Stability and substrate specificity of a beta-glucosidase from the thermophilic bacterium Tp8 cloned into Escherichia coli.
A facile isolation of beta-glucosidase (EC 3.2.1.21) from Escherichia coli containing the recombinant plasmid pNZ1001 carrying a beta-glucosidase gene from the extremely thermophilic anaerobic bacterium Tp8 is reported. The enzyme was purified to homogeneity by anion-exchange chromatography and steric exclusion HPLC following thermal denaturation/precipitation of heat-labile E. coli proteins. The enzyme had a broad specificity for beta-D-glucosides, galactosides, fucosides, and xylosides. Action on aryl-beta-D-glycosides of glucose, galactose, and fucose was characterized by low Km and high Kcat/Km values compared with disaccharide substrates for which specificity decreased in the order laminaribiose, sophorose, cellobiose, beta-gentiobiose, lactose. Galactono-1-4-lactone, glucono-1-5-lactone, and 1-O-methyl-beta-D-glucose were competitive inhibitors with Ki values of 1.6, 0.09, and 17.5 mM, respectively. The enzyme was remarkably stable to detergents, urea, and organic solvents. Thermostability was greatest at the pH activity optimum (pH 6.0-6.5) and half-life (t1/2) values were 11 min at 90 degrees C, 105 min at 85 degrees C, and 900 min at 80 degrees C. Activity was destabilized by Sr2+, Co2+, Ca2+, Mg2+, and Mn2+, but t1/2 increased in the presence of substrates or competitive inhibitors. Activation energy, Ea, was 54.3 kJ.mol-1. A free thiol group(s) was required for full activity, this being rapidly lost in the presence of Hg2+ or N-ethyl maleimide.
D001421:Bacteria, Anaerobic / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D005959:Glucosidases / Q000378:metabolism*; D006207:Half-Life; D010957:Plasmids; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D005959:Glucosidases; D001617:beta-Glucosidase; D005033:Ethylmaleimide