Mechanism-Based Inhibition of GH127/146 Cysteine Glycosidase by Stereospecifically Functionalized L-Arabinofuranosides

Bifidobacterial GH146 β‐l‐Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline‐Rich Glycoproteins

“…The optimal temperature and pH for Bll4HypBA1 hydrolysis were estimated as 35 °C and 3.5, respectively (Figure 3). Additionally, the hydrolysis of the substrate with Bll4HypBA1 was inhibited by α/β‐ l ‐Ara f bromoacetamide [15b,c] (Figure SI‐4 and SI‐5), probably through a mechanism similar to that of Bll1HypBA1. Since Bll4HypBA1 did not show activity against any β‐ l ‐Ara f (1→2)‐ l ‐Ara f motif, Bll4HypBA1 was hypothesized to act after stepwise degradation with other bifidobacterial enzymes, such as HypAA and HypBA2.…”

“…TLC and HPAEC‐PAD analysis : For thin‐layer chromatography (TLC), silica gel 60 aluminum plates (Merck) developed with a propan‐1‐ol/EtOH/water (7 : 1 : 2, v / v / v ) solvent mixture, and the separated sugars were visualized by spraying orcinol‐sulfate reagent on the plates [20] . The conversions were calculated by image J using TLC results as shown previously [12c] . For high‐performance anion‐exchange chromatography with pulsed amperometric detection (HPAEC‐PAD), the oligosaccharides were analyzed with a CarboPac PA‐1 column (4 mm internal diameter × 250 mm; Dionex Corp., Sunnyvale, CA, USA).…”