Microbial sensor for new-generation cephalosporins based in a protein-engineered beta-lactamase.
A protein-engineered beta-lactamase, constructed by site-directed mutagenesis in Escherichia coli (E104M/G238S), and having broadened specificity, was able to degrade cephalosporins of first, second, and third generations. Manipulations of culture conditions allowed an increase in beta-lactamase specific activity by up to twofold. The resultant bacteria were used to construct an immersable whole-cell biosensor for the detection of new-generation cephalosporins. Cells were immobilized on agar membranes, which in turn were attached to the surface of a flat pH electrode, thus constituting a biosensor based on the detection of pH changes. The sensor was able to detect second- and third-generation cephalosporins: cefamandole (0.4-4 mM), cefotaxime (0.4-3.5 mM), and cefoperazone (0.3-1.85 mM). Response times were between 3.5 and 11 min, depending on the kind of cephalosporin tested. The biosensor was stable for at least 7 d, time during which up to 100 tests were performed.
García
J L
JL
Department of Bioengineering, National University of Mexico, Cuernavaca, Morelos, México.
Nuñez
C J
CJ
González
E G
EG
Osuna
J
J
Soberón
X
X
Galindo
E
E
D015374:Biosensing Techniques; D002511:Cephalosporins / Q000032:analysis*; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D002511:Cephalosporins; D001618:beta-Lactamases