Overexpression system and biochemical profile of CTX-M-3 extended-spectrum β-lactamase expressed in Escherichia coli

• Published in: FEMS microbiology letters 2004-12, Vol.241 (2), p.229-232
• Main Authors: Perilli, Mariagrazia, Ettorre, Daniela, Segatore, Bernardetta, Caporale, Bibiana, De Santis, Francesca, Pochetti, Giorgio, Brigante, Gioconda, Mazza, Fernando, Tavìo, Marìa M., Amicosante, Gianfranco
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Amino acids; Aztreonam; Bacteriology; beta-Lactamases - genetics; beta-Lactamases - isolation & purification; beta-Lactamases - metabolism; beta-Lactams - metabolism; Biological and medical sciences; Biotechnology - methods; Cefotaxime - metabolism; Ceftazidime; Cephalosporins; Citrobacter freundii - enzymology; Citrobacter freundii - genetics; Clavulanic acid; CTX‐M‐3; E coli; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - growth & development; ESβL; Fundamental and applied biological sciences. Psychology; Kinetics; Metabolism. Enzymes; Microbiology; Overexpression; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity; Sulbactam; Tazobactam; Up-Regulation
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/j.femsle.2004.10.031

Abstract An efficient over-expression system was developed for CTX-M-3 extended-spectrum-β-lactamase. The recombinant enzyme was purified from 1 l of culture to yield 22 mg of pure enzyme. The N-terminal amino acid sequence was determined to be NH2-QTADVQ… Determination of kinetic parameters with the purified CTX-M-3 revealed efficient hydrolysis of penicillins and cephalosporins, while ceftazidime and aztreonam were very poor substrates. Clavulanic acid, sulbactam and especially tazobactam inhibited the CTX-M-3 enzyme.