Basic Studies to Clarify the Role of the β-Primeverosidase in Tea Leaves

澄清茶叶中 β-Primereverosidase 作用的基础研究

• 批准号: 09660112
• 负责人: SAKATA Kanzo
• 金额: $ 2.11万
• 依托单位: KYOTO UNIVERSITY (1998-1999)Shizuoka University (1997)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660112/
• 关键词: Primeverosidase; Glycosidase; Inhibitor; Affinity Choromatography; Disaccharide glycoside; Tea; Amidine; 1-Amino sugar; Camellia sinensis; 茶; グリコシダーゼ阻害剤; アミジン; アフィニティー; 配糖体; β-プリメベロシダーゼ; p-aminophenyl 1-thio-β-primeveroside; グルコシダーゼ

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 … More 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

为了研究我们发现的与乌龙茶和红茶加工过程中花香形成有关的β-Primeveroside酶在茶树中的真实作用以及它在其他植物中的存在，本研究计划启动了这项研究计划。首先，我们尝试制备酶的亲和吸附剂，建立一种简便的酶的纯化方法。1)以ρ-氨基苯基-1-硫代-β-糖苷为配体制备亲和吸附剂，并将其应用于茶叶糖苷酶的亲和层析。按照已报道的方法合成了两种ρ-硝基-1-硫代-β-葡萄糖苷和1-硫代-β-Primeveroside。它们被催化还原并连接到商业上可获得的基质甲酰-纤维素钠。一种含有β-Primeveroside酶和β-葡萄糖…的粗糖苷酶用每种亲和吸附剂处理更多的Dase。2)以1-氨基糖为配基的亲和吸附剂的制备及其对茶叶糖苷酶亲和层析的评价1-β-氨基糖以结晶形式存在。它们与丁二酸酐和3.3-二甲基戊二酸酐反应得到酰基产物。对这些氨基糖及其酰基衍生物进行了茶叶粗糖苷酶的抑制试验。每种氨基糖都表现出很强的抑制活性，但它们的酰基衍生物不再具有活性。根据已有的知识，我们所做的所有努力都是徒劳的，都是徒劳的。因此，我们决定从1-β-氨基糖开始开发一种新的配体。1-β-氨基糖被证实对糖苷酶具有很强的竞争抑制活性，并且易于高产率获得。3)开发一种新型的以1-氨基糖为起始的亲和吸附剂的配体。1-DNA-氨基葡萄糖、D-半乳糖和D-木糖与苯硫代乙酰亚氨酸甲酯反应，以高产率(83-95%)得到新的氨基氢溴胺(1，2和3)。这些化合物对相应的糖苷酶均表现出较强的竞争性抑制作用(Ki=9.4×10ィイd1~8ィエd1，2.4×10ィイd1~6ィエd1和2.5×10ィイd1~6ィエd1M)。这些活动也是非常有选择性的。这些配体是非常有前途的糖苷酶亲和层析吸附剂。较少

项目成果

Ijima,Y.et al.,: "Characterization of β-primeverosidase, being concernde with alcoholic aroma formation in tea leaves to be processed into・・・・"J.Agric.Food Chem.. 46. 1712-1718 (1998)

Ijima, Y. 等人：“β-primeverosidase 的表征，与待加工的茶叶中酒精香气的形成有关”J.Agric.Food Chem.. 46. 1712-1718 (1998)

Sakata, K.: "β-Primeverosidase concerned with the floral tea aroma formation during processing of oolong tea and black tea.""Chemistry of Caffeinated Beverages. ACS Symposium Series," ed. by Ho, C. and Parliament, T. H., American Chemical Society, Anaheim

Sakata, K.：“β-Primeverosidase 与乌龙茶和红茶加工过程中花茶香气的形成有关。”“含咖啡因饮料的化学。ACS 研讨会系列”，Ho, C. 和Parliament, T. H. 编，美国。化学会，阿纳海姆

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).

郭，W.，平武，J.，小川，K.，山本，M.，马，S-J.，臼井，T.和坂田，K：“N-β-糖基脒作为β-糖苷酶的有效和高选择性抑制剂“J. Amer. Chem. Soc.（准备中）。

K.Sakata,N.Watanabe and T.Usui: "Food for Health in the Pacific Rim"Food & Nutrition Press,Inc.,Trumbell,Conneticat,USA. 93-105 (1999)

K.Sakata、N.Watanabe 和 T.Usui：“环太平洋地区的健康食品”食品

Sakata,K..: ""Chemistory of Caffeinated Bevarages, ACS Symposium Siries"β-Primeverosidase concerned with the floral tea aroma・・…"American Chemical Society(in press). (2000)

Sakata, K..：““含咖啡因饮料的化学，ACS 研讨会系列”与花茶香气有关的β-Primeverosidase...”美国化学会（2000 年出版）。