Enzyme kinetics of cefotaxime hydrolysed by beta-lactamases extracted from Gram-negative bacilli

The enzyme kinetics of cefotaxime and cefoperazone against plasmid-and chromosomal-mediated beta-lactamases were studied with double-wavelength and double-beam spectrophotometer. beta-Lactamases were extracted from Gram-negative bacilli and identified by isoelectric focusing method with the special reagent nitrocefin. Michaelis constant (Km), maximal velocity (Vmax) and inhibition constant (Ki) values were obtained from Lineweaver-Burk plots. The results showed that cefotaxime was more stable than cefaloridine and cefoperazone against the hydrolysis of (3-lactamases. Vmax of cefotaxime to TEM-1, SHV-1, P99 enzymes were<0.10%. Cefotaxime demonstrated a high affinity to chromosomal-mediated P99 enzyme. The Ki of cefotaxime was 0.036 vmol/L. Cefoperazone exhibited less enzyme inhibitory activity and was unstable to most of the beta-lactamases tested.