Synthesis of rutinosides and rutinose by reverse hydrolysis catalyzed by fungal α-L-rhamnosidases

The synthesis of alpha-L-rhamnosyl(16)-beta-D-glucosides (rutinosides) and alpha-L-rhamnosyl(16)-beta-D-glucose (rutinose) catalyzed by several fungal alpha-L-rhamnosidases was studied by the reverse hydrolysis reaction between rhamnose plus naringenin 7-beta-D-glucoside (prunin) and rhamnose plus glucose, respectively. The products of the reaction were determined by HPLC with some available standards. As expected, the major product of prunin rhamnosylation was always narirutin (naringenin-7-beta-D-rutinoside), which originated from the glycosylation of the primary alcoholic group of the glucoside. Whereas, Aspergillus terreus, Penicillium decumbens and P. ulaiense alpha-L-rhamnosidases gave other minor derivatives besides narirutin, two commercial A. niger enzymes synthesized it as the unique reaction product. Initial rate kinetics of narirutin synthesis catalyzed by the purified A. niger alpha-L-rhamnosidase did not permit distinction between sequential and ping pong mechanisms, but showed strong inhibition by the substrate l-rhamnose. Equations derived for competitive inhibition together with non-exclusive inhibition by this substrate, gave the best fit to the kinetic experimental data. When glucose was rhamnosylated by the A. niger enzyme, two other minor products appeared together with the disaccharide rutinose. Conditions for obtaining maximum rutinose yield were determined.