KnowVolution Campaign of an Aryl Sulfotransferase Increases Activity toward Cellobiose

Sulfated polysaccharides such as cellulose can mimic the functionalities of pathophysiologically important glycosaminoglycans. Enzymatic sulfation offers a green chemistry route to selective (mono)sulfation of oligosaccharides (e.g., cellobiose as a building block of cellulose) in aqueous solution, at ambient temperature, and high chemoselectivity. Here, we report the first KnowVolution campaign for the aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense to advance ASTB toward a synthetically attractive biocatalyst. The generated final recombination variant (ASTB‐M5) carries two amino acid substitutions (Leu446Pro and Val579Lys) leading to an up to 7.6‐fold increase in specific activity (6.15 U mg −1 ) that was obtained with one round of KnowVolution. Mass spectrometry analysis confirmed a monosulfated product of cellobiose and structure elucidation by NMR confirmed the sulfation at the positions C‐3 or C‐4 of GlcNAc‐linker‐ t Boc as opposed to the preferred C‐6 by chemical means. Computational analysis suggested an important role of Leu446Pro in substrate‐binding and recognized Val579Lys as a distal substitution.