Construction of chimeric β-glucosidases with improved enzymatic properties

Abstract

The amino acid sequences of β-glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show about 40% similarity. The pH/temperature optima and stabilities and substrate specificities 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. The substrate specificity of A. tumefaciens enzyme toward different aryl glycosides is broader than C. gilvus enzyme. To analyze these properties further, three chimeric β- glucosidases were constructed by substituting segments from the C-terminal homologous region of C. gilvus β-glucosidase gene with that of A. tumefaciens. The chimeric enzymes were characterized with respect to pH/temperature activity and stability and substrate specificity. Chimeric enzymes exhibited chromatographic behavior similar to that of C. gilvus enzyme. However, enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45-50 °C and pH 6.6-7.0. K(m) values of chimeric enzymes for the various saccharides were admixtures of both parental enzymes. These results suggest that the two domains of C. gilvus and A. tumefaciens enzymes probably can fold independently. The homologous C-terminal region in β-glucosidase appears to play an important role in determining enzyme characteristics. Changes in the properties on substitution of segments in this region might be related to the enzyme specificity, and β-glucosidases with improved properties can be prepared by manipulating this region.