Extended Data Fig. 5: Analysis of the activity and stability of BT16246S-Gal/GalNAc and its mutant variants.
From: Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota
a, Thin layer chromatography (TLC) analysis of wild-type (WT) BT16246S-Gal/GalNAc and its mutants. Asterisks are placed above lanes where activity is observed. b, High pressure anion exchange chromatography (HPAEC) of wild-type BT16246S-Gal/GalNAc WT and its mutants. The desulfated product is highlighted by a grey box. All TLC (a) HPAEC (b) reactions utilised 6 mM substrate and 5 μM enzyme, with 3 mM HEPES, 45 mM NaCl, and 5 mM CaCl2. Reactions incubated for 48 h at 37 °C. Control represents the substrate incubated in same conditions without adding enzyme. c, DSF analysis showing relative thermostability of BT16246S-Gal/GalNAc mutant proteins with respect to the WT enzyme. d, DSF analysis of the effects of alanine scanning on the ability of BT16246S-Gal/GalNAc to bind galactose, with the Tm of the protein shown above the bar. The experiments were performed using 5 μM of protein and 324 mM of galactose in 100 mM BTP pH 7.0 and 150 mM NaCl. Experiments are technical triplicates and error bars represent SEM.
