Construction and characterization of chimeric enzymes of the Agrobacteriumtumefaciens and Thermotoga maritima beta-glucosidases

The beta -glucosidases of Agrobacterium tumefaciens and Thermotoga maritima display quite distinct characteristics, both in terms of their pH optima, temperature optima and substrate specificities. Despite such differences, t he encoded enzymes show homologies of approximately 45 and 37% in the amino acid sequences of the N- and C-terminal regions, respectively. Based on th e amino acid alignment of these two enzymes, three chimeric beta -glucosida se genes were constructed by shuffling selected DNA regions. The chimeric g enes were expressed in Escherichia coli BL 21(DE3) and two catalytically ac tive enzymes were obtained. Unlike other chimeras which display profiles in termediate to those of their respective parents, these chimeric enzymes dis played quite different profiles. The observed thermal stability of the more catalytically active chimeric enzyme was lower than that observed for eith er parental enzyme, however, the kinetic parameters were more similar to th ose of the T. maritima beta -glucosidase. (C) 2001 Elsevier Science B.V. Al l rights reserved.