jf0c02316_si_001.pdf (1.06 MB)
Effect of Polyethylene Glycol-Induced Molecular Crowding on the Enzymatic Activity and Thermal Stability of β‑Galactosidase from Kluyveromyces lactis
journal contributionposted on 2020-08-06, 22:21 authored by Verónica Nolan, Alejandro Collin, Carolina Rodriguez, María A. Perillo
Here, we report the effect of polyethylene glycol (PEG6000)-induced molecular crowding (MC) on the catalytic activity and thermal stability of Kluyveromyces lactis β-galactosidase (β-Gal). The β-Gal-catalyzed hydrolysis of o-nitrophenyl-β-d-galactopyranoside followed a Michaelian kinetics at [PEG6000] ≤ 25% w/v and positive cooperativity at higher concentrations (35% w/v PEG6000). Compared with dilute solutions, in the MC media, β-Gal exhibited stronger thermal stability, as shown by the increase in the residual activity recovered after preincubation at high temperatures (e.g., 45 °C) and by the slower inactivation kinetics. Considering the effects of water thermodynamic activity on the reaction kinetics and protein structure and the effect of the exclusion volume on protein conformation, we suggest that changes in the protein oligomerization state and hydration could be the responsible for the behavior observed at the highest MC levels assayed. These results could be relevant and should be taken into account in industrial food processes applying β-Gal from K. lactis.