Construction and characterization of chimeric enzymes of the Agrobacterium tumefaciens and Thermotoga maritima β-glucosidases

Abstract

The β-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, the encoded enzymes show homologies of approximately 45 and 37% in the amino acid sequences of the N- and C-terminal regions, respectively. Based on the amino acid alignment of these two enzymes, three chimeric β-glucosidase genes were constructed by shuffling selected DNA regions. The chimeric genes were expressed in Escherichia coli BL 21(DE3) and two catalytically active enzymes were obtained. Unlike other chimeras which display profiles intermediate to those of their respective parents, these chimeric enzymes displayed quite different profiles. The observed thermal stability of the more catalytically active chimeric enzyme was lower than that observed for either parental enzyme, however, the kinetic parameters were more similar to those of the T. maritima β-glucosidase.