Synthesis of fisetin glycosides by cyclodextrin glycosyltransferase from paenibacillus sp. Rb01

Abstract

Fisetin is a flavonoid which was widely exerted in pharmacological industries. However, fisetin has low water solubility and unstable in aqueous solution. So the uses of fisetin have been limited. The purpose of this study was to increase water solubility of fisetin by transglycosylation reaction using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01. The reaction had to perform in co-organic solvent system and DMSO at 40% (v/v) was proved to be the most suitable co-organic solvent since it provided the high enzyme stability and high production yield. Moreover, the types of glycosyl donor were also determined and β-cyclodextrin was found to be an appropriate glycosyl donor. The effects of fisetin, β-cyclodextrin, and enzyme concentration and incubation time on the fisetin glycoside synthesis were also determined. It was found that the optimum condition was to incubate 0.25% (w/v) of fisetin and 1.0% (w/v) of β-CD with 200 U/mL of CGTase at 40ºC for 24 hours. Under optimum condition, the percent conversion of fisetin (17.1% ± 4.21%) was significantly increased when compared to that of non-optimized condition (10.1% ± 1.15%). HPLC analysis showed that at least 5 fisetin glycosides were synthesized by CGTase. Structural analysis by glucoamylase and α-glucosidase revealed that fisetin linked with glucose through alpha-glycosidic bonds, while glucose molecules joined together by α-1, 4 glycosidic bond. The obtained fisetin glycoside products were purified by n-butyl acetate extraction and further refined by HPLC. LC-MS/MS revealed that CGTase synthesized two types of fisetin monoglucosides (m/z of 447), two types of fisetin diglucosides (m/z of 609) and one type of fisetin triglucoside (m/z of 771). Fragmentation patterns suggested two possible structures of fisetin monoglucoside products. The water solubility of three purified products were investigated. It was found that water solubility of fisetin glycosides increased at least 500 times higher than the parent fisetin. DPPH assay showed that the antioxidant activity of synthesized fisetin glycosides was not different when compared with fisetin.