| Respective to the increasing demands for efficient screening systems for beta-lactam compounds, in our study, a class C beta-lactamase from Enterobacter cloacae P99 has been engineered by rational design into a fluorescent biosensor designated as V211Cf for sensing beta-lactam antibiotics and beta-lactamase inhibitors. This novel V211Cf was a fluorescein-labelled beta-lactamase prepared by tethering a fluorescein molecule to this unique cysteine-containing mutant at the 211 position that is in close proximity to the active site via a maleimide linker. Kinetic analysis of V211Cf indicated that there was no loss of hydrolytic capability of V211Cf with a fluorophore attached in vicinity to the active site. As revealed by fluorometric studies, addition of the two main classes of beta-lactam antibiotics, including penicillins and cephalosporins, triggered fluorescence enhancements of V211Cf. Such increased signals then disappeared according to the rate of the hydrolysis of the substrates by V211Cf. In addition, V211Cf displayed distinct signal patterns for the beta-lactamase inhibitors, including clavulanic acid, sulbactam, tazobactam, and transition-state analogs. These results indicated that V211Cf can probe for the presence of the beta-lactam antibiotics and beta-lactamase inhibitors. Furthermore, Y150S/V211Cf, a catalytically-impaired derivative of V211Cf has been constructed to optimize the performance of V211Cf in the detection for beta-lactam antibiotics. Y150S/V211Cf showed improved signal stability than V211Cf. Besides, Y150S/V211Cf only exhibited fluorescence signals in the presence of cephalosporins and not penicillins, thus it was specific in sensing cephalosporin-type substrates. When comparing the performance among the class C labelled-beta-lactamases, V211Cf and Y150S/V211Cf, and the previously reported class A beta-lactamase-based E166Cf in detecting cephalothin, E166Cf was superior to the fluorescent class C beta-lactamases in sensitivity whereas the fluorescein-modified class C beta-lactamases demonstrated a more rapid response time than E166Cf. To conclude, our results demonstrated for the first time the feasibility of the fluorescein-labelled class C beta-lactamases in the application for sensing beta-lactam antibiotics and beta-lactamase inhibitors. Furthermore, our study further supported the applicability of utilizing a non-allosteric protein modified with a fluorophore nearby the active site as a sensing system for its ligands. This probably illustrates an innovative approach for the future development of biosensor. |
