학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부(식품생명공학전공), 2013. 2. 장판식.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.1. Introduction 1
2. Materials and Methods 5
2-1. Materials 5
2-2. Preparation of crude enzyme 6
2-3. Purification using fast protein liquid chromatography 7
2-4. Analysis of β-glucosidase activity and protein concentration 9
2-5. Polyacrylamide gel electrophoresis 11
2-6. Determine of molecular mass 12
2-7. Effects of temperature and pH on the enzyme 12
2-8. Enzyme kinetics 13
2-9. Zymography 14
2-10. Amino acid analysis 14
2-11. Circular dichroism 15
3. Results and Discussion 17
3-1. Purification of β-glucosidase seeds of pumpkin (Cucurbita moschata) 17
3-2. Characterization of β-glucosidase from seeds of pumpkin (Cucurbita moschata) 22
3-2-1. Determination of molecular mass 22
3-2-2. Effects of pH and temperature on the enzyme 24
3-2-3. Enzyme kinetics 30
3-3. Effects of interaction between subunits on the enzyme activity 32
3-4. Structural characteristics of β-glucosidase from seeds of pumpkin (Cucurbita moschata) 35
4. Conclusion 42
5. References 44
국문초록 47Maste
