Characterization of a α-L-rhamnosidase from Bacteroides thetaiotaomicron with high catalytic efficiency of epimedin C

In this study, a alpha-L-rhamnosidase gene from Bacteroides thetaiotaomicron VPI-5482 was cloned and expressed in Escherichia coli. The specific activity of rhamnosidase was 0.57 U/mg in LB medium with 0.1 mM Isopropyl beta-D-Thiogalactoside (IPTG) induction at 28 degrees C for 8 h. The protein was purified by Ni-NTA affinity, which molecular weight approximately 83.3 kDa. The characterization of BtRha was determined. The optimal activity was at 55 degrees C and pH 6.5. The enzyme was stable in the pH range 5.0-8.0 for 4h over 60%, and had a 1-h half-life at 50 degrees C. The Kcat and Km for p-nitrophenyl-alpha-L-rhamnopyranoside (pNPR) were 1743.29 s(-1) and 2.87 mM, respectively. The alpha-L-rhamnosidase exhibited high selectivity to cleave the alpha-1,2 and alpha-1,6 glycosidic bond between rhamnoside and rhamnoside, rhamnoside and glycoside, respectively, which could hydrolyze rutin, hesperidin, epimedin C and 2 ''-O-rhamnosyl icariside II. Under the optimal conditions, BtRha transformed epimedin C (1 g/L) to icariin by 90.5% in 4h. This study provides the first demonstration that the alpha-L-rhamnosidase could hydrolyze alpha-1,2 glycosidic bond between rhamnoside and rhamnoside.