Conferencia Plenaria: In situ/operando studies of enzymes by FTIR-ATR spectroscopy

COLLINS, SEBASTIAN

Santiago del Estero

Simposio; Actas Reunión Anual Biofisica (SAB2015); 2015

Infrared spectroscopy inattenuated total reflection (ATR) mode allows the study of processes at theliquid interfaces. In this talk, the theoretical basis and the technical and practicalaspects of its implementation to perform investigations under in situ andoperando conditions, including the design of micro-reactors cells andtechniques for data acquisition, will be discussed. Particularly, a newtechnique based on experiments of modulation excitation spectroscopy (MES) willbe presented.Examples of works performed byour group investigating pure and supported lipases will be shown. ATR allowsthe study of conformational changes of enzymes from the band distribution ofthe Amide I signal. To achieve this, protocols of isotopic exchange with D2Omust be developed in order to remove spectral interferences. Using thesemethods, the synthesis, characterization and reactivity of Rhizomucor meiheilipase supported on pure chitosan and functionalized with glutaraldehyde wasinvestigated. Operando ATR showed the activity of these biocatalysts in thehydrolysis reaction of ethyl stearate. Additionally, we were able to identifythe acyl intermediate adsorbed in the active site of lipase. In other study,the stability of papain from Carica papaya was investigated. The effect on thesecondary structure of extraction solvents for this enzyme was shown. Finally,the effect of hydrophobic/hydrophilic solvents on the secondary structure andthe active site of lipases and its correlation with enzyme activity will bepresented.