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.

ROSALES, J; PERILLO, M A; NOLAN, V.

Buenos Aires

Congreso; Reunion Anual SAB; 2021

Sociedad Argentina de Biofísica

The yeast β-galactosidase or lactase [EC 3.2.1.23] (β-Gal) is a soluble enzyme capable ofcatalyzing lactose hydrolysis into its constitutive monosaccharides: glucose and galactose.In addition, and depending on the conditions of the environment, fundamentally highlactose concentration, β-Gal catalyzes the transglycosylation reaction whose products willbe the Galacto-oligosaccharides (GOS). These molecules are considered prebioticsbecause they are not degraded in the digestive tract, reaching the intestine where theyare a substrate for the growth of beneficial bacteria. GOS production is favored by: highlactose concentration, high reaction temperature and low water availability. Theseexperimental conditions can be achieved if macromolecular crowded media (MCM) areused as the reaction medium. In this work we investigate the effect that molecularcrowding induces on the activity and thermal stability of β-galactosidase fromKluyveromices lactis. PEG6000, a non-charged highly water-soluble polymer with wellknowneffects 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 affinityincreased while the Vmax remained unchanged. Temperature-dependent β-Gal activityprofile was studied both in the absence or in the presence of molecular crowded agent ina range from 37 to 50 °C. Results obtained showed that β-Gal thermal activity profile wasenhanced in molecular crowded environment. The enzyme maintained its activity when itwas incubated at temperatures 5 degrees higher in the presence than in the absence ofmolecular crowding agent. Thermal inactivation kinetic was also studied: in this type ofexperiments, the enzyme was pre-incubated at 37 and 50 °C during different periods oftime and after that, the enzymatic activity was measured in optimal conditions. Resultsobtained show again that molecular crowding conditions protect the enzyme from heatdenaturation. In this case, it was observed that the enzyme maintains its activity evenwhen it is subjected for a considerable period of time at high temperature when it is in thepresence of the molecular crowding agent.