Effect of PEG-induced molecular crowding on β-Gal transgalactosilation activity

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 kinetic parameters and transgalactosilation activity of βgalactosidase from Kluyveromices lactis. PEG6000, a non-charged highly water-soluble polymer with well-known 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 modulated by in MCM. At low molecular crowding agent concentration, an improvement on enzymatic activity was observed: KM decreases (affinity improves) and Vmáx enhances when MCM is increased from 0 to 25 %W/V. On the other hand, at high crowding agent concentration (35% W/V), a qualitative change from a michaelian to a sigmoidal behavior was observed. Temperature-dependent β-Gal transgalactosilation activity was studied at different initial substrate concentration and in the absence or in the presence of molecular crowded agent. Results obtained showed that, at low substrate concentration, MCM protects the enzyme against denaturation and at higher substrate concentration, a synergic effect between crowding agent and substrate occurs. At the highest temperature assayed it was found that the enzyme maintains its activity when it is in the presence of both high substrate and crowder, but it lost its activity in dilute conditions.