Superactive β-galactosidase inclusion bodies.

Graphical abstract Highlights • Distinct kinetic features of IB β-Gal at in vitro conditions were described. • IB β-Gal was more efficient than S β-Gal to catalyse lactose hydrolysis. • Protein-protein interactions in IB β-Gal preserved β-Gal activity at extremes pH and T. • The structure-function relationship of D β-Gal was similar to that of S β-Gal. Abstract Bacterial inclusion bodies (IBs) were historically considered one of the major obstacles in protein production through recombinant DNA techniques and conceived as amorphous deposits formed by passive and rather unspecific structures of unfolded proteins aggregates. Subsequent studies demonstrated that IBs contained an important quantity of active protein. In this work, we proved that recombinant β-galactosidase inclusion bodies (IB β-Gal) are functional aggregates. Moreover, they exhibit particular features distinct to the soluble version of the enzyme. The particulate enzyme was highly active against lactose in physiological and in acid pH and also retained its activity upon a pre-incubation at high temperature. IB β-Gal washing or dilution induced the spontaneous release of active enzymes from the supramolecular aggregates. Along this process, we observed a continuous change in the values of several kinetic parameters, including specific activity and Michaelis–Menten constant, measured in the IB β-Gal suspensions. Simultaneously, IB β-Gal turned into a more heterogeneous population where smaller particles appeared. The released protein exhibited secondary structure features more similar to those of the soluble species than to the aggregated enzyme. Concluding, IB β-Gal represents a reservoir and packed source of highly active and stable enzyme. [ABSTRACT FROM AUTHOR]