Overproduction and biochemical characterization of the Chryseobacterium meningosepticum BlaB metallo-beta-lactamase.
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.
Vessillier
Sandrine
S
Dipartimento di Scienze e Tecnologie Biomediche, Università di L'Aquila, I-67100 L'Aquila, Italy.
Docquier
Jean-Denis
JD
Rival
Sandrine
S
Frere
Jean-Marie
JM
Galleni
Moreno
M
Amicosante
Gianfranco
G
Rossolini
Gian Maria
GM
Franceschini
Nicola
N
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
D015780:Carbapenems; D002511:Cephalosporins; D003470:Culture Media; D004274:DNA, Recombinant; D010406:Penicillins