CONICET | Buscador de Institutos y Recursos Humanos

β-D-galactosidase[EC 3.2.1.23] (β-Gal)is a soluble enzyme capable of catalyzing lactose hydrolysis into itsconstitutive monosaccharides: glucose and galactose. β- Gal has been extensively studied because ofits nutritional, biotechnological and therapeutic impact. Not only in thecellular milieu but also in situations of technological interest like duringencapsulation, the activity of β-Gal has to be evaluated in crowded systems. β-Gal from kluyveromices lactis was historicallyisolated as a dimeric active protein of about 250 kDa. But, recent researcheshave described that in some conditions, like those generated by the presence ofcrowding conditions in the interior of a non-denaturing electrophoresis gel, atetrameric form appear. This tetramer has lower activity than the dimericstate. The objective of the present work was to evaluate the effect ofmolecular crowding on β-Galstructure and the relationship with its enzymatic activity modulation. PEG6000,a non-charged highly water-soluble polymer with well-known effects on waterdynamics was used to produce the crowded environment. The enzymatic reactionwas evaluated by measuring kinetic parameters of β-Gal against an artificial substrate(ONPG).Protein conformation and thermal stability were analysed by fluorescencespectroscopy. Results showed that molecular crowding induced changes on kineticparameters of β-Gal: atlow molecular crowding agent concentration, an enhancement on enzymaticactivity was observed, while at high crowding agent concentration, aqualitative change from a michaelian to a sigmoidal behavior was observed. Thestudies on protein conformation showed that molecular crowding affected β-Gal structure: changes onfluorescence emission spectra and protein calorimetric profile were observed.Changes in protein compactness and hydration could be the responsible for thequalitative change behaviour observed at the highest molecular crowdingassayed.