Superactive beta;-galactosidase inclusion bodies

FLORES, SANDRA S.; NOLAN, VERÓNICA; PERILLO, MARÍA A.; SÁNCHEZ, JULIETA M.

COLLOIDS AND SURFACES B-BIOINTERFACES

ELSEVIER SCIENCE BV

Año: 2018 vol. 173 p. 769 - 775

0927-7765

Bacterial inclusion bodies (IBs) were historically considered one of the major obstacles in protein productionthrough recombinant DNA techniques and conceived as amorphous deposits formed by passive and rather unspecificstructures of unfolded proteins aggregates. Subsequent studies demonstrated that IBs contained animportant quantity of active protein. In this work, we proved that recombinant β-galactosidase inclusion bodies(IBβ-Gal) are functional aggregates. Moreover, they exhibit particular features distinct to the soluble version ofthe enzyme. The particulate enzyme was highly active against lactose in physiological and in acid pH and alsoretained its activity upon a pre-incubation at high temperature. IBβ-Gal washing or dilution induced the spontaneousrelease of active enzymes from the supramolecular aggregates. Along this process, we observed acontinuous change in the values of several kinetic parameters, including specific activity and Michaelis?Mentenconstant, measured in the IBβ-Gal suspensions. Simultaneously, IBβ-Gal turned into a more heterogeneous populationwhere smaller particles appeared. The released protein exhibited secondary structure features more similarto those of the soluble species than to the aggregated enzyme. Concluding, IBβ-Gal represents a reservoirand packed source of highly active and stable enzyme.