Purification, biochemical characterization and antioxidant property of ZCPG, a cysteine protease from Zingiber montanum rhizome.

Zingiber montanum cysteine protease glycoprotein (ZCPG) was purified to homogeneity by DEAE- cellulose and Sephadex G50 resulting in sixteen fold purification and total activity of 39.4U/mg. ZCPG presented a prominent single peak in HPLC chromatogram with an estimated molecular weight of 48kDa on native PAGE. SDS-PAGE gave two subunits of ∼24.3 and ∼24.6kDa showing its heterodimeric form. Protein sequencing was studied by MALDI-TOF MS/MS. Isoelectrofocusing exhibited two isoforms with pI values of 4.8 and 5.1. Analysis of the total carbohydrate by GC-MS/MS showed the presence of glucose, mannose, fucose and xylose. The pH and temperature optimum were 9 and 60°C respectively while Km and Vmax values were 0.5±0.03μg and 13.73±2.07U/ml respectively. ZCPG was strongly inhibited by NEM indicating the cysteine-type. Substrates such as casein, azocasein, gelatin, BSA and haemoglobin showed high relative activity. Metal ions of CuCl2, CoCl2, HgCl2 and ZnCl2 showed partial inhibition at 1mM concentration. Furthermore, ZCPG exhibited promising antioxidant activity in biochemical systems as well as THP-1 cells. These findings suggested, ZCPG with significant antioxidant activity might have potential applications in therapeutic and food industry.