以蛋白質體學鑑定嗜熱放線菌
Thermomonospora fusca生長於纖維素之分泌蛋白質

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許琇雁(Hsiu-Yen Hsu) 查詢紙本館藏

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以蛋白質體學鑑定嗜熱放線菌 Thermomonospora fusca生長於纖維素之分泌蛋白質
(Proteomic identification of proteins secretedfrom Thermomonospora fusca grown on cellulose )

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摘要(中)

嗜高溫放線菌(Thermomonospora fusca YX)可分泌多種胞外熱穩定蛋白質，特別是纖維素之分解酵素。此菌株在1957年於德國由Henssen等人從馬糞堆肥中分離；在本研究進行中時，2007年三月由Wilson等人完成基因體定序，共有3117個ORFs，並於同年九月發表首篇以蛋白質體學研究T. fusca YX生長於纖維雙糖(cellobiose)之胞外蛋白質。
本研究之目的為探討T. fusca YX生長在以纖維素(cellulose)為唯一碳源的環境下之胞外蛋白質，菌株培養在最適溫度(55oC)，並於穩定期收集胞外蛋白質體，經由蛋白質體學方法(一維、二維電泳分離蛋白質經膠體內胰蛋白酶消化及直接在分泌體中進行消化)，續以兩種不同質譜儀(LC-Q-TOF-MS/MS及MALDI-Q-TOF-MS/MS)鑑定蛋白質，鑑定到的蛋白質以蛋白質家族(Pfam)及兩種胞內外蛋白質預測工具(CELLO及PSORTb)判斷蛋白質於細胞的位置(subcellular location)。研究中共鑑定到93個蛋白質，其中32個(34.4%)為胞外蛋白質，8個( 8.6%)為膜蛋白質，53個(57.0%)為細胞內蛋白質。32個已鑑定胞外蛋白質以COG進行功能分類，與醣類代謝相關蛋白質佔30%。本研究已鑑定12個與醣苷水解酶有關的酵素(如cellulase, endoglucanase, xylanase)，5個具醣類結合結構的蛋白質(如cellulose-binding protein)，2個與多醣分解相關蛋白質(pectate lyase、parallel beta-helix repeat-containing ricin B lecctin)，4個蛋白質分解酶(serine peptidase、aminopeptidase Y、hypothetical protein Tfu 0745、hypothetical protein Tfu 0746)，4個酯/脂解酶(acetyl xylan esterase、triacylglycerol lipase、hypothetical protein Tfu 2387)，1個抗氧化酶(superoxide dismutase)，2個與細胞壁上結合相關蛋白質(surface protein、hypothetical protein Tfu 2615)，1個抗生素(bacteriocin)及1個熱休克蛋白質(heat shock protein DnaJ)。
本研究與2007年Wilson研究團隊於研究方法上有以下3點不同: (1)菌株生長所選用的唯一碳源分別為cellulose與cellobiose；(2)收集分泌體的時間點上，本研究於可分泌大量蛋白質的穩定期，Wilson則為對數期；(3)胞外蛋白質的分析方法上，我們以一維、二維電泳及四種不同條件之溶液內消化進行分析，使分析複雜蛋白質體更加完整，而Wilson僅以二維電泳進行分析。將Wilson鑑定到15個胞外蛋白質與本研究鑑定到32個胞外蛋白質進行比較，有11個相同(Tfu 0620、Tfu 1074、Tfu 1627、Tfu 1959、Tfu 2176、Tfu 0901、Tfu 2923、Tfu 2130、Tfu 1268、Tfu 1665、Tfu 2403)，有4個於本研究中未發現: putative secreted xylanase or chitin deacetylase (Tfu 2789)、putative secreted protein containing lytic transglycosylase and goose egg white lysozyme domain (Tfu 0458)、putative secreted proteolytic enzyme containing NLPC_P60 domain (Tfu 1029、putative secreted proteolytic enzyme containing NLPC_P60 domain (Tfu 2262)，而我們多鑑定到21個胞外蛋白質，如(1)多醣類分解酶: cellulase (Tfu 2712)、2種xylanase (Tfu 1213、Tfu 2791)、pectate lyase (Tfu 2168)、mannan endo-1,4-beta-mannosidase (Tfu 0900)；(2)具醣類結合結構的蛋白質: 3種cellulose binding protein (Tfu 2990、Tfu 2788、Tfu 1612)、parallel beta-helix repeat-containing ricin B lectin(Tfu 1643)；(3)可將環境中大分子有機物分解為小分子的分解酶: serine peptidase (Tfu 0484)、acetyl xylan esterase (Tfu 0082)、2種triacylglycerol lipase (Tfu 0882、Tfu 0883)、hypothetical protein Tfu 0745、hypothetical protein Tfu 0746、hypothetical protein Tfu 2387；(4)可清除細胞外會造成酵素受到氧化損害的超氧化物或自由基之抗氧化酵素: superoxide dismutase (Tfu 0957)；(5)其他: bacteriocin (Tfu 0814)、heat shock protein DnaJ (Tfu 0198)、surface protein (Tfu 0607)、hypothetical protein Tfu 2615。
本研究希望以T. fusca YX建立鑑定環境微生物分泌蛋白體學之平台，尤其是針對嗜熱性纖維素分解菌，以助於生質酒精的開發；此外，比較兩種蛋白質細胞位置預測工具的準確性與效能，PSORTb (Precision 100%，Recall 0.89)略佳於CELLO (Precision 92%，Recall 0.83)，但CELLO針對胞外蛋白質的預測則較為準確，研究中鑑定到的32個胞外蛋白質，CELLO預測結果有31筆為True positive (TP)，因此，以CELLO較適用於T. fusca YX之胞外蛋白質的研究，將來可用於預測其他放線菌的胞外蛋白質。

