In vitro and in vivo beta-Gal self-assembling improve the enzymatic activity and stability

FLORES, SANDRA SOLEDAD; PEDRO D. CLOP; MARIA A. PERILLO; VERÓNICA M. NOLAN; SANCHEZ, JULIETA MARÍA

Córdoba

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

In our laboratory we are interested in describing the functional plasticity of proteins intheir self assembled states driven by oligomerization and/or aggregation.The objective of this work is to study how the oligomeric/aggregated states of E. colibeta galactosidase (β-Gal) modulate the enzymatic activity with respect to themonomeric state.We achieved self-assembled β-Gal by two strategies: In vitro; controlling the presence ofMgCl2 in the β-Gal solution, and in vivo: isolating aggregate protein in the form ofinclusion bodies (IBs) from E. coli. The presence of oligomers and/or aggregates wereevidenced by means of analytical ultracentrifugation and by quasi-elastic lightscattering.In all the cases the activity was evaluated using lactose as a substrate and quantifyingglucose as the hydrolysis product. The reaction conditions were 37° C and pH 6.8maintained by 0.1 M phosphate buffer. In all self-assembled states, we measured thekinetic parameters by non-linear regression of the experimental data using the minimumsquare method. Besides we also evaluated the optimum temperature and pH of theenzyme in each format.We prove that Mg+2 is able to stabilize the tetrameric forms of the enzyme but nothigher oligomeric states. We could produce and isolate functional IBs of β-Gal of around1000 nm. We also demonstrate that the catalytic activity increases when the enzyme isself-assembled. On the other hand, studies of β-Gal activity against pH and temperatureare similar in the absence or in the presence of MgCl2. However, when we study the pHand temperature profiles of the β-Gal in IBs, we observe their higher stability comparedto the soluble enzyme.Our results contribute to highlighting the effect of protein-protein interaction on theprotein functionality beyond the origin and dimension of the protein supramolecularstructure.