Aqueous Two-Phase Purification, In Situ Immobilization And Application Of Papain

Papain was purified from spray-dried Carica papaya latex using aqueous two-phase system (ATPS). Then it was recovered from PEG phase by in situ immobilization or preparing cross-linked enzyme aggregates (CLEAs). The Plackett-Burman design and the central composite design (CCD) together with the response surface methodology (RSM) were used to optimize the APTS processes. The highly purified papain (96-100%) was achieved under the optimized conditions:40%(w/w) 15mg/ml enzyme solution,14.33-17.65%(w/w) PEG 6000,14.27-14.42%(w/w) NaH2PO4/K2HPO4 and pH 5.77-6.30 at 20℃. An in situ enzyme immobilization approach, carried out by directly dispersing aminated supports and chitosan beads into the PEG phase, was investigated to recover papain, in which a high immobilization yield (>90%) and activity recovery (>40%) was obtained. Moreover, CLEAs were successfully used in recovering papain from PEG phase with a hydrolytic activity hundreds times higher than the carrier-bound immobilized papain (360.0 nkat/g for CLEAs, 27.5 nkat/g for ZH-HA,16.9 nkat/g for LH-HA,10.9 nkat/g for BB-A,5.0 nkat/g for CH). In addition, the immobilized papain was applied to digest monoclonal antibody IgG to produce Fab fragment successfully.