摘要(英)

Thermophilic actinomycete (Thermomonospora fusca YX) is able to secrete many different extracellular thermophilic proteins, especially the cellulose degrading enzymes. This bacterium was isolated from horse compost by Henssen et al. in Germany 1957. In March of 2007, Wilson et al. completed the genome sequence of YX with a total of 3117 ORFs; furthermore, in the same year of 2009, T. fusca YX was published for the identification of cellobiose using the study of proteomics.
In our study, we want to find the extracellular proteins secreted from T. fusca YX using cellulose as the sole carbon source grown at 55oC. The extracellular proteins are collected when bacteria are harvested at stationary phase. This study uses proteomics (1D and 2D SDS-PAGE using in-gel and in-solution digestion), followed by two different mass analysis (LC-Q-TOF-MS/MS and MALDI-Q-TOF-MS/MS). The enzymes identified are analyzed using Pfam and extracellular protein prediction tools (CELLO and PSORTb) to delicately predict the proteins in the subcellular location. The 93 proteins were identified and among them, 32 (34.4%) were extracellular proteins, 8 (8.6%) were membrane proteins, 53( 57%) were cytosolic proteins. The 32 extracellular proteins were further using COG for function classification. There were 30% of proteins related to glycolysis and most are either slightly acidophilic proteins (the pIs lower than 7 are 82%). This study also identified 12 glycoside hydrolyzing enzyme (for example, cellulase), 5 glucose binding proteins such as cellulose binding proteins, 2 were polysaccharide related degrading enzymes (pectate lyase, parallel beta-helix repeat-containing ricin B lecctin), 4 were protein protease (serine peptidase, aminopeptidase Y, hypothetical protein Tfu 0745, hypothetical protein Tfu 0746), 4 were esterase/lipase (such as acetyl xylan esterase and triacylglycerol lipase), 1 antioxidant enzymes (superoxide dismutase), 2 cell wall/solute-binding proteins (the surface protein, hypothetical protein Tfu 2615), 1 antibiotic (bacteriocin) and 1 heat shock proteins (heat shock protein DnaJ).
In 2007, the Wilson group uses only 2D SDS-PAGE to analyze extracellular proteomes and the 15 proteins identified in the literature were compared with 32 proteins found in our lab. There were 11 matched proteins with the additional 21 thermostable secretomes that were not identified in Wilson’s study. The 21 proteins are for example, the glucose degrading enzymes such as cellulose, xylanase, pectate lyase, and mannan endo-1,4-beta-mannosidase. The protease that is able to degrade large molecules of organic materials into smaller ones in the environment like serine peptidase, aminopeptidase Y, acetyl xylan esterase and triacyoglycerol lipase; proteins like superoxide dismutase that is able to act again the toxins from hydrogen peroxide or free radicals in order to protect cells from damage. And finally, antibiotic like bacteriocin for inhibit or terminate bacterial infection.
We selected a growth phase and carbon source (cellulose) that is able to secrete a great amount of secretome, which is different from Wilsons that use cellobiose as the sole carbon source and harvest bacteria at the log phase. Other than 2D SDS-PAGE for protein separation, 1D SDS-PAGE and 4 different conditions of Gel-free digestion are also used in order to increase the protein amount. The three different isolation methods can provide a full report for complex proteomic research. We use T. fusca YX to as a model and built a research platform for studying the secretome of the environmental microorganisms, especially this thermostable cellulolytic bacterium is helpful in the exploration of bioethanol research; in addition, when comparing the above tools for predicting cellular localization in terms of precision and performance, CELLO is the most suitable prediction tool for the cell localization of T. fusca YX in the proteomic study of actinomycetes.

