Purification and Characterization of β-Glucosidase from Seeds of Pumpkin (Cucurbita moschata)

Abstract

The objective of this study was purification and characterization of β-glucosidase (EC 3.2.1.21), catalyzing the hydrolysis of β-glucosidic bonds, from Pumpkin seed (Cucurbita moschata). The β-glucosidase was purified by fast protein liquid chromatography using Hitrap DEAE-sepharose FF, Hitrap Q-sepharose XL, and HiPrep 16/60 Sephacryl S-100 Hiresolution column. The 8.24-fold purified enzyme had a specific activity of 16.62x10^(-2) unit/mg protein against p-nitrophenyl-β-D-glucopyranoside (pNPG). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis showed β-glucosidase from pumpkin seed was dimeric structure with molecular mass of 48.1 kDa, the sum of 28.8 and 19.3 kDa. The molecular mass estimated by SDS-PAGE was analogous with that of 42.8 kDa estimated by gel permeation chromatography using Sephacryl S-100 Hiresolution column. The optimum temperature and pH of the enzyme were 70°C and pH 4.0, respectively. The enzyme was stable in the range of pH 2.0 to 10.0 and under 60°C, respectively. The Km, Vmax, and kcat of β-glucosidase employing p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrate were 2.22 mM, 0.078 unit/mg protein, and 13.29 min^(-1), respectively. From the result of zymogram analysis, the enzyme had the activity in the condition not that subunits were departed but that subunits were associated. It was composed of 364 amino acids, and the secondary structure of β-glucosidase from seeds of pumpkin (Cucurbita moschata) consists of α-helix (26.10%), antiparallel β-sheet (20.17%), parallel β-sheet (8.22%), β-turn (18.16%), and random coil (27.34%) in the stable condition and it was affected by temperature and pH. There is a possibility that the enzyme have the characteristics of pH reversible inactivation according to the study of the effect on the enzyme activity and stability. The purified β-glucosidase could cleave β-glucosidic bonds in phytochemicals and enhance the bioavailability of phytochemicals. Moreover, it was found that pumpkin seed-derived β-glucosidase has exceptional stability at wide range of pH in pre-experiment. Therefore the purified β-glucosidase could be applied to the food industry and pharmaceutical industry.