title: Overproduction and biochemical characterization of the Chryseobacterium meningosepticum BlaB metallo-beta-lactamase.
abstract: The BlaB metallo-beta-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k(cat)/K(m) ratios of >10(6) M(-1) s(-1)) toward most penam and carbapenem compounds, with the exception of the 6-alpha-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-beta-lactamase inhibitors, including beta-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.
authors.affiliation: Dipartimento di Scienze e Tecnologie Biomediche, Università di L'Aquila, I-67100 L'Aquila, Italy.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Vessillier
authors.forename: Jean-Denis
authors.initials: JD
authors.lastname: Docquier
authors.forename: Sandrine
authors.initials: S
authors.lastname: Rival
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Frere
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Amicosante
authors.forename: Gian Maria
authors.initials: GM
authors.lastname: Rossolini
authors.forename: Nicola
authors.initials: N
authors.lastname: Franceschini
mesh_terms: D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D015780:Carbapenems / Q000378:metabolism; D002511:Cephalosporins / Q000378:metabolism; D003470:Culture Media; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005285:Fermentation; D005822:Genetic Vectors; D007700:Kinetics; D010406:Penicillins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics
chemical_list: D015780:Carbapenems; D002511:Cephalosporins; D003470:Culture Media; D004274:DNA, Recombinant; D010406:Penicillins