Table 2. Characterization of PenP-E166Cf, PenP-E166Cf/N170Q, and PenPC-E166Cf in Terms of Biosensor Performance and Thermostability.
| PenP-E166Cf | PenP-E166Cf/N170Q | PenPC-E166Cfa | |||
|---|---|---|---|---|---|
| biosensor performance | response time (s)b | ampicillin | NDg | ND | NDi |
| penicillin G | ND | ND | ND | ||
| penicillin V | ND | ND | NDi | ||
| cefotaxime | ND | ND | NAj | ||
| cefoxitin | 133 | 125 | 1000 | ||
| moxalactam | 688 | 600 | 1385 | ||
| maximum fluorescence change (%)c | ampicillin | 34 | 31.6 | 59.2i | |
| penicillin G | 35.6 | 34 | 60 | ||
| penicillin V | 30 | 33 | 56.3i | ||
| cefotaxime | 22.5 | 20 | NA | ||
| cefoxitin | 29.1 | 25 | 23.8 | ||
| moxalactam | 25.8 | 29.1 | 44 | ||
| duration of signal (s)d | ampicillin | 842 | 1947 | 1781i | |
| penicillin G | 1867 | 2353 | 1286 | ||
| penicillin V | 2474 | >3600; NMh | 1375i | ||
| cefotaxime | >3600; NM | >3600; NM | NA | ||
| cefoxitin | >3600; NM | >3600; NM | >3600; NM | ||
| moxalactam | >3600; NM | >3600; NM | >3600; NM | ||
| limit of detection (μM)e | ampicillin | 0.01 | 0.01 | 0.1i | |
| penicillin G | 0.01 | 0.01 | 0.1 | ||
| penicillin V | 0.01 | 0.01 | 0.1i | ||
| cefotaxime | 0.01 | 0.01 | NA | ||
| cefoxitin | 0.01 | 0.01 | 0.1 | ||
| moxalactam | 0.01 | 0.01 | 0.1 | ||
| thermostability | Tm (°C)f | 57 | 57 | 40 | |
| residual activity after 1 h incubation at 50 °C(%) | 100 | 100 | 0 |
Fluorescence data of PenPC-E166Cf in the presence of penicillin G, cefoxitin, and moxalactam are shown in Figure S5.
Response time: time taken by the biosensor to give a signal reaching a stable maximal level after the addition of a minimal saturating concentration of antibiotics. The lowest antibiotic concentration that saturates PenP biosensors and the PenPC biosensor were 1 and 10 μM, respectively.
Maximum fluorescence change (%) is determined in the presence of antibiotics at a saturating concentration.
Duration of the signal is assessed in the presence of a minimal saturating concentration of antibiotics (for PenP biosensors: 1 μM and for PenPC biosensor: 10 μM) and indicated by the time at which a stable maximal level of the fluorescence signal starts to decline.
Limit of detection: the lowest antibiotic level that can be detected by the biosensor.
Tm (oC): temperature at the relative ellipticity at 222 nm of a protein is dropped to 50% as observed by CD spectroscopy.
ND: not detectable.
NM: not measured.
values obtained by analyzing the results obtained from Chan et al.29
NA: not available.