Figure 3

NeuNAc synthesis in vitro in an enzymatic assay. (a) EICs of the HPLC-MS analysis at 222.098 atomic mass units (amu) corresponding to the mass of the [GlcNAc+H]⁺ ion and the [ManNAc +H]⁺ ion. Retention times (RTs) of ManAc (12.288 min) and GlcNAc (12.988 min) are indicated. (1) Chromatogram of the in vitro assay using GST fusion proteins of GlcNAc-2-epimerase and NeuNAc synthase. (2) Chromatogram of the in vitro assay using a cell-free extract of the PEC/PSC1 strain. (3) Chromatogram of the in vitro assay using a cell-free extract of the parental strain. (b) EICs at 310.1134 amu, corresponding to the mass of the [NeuNAc+H]⁺ ion. RT of NeuNAc (8.348 min) is indicated. (1) Chromatogram of the in vitro assay using GST fusion proteins of GlcNAc-2-epimerase and NeuNAc synthase. (2) Chromatogram of the in vitro assay using a cell-free extract of the PEC/PSC1 strain showing an 8-fold amplification compared to (1). (3) Chromatogram of a cell-free extract of the parental strain showing a 250-fold amplification compared to (1). (ad 1 MS) and (ad 2 MS) are MS spectra of chromatograms 1 and 2, respectively, at a RT of 8.348 min. (c) Cell-free extracts (active) of the PEC/PSC1 strain obtained from cultivation on chitin were mixed with NeuNAc and incubated for 0 and 24 h. A heat-inactivated cell-free extract was similarly treated. An active cell-free extract without NeuNAc (-NeuNAc) was also incubated, DMB-derivatized and analyzed. Values are means of biological duplicates derivatized in duplicate. Error bars indicate standard deviations.