Construction and characterization of a chimeric β-glucosidase

Abstract

The amino acid sequences of β-glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show significant similarity in most of the parts. However, the pH/temperature optima and stabilities of the two enzymes are quite different. C. gilvus β-glucosidase exhibits an optimum pH of 6.2-6.4 and temperature of 35°C, whereas the corresponding values for A. tumefaciens are 7.2-7.4 and 60°C respectively. To analyse these properties further, a chimeric β-glucosidase was constructed by replacing a segment from the C-terminal region of C. gilvus β-glucosidase gene with that of A. tumefaciens. The partially purified chimeric enzyme was characterized with respect to pH/temperature activity and stability and substrate affinity. Our results suggest that C-terminal segment(s) might be important in β-glucosidase specificity, and shuffling of even a small segment of gene in this region might significantly alter or improve the enzymic properties such as thermal stability.