fl-Lactam antibiotics and the crude enzyme were mixed in deuterium oxide and placed in a nuclear magnetic resonance tube. The change of the nuclear magnetic resonance spectrum during the enzymatic reaction was then analyzed to determine 8i-lactamase activity. By using fl-lactam antibiotics such as penicillins, cephalosporins, and cephamycins as substrates, a comparison of the fl-lactamase activities was made between the nuclear magnetic resonance spectrometric assay and the iodometric assay. There was a close correlation between these two methods.For the determination of ,B-lactamase (EC 3.5.2.6), many methods such as the iodometric assay (8, 11), the Sargent method (13), the hydroxamate method (1, 2), the pH-stat alkalimetric titration method (3, 5), the ultraviolet adsorption method (10, 14), the red decomposition product method (9), and the microbiological assay have been used. We reported previously that enzymatic degradation of cephalothin was observed by the change of nuclear magnetic resonance (NMR) spectra during enzymatic hydrolysis, and we indicated that this NMR spectrometric assay might be applicable for the kinetic study of a hydrolyzing enzyme (7). This paper deals with the kinetics of the enzymatic reaction with the crude enzyme and compares the data of f8-lactamase activity from the NMR spectrometric assay and the iodometric assay.

Cephalothin (CET)-susceptible Escherichia coli, which can degrade CET after prolonged incubation in broth containing a concentration of the drug greater than the minimum inhibitory concentration, was found in a clinical specimen. The substrate specificity of the partially purified enzyme to cephalosporin analogs strongly indicated the occurrence of CET-specific degradation. Nuclear magnetic resonance analysis of the degradation reaction demonstrated the appearance of two new signals attributed to deacetyl CET. This suggests the possibility of the presence of acylesterase.

An application of a nuclear magnetic resonance (NMR) spectrometer for the measurement of β‐lactamase activity in clinical material containing bacteria is presented. By means of proton (1H)‐NMR, it was easy to measure quantitatively β‐lactamase activity in human bacteriuria, without performing any such pretreatment as isolation of bacteria or extraction of crude enzymes and without preparing special reagents for the detection. This is the first report on the application of 1H‐NMR analysis of structural changes for determining hydrolysis of β‐lactam antibiotics with β‐lactamase‐producing bacteria in aqueous solution.

No abstract