53d] β-Lactamase (Escherichia coli RTEM+)

Abstract

Publisher Summary This chapter discusses the purification procedure and properties if β-lactamase (Escherichia coli, gram negative ). The β-lactamases of gram-negative bacteria present a sharp contrast in properties when compared with similar enzymes from gram-positive species. First, these enzymes are invariably cell-associated, and second, their synthesis is usually constitutive. This means that β-lactamase purification from enteric bacteria involves the isolation of a protein from the bacterial cells themselves without induction or the need to isolate constitutive mutants. The purification procedure involves ultrasonic disintegration, centrifugation, dialysis, adsorption on DEAE-cellulose, and elution from DEAE-cellulose Sephadex G-75. The β-lactamases of gram-negative bacteria has a broad specificity encompassing both penicillins and cephalosporins. However, in practice the enzyme is much more effective against penicillins because, in general, the Km values for cephalosporin substrates are high. This fact probably accounts for the greater ability of this enzyme to protect gram-negative bacteria against penicillin than against cephalosporin.