Preparative purification of bromelain (EC 3.4.22.33) from pineapple fruit by high-speed counter-current chromatography using a reverse-micelle solvent system.

The crude protein of pineapple fruit was purified by high-speed counter-current chromatography (HSCCC). Excellent separation was achieved after careful investigation as follows: cetyltrimethylammonium bromide (CTAB) was selected to prepare the reverse micelle phase at the concentration of 0.10g/mL. The mobile phase was 0.05M sodium phosphate buffer solution, including mobile phase A (pH 9.5, containing 0.2M KCl) used for equilibration and mobile phase B (pH 7.0, containing 0.4M KCl) used for elution. The flow rate of the mobile phase was set at 1.5mL/min and the effluent was monitored at 280nm. Under these conditions, 127.3mg bromelain (EC 3.4.22.33) was produced from 200mg crude sample. A large-scale procedure was then carried out, and 3.01g bromelain was obtained from 5.00g crude extract in a 200min run. The separated protein was analysed by SDS-PAGE, compared with the standard and then identified by TOF/TOF-MS.