Abstract: In this experiment, salting-out followed by aqueous two-phase extraction was applied to purify papain from Hainan-grown papaya fruit. The crude enzyme was precipitated by a two-step salt-outing method with optimized saturation degree of (NH4)2SO4 of 20% and 45%, respectively. Using one-factor-at-a-time method and response surface methodology with a three-factor and three-level Box-Behnken design, the optimized conditions for aqueous two-phase extraction were determined as follows: [C4mim]N(CN)2 concentration 29.76%, (NH4)2SO4 concentration 16.08% and pH 8.18. Under these conditions, the extraction efficiency of papain was 89.01% and the specific activity of the purified enzyme was 1 056 U/mg with 86.72% recovery and 10.6-fold purification. The purified enzyme contained a significantly reduced number of impurity bands, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), suggesting good purification efficiency.

Key words: papaya, papain, salting-out, ionic liquids, aqueous two-phase system, sodium dodecyl sulfate-polyacrylamide gel electrophoresis