Calorimetric analysis of cephalosporins using an immobilized TEM-1 β-lactamase on Ni²⁺ Chelating Sepharose Fast Flow

Two β-lactamases, penicillinase type I from Bacillus cereus and TEM-1 β-lactamase from Haemophilus ducreyi, were immobilized on a Chelating Sepharose Fast Flow column loaded with Ni²⁺ in an active form. Flow-injection analysis of β-lactams was performed by using an enzyme column reactor fitted into the enzyme thermistor. With both enzymes it was possible to monitor both penicillins and cephalosporins. Moreover, Michaelis constants of the TEM-1 β-lactamase were markedly increased upon immobilization for all substrates, especially carbenicillin, cephaloridine, and cefoperazone.