Partial Purification of Mulberry (Morus rotunbiloba) Peroxidase Using Aqueous Two-Phase Extraction Coupling with Ion-exchange and Gel-filtration Chromatography

บทคัดย่อ

The third to fifth leaves of mulberry plant were selected for peroxidase extraction due to its high specific activity comparing to other sets of leaf. The initial enzyme isolation included homogenization and extraction of phenolic compound using aqueous two-phase extraction system consisting of 20/8.9 %(w/v) PEG/(NH)₂SO₄. This system gave a satisfactorily less partition coefficient (0.018) and also less peroxidase volume (34% of total volume). The purification was performed on a DEAE-Cellulose column and double chromatography of Sephadex G-75. The overall result of mulberry peroxidase purification gave 157.4-folds with 43.4% recovery. The native molecular weight of mulberry calculated from the relative fraction of the standard curve from gel filtration was found to be 29 kDa as which agreed with one band of the molecular weight identified by SDS-PAGE.