Our team identified that a crucial step in researching and battling the problem with the antibiotic meropenem is to see its degradation in real time. This would provide us with a wealth of valuable information for our upcoming experiments and steer us toward the ideal selection of a metallo-β-lactamase for our enhanced CADABRA cocktail. For this exact reason we decided to perform UV-Vis spectrophotometric measurements and determine the exact enzymatic activity of our MBL constructs.
Firstly, we prepared cell-free supernatants of several VIM-2, NDM, IMP clones and DH10B cells with pSB1C3 used as our control sample. Afterwards we started with mixing 1 µl of meropenem (50 mg/ml) with various amounts of our MBL supernatant.
Optimization of the measurement’s parameters
Using the trial and error approach, we found that 100µl of cell free supernatant supplemented with 1 µl of meropenem and diluted in 900 µl of distilled water shows the most optimal results. Under these conditions, we let the spectrophotometer to take measurements for 600 seconds at wavelength of 298 nm (the absorption maximum of meropenem) and observed curves that had a great consistent incline, which showed our VIM-2 and NDM enzymes worked perfectly. We did not observe such consistent results with our clones of the IMP enzyme.
Then, to further confirm that the observed reduction is due to a MBL enzyme activity, we repeated the measurements but this time adding also a commonly used inhibitor -Ethylenediaminetetraacetic acid (EDTA). Upon introducing 50µl of EDTA into our reactions, we observed a linear relationship with a slope approaching zero.
With these experiments our team managed to conduct a real-time spectrophotometric protocol for the degradation of the antibiotic meropenem. It gave us a lot of information to consider for the future improvement of our CADABRA cocktail. An evaluation like this one helped us visualize exactly the properties, mechanisms and speed in which the MBL enzymes work.