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 0.10 g/mL. The mobile phase was 0.05 M sodium phosphate buffer solution, including mobile phase A (pH 9.5, containing 0.2 M KCl) used for equilibration and mobile phase B (pH 7.0, containing 0.4 M KCl) used for elution. The flow rate of the mobile phase was set at 1.5 mL/min and the effluent was monitored at 280 nm. Under these conditions, 127.3 mg bromelain (EC 3.4.22.33) was produced from 200 mg crude sample. A large-scale procedure was then carried out, and 3.01 g bromelain was obtained from 5.00 g crude extract in a 200 min run. The separated protein was analysed by SDS–PAGE, compared with the standard and then identified by TOF/TOF-MS.