One-pot bi-enzymatic cascade synthesis of puerarin polyfructosides

Enzymatic glycosylation is an efficient way to increase the water solubility and the bioavailability of flavonoids. Levansucrases from Bacillus subtilis (Bs_SacB), Gluconacetobacter diazotrophicus (Gd_LsdA), Leuconostoc mesenteroides (Lm_LevS) and Zymomonas mobilis (Zm_LevU) were screened for puerarin (daidzein-8-C-glucoside) fructosylation. Gd_LsdA transferred the fructosyl unit of sucrose onto the glucosyl unit of the acceptor forming beta-D-fructofuranosyl-(2 -> 6)-puerarin (P1a), while Bs_SacB, Lm_LevS and Zm_LevU synthesized puerarin-4'-O-beta-D-fructofuranoside (P1b) and traces of P1a. The Gd_LsdA product P1a was purified and assayed as precursor for the synthesis of puerarin polyfructosides (PPFs). Bs_SacB elongated P1a more competently forming a linear series of water-soluble PPFs reaching at least 21 fructosyl units, as characterized by HPLC-UV-MS, HPSEC and MALDI-TOF-MS. Simultaneous or sequential Gd_LsdA/Bs_SacB reactions yielded PPFs directly from puerarin with the acceptor conversion ranging 82-92 %. The bi-enzymatic cascade synthesis of PPFs in the same reactor avoided the isolation of the intermediate product P1a and it is appropriate for use at industrial scale.