1999 Fiscal Year Final Research Report Summary
Basic Studies to Clarify the Role of the β-Primeverosidase in Tea Leaves
| Project/Area Number |
09660112
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | KYOTO UNIVERSITY (1998-1999)
Shizuoka University (1997) |
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Principal Investigator |
SAKATA Kanzo Kyoto University, Institute for Chemical Research, Professor, 化学研究科, 教授 (20087563)
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| Co-Investigator(Kenkyū-buntansha) |
坂田 完三 京都大学, 化学研究科, 教授 (20087563)
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| Project Period (FY) |
1997 – 1999
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| Keywords | Primeverosidase / Glycosidase / Inhibitor / Affinity Choromatography / Disaccharide glycoside / Tea / Amidine / 1-Amino sugar / Camellia sinensis |
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| Research Abstract |
To investigate the real role of the β-primeverosidase, which was found by us to be concerned with the floral tea aroma formation during the processing of oolong tea and black tea, in tea plants as well as its occurrence among other plants, this research plan was launched. First of all, we tried to prepare affinity adsorbent fortheenzyme to establish a facile purification method for the enzyme. We started with application of the affinity ligands previously reported for affinity chromatography of a few kinds of glycosidases.
1) Preparation of affinity adsorbents with ρ-aminophenyl 1-thio-β-glycosides as ligands and their evaluation for affinity chromatography of glycosidases from tea leaves.
According to the reported procedures two kinds of ρ-nitrophenyl 1-thio-β-glucoside and 1-thio-β-primeveroside were synthesized. They were catalytically reduced and connected to commercially available matrix Formyl-Cellulofine. A crude glycosidase fractions containing the β-primeverosidase and β-glucosi
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dase was treated with each of the affinity adsorbents. Selective adsorption was not observed.
2)Preparation of affinity adsorbents with 1-amino sugars as ligands and their evaluation for affinity chromatography of glycosidases from tea leaves.
1-β-Amino sugars were found to be easily obtained as crystals using methanolic ammonia. They were reacted with succinic anhydride and 3.3-dimethylglutaric acid anhydride to give acyl products. These amino sugars and their acyl derivatives were subjected to the inhibition test for the crude glycosidase from tea leaves. Each amino sugar showed potent inhibitory activity, but their acyl derivatives were not active anymore. They were judged not to be suitable for the ligands for the affinity chromatographic adsorbent.
All our efforts to prepare affinity adsorbents for the primeverosidase based on the previous knowledge have resulted in vain. So we decided to develop a new ligand for our purpose starting with the 1-β-amino sugars which were confirmed to show potent competitive inhibitory activities against glycosidases and to be easily obtained in high yield.
3)Development of a new type of ligand for affinity adsorbents starting with 1-amino sugars.
1-β-Amino sugars of D-glucose, D-galactose and D-xylose were reacted with methyl phenythioacetimidate hydrobromide to yield new amidinium hydrobomide (1,2 and 3) in high yield (83-95%). These compounds showed high competitive inhibition (ki=9.4×10ィイD1-8ィエD1, 2.4×10ィイD1-6ィエD1 and 2.5×10ィイD1-6ィエD1 M) against each corresponding glycosidases. These activities were also very selective. These ligands are very promising ones for the affinity chromatographic adsorbents for glycosidases. Less
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[Publications] Ijima, Y., Ogawa, K., Watanabe, N., Usui, Ohnishi-Kameyama, M., Nagata, T. and Sakata, K: "Characterization of β-primeverosidase, being concerned with alcoholic aroma formation in tea leaves to be processed into black tea, and preliminary observations on its substrate specificity."J. Agric. Food Chem. 46(5). 1712-1718 (1998)
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「研究成果報告書概要(欧文)」より
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[Publications] Sakata, K., Watanabe, N., and Usui, T: "Molecular basis of alcoholic aroma formation during tea processing"Food for Health in the Pacific Rim, "Proceedings of the 3rd International Conference of Food Science and Technology,(ed.by J. R. Whitaker, et al)., Davis, California, Foods Nutrition Press, Inc., Trumbull, Connecticut. 93-105 (1999)
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「研究成果報告書概要(欧文)」より
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[Publications] Guo, W., Hiratake, J., Ogawa, K., Yamamoto, M., Ma, S-J., Usui, T. and Sakata, K: "N-β-Glycosylamidines as potent and highly selective inhibitors of β-glycosidases"J. Amer. Chem. Soc. (in preparation).
Description
「研究成果報告書概要(欧文)」より
