Effect of PEG-induced molecular crowding on β-Gal activity and thermal stability: Optimization of beta galactosidase function for GOS production using milk lactose as substrate

Abstract

The yeast β-galactosidase or lactase [EC 3.2.1.23] (β-Gal) is a soluble enzyme capable of catalyzing lactose hydrolysis into its constitutive monosaccharides: glucose and galactose. In addition, and depending on the conditions of the environment, fundamentally high lactose concentration, β-Gal catalyzes the transglycosylation reaction whose products will be the Galacto-oligosaccharides (GOS). These molecules are considered prebiotics because they are not degraded in the digestive tract, reaching the intestine where they are a substrate for the growth of beneficial bacteria. GOS production is favored by: high lactose concentration, high reaction temperature and low water availability. These experimental conditions can be achieved if macromolecular crowded media (MCM) are used as the reaction medium. In this work we investigate the effect that molecular crowding induces on the activity and thermal stability of β-galactosidase from Kluyveromices lactis. PEG6000, a non-charged highly water-soluble polymer with wellknown effects on water dynamics was used to produce the crowded environment. The effect of PEG6000 on β-Gal kinetic parameters was studied using lactose as substrate. Results obtained showed that enzymatic activity is improved in MCM: the affinity increased while the Vmax remained unchanged. Temperature-dependent β-Gal activity profile was studied both in the absence or in the presence of molecular crowded agent in a range from 37 to 50 °C. Results obtained showed that β-Gal thermal activity profile was enhanced in molecular crowded environment. The enzyme maintained its activity when it was incubated at temperatures 5 degrees higher in the presence than in the absence of molecular crowding agent. Thermal inactivation kinetic was also studied: in this type of experiments, the enzyme was pre-incubated at 37 and 50 °C during different periods of time and after that, the enzymatic activity was measured in optimal conditions. Results obtained show again that molecular crowding conditions protect the enzyme from heat denaturation. In this case, it was observed that the enzyme maintains its activity even when it is subjected for a considerable period of time at high temperature when it is in the presence of the molecular crowding agent.