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On Column Binding a Real-Time Biosensor for ?-lactam Antibiotics Quantification.

ABSTRACT: This work aimed to develop accurate, quick, and practical tools for the detection of residues of penicillin G antibiotic in biological and non-biological samples. The assays were developed based on the binding mechanism of ?-lactam to penicillin-binding proteins; samples of different concentrations of penicillin G were incubated with in vitro expressed 6X-Histidine-tagged soluble penicillin-binding protein (PBP2x*) of Streptococcus pneumoniae (S. pneumoniae), whereby penicillin G in samples specifically binds to PBP2x*. The fluorescent-labeled ?-lactam analogue Bocillin FL was used as a competent substrate, and two different routes estimated the amounts of the penicillin G. The first route was established based on the differences in the concentration of non-bounded Bocillin FL molecules within the reactions while using a real-time polymerase chain reaction (PCR)-based method for fluorescence detection. The second route depended on the amount of the relative intensity of Bocillin FL bounded to Soluble PBP-2x*, being run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-page), visualized by a ChemiDoc-It®2 Imager, and quantified based on the fluorescence affinity of the competent substrate. While both of the methods gave a broad range of linearity and high sensitivity, the on column based real-time method is fast, non-time consuming, and highly sensitive. The method identified traces of antibiotic in the range 0.01-0.2 nM in addition to higher accuracy in comparison to the SDS-based detection method, while the sensitivity of the SDS-based method ranged between 0.015 and 2 µM). Thus, the on column based real time assay is a fast novel method, which was developed for the first time based on the binding inhibition of a fluorescence competitor material and it can be adapted to screen traces of penicillin G in any biological and environmental samples.

SUBMITTER: Abdullah SM

PROVIDER: S-EPMC7179420 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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On Column Binding a Real-Time Biosensor for β-lactam Antibiotics Quantification.

Abdullah Shahla M SM Rachid Shwan S

Molecules (Basel, Switzerland) 20200310 5

This work aimed to develop accurate, quick, and practical tools for the detection of residues of penicillin G antibiotic in biological and non-biological samples. The assays were developed based on the binding mechanism of β-lactam to penicillin-binding proteins; samples of different concentrations of penicillin G were incubated with in vitro expressed 6X-Histidine-tagged soluble penicillin-binding protein (PBP2x*) of Streptococcus pneumoniae (S. pneumoniae), whereby penicil ...[more]

PMID: 32164217

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Project description:Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic-enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their beta-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its beta-lactam ring. In Tris-NaCl-MgCl(2) buffer at pH7.7 and 37 degrees C, the rate constants for the dissociation of the antibiotic-enzyme complexes were 2.8x10(-6), 1.5x10(-6) and 0.63x10(-6)s(-1) (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [(14)C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a beta-lactam-antibiotic-destroying enzyme but did not function as a beta-lactamase. Incubation at 37 degrees C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [(14)C]benzylpenicillin-enzyme complex. The rate constants were 1.6x10(-5)s(-1) and 0.8x10(-4)s(-1) respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.

Dataset Information

On Column Binding a Real-Time Biosensor for ?-lactam Antibiotics Quantification.

Publications

On Column Binding a Real-Time Biosensor for <i>β</i>-lactam Antibiotics Quantification.

Similar Datasets

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