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研究生:蘇琬婷
研究生(外文):Wan-Ting Sue
論文名稱:鹽濃度對於嗜熱性肝醣支切酶的活性及構形之影響
論文名稱(外文):Effects of Salt Concentration on the Activity and Conformation of Thermophilic Glycogen Debranching Enzyme
指導教授:方翠筠
指導教授(外文):Tsuei-Yun Fang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:85
中文關鍵詞:鹽濃度肝醣支切酶構形
外文關鍵詞:SaltGlycogen Debranching EnzymeConformation
相關次數:
  • 被引用被引用:3
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摘要
利用已選殖於大腸桿菌表現載體的 Sulfolobus solfataricus 的 treX 基因,由大腸桿菌來生產肝醣支切酶 (glycogen debriching enzyme, GDE),此肝醣支切酶於200 mM NaCl 下酵素穩定性及活性都較高,於無NaCl存在下酵素失去活性,因此本研究探討鹽濃度對於酵素活性與構形造成之影響。利用HPLC來分析發現,具有活性的GDE是由三個單體所組成,三聚體的分子量約為249 kDa,經脫鹽處理或降低鹽濃會使酵素構形由三聚體變成單體,而單體則不具有活性,所以鹽濃度的差異會對活性與構形造成影響。利用Circular dichroism spectra (CD spectra) 來分析重組原生型GDE的二級結構,於200 mM NaCl緩衝溶液下,三聚體有較多的α-螺旋和β-摺板二級結構,雖然單體存於200 mM 的鹽濃度下,橢圓率甚至接近三聚體,其二級結構比無鹽狀態下增加,但仍不具有活性。將三聚體隨著鹽濃度增加至200 mM NaCl時,其二級結構α-螺旋、β-摺板也增加,所以NaCl能穩定此酵素之二級結構構形。再以SELCON3、CDSSTR和CONTIN-LL三種CD光譜分析軟體分析GDE的二級結構組成,當三聚體鹽濃度由200 mM下降至10 mM時,其部分的螺旋結構會轉變成摺板。將重組原生型GDE三聚體和單體置於10-200 mM NaCl,並激發位於親水區域表面Trp和Tyr的螢光,單體和三聚體鹽濃度增加時,都能增加其螢光強度,顯示NaCl的增加有助於穩定單體及三聚體其二級結構。另外取三聚體和單體分別與ANS作用,分析疏水性區域與ANS結合的情形,發現隨著鹽濃度的降低,部分GDE可能變性,ANS與局部暴露的疏水性區域氨基酸結合,所以三聚體於10 mM NaCl緩衝溶液之螢光強度最大。而單體全部的螢光強度都很低,大部分疏水性區域可能因變性程度過大,所以螢光強度較低。由以上顯示,鹽濃度可影響GDE的構形。
Abstract
The recombinant GDEs were produced from E.coli by expressing the cloned treX gene of Sulfolobus solfataricus ATCC35092. The enzymes had higher stability and activity in presence of salt, while they lost their activities in the absence of salt. The goal of tHis study is to elucidate whether the salt concentrations affect the activity and conformation of the enzyme. After using HPLC to analyze the molecular weight under the native condition, GDE’s active form was trimer, and its molecular weight was about 249 kDa. We also found that desalting or lower the salt concentration caused the active enzyme changing its conformation from trimer to monomer, and the monomer did not have activity; therefore, the salt concentrations affect enzyme activity and conformation. To study the secondary structures of Wild-type GDE, the CD scpectra analysis was conducted. We found that trimer had more α-helixs and β-sheets in the presence of 200 mM NaCl. In the presence of 200 mM NaCl, the ellipticity of the monomer increased to nearly as same as trimer, and the secondary structures of the monomer also increased compared with that in the absence of salt. However, This monomer still had no activity in the presence of 200 mM NaCl. In different concentrations of NaCl, the α-helixs and β-sheets of trimer increased when the salt concentration increased; therefore, NaCl can stabilize enzyme’s secondary structures. Using softwares SELCON3, CDSSTR and CONTIN-LL to analyze GDE’s secondary structures, the α-helixs of trimer transform to β-sheets, when the salt concentration decreased from 200 mM to 10 mM. In addition, 200 mM salt could not convert the monomer to trimer. In the presence of 10-200 mM NaCl, the Trp and Tyr on the hydrophilic surface areas of GDE trimer and monomer were excited with 295 nm UV light, both monomer and trimer had enhanced fluorescence intensities when the salt concentration increased. This result indicated that the addition of salt can stabilize the secondary structures of monomer and trimer. In addition, ANS was used to react with trimer and monomer, respectly, to analyze the binding of ANS with hydrophobic regions of the enzyme. When the salt concentration decreased, ANS bound to the amino acids of partial exposed hydrophobic regions; therefore, trimer had maxium fluorescence intensity in 10 mM NaCl. Since most hydrophobic regions in monomer were probably disappeared due to protein denaturation, the monomers had low fluorescence intensity under various salt concentrations. The ANS analysis also showed the salt concentrations affect the GDE conformations.
目錄

