Fig. 3

Purification and activation of prenisin produced by C. glutamicum. A Relative mCherry fluorescence normalized to OD (RFU/OD) of L. lactis NZ9000/pNZ-Pnis-mcherryLl grown o/N in the presence of supernatants (SN) of C. glutamicum CR099/pXMJ-nisZBTCCg. The producer was grown o/N in 2xTY with 2% Glc and 0.2 mM IPTG. B Purification of ammonium sulphate-precipitated SN proteins by cation exchange (CIEX) and subsequent reverse phase (RP) chromatography on the CIEX peak fraction. Indicated is absorbance at 214 nm (red) and conductance (mS/cm; black, in CIEX) or % of elution buffer (%B, black, in RP) over the elution volume. Boundaries of the peak fractions further analysed are marked with blue broken lines. CRFU/OD of L. lactis NZ9000/pNZ-Pnis-mcherryLl grown o/N in the presence of samples obtained at different steps during the purification of prenisin from SN shown in (A). prec: ammonium sulphate-precipitated SN proteins resuspended in pure H2O; CIEX and RP peak: peak fraction of the CIEX and RP chromatography. Prior to assays, samples were activated by incubation with trypsin (0.5 mg/ml for 3.5 h) and diluted 1:1000. As positive controls, the biosensor was grown in the presence of nisin Z at the indicated concentration. As negative controls, SN without trypsin treatment were included. D Mass spectrometry of the peak fraction obtained in RP chromatography in B with arbitrary peak intensity units (intensity [AU]) over mass/charge ratio (m/z). Values in A and C are mean ± SD of n = 3 independent cultures (A) or technical triplicates of one representative preparation (C)