Characterization and Purification of TEM-1 β-Lactamase

One method of antibiotic resistance in bacteria to the β-lactam class of antibiotics is through the production of β-lactamase enzymes. One of the β-lactamases is an enzyme from Escherichia coli (TEM-1) that plays a role in the hydrolysis of β-lactam antibiotics by using a serine residue at the active site. To better understand the mechanism of the enzyme by kinetic analyses, TEM-1 has been cloned, overexpressed, and purified. The effects of different buffers and changes in ionic strength were tested to determine if changes in these conditions had an effect on enzyme activity. As a result, we found that the Km value was 20.2 µM and the Vmax was 608.8 µM/min (30 mM Tris, 250 mM NaCl, pH 7.5). The pH dependence of TEM-1 was determined as the pH versus the log of kcat/Km with two pKa values of 5.5 and 9.0. These studies will provide an avenue for better understanding the acid base mechanism of TEM-1 β-lactamase.