed upon prolonged incubation at 60 degreesC. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues i n the native structure have an activation energy of 1.9 kcal mol(-1) for te mperature-dependent non-radiative deactivation of the excited state. A stru ctural change occurs at approximately 66.7 degreesC and the activation ener gy increases to 10.2 kcal mol(-1). This structural change is not characteri zed by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation a t 60 degreesC were assessed by fluorescence, CD and SLS measurements. CD sp ectra show that both secondary and tertiary structures remain native-like a fter incubation at 60 degreesC in spite of the fluorescence changes observe d (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurement s together with the CD data show that deactivation may be due to protein as sociation between native molecules. Deactivation and the decrease on the fr action of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an un derestimation of the deactivation. Reactivation upon dilution during the ac tivity assay and substrate-induced reactivation due to lipase interfacial a dsorption are possible causes for this underestimation. (C) 2000 Elsevier S cience S.A. All rights reserved.