Erschienen:
[Erscheinungsort nicht ermittelbar]: Faculdade de Ciências e Tecnologia, 2012
Sprache:
Englisch
Identifikator:
Entstehung:
Hochschulschrift:
Dissertation, Faculdade de Ciências e Tecnologia, 2012
Anmerkungen:
Beschreibung:
Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica ; Cutinase from Fusarium solani pisi was entrapped in sol-gel matrices of composition 1:5 tetramethoxysilane:n butyltrimethoxysilane (TMOS/BTMS), and its specific activity was measured as a function of enzyme loading in the range of ca. 0.1 % to 7 %. To elucidate the pronounced increase in specific activity that was observed as enzyme loading decreased, an active site titration technique was applied, based on enzyme inactivation by the inhibitor paraoxon. It was found that the number of available enzyme activate sites decreased as enzyme loading in the matrices increased, suggesting that enzyme aggregation occurred in the more heavily loaded sol-gel supports. The impact of enzyme loading on the packing of cutinase inside the matrices was studied by fluorescence spectroscopy based on the single tryptophan residue of cutinase. Fluorescence anisotropy decay measurements led to relatively low order parameters, indicating that the restrictions imposed on tryptophan by its microenvironment are small, which is consistent with the fact that tryptophan is only partially exposed on the cutinase molecule. The order parameter increased as enzyme loading increased, indicating loss of mobility of the tryptophan in the more heavily loaded sol-gel matrices, which is consistent with enzyme aggregation. The effect of enzyme loading on solvent mobility within the matrix was assessed by determining the self-diffusion coefficient of the solvent using High Resolution Magic Angle Spinning (HR-MAS) Pulsed Field Gradient Spin Echo (PFGSE) NMR spectroscopy. The diffusion coefficients determined indicate that enzyme loading has a negligible effect on solvent mobility. We also studied the thermal stability of cutinase entrapped in 1:5 TMOS/BTMS sol-gel matrices, at enzyme loadings in the range of ca. 0.1 % to 4 %. We found that submitting the entrapped enzyme to 40 ºC for 24 h had a positive effect on enzyme specific activity and that thermal treatment for the same length of time at higher temperatures no longer brought about any activity enhancements. The impact of temperature on enzyme specific activity was more pronounced in the case of more dilute matrices, whose activity nonetheless became very similar to that of the more heavily loaded matrices after treatment at 100 ºC. Steady-state fluorescence measurements did not reveal an increase in fluorescence emission with exposure to temperature, and time resolved fluorescence showed that the fluorescence decay times of the single tryptophan of cutinase did not increase as the temperature of incubation of the sol-gel matrices increased. Both findings indicate that unlike what happens with cutinase in solution, entrapped cutinase submitted to higher temperatures does not suffer a denaturation process characterized by ample conformational mobility in the region of the tryptophan residue. This protective effect is not mediated by enzyme packing, known to occur at higher enzyme loadings, because not even the more dilute matrices showed evidence of enzyme denaturation. The matrices are hydrophobic and have little water. A certain extent of enzyme dehydration upon thermal treatment at moderate temperatures could be beneficial for enzyme conformational mobility and hence enzyme activity, as it was also observed in this study, and become detrimental to enzyme function at higher temperatures. This would explain the similarity of enzyme specific activity values after thermal treatment at the highest temperature tested, irrespective of enzyme loading. ; Fundação para a Ciência e a Tecnologia