Synthesis of epicatechin glycosides via specific transglycosylation of cyclodextrin glycosyltransferase

Abstract

Transglycosylation of catechins by partially purified cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp.RB01 was investigated. Three to four glucoside products were obtained by TLC analysis when used (+) catechin, (-) epicatechin, or (-) epigallocatechin gallate as acceptor and beta-cyclodextrin as glucosyl donor. The enzyme showed higher acceptor specificity towards epigallocatechin gallate whereas the same specificity for donor substrate (starch, beta-cyclodextrin, maltoheptaose) was demonstrated. When the occurred products in the reaction were analyzed by HPLC, the optimum condition for transglucosylation with epicatechin as an acceptor was: incubation of 80 U/ml CGTase with 1.0% (w/v) beta-CD and 0.5% (w/v) epicatechin in 10 mM acetate buffer, pH 6.0 at 50℃ for 24 hours. The 18.07 % of overall product yield was obtained. The amounts of each product at Rt 27, 30, 45, and 48 minutes were 1.44%, 3.39%, 4.18%, and 8.33%, respectively. During optimization of transglucosylation reaction, epimerization of epicatechin to catechin was observed at alkaline pH, high temperature and longer incubation time. Reaction products were then prepared in larger scale and were isolated using Sephadex LH-20 and C18-reversed phase HPLC column. The 5.6% yield of major product at Rt48 min was obtained. The structures of the two major glucoside products elucidated by MS and NMR techniques were epicatechin-3́ -O-α-D-glucopyranoside (for Rt48 min) and epicatechin-3́-O-α-D-diglucopyranoside (for Rt45 min). The water solubility of epicatechin, the monoglucoside and the diglucoside in water were 4.9, 95 and 2.0 mg/ml, respectively. While the browning resistance to UV irradiation of epicatechin monoglucoside was higher than epicatechin diglucoside and epicatechin. The antioxidant activity of the products was also determined by DPPH scavenging assay. It was found that the inhibitory concentrations at which the absorbance of DPPH was reduced by 50% (IC₅₀) of epicatechin, the monoglucoside and the diglucoside were 25, 36 and 46 µM, respectively.