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.
Utilization of plant resources for treatment of Helicobacter pylori infections is one of the appealing approaches as rapid emergence of antibiotic-resistant strains is occurring throughout the world. Ethanol extract and its fractions from Hibiscus rosa-sinensis red flower were assessed for antibacterial and urease inhibitory activities towards forty-three clinical strains and two reference strains of H. pylori. The ethyl acetate fraction exhibited the most potent bacteriostatic activity with minimum inhibitory concentrations (MICs) of 0.2-0.25 mg/mL and minimum bactericidal concentrations (MBCs) of 1.25-1.5 mg/mL against all test strains, including forty-three strains resistant to one to four antibiotics, azithromycin (MICs, 8-256 mg/mL), erythromycin (MICs, 8-128 mg/mL), levofloxacin (MICs, 8-256 mg/mL), and/or metronidazole (MICs, 8-256 mg/mL). The fraction had similar antibacterial activities toward these test strains suggesting the preparation and the antibiotics do not have a common mechanism of anti-H. pylori activity. The fraction also had stronger effects on biofilm formation, morphological conversion, and urease activity of H. pylori than the other fractions and the ethanol extract. These flower preparations were non-toxic to three human cell lines, and nine compounds were also isolated and identified from the ethyl acetate fraction. In vivo research needs to be conducted to confirm the potential usefulness of H. rosa-sinensis flower and its constituents for effective prevention and treatment of H. pylori disease.
Based on homology search in the amino acid sequences, a chimeric ß-glucosidase with enzyme activity has been prepared by replacing C-terminal region of Thr509-Arg646 from Cellvibrio gilvus with N-terminal region of Arg160-Gly283 from Ruminococcus albus. The chimeric enzyme showed broader optimum pH and slightly higher heat stability compared with original C. gilvus enzyme.
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