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-tBoc 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.

DWI Leibniz Institute for Interactive Materials Forckenbeckstraße 50 52056 Aachen Germany

Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 14220 Prague Czech Republic

Laboratory for Biomaterials Institute of Biotechnology and Helmholtz Institute for Biomedical Engineering RWTH Aachen University Pauwelsstraße 20 52074 Aachen Germany

Citace poskytuje Crossref.org

New Bacterial Aryl Sulfotransferases: Effective Tools for Sulfation of Polyphenols

Sulfated Phenolic Substances: Preparation and Optimized HPLC Analysis