Kinetic parameters for hydrolysis of β-lactams by the purified OXA-830 β-lactamase were examined using a UV-VIS spectrophotometer (U-3900, HITACHI, Japan) at 30°C in 10 mM phosphate buffer (pH 7.0) in a final reaction volume of 300 μL. The steady-state kinetic parameters (kcat and KM) were determined by non-linear regression of the initial reaction rates with the Michaelis–Menten equation in Prism (version 7) software (GraphPad Software, San Jose, CA, United States).
β-lactamase inhibition was studied with benzylpenicillin (500 μM) as the substrate. The β-lactamase inhibitors sulbactam, tazobactam and clavulanic acid at various concentrations were preincubated with the purified OXA-830 β-lactamase for 3 min at 30°C before addition of substrate. The inhibitor concentration required to reduce the hydrolysis of 500 μM benzylpenicillin by 50% was determined by non-linear regression with the log(inhibitor) vs. response – Variable slope equation in Prism (version 7) software (GraphPad Software, San Jose, CA, United States).
β-lactamase inhibition was studied with benzylpenicillin (500 μM) as the substrate. The β-lactamase inhibitors sulbactam, tazobactam and clavulanic acid at various concentrations were preincubated with the purified OXA-830 β-lactamase for 3 min at 30°C before addition of substrate. The inhibitor concentration required to reduce the hydrolysis of 500 μM benzylpenicillin by 50% was determined by non-linear regression with the log(inhibitor) vs. response – Variable slope equation in Prism (version 7) software (GraphPad Software, San Jose, CA, United States).
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