Interfasial behavior of β-galactosidases

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

Rosario

Congreso; Reunion Anual de la Sociedad Argentina de Biofisica; 2022

Sociedad Argentina de Biofísica

β-galactosidase (β-Gal) is an enzyme that hydrolyzes lactose, its natural substrate. Thislactase is of great biotechnological interest and is used in the dairy industry,pharmacology and molecular biology. In our laboratory we are interested in finding theoptimal environmental condition to improve the structure-function of the protein.Currently, we produce a recombinant β-Galactosidase (β-GalHis) with 6 histidine residuesin the carboxyl terminus that facilitates its subsequent purification by metal ion affinitychromatography.In this work we tested the protein in heterogeneous systems such as multilamellar vesicles(MLVs) dispersions and monomolecular layers of different lipid composition (pure Egg-PC,DOPG and in a mixture of Egg-PC-DOPG (ratio 80:20)).We compare the catalytic activity against lactose and the thermal stability of β-GalHis andits native counterpart (β-GalWT) in the presence and absence of multilamellar vesicles(MLVs) dispersion. In addition, we evaluated the ability of the enzyme to penetrate to thelipid monomolecular layers.β-GalHis present lower activity and thermal stability than β-GalWT that could be explainedby a particular conformational evidenced by the intrinsic fluorescence of the protein. Ourresults demonstrated that both enzymes, but mainly the β-GalHis, tended to be activatedand thermoprotected up to 50ºC by MLVs. These results are in accordance with thedetermination of the Tm values. The fact that β-GalHis was highly activated by neutral PC(Egg-PC) could be related with the highest penetration of β-GalHis to Egg-PCmonomolecular layers.