Purification and structure analysis of yam proteins from different species

• Main Authors: Liao, Yu-Hsiu, 廖育秀
• Other Authors: Tseng, Chin-Yin
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/46332899894187731272

碩士 === 國立嘉義大學 === 食品科學系碩士班 === 92 === Yam, the tuber of Dioscorea spp., is rich in proteins and shows diversified enzymatical, biological and physiological functions. Although yams have been widely studied, little is known about the structure-related properties. Thus, in this study will be carried out by near infrared FT-Raman spectroscopy. First of all, yam proteins isolated from various yam cultivars including Dioscorea alata L., D. alata L.var. purpurea, and D. Japonica are assayed with HPLC and SDS-PAGE, and structural analysis is performed by FT-Raman spectroscopy. Raman results clearly indicate that the secondary structure of Dioscorea alata L. is mainly in α-helix, while D. alata L. var. purpurea was mostly in anti-parallel β-sheet. In contrast, D. Japonica, which belongs to a different species, exhibited explicit differences in amino acid compositions and molecular structure of which conformation is a mixed form of α-helix and anti-parallel β-sheet. FT-Raman also directly proved the existence of S-S in yam proteins, implying that oligomers formation in yam proteins might be due to disulfide linking of dioscorin. Second, FT-Raman is employed to probe the structural change of yam proteins against different environmental impacts such as heating, UV irradiating, pH changing, and disturbing agents stressing. It indicates that yam proteins from different cultivars behaved differently in various environments. Dioscorea alata L. is the most sensitive to external impacts, and D. Japonica is the least. Upon heat aggregation, the secondary structure of all three cultivars is predominant in anti-parallel β-sheet. D. alata L.var. purpurea exhibits most heat resisting, while D. alata L. is most labile to heating. Upon pH value changing, the three yam cultivars show similar trend as the temperature effect, i.e. pH accessible is in the order of D. alata L., D. alata L.var. purpurea, and D. Japonica. The results according to UV irradiation also reveal that D. alata L. is most sensitive to UV irradiation. Based on the analysis of Raman spectra of yam proteins against denaturing agent such as SDS, DTT, NEM, 2-ME, and EG, it implies that hydrophobic interact, ionic interaction, and disulfide bonding play important roles in maintaining the structural stability of yam proteins. Finally, the major storage protein of yam, dioscorin, is separated, purified and analyzed with IEC, HPLC, and SDS-PAGE, and its structure is characterized by FT-Raman. It demonstrates that dioscorin is made up of dioscorin A and dioscorin B with molecular mass 31 kDa and 29 kDa, respectively. Both dioscorin A and B might aggregate into a dimmeric molecule with molecular mass 64 kDa. According to FT-Raman spectra, the secondary structure of dioscorin A is mainly in α-helix and dioscorin B is in anti-parallel β-sheet In summary, in these studies FT-Raman is used to investigate the molecular structure of yam proteins, which will provide a conspicuous assistance for clarifying the functional properties and processing potentiality of yam proteins.