Fig. 4: Increased enzymatic activity by controlled loading of a synthetic two-enzyme cascade.

a Schematic overview of the reaction scheme to produce Indigo from l-Trp by TnaA and FMO. b Analysis of the purified enzyme variants by SDS-PAGE gel electrophoresis. Left to right: marker (M), TnaA, FMO, M. c Confocal micrographs of coacervates loaded with 100 nM Sulfo-Cy5-NHS-labeled TnaA + 200 nM unlabeled FMO (top) or 200 nM FMO + 100 nM unlabeled TnaA (bottom). Membrane stained with Nile red (purple), scale bar: 10 µm. Uncropped images are to be found in Supplementary Fig. 18. d Box plot analysis of the loading of fluorescently labeled enzymes inside the coacervates. For all cases, n > 50. e, f NADPH consumption measured by the absorbance at 340 nm. 1 mM l-Trp and 0.5 mM NADPH was added prior to the measurement. Orange circles represent enzymes inside the coacervates, gray circles show enzymes in solution. Three individual batches of coacervates were made for each condition, the standard deviation of three individual measurements is represented by the shaded area. For clarity of the graph, only 1/3rd of all collected data points are shown. e 250 nM TnaA + 500 nM FMO. f 125 nM TnaA + 250 nM FMO.