Extended Data Fig. 3: Activity and stability analysis of S1_16 sulfatases and their mutant variants.
From: Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota
a, DSF analysis of the effects of galactose and N-acetylgalactosamine on thermostability, with a positive-shift indicative of substrate binding. b, Normalised DSF melt curves of BT30574S-Gal/GalNAc and BT37964S-Gal/GalNAc. c, DSF melt curves of purified monomer and dimer species (left), monomer species in the presence of galactose and N-acetylgalactosamine (middle), or dimer species in the presence of galactose and N-acetylgalactosamine (right). d, Thin layer chromatography (TLC) analysis of wild-type (WT) and mutant S1_16 sulfatases. Asterisks are placed above lanes where sulfatase activity is observed. e, High pressure anion exchange chromatography (HPAEC) of WT and mutants. A grey block highlights the desulfated product. Both TLC and HPAEC reactions utilised 6 mM substrate and 1 μM enzyme, except for W109A variants where 10 μM was used, with 3 mM HEPES, 45 mM NaCl, and 5 mM CaCl2. Reactions incubated at 37 °C for 48 h. Control represents the substrate incubated in same conditions without adding enzyme. Experiments are technical triplicates and error bars represent SEM.
