Thermal storage of beta-galactosidase immobilized in Inclusion Bodies

FLORES, SANDRA; PERILLO, MARÍA A.; NOLAN, VERONICA; SANCHEZ, JULIETA M

La Plata

Congreso; Reunion Anual de la Sociedad Argentina de Biofisica; 2018

Sociedad Argentina de Biofisica

Thermal storage of -galactosidase immobilized in Inclusion Bodies Flores, SS; Perillo, MA, Nolan, V y Sanchez, JM1,21Universidad Nacional de Córdoba, Facultad de Ciencias Exactas, Físicas y Naturales. ICTA y Departamento de Química, Cátedra de Química Biológica. Córdoba. Argentina. 2CONICET, Instituto de Investigaciones Biológicas y Tecnológicas (IIBYT), Córdoba, Argentina. jsanchezqa@gmail.comIn 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, storage temperature of IBsβ-Gal is critical in the reusability achievement. In this work we evaluate three storage temperatures (ST) on the structure-function of IBsβ-Gal. We demonstrate that the ST modulates the recovery of IBβ-Gal catalytic activity. At ST=4ºC the activity decrease, 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 was obtained. In full hydrated conditions (4ºC) the enzyme desorbed from IBs. In contrast, the non-hydrated (frozen) conditions prevent desorption process and consequently the activity lost. Moreover, the freezing rate (dehydration rate) seems to determine the enzyme integrity. At ST=-20ºC the lower dehydration rate could also allow unfolding/desorption of a protein population within IBs. Conversely, the fast dehydration (ST=-80ºC) preserved a high-quality conformation concomitant with an IBβ-gal restructuration in each dehydration process. The intrinsic fluorescence parameters demonstrate that max moves to lower values as the ST decrease from de desorbed β-Gal (max =349 nm) to the IBsβ-Gal sample conserved at -80ºC (max,-80 =341 nm). We propose that the best storage condition is at -80ºC where the microorganism contamination is prevented and the speedy of freezing would improve -Gal catalytic activity. Acknowledgment: to SeCyT, CONICET and Foncyt for financial support. MVN, MAP and JMS are members of CIC from CONICET.