壹、前言 7
貳、文獻整理 9
一、熱穩定海藻糖生成相關酵素之簡介 9
二、熱穩定肝醣支切酶 9
三、蛋白質的構形 11
四、蛋白質構形和鹽濃度的關係 12
五、分析蛋白質構形的方法 13
肆、材料方法 19
一、實驗材料 19
二、實驗方法 24
(一)重組原生型GDE的生產與純化方法 24
(二)重組蛋白質的生產與純化方法 26
(三)蛋白質電泳分析 28
(四)肝醣支切酶的酵素活性測定方法 31
(五)高效液相層析儀分析純化後之重組原生型與重組重組型GDE 34
(六)脫鹽處理 37
(七)原態梯度電泳分析與活性染色 37
(八)Circular Dichroism Spectra (CD spectra) 39
(九)螢光儀分析分析 41
(十)高效液相層析儀分析不同鹽濃度下之GDE三聚體及單體 41
(十一)復性實驗 42
伍、結果與討論 43
柒、參考文獻 80

圖表目錄

表一、重組原生型和重組重組型GDE純化表 55
表二、重組原生型和重組重組型GDE以PD-10管柱脫鹽處理的相對活性與三聚體和單體比例 56
表三、分析重組原生型GDE的二級結構組成 57
圖一、重組原生型GDE之離子交換管柱層析圖 58
圖二、重組重組型GDE親和性管柱層析圖 59
圖三、以SDS-PAGE分析重組原生型和重組重組型GDE的純化過程 60
圖五、以Bio-SEP-S-4000管柱HPLC分析重組重組型GDE 62
圖六、以BioSep-SEC-S-4000管柱HPLC分析重組原生型和His-tagged GDE分子量 63
圖七、以BioSep-SEC-S-2000管柱HPLC分析重組原生型和重組重組型GDE分子量 64
圖八、HPLC分析PD-10管柱脫鹽處理的重組原生型GDE 65
圖九、HPLC分析PD-10管柱脫鹽處理的重組重組型GDE 66
圖十、HPLC分析PD-10管柱脫鹽處理控制組的重組原生型GDE 67
圖十一、以HPLC分析PD-10管柱脫鹽處理控制組重組重組型GDE 68
圖十二、原態梯度電泳分析重組原生型和重組重組型GDE 69
圖十三、重組原生型GDE Sephacryl S-200膠體層析圖 70
圖十四、HPLC分析經Sephacryl S-200膠體層析的重組原生型GDE三聚體 71
圖十五、CD spectra分鹽對重組原生型GDE三聚體和單體的作用 72
圖十六、CD spectra分析鹽對重組原生型GDE三聚體的作用 73
圖十七、螢光分析鹽濃度對重組原生型GDE三聚體之影響 74
圖十八、螢光分析鹽濃度對重組原生型GDE單體之影響 75
圖十九、螢光分析ANS存在時鹽濃度對重組原生型GDE三聚體和單體之影響 76
圖二十、HPLC分析不同鹽濃的重組原生型GDE 77
圖二十一、鹽濃度對重組原生型GDE三聚體活性之影響 78
柒、參考文獻
黃幸光,2002,嗜高溫海藻糖生成相關酵素之基因選殖以及海藻糖苷糊精生成酶的生產與特性探討,國立海洋大學食品科學系碩士學位論文,基隆。
游靜茹,2004,嗜高溫古細菌之重組肝醣支切酶的純化及其特性探討,國立海洋大學食品科學系碩士學位論文,基隆。
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NCBI Conserved Domain Summary
http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=precalc&SEQUENCE=13815379

CD Pro
http://lamar.colostate.edu/-sreeram/CDPro/main.html

Genetics Computer Group
(http://gcg.nhri.org.tw/)
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