CONICET | Buscador de Institutos y Recursos Humanos

In our laboratory we produce a recombinant β-Gal in E.coli. In controlled conditions we favor the overexpresion of β-Gal rearranged in the formed of inclusion bodies (IBsβ-Gal). Interestingly, the IBsβ-Gal exhibits not only higher activity but also higher resistance to temperature and pH inactivation with respect to the soluble β-Gal. As IBsβ-Gal represent an immobilized protein sample we are studying the properties of this naturally occurring structures as a reusable catalytic device. Therefore, the storage temperature of IBsβ-Gal is critical in its reusability achievement. In this work we evaluate three storage temperatures (ST) on the structure-function relationship of IBsβ-Gal. We demonstrate that the ST modulates the recovery of IBβ-Gal catalytic activity measured at 37ºC. At ST=4ºC the activity decreases, but in frozen conditions the activity grows up vs the conservation time. At ST=-20ºC we measured the lowest activities. Surprisingly at ST=-80ºC the highest IBβ-Gal activities were obtained. In full hydrated conditions (4ºC) the enzyme desorbed from IBs. In contrast, the non-hydrated (frozen) conditions prevent desorption process and consequently the enzyme activity lost. Moreover, the freezing rate, which affects the crystal structure of water, seems to determine the enzyme integrity. At ST=-20ºC the bigger size of ice crystals could be related to certain unfolding of a protein population within IBs. Conversely, the small crystal structures (ST=-80ºC) preserved a high-quality conformation concomitantly with a IBβ-gal restructuration, The intrinsic fluorescence parameters demonstrate that max shifts to lower values as the ST decrease from de desorbed β-Gal (max =349 nm) to the IBsβ-Gal sample kept at -80ºC (max,-80 =341 nm). We propose that the best storage condition is at -80ºC where the microorganism contamination is prevented and the freezing rate would improve -Gal catalytic activity.