CONICET | Buscador de Institutos y Recursos Humanos

Previously we demonstrated that the activity of a soluble wild-type E. coli β-galactosidase (b-Galwt) against both lactose (the natural substrate) as ortho-nitrophenilgalactopyranoside (ONPG, artificial substrate) increases in the presence of multilamellar vesicles (MLVs) composed of neutral and charged phospholipids. The aim of this study was to compare the activity of a recombinant b-Gal (b-Gal-His6) against two different substrates in the presence of MLVs of different lipid composition. b-Gal-His6 was overexpressed in E.coli, and the six histidine residues (His-tag) fused to the carboxyl terminus facilitated purification by ion metal affinity chromatography (IMAC). The enzyme activity was measured by visible spectrophotometry, in the absence or presence of MLVs of pure egg phosphatidylcholine (EPC interface zwitterion) or at 80:20 molar ratio with dioleoyl phosphatidyl glycerol (EPC80/DOPG20) (negative interface). Kinetic parameters were determined by fitting the michaelian model to the experimental data using nonlinear regression. Our results showed that the enzyme activity was more efficient against the artificialsubstrate, ONPG compared to lactose (kcat/KMONPG > kcat/KMLactosa ) as previously described for other beta galactosidase. An activation of the enzymatic activity was observed in the presence of lipid interfaces against both substrates. Moreover, the presence ofMLVs negatively affected the substrate affinity (KMMLVs > KMcontrol). This last effect and the interfacial activation were enhanced by the presence of charged interfaces favored by electrostatic interactions mediated by the presence of His residues from the enzyme. The nature of the substrate not qualitativelyaffected the kinetics of the reaction catalyzed by b-Gal-His6 in the presence of lipid interfaces.