A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase.
The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60 degrees C. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol(-1) for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7 degrees C and the activation energy increases to 10.2 kcal mol(-1). This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60 degrees C were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60 degrees C in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction 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 underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation.
Melo
E P
EP
Unidade de Ciências e Tecnologias Agrárias, Universidade do Algarve, Faro, Portugal. emelo@ualg.pt
Taipa
M A
MA
Castellar
M R
MR
Costa
S M
SM
Cabral
J M
JM
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002861:Chromobacterium / Q000201:enzymology*; D002942:Circular Dichroism; D004795:Enzyme Stability; D008027:Light; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D014364:Tryptophan; D008049:Lipase