關鍵字(中)

★ 纖維素分解酶
★ 纖維素
★ 胞外蛋白質
★ 嗜高溫放線菌
★ 蛋白質體

關鍵字(英)

★ cellulose
★ cellulase
★ Proteomic
★ extracellular protein
★ Thermophilic actinomycete

論文目次

摘要……………………...…………………………...…………………..I
Abstract....................................................IV
目錄……………………...…………………………..…………………..VII
圖目錄……………………………………………..…………….….........X
表目錄……………………………………………..………….……….....XII
附錄目錄…………………………………………..……………...….......XIII
縮寫與全名對照………………………………...…………………….....XIV
壹、緒論…………………………………………...……….……………..1
一、熱穩定性酵素及其應用價值……….........…………………….…1
二、嗜熱菌之特性及其嗜熱機制……........…………………….…….3
2-1 生理特性….....…………..........................………..3
2-2 嗜熱機制….................………............………..…4
三、嗜高溫放線菌在生物技術產業之應用..............................6
四、Thermomonospora fusca YX簡介...............................7
五、蛋白質體學於T. fusca YX.…….......……..........…….8
5-1 蛋白質體學之介紹.......................................8
5-2 T. fusca YX之蛋白質體學..................................9
六、研究動機與目的........................................10
貳、材料與方法……………………………......…………………………12
一、菌株與培養基............................................12
二、生長.......................................................13
三、胞外蛋白質樣品製備....................................14
四、蛋白質定量.................................................15
五、蛋白質一維電泳分析........................................16
六、蛋白質二維電泳分析........................................18
七、電泳膠體軟體分析.........................................21
八、膠體內消化(In-gel digestion)......................21
九、溶液內消化（Gel-free digestion）.....................23
十、蛋白質鑑定...........................................24
十一、次細胞位置........................................24
十二、實驗儀器與化學藥品.................................25
叁、結果………………………............................….30
一、T. fusca YX之生長……...........................………..30
二、胞外蛋白質體之膠體電泳分析…………….......………………...30
三、胞外蛋白質體之溶液內消化.................................33
四、不同方法鑑定之熱穩定胞外蛋白質........................34
五、蛋白質之功能分類…………………….......................34
肆、討論……………………………………………….....………………37
一、胞外蛋白質總量......................................37
二、胞外蛋白質分子量及等電點分析........................38
三、胞外蛋白質功能分析.......................................39
四、以三種方法研究胞外蛋白質之結果探討......................41
五、以蛋白質體學研究胞外蛋白質之文獻比較.....................42
六、生物資訊應用於胞外蛋白質之預測..........................44
伍、結論與建議……………………………...…………………………..46
陸、參考文獻…………………………………...………………………..48
柒、圖…………………………………………...………………………..53
捌、表……………………………………...……………………………..73
玖、附錄……………………………………...…………………………..91

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2009-2-3

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