Biochemical characterisation of the subclass B2 metallo-b-lactamase SfhI
Abstract number: P908
Fonseca F., Correia A., Spencer J.
Objectives: Metallo-b-lactamases are characterised by their ability to hydrolyse almost all b-lactam antibiotics and are not inhibited by commonly used inhibitors (e.g. clavulanate, tazobactam). The b-lactamase SfhI was detected in strain Serratia fonticola UTAD54, an environmental isolate obtained from untreated drinking waters in the Northeast of Portugal. The aim of the present study was to purify this class B carbapenemase and determine its biochemical properties.
Methods: The blaSfhI gene was cloned into expression vector pET-26b(+) after PCR amplification using specific primers and the accuracy of the DNA construction verified by sequencing. The recombinant plasmid was transformed into Escherichia coli BL21(DE3). Metallo-b-lactamase SfhI was purified from induced cultures of E. coli BL21(DE3)(pSfhI) grown in M9Y medium supplemented with kanamycin at 25°C. Purification of SfhI was achieved by means of two chromatographic steps, anion-exchange at pH 8.5 followed by size exclusion at pH 7.0. Molecular mass and metal content of SfhI were determined by mass spectrometry. Initial hydrolysis rates for a range of b-lactam antibiotics were measured in 50 mM Hepes buffer pH 7.0 at 25°C.
Results: High level production of recombinant SfhI was achieved. Approximately 80 mg purified protein per liter of culture were obtained. By SDS-PAGE, protein was estimated to be >95% pure. On SDS-PAGE gels the purified SfhI migrates as a single band of approximately 26 kDa. Molecular mass of SfhI was determined as 26136 Da by mass spectrometry, in agreement with values predicted from the deduced amino acid sequence. The purified protein was shown to bind one equivalent of zinc, consistent with the assignment, on the basis of amino acid sequence, of SfhI to the B2 metallo-b-lactamase subgroup. Preliminary kinetic data suggest that SfhI hydrolyses penicillins, cephalosporins and carbapenems, exhibiting high affinity for carbapenems and low affinity for penicillins and third generation cephalosporins. Hydrolysis of cefoxitin and moxalactam was also detected.
Conclusion: The overexpression of blaSfhI allowed us to obtain a large amount of SfhI enzyme, enabling effective protein purification. Although the SfhI sequence is the most divergent (c.50% identity) of the known B2 metallo-b-lactamases, kinetic studies showed a substrate range that generally resembles those of other subgroup members, but includes cefoxitin and moxalactam, known inhibitors of these related enzymes.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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