Comparison of four purification methods to purify cysteine protease from Asian pear fruit (Pyrus pyrifolia)

Pear fruit is one of the most popular fruits, extensively exploited for its protease activity commercially in the food, cosmetic, and pharmacological industries. The aim of this study was to compare four different purification methods for protease purification from an Asian pear cultivar. Pear proteases were purified by ethanol precipitation, two phase partitioning, or ion exchange chromatography (DEAE Sepharose alone or followed by Mono Q). Purified proteases from each method were further evaluated for purification efficiency by SDS-PAGE analysis and enzyme activity with succinlyated casein substrate. All methods produced two protease bands with molecular weights of 36 kDa and 38 kDa. Among the methods, 15% PEG 1000-20% MgSO4 system and DEAE followed by Mono Q chromatography showed efficient separation of protease with high recovery of enzymatic activity (91-98%). DEAE followed by Mono Q chromatography achieved the highest purification fold (15). These result suggested that a two phase partitioning technique is effective as a single purification step for pear protease.