Generation of an alpha-rhamnosidase library and its application for the selective derhamnosylation of natural products

A screening of 16 different fungal strains was performed under different cultivation conditions, using L-rhamnose or L-rhamnose-containing flavonoid glycosides (rutin, hesperidin, and naringin) as specific inducers. No significant constitutive production of a-L-rhamnosidases was detected in noninduced cultures, while high levels of these glycosidase activities were obtained using different inducers. New species, so far unknown for the production of a-L-rhamnosidases, were identified. More than 30 different a-L-rhamnosidase samples were prepared by ammonium sulfate precipitation. Substrate specificity of this a-L-rhamnosidase library was tested with various L-rhamnose-containing natural compounds (flavo- noids, terpenoids, and saponins). Most of the enzymatic preparations showed broad substrate specificity, and some of them were also acting on sterically hindered substrates (e.g., quercitrin). The screening of the library under different reaction conditions showed the coexis- tence, in the same preparation, of more than one a-L- rhamnosidase activities with different substrate specificity and different stability towards organic cosolvents. To exploit this enzymatic library for synthetic applica- tions, the presence of contaminating a-L-arabinosidases and h-D-glucosidases was investigated. The latter en- zymes were observed in several preparations, while a-L- arabinosidase content was generally quite low. The selective derhamnosylation of the saponin desglu- coruscin was performed on a preparative scale. The enzyme obtained by rhamnose induction of the Aspergillus niger K2 CCIM strain showed high activity towards this substrate and negligible a-L-arabinosidase contamination. Therefore, it was chosen as a catalyst for the selective derhamnosylation reaction, which provided the desired product in 70% yield.