Project Title: "Enzyme kinetics studies under pressure"

Author: Eduardo Breno Ribeiro dos Santos, Universidade Estadual Paulista “Júlio de Mesquita Filho” UNESP, Brazil

Host Supervisor: Isabel Belo, PhD, Department of Biological Engineering, University of Minho

The enzymes are biocatalysers that speed up the reactions, are not destroyed by the effect of the reaction and they do not modify the position of chemical balance in the reactions where they participate. For beyond these capacities, the enzymes have the capacity to lower the activation energy necessary to the reaction, due to its protein nature, are extremely specific and have still the advantage to have its control activity.

While in the last decades, the effect of the high pressure in the bioprocess has been white of many studies, is few and dispersed the works on the influence of moderate pressures in the bioprocess. In the sequence of previous works on the effect of moderate pressures on the microbial activity, it was intended to study in this work the effect of moderate pressures (up to 6 bar) in the activity and in the thermo-stability of ß-galactosidase of Kluyveromyces fragilis, using the commercial commodity Lactozyme®

The enzymatic hydrolysis of the lactose for ß-galactosidase is one of the most important biotechnological processes in the industrial application for the potential of the beneficial effect in the food use that contains lactose, as well as, the technological possibilities and environmental advantages in the milkmaid industry including: elimination of the lactose intolerance; formation of galacto-oligosaccharides during hydrolysis of the lactose to favour the growth of the intestinal bacterial micro flora, what it is desirable in foods; to increase the solubility and greater sweetening power and thus lower caloric content and to increase the biodegradability of the ways in which the lactose has been hydrolysed.

In the present work, a stainless steel reactor was used to carry out discontinuous assays where the reaction mixtures were submitted to air pressures up to 6 bar, comparing the kinetics under pressure with the kinetic at atmospheric pressure. The effect of the pressure in the kinetic degradation of the lactose was studied varying lactose and galactose concentrations.

According to the results, no significant variation of the kinetic activity of ß-galactosidase for the pressures studied (1 bar - 6 bar) was detected for an initial lactose concentration of 50 g/L. However, the concentration of the lactose affects the effect of the pressure as this concentration get lower. Moreover, the effect of galactose concentration on the enzyme kinetics was not affect by pressure rise. In the thermo-stability assays a greater stability of the enzyme to the pressure of 6 bar was observed than that of 1 bar for the temperature of 37 ºC, but at 50 ºC a fast deactivation (30 min) was verified to both pressures tested.