Activity over lactose and ONPG of a genetically engineered beta-galactosidase from Escherichia coli in solution and immobilized: Kinetic modelling

The kinetic study of the hydrolysis of lactose and o-nitrophenol-beta -D-ga lactoside (ONPG) with a beta -galactosidase from Escherichia coli, both in solution and covalently immobilized on a silica-alumina, is presented. The enzyme employed in this work had been modified previously by genetic engine ering and purified to homogeneity by affinity chromatography. Firstly, the influence of pH and temperature on the activity and the stability of the en zyme, both free and immobilized, have been studied. Secondly, hydrolysis ru ns of lactose and ONPG with both forms of the enzyme were carried out in a wide experimental range of temperature and concentrations of substrates, pr oducts and enzyme. Data obtained were fitted to several kinetic models base d on the Michaelis-Menten mechanism by non-linear regression. Finally, the models and their parameters were compared to determine the influence of the immobilization process and the substrate on the activity of the enzyme. In the hydrolysis of lactose and with both forms of the enzyme, acompetitive inhibition due to glucose was observed while the most common inhibition by galactose (which is usually a competitive inhibitor of beta -galactosidases ) was not observed. Curiously, when the immobilized enzyme was the catalyst employed, lactose was an acompetitive inhibitor of the hydrolysis. When th e substrate hydrolysed was the o-nitrophenol-beta -D-galactoside (ONPG), th e galactose acted as a competitive inhibitor and the o-nitrophenol (ONP) wa s an acompetitive inhibitor for the free enzyme, being the immobilization p rocess able to avoid the interaction between the ONP and the enzyme. (C) 20 01 Elsevier Science Inc. All rights reserved.