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Title: | 甘藷塊根Sporamin之胰蛋白抑制因數生化活性分析及其分子結構模擬 The biochemical analysis of sporamin for trypsin inhibitor activity and its homology modeling. |
Authors: | 林雅慧 |
Publication Year : | 1998 |
Degree: | 碩士 |
Abstract: | 甘藷塊根內主要的水溶性蛋白質sporamin,過去被定義為一儲藏性蛋白質,然經活性分析結果得知具有胰蛋白抑制因數活性,而將此蛋白之基因轉殖於菸草內,所得之轉殖株以斜紋夜盜幼蟲(Spodopetra litura)進行餵蟲試驗得知其具抗蟲效力,故本實驗進而探討sporamin基本生化特性及可能影響其抑制作用的活性作用區域。 將sporamin基因構築於表現載體,送入大腸桿菌中進行蛋白質的大量表現,並純化此融合蛋白進行生化特性分析:在酵素動力學的分析中,得知sporamin屬於混合型的抑制反應(Mix-type inhibition),其熱安定性(Heat stability)可達65℃ 10分鐘;其最適反應溫度(Optimal reaction temperature)為35-45℃;其最佳反應pH值(Optimal pH)為pH 7-10;外在因數對此sporamin的胰蛋白脢抑制因數活性之影響的探討研究中,得知30%以上的酒精才會降低其對抑制胰蛋白的能力,而低濃度(<7.5 mM)的鎘和鎂離子則會增加其抑制的能力;低濃波(<7.5 mM)的EDTA會降低其抑制胰蛋白的能力;然隨著鈣離子濃渡的增加會使其抑制胰蛋白的能力隨之下降。 Sporamin經序列分析比對結果發現與其他胰蛋白抑制因數具約30%同一性(Identity)及50%同源性(Homology),經序列排列(Sequence alignment)、二級結構預測(Secondary structure prediction)及蛋白質資料庫(PDB, Protein Data Bank)比對分析,推測其與Erythrina caffra的ETI結構類似,故以ETI結構為範本進行同源模擬(Homology modeling),結果顯示sporamin亦具12個β-sheet,其活性作用區推論為一loop,然此loop之胺基酸特性偏屬帶負電性電荷與ETI及其他同功能的胰蛋白抑制因數相對應之帶正電胺基酸特性不相似。 Sporamin, the major storage protein in the tuberous root of sweet potato, had been found to have tryspin inhibitor activity. The expression of sporamin gene in transgenic tobacco can confer resistance against Spodopetra litura. This thesis is focused on the biochemical analysis of sporamin for its trypsin inhibitor activity and on searching for its possible reactive sites. Sporamin is expressed in E. coli and then purified for the biochemical analysis. In the kinetics analysis, sporamin belongs to mix-type inhibition. The thermo-stability of sporamin is up to 65℃ for 10 minutes; its optimal reaction temperature is in the range of 35-45℃; its optimal reaction pH ranges from pH7-10. Sporamin can tolerence alcohol up to 30%. Low concentration (<7.5mM) of Cd++ and Mg++ can enhance its ability to inhibit trypsin activity; low concentration (<7.5mM) of EDTA can decrease its ability to inhibit trypsin activity and the higher concentration of Ca++ can lower the trypsin inhibitory activity. Sporamin have 30% identity and 50% homology with soybean Kunitz type trypsin inhibitor. Based on sequence alignment, secondary structure prediction, and protein data bank (PDB) three-dimensional structure analysis, it is predicted that sporamin has a similar structure to Erythrina caffra trypsin inhibitor DE-3 (ETI). Using the ETI structure as a template, the conformation of sporamin was predicted by homology modeling. The result indicates that sporamin also has 12 β-sheet like other Kunitz type trypsin inhibitors and the putative reactive site is located in a loop. However, the reactive loop is composed of negative charge amino acid residues which is quite different with other trypsin inhibitors. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76372 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 植物科學研究所 |
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