Supplementary Figure 35, 36, and 37 Enzymatic activity of the three-component atrazine-degradation assembly and comparison of fractal vs globular assembly

Supp Figure 35: (A) Atrazine degradation pathway, enzymatic conversion of atrazine to cyanuric acid, and further enzymatic conversion to NH3 and CO2. (B) AtzB was added as an SH2-domain fusion to the two-component (AtzA-AtzC) assembly. c and d, Three-component assembly formation was validated using HIM (C), and the incorporation of AtzB was confirmed with fluorescence microscopy (using an Alexa-658-labeled AtzB) (D). (E) and (F), Assemblies were found to be more thermotolerant, as detected by incubation at a given temperature for 30 min followed by activity assays, and more robust to mechanical shearing forces, as detected by ability to withstand shaking. (G), Assemblies and free enzymes were incorporated into a Basotect® polymer foam with different TEOS % layers, to trap proteins, and assayed for cyanuric acid production. Proteins can be lost during the wash step after crosslinking and the % of protein lost under each condition is indicated on top of the bars. Supp Figure 36: Phase contrast micrographs of the Basotect® polymer foam with and without assemblies for the AtzAM1, AtzBSH2, and AtzCM1 components. (A and B) The microporous polymer foam with no assemblies. (C and D) The assemblies have been immobilized into the polymer foam, red arrows depict locations with assemblies. Images were taken with a Leica DM4000 B LED microscope, 10X objective (100X total magnification). Supp Figure 37: The fractal-like assemblies (Reg-Assembly) and the extended linker globular assemblies (ExtLinker-Assembly) enzymatic conversion of atrazine to cyanuric acid demonstrates no enzymatic benefit of a globular assembly. (A) AtzB was incorporated into the two-component assembly as an SH2-domain fusion as previously described to create the three-component assembly for both the fractal and globular assemblies. The activity of the fractal assembly was similar than the extended linker assemblies (GS linker) under different shaking speeds. Note: All enzymatic data from excel is then taken into graph pad for analyses, converted to Relative Yield %, addition of error bars, exported in vector format, and edited in Illustrator for final figure.