Fig. 2

Cutinase secretion by C. glutamicum K9. Cells of C. glutamicum K9 possessing the pEKEx2 empty vector (EV) or pEKEx2-NprE-cutinase were inoculated to an OD₆₀₀ of 0.5 in 750 µl CGXII medium in a 48-well FlowerPlate and subsequently cultivated in a BioLector for 24 h at 30 °C, 1200 rpm and constant 85% relative humidity. After 4 h of cultivation, IPTG was added at the indicated final concentrations. a Growth of the respective cultures was monitored as backscattered light in 15 min intervals starting at the beginning of the cultivation. The growth curves show one representative experiment of three independent biological replicates. Standard deviations are given for selected time points. b Specific fluorescence of the respective cultures during the BioLector cultivation. Also here, one representative experiment of three independent biological replicates is shown and the standard deviations are given for selected time points. c Cutinase activity in the supernatant (black symbols) and specific fluorescence values (green symbols) after 24 h of cultivation of C. glutamicum K9 (pEKEx2-NprE-cutinase) induced by different IPTG concentrations. d After 24 h of growth, samples of the culture supernatant corresponding to an equal number of the respective C. glutamicum K9 cells that had been induced by the IPTG concentrations indicated below the lanes were analyzed by SDS-PAGE and proteins were visualized by Coomassie Brilliant Blue staining. EV, C. glutamicum K9 (pEKEx2 empty vector) as negative control; NprE-cutinase, C. glutamicum K9 (pEKEx2-NprE-cutinase); M, molecular weight protein markers in kDa. The position of the secreted cutinase protein is indicated by an asterisk. AU arbitrary units