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 39.4 U/mg. ZCPG presented a prominent single peak in HPLC chromatogram with an estimated molecular weight of 48 kDa on native PAGE. SDS-PAGE gave two subunits of ∼24.3 and ∼24.6 kDa 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 ± 0.03 μg and 13.73 ± 2.07 U/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 1 mM 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.