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.
Vessilier, S., Docquier, J.D., Rival, S., Frère, J.M., Galleni, M., Amicosante, G., et al. (2002). Overproduction and biochemical characterization of the Chryseobacterium meningosepticum BlaB metallo-beta-lactamase. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 46(6), 1921-1927 [10.1128/AAC.46.6.1921-1927.2002].
Overproduction and biochemical characterization of the Chryseobacterium meningosepticum BlaB metallo-beta-lactamase
DOCQUIERJ. D.;
2002-01-01
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
https://hdl.handle.net/11365/19293
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