TY - JOUR
T1 - Overproduction and biochemical characterization of the Chryseobacterium meningosepticum B1aB metallo-β-lactamase
AU - Vessillier, Sandrine
AU - Docquier, Jean Denis
AU - Rival, Sandrine
AU - Frere, Jean Marie
AU - Galleni, Moreno
AU - Amicosante, Gianfranco
AU - Rossolini, Gian Maria
AU - Franceschini, Nicola
PY - 2002
Y1 - 2002
N2 - The BlaB metallo-β-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 (kcat/Km ratios of > 106 M-1 s-1 toward most penam and carbapenem compounds, with the exception of the 6- α-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-β-lactamase inhibitors, including β-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.
AB - The BlaB metallo-β-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 (kcat/Km ratios of > 106 M-1 s-1 toward most penam and carbapenem compounds, with the exception of the 6- α-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-β-lactamase inhibitors, including β-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.
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U2 - 10.1128/AAC.46.6.1921-1927.2002
DO - 10.1128/AAC.46.6.1921-1927.2002
M3 - Article
C2 - 12019109
AN - SCOPUS:0036090517
VL - 46
SP - 1921
EP - 1927
JO - Antimicrobial Agents and Chemotherapy
JF - Antimicrobial Agents and Chemotherapy
SN - 0066-4804
IS - 6
ER -