Structure changes of the enzime β-Galactocidase induced by the encapsulation in a mesoporous silicate-gel matrix

OCHOA A; BURGOS I; PERILLO MA

Congreso; III LAFeBS- IX IberoAmerican Congress of Biophysics ? SAB2016; 2016

Thesynthesis of silicate gels by the sol-gel method under mild conditions of pHand temperature, enables the entrapment of proteins with biological activitywhile allows the diffusion of small molecules through the mesoporous matrix. Inprevious studies, we determined that the encapsulated enzyme b-Galactosidase (Eb-Gal) has equal or higher catalytic activitythan the soluble enzyme, and we proposed that the structuring of watermolecules within the gel nano-pores have significant importance in thedifferences observed for the hydrolysis of ONPG in fresh and aged gels fordifferent periods of time1. This observation was supported by 1H-NMRexperiments. In the present work, we studied the changes in the conformationalstructure of Eb-Galconfined in the silicate gel employing fluorescence spectroscopy and circulardichroism (DC). The emission spectrum of fluorescence Eb-Gal evidenced a shift of the center of spectralmass of 3 nm towards longer wavelengths compared to the soluble enzyme. Thiseffect indicates that the tryptophans of the encapsulated protein are in a lesshydrophobic environment (higher dielectric constant)  which could be due to a loss of compactionoverall the tertiary structure. Furthermore, DC data evidenced changes in thesecondary structure of Eb-Galwith respect to the soluble b-Galmainly reflecting an increased proportion of amino acids in a-helix structure. Concluding, the presentresults suggest that the effects of confinement on Eb-Gal activity might be at least partly due tochanges in the protein conformation.