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

为了研究β-primeverosida酶的真正作用，我们发现了哪些是与在加工过程中形成的花卉茶芳香相结合的，在茶叶植物中，作为其他植物中的生长情况良好，本研究计划已启动。首先，我们已经尝试过准备好用酶来吸引亲和力，以建立一种用于酶的表面纯化方法。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\-aminophenyl1-thio-\-glycosides as ligands and their evaluation for affinity chromatography of glycosidases from tea leaves。According to the reported？two kinds of？-thio-glycoside and 1-thio-primeveroside were synthesized。它们已被分类地减少并连接到商业上可用的矩阵公式-蜂窝式。crude glycosidase fractions含有β-primeverosidase和β-glucosi ... More 这个日期被每一个亲和力的吸收者治疗了。选择性吸收没有被观察到。2）与1-氨基糖作为配体及其对从茶叶中糖基化酶的亲和色谱的制备。1-β-氨基糖基化酶发现很容易被发现为晶体，而使用甲基氨酰胺。它们与succinic anhydride和3.3-二甲基glutaric acid anhydride反应以给予acyl产品。这些氨基糖及其乙酰衍生物被提交到茶叶中的crude glycosidase的抑制试验中。每个氨基糖都显示出潜在的抑制剂活动，但他们的乙酰衍生物不再起作用。他们并不被评判为适合于该配体的亲和力色谱吸收;所有我们的努力都要在基于先前知识的基础上准备亲和力吸收酶。因此，我们决定开发一种新的配体，以启动我们的目的，以确定如何展示潜力对抗糖基化酶的活性，并在高年级容易获得.3）开发一种新的配体，以启动1-氨基糖的D-葡萄糖的氨基糖，D-半乳糖和D-木糖与甲基苯甲酸甲酯羟基反应，导致新的羟基氨基（1、2和3）在高产量（83- 95%）。These compounds showed high competitive inhibition （ki=9.4×10型D1-8型D1， 2.4×10型D1-6型D1 and 2.5×10型D1-6型D1 M） against each corresponding glycosidases。这些活动也很有选择性。这些配体对糖基化酶的亲和力色谱吸附剂有很大的承诺。Less（低）

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