Separation And Purification Of Astragaloside ? Biotransformed By Enzyme

In this paper we studied the purification and characters of Astragaloside glycosidase from microorganism Absida sp.A3r,and mainly focused on the separation and purification of Astragaloside ? and other Astragalosides with fewer glycosyl from the enzyme reaction.Initially the theory of the enzyme reaction was studied.The enzyme from Absida sp.A3r had the activity to hydrolyze Astragaloside into Astragaloside ?.The yield is 1.2%.We studied the methods to detect enzyme activity and researched the enzyme character.The enzyme was purified by DEAE-cellulose column.The purity of astragaloside glycosidase was obtained at the 120mmol/L concentration of KCl liquor.The pure enzyme showed a single protein band,and the protein molecular weight was estimated to be about 50kDa by SDS-PAGE.It was found that the optimum temperature of enzyme reaction was 35? within the stability of enzyme reaction temperature which is from 20? to 50?,the optimum concentration of substrate was 20mg/mL,the optimum pH was 5.0 within the limit of pH which was from 4.0 to 7.0,and the optimum time for reaction was 28 hours.So this enzyme was a special hydrolase.The products of enzyme reaction were firstly depurated by macroporous adsorptive resins AB-8 and D-280.The concentration of eluant was detected at 60%?70%ethanol.And after decolorization by D-280 macroporous adsorptive resins,the yield was 12.70%.Then the products were separated and purified by silica gel column through gradient elution with the solvent of chloroform and methanol(at volume proportion of 9:1).The content of Astragaloside ? after biotransformation has increased to be 6.91%.The Astragaloside ?,purified by silica gel column,was identified by HPLC,with the purification of 62.92%.The principle of Astragaloside glycosidase was initially studied to be that Astragaloside with three glycosyl was get rid of Rhamnegin by enzyme to be Astragaloside IV.