Effect of Polyethylene Glycol-Induced Molecular Crowding on the Enzymatic Activity and Thermal Stability of β-Galactosidase from Kluyveromyces lactis

Abstract

Here, we report the effect of polyethylene glycol (PEG 6000 )-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-Dgalactopyranoside followed a Michaelian kinetics at [PEG 6000 ] ≤ 25% w/v and positive cooperativity at higher concentrations (35% w/v PEG 6000 ). 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.

A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor

“…[42][43][44] There are, of course, additional mechanisms by which such crowding can affect activity that are electrostatic in nature. 45,46 This current study expands upon a recently reported method of semi-random chemical crosslinking to mimic the crowding of proteins by other proteins. 47 Although crosslinking is widely used to trap specific proteinprotein interactions, [48][49][50][51] we found that commercial crosslinkers can link noninteracting proteins (of various net charge) in a quasi-random fashion.…”

“…One subsite, B1, is composed of Thr45, Asp83, Phe120, and Ser123 which utilize hydrogen bonding and van der Waals forces to selectively bind only pyrimidine bases 42–44 . There are, of course, additional mechanisms by which such crowding can affect activity that are electrostatic in nature 45,46 …”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…Addition of DMSO but also of glycerol or ethylene glycol serve as cryoprotectants preventing protein solutions from freezing at −20 • C and allowing multiple use of a unique sample without freeze-thaw cycles [38,42]. Adding polymers in solution such as poly(ethylene glycol) (PEG), alginate, or chitosan maintains a hydration shell around the protein according to the exclusion mechanism [38,[43][44][45]. Addition of polymers also prevents protein aggregation by modifying protein-protein interactions and increasing medium viscosity, thus decreasing enzyme motion.…”

Atomistic Simulation of Lysozyme in Solutions Crowded by Tetraethylene Glycol: Force Field Dependence

“…A number of factors such as shape, size, concentration, and composition of molecular crowders were reported to be influential [ 18 , 24 , 25 ]. Polyethylene glycol (PEG) was often used as a crowder to mimic the cell-like environment [ 6 , 26 , 27 , 28 , 29 , 30 ]. For instance, Nolan and coworkers reported a noticeable increase in the thermal stability and catalytic activity of β -galactosidase in the presentence of PEG with a concentration of 25% and 35% ( w/v ) [ 29 ].…”

“…Polyethylene glycol (PEG) was often used as a crowder to mimic the cell-like environment [ 6 , 26 , 27 , 28 , 29 , 30 ]. For instance, Nolan and coworkers reported a noticeable increase in the thermal stability and catalytic activity of β -galactosidase in the presentence of PEG with a concentration of 25% and 35% ( w/v ) [ 29 ]. Wang and coworkers indicated that the increase in the residual activity of lysozyme was ascribed to the fact that the addition of PEG suppressed enzyme aggregation via a strong PEG–protein interaction and stabilized the protein secondary structure somewhat [ 6 ].…”