Fig. 4
The MHETase whole-cell catalysts follow Michaelis–Menten kinetics. A–I Michaelis–Menten plots for the MHETase chimeras and recombinant MHETase. For the displayed MHETase chimeras (A–G), cells were induced for 4 h in YPD, rinsed twice and resuspended in 100 mM phosphate buffer pH 7.5 prior to assaying MHETase activity by incubating with MpNPT at the indicated concentrations for 10 min at 24 °C, followed by measuring absorbance at 405 nm. For the recombinant and secreted enzyme (H, I), assays were performed under the same buffer and temperature conditions in the presence of the indicated MpNPT concentrations. Michaelis–Menten curves were fitted to the data. J Seven biological replicates of the MHETase-Tip1 fusion were assayed in parallel. Michaelis–Menten curves were fitted to each replicate (black lines). Vmax and Km were calculated from the fitted curves (inset). K Representative chromatograms from HPLC of MHETase reaction products for MHETase-Tip1 (M + Tip1) and intracellular MHETase (intra-M) after 1 h or 24 h at 24 °C (n = 3). L Quantification of TPA produced by the M + Tip1 and intra-M whole-cell catalysts after 1 h and 24 h at 24 °C with 400 nmol MHET. Horizontal bars indicate the means of the replicates. n = 3