Abstract:This paper explored the separation and purification of β-glucosidase from abalone viscera by twotime aqueous two-phase extraction combined with chitosan flocculation and precipitation and investigated the influence of factors on purification efficiency，such as aqueous two-phase composition，chitosan addition，pH during complexation，and shaking time. Finally，a highly efficient separation and purification technology combining aqueous two-phase extraction with chitosan precipitation was successfully constructed. In the primary aqueous two-phase extraction process，the total system was 10 g with natural pH conditions，and the optimal composition was PEG600/（NH4）2SO4，with mass fractions of 18% and 14%. The purification factor was 5.8. By taking the upper phase 5 g of the primary extraction system and adding NaH2PO4 powder to 17% （mass fraction） of the total extraction system for secondary extraction，the secondary aqueous two-phase extraction system was developed，with a total purification factor of 7.9. The optimal conditions for the chitosan precipitation method were chitosan addition of 1.5 mg/mL，pH of 5.8 during complexation，and a shaking time of 30 min.Through the above process，β-glucosidase with higher enzyme activity recovery and purification factor was obtained，with the specific activity of 8.4 U/mg and the purification factor of 42.2.