AbstractWe report the first heterologous production of a fungal rutinosidase (6‐O‐α‐L‐rhamnopyranosyl‐β‐D‐glucopyranosidase) in Pichia pastoris. The recombinant rutinosidase was purified from the culture medium to apparent homogeneity and biochemically characterized. The enzyme reacts with rutin and cleaves the glycosidic linkage between the disaccharide rutinose and the aglycone. Furthermore, it exhibits high transglycosylation activity, transferring rutinose from rutin as a glycosyl donor onto various alcohols and phenols. The utility of the recombinant rutinosidase was demonstrated by its use for the synthesis of a broad spectrum of rutinosides of primary (saturated and unsaturated), secondary, acyclic and phenolic alcohols as well as for the preparation of free rutinose. Moreover, the α‐L‐rhamnosidase‐catalyzed synthesis of a chromogenic substrate for a rutinosidase assay – para‐nitrophenyl β‐rutinoside – is described.magnified image