Construction and characterization of a chimeric beta-glucosidase.
The amino acid sequences of beta-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 beta-glucosidase exhibits an optimum pH of 6.2-6.4 and temperature of 35 degrees C, whereas the corresponding values for A. tumefaciens are 7.2-7.4 and 60 degrees C respectively. To analyse these properties further, a chimeric beta-glucosidase was constructed by replacing a segment from the C-terminal region of C. gilvus beta-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 beta-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.
Biomaterials Conversion Laboratory, National Food Research Institute, Ibaraki, Japan.
A
A
Singh
K
K
Hayashi
T T
TT
Hoa
Y
Y
Kashiwagi
K
K
Tokuyasu
D016960:Agrobacterium tumefaciens / Q000201:enzymology; D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D001617:beta-Glucosidase