Properties of Sepharose-bound beta-lactamase from Enterobacter cloacae.
beta-Lactamase from Enterobacter cloacae P99 was immobilized onto Sepharose by the cyanogen bromide activation method and the properties of the Sepharose-bound enzyme were compared with those of soluble and cell-bound enzyme. The immobilized beta-lactamase showed enhanced stability to storage at 4 degrees C (approximately 1 year) in respect to the free enzyme in solution (few days). The optimum pH for activity is similar for both Sepharose- and cell-bound beta-lactamase and extends over a broader pH range (pH 6-9) than the soluble enzyme (pH 8-9). Immobilization leads also to significant enhancement of thermal stability. Effective enzyme inhibition by flucloxacillin occurs with both soluble and Sepharose-bound beta-lactamase, whereas the cell-bound enzyme is much less (10(-5) times) inhibited. These results indicate that immobilized beta-lactamase could be usefully employed as a tool for investigating the properties of newly designed beta-lactamase inhibitors.
D003488:Cyanogen Bromide; D004355:Drug Stability; D004754:Enterobacter / Q000201:enzymology*; D004755:Enterobacteriaceae / Q000201:enzymology*; D004800:Enzymes, Immobilized; D005436:Floxacillin / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012685:Sepharose; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000032:analysis*
D004800:Enzymes, Immobilized; D065093:beta-Lactamase Inhibitors; D005436:Floxacillin; D012685:Sepharose; D001618:beta-Lactamases; D003488:Cyanogen Bromide