PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
RESULTS AND DISCUSSION
It has been assumed that the enzyme inside the vesicle follows Michaelis-Menten
kinetics identical to the behavior outside of the vesicle. 8 In order to study the kinetic
effect of microencapsulation of glucose oxidase in liposomes, the initial rates of
glucose oxidase reaction by the free and liposomal enzyme were measured at
various glucose concentrations (Fig. 1). At glucose concentration higher than 0.28
mol dm -3 , liposomal glucose oxidase did no follow pure Michaelis-Menten kinetics and
the form of the plot is typical of enzymes exhibiting substrate inhibition 20 . This
behaviour was reaffirmed fitting the experimental data to the reaction equation for
substrate inhibition. The data was also fitted to reaction equation for product inhibition
and the fit was not good. All of the above considerations indicate that the liposomal
glucose oxidase suffer an apparent inhibition by glucose accumulating inside the
vesicles with a substrate inhibition constant of the 0.95 ± 0.12 mol dm -3 .
The Km and Vmax values were (i) 0.14 ± 0.02 mol dm -3 and 5.70 ± 0.52 U cm -3 ,
respectively, using soluble enzyme and (ii) 0.19 ± 0.02 mol dm -3 and 2.01± 0.13 U
cm -3 , respectively, when the entrapped enzyme was assayed. The apparent Km of the
encapsulated enzyme was higher than of the enzyme in solution suggesting that the
lipid vesicles limited the permeation rate of substrate through the semi-permeable
vesicle membrane. This difference could be also due to chemical and/or
conformational changes in the enzyme structure provoked by an association of
glucose oxidase with the lipid vesicles.