Hydrolysis of lactose by free and immobilized beta-galactosidase from Thermus sp. strain T2

Abstract

The hydrolysis of lactose by a beta-galactosidase from the thermophilic microorganism Thermus sp. strain T2, both in solution and immobilized on a commercial silica-alumina, has been studied. The enzyme has been previously produced by Escherichia coli JM101 harboring the plasmid pBGT1, which contains the codifying gene under the promoters lpp(P) and lac(PQ). The enzyme was immobilized on the support activated with tris-hydroxymethylphosphine (THP). Activity and stability of the free and the immobilized enzyme towards pH and temperature were tested. To study the activity at different pH and temperature values, lactose was used as substrate. To check the stability, the enzyme was incubated either in buffer BP or in a solution of lactose in buffer BM at different pH and temperatures, being the remaining activity tested by withdrawing samples and determining their activity toward ONPG at 70 degrees C in buffer BP. Afterward, runs were performed to obtain kinetic models adequate for the description of the hydrolysis of lactose by the free and the immobilized enzyme. These data were fitted to the kinetic models proposed (all based on the Michaelis-Menten mechanism) by non-linear regression, being the models and their parameters compared to determine the effect of the immobilization on the kinetic behavior of the enzyme. Both the free and the immobilized enzyme are competitively inhibited by galactose, while glucose inhibited only the action of the free enzyme, in an uncompetitive way. The immobilization step seems to eliminate the inhibition by glucose. Moreover, the immobilization reduced to a half the inhibitory action of galactose. In general, the immobilization reduced the activity of the enzyme, but increased its thermal stability. Finally, a comparison between the kinetic behavior of this thermophilic enzyme and enzymes of mesophile microorganisms previously studied by us (E. coli and K. fragilis) and by other authors (Aspergillus niger) is performed.