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Antimicrobial Agents and Chemotherapy, June 2002, p. 1921-1927, Vol. 46, No. 6
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.6.1921-1927.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Overproduction and Biochemical Characterization of the Chryseobacterium meningosepticum BlaB Metallo-ß-Lactamase

Sandrine Vessillier,¹ Jean-Denis Docquier,² Sandrine Rival,³ Jean-Marie Frere,⁴ Moreno Galleni,⁴ Gianfranco Amicosante,¹ Gian Maria Rossolini,² and Nicola Franceschini^1*

Dipartimento di Scienze e Tecnologie Biomediche, Università di L'Aquila, I-67100 L'Aquila,¹ Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, I-53100 Siena, Italy,² Institut für Biochemie, Gebande 6, Universität des Saarlandes, 66123 Saarbrücken, Germany,³ Laboratoire d'Enzymologie and Centre d'Ingénierie des Protéines, Institut de Chimie, Université de Liège, Sart Tilman, B-4000 Liège, Belgium⁴

Received 31 July 2001/ Returned for modification 23 November 2001/ Accepted 16 March 2002

The BlaB metallo-ß-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 >10⁶ M^-1 s^-1) toward most penam and carbapenem compounds, with the exception of the 6--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-ß-lactamase inhibitors, including ß-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.

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* Corresponding author. Mailing address: Dipartimento di Scienze e Tecnologie Biomediche, Università dell'Aquila, Loc. Coppito, 67100-L'Aquila, Italy. Phone: 39 0862 433456. Fax: 39 0862 433433. E-mail: nicola.franceschini{at}cc.univaq.it.

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Antimicrobial Agents and Chemotherapy, June 2002, p. 1921-1927, Vol. 46, No. 6
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.6.1921-1927.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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