嗜熱菌Geobacillus kaustophilus ATCC8005之蛋白體與熱穩定酵素

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廖嘉欣(KA-IAN LIO) 查詢紙本館藏

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論文名稱

嗜熱菌Geobacillus kaustophilus ATCC8005之蛋白體與熱穩定酵素
(Proteome and thermal stable enzymes from Geobacillus kaustophilus ATCC8005)

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摘要(中)

本研究以一株格蘭氏陽性嗜高溫細菌Geobacillus kaustophilus ATCC8005為材料，在最適生長條件(55oC、pH 7)，探討對數時期之胞內和胞外所表達之蛋白質，經三種方法(一維或二維電泳分離之膠體內胰蛋白脢消化或不經膠體分離直接在溶液中消化)和電噴霧四極桿飛行時間質譜儀，分析蛋白質胜?序列，以鑑定蛋白質。已完成基因體定序的G. kaustophilus HTA426則提供我們在資料庫比對時，>90%蛋白質身份鑑定之依據。
由胞外蛋白體中，共鑑定49個蛋白質，分別有5個(10%)和11個(22%)為細胞外和膜上的蛋白質，包括數個能夠與胺基酸和寡胜?鏈結合的抗熱性運送蛋白質、兩個與水解胞外核?酸利用有關的蛋白質和一個假設蛋白質(GK2383)。胞內蛋白體一共鑑定出517個蛋白質。這些被鑑定到的胞外與胞內的蛋白質共528個，佔HTA426 ORFs的15%。這些被鑑定到的蛋白質資料將提供我們(1)探討高溫菌在高溫逆境生長的角色上，(2)建立熱穩定蛋白質預測工具的實驗數據，(3)並能應用於搜尋新穎並具有應用性的熱穩定蛋白質上。
把本實驗所鑑定之胞內蛋白體與前人55oC熱處理30分鐘的可溶性胞內次蛋白體比較，發現只有68%的蛋白質在體外仍然保有熱穩定性，並發現與胺基酸運送和代謝相關種類的蛋白質及酵素種類的蛋白質較具有熱穩定性；另外，為了驗證前人篩選熱穩定蛋白質之方法，本研究從前人所鑑定的熱穩定蛋白質中，挑選出不同溫度範圍且具有工業應用潛力的酵素，分別為55~100oC熱穩定之三個抗氧化酵素superoxide dismutase (SOD)、peroxiredosin (PRX)和thioredoxin (TXN)；55~85oC和75~85oC熱穩定之兩個未知功能蛋白質，其序列經BLAST分析比對後，發現與Bacillus halodurans之endo-1, 4 beta-glucanase相似，可能具有纖維酵素的活性；55~75?C和55~65oC熱穩定酵素分別選取了cephalosporin acylase與monoacylglycerol lipase (MGLP)，將以傳統的生化方法來驗證其熱穩定性。本論文已完成了其中兩個純化的酵素(SOD, MGLP)的熱穩定性測定，SOD的Tm值為94oC，MGLP的最適催化溫度和Tm值分別為65oC和82oC，這些在特定溫度下具有活性與穩定性驗證了，本實驗室所建立篩選熱穩定蛋白質的平台之可用性。另外，與已發表之文獻相比較，G. kaustophilus ATCC8005之SOD的熱穩定性最佳，在94oC加熱30分鐘仍然保有一半的活性，已獲申請中美智慧財產的保護。

摘要(英)

In this study, G. kaustophilus ATCC8005, a Gram-positive, aerobic, and thermophilic bacteria, was grown at its optimal growth conditions, 55oC, pH 7. Extracellular proteins from the culture medium (secretome) and whole intracellular proteins in cellular extracts (cytosol proteome) were collected at mid-log phase. By using 1D SDS-PAGE/in-gel digestion, 2D SDS-PAGE/in-gel digestion and in-solution digestion, proteins were identified by ESI-Q-TOF in combination with Mascot search program. The genome sequence from G. kaustophilus HTA426 allows us to accurately identify (>90%) the target proteins in a high-throughput format.
In total, there were 49 proteins identified from secretome. Among them, 5 (10%) and 11 (22%) of them were found to be extracellular and located in membrane part localization, respectively. Among the extracellular proteins, they are thermostable amino acid and oligopeptide binding proteins, nuclease related proteins and one is hypothetical protein. Plus the 517 unique proteins identified from cytosol proteome, there is ~15% ORFs (HTA426) identified in log-phase. These identified proteins will be helpful to explore the growth mechanism of the thermophiles in adverse environment such as high temperature. Futhermore, it can also be used to build a prediction tool base on the experimental data of the heat-stable proteins for searching novel applicable thermostable proteins.
By comparing to a 55oC-treated cell-free extract of Geobacillus kaustophilus ATCC8005, only 68% of proteins is heat stable in vitro. Proteins belong to amino acid transport and metabolism and enzyme category are more thermostable. Furthermore, in order to verify the method for screening the thermostable proteins, we select the enzymes with different temperature range and its importance to the industrial application from those identified proteins. We found three antioxidant enzymes from 55~100oC: superoxide dismutase (SOD), peroxiredosin (PRX) and thioredoxin (TXN). When identify two unknown function proteins from 55~85oC and 75~85 oC, and sequences are compared using BLAST, we found endo-1, 4 beta-glucanase of Bacillus halodurans is similar to the hypothetical proteins we found. But the cellulase activity is still waiting for further research. Under the temperatures of 55~75oC and 55~65oC, two thermostable enzymes, cephalosporin acylase and monoacylglycerol lipase (MGLP), respectively, were identified. We will use traditional biochemical approach to verify these enzymes’ thermostability. In this thesis, we completed the thermostability assay of two thermostable proteins, which are SOD and MGLP. The Tm of SOD is 94oC, and the Topt. and Tm of MGLP is 65oC and 82oC. Thus, we have proved past students’ platform for screening thermostable proteins. In addition, when comparing with the published paper, the SOD found in this experiment has the highest thermostability. After heating for 94oC for 30 minutes, there still has half of the activity, thus we applied the patent to protect our finding.

關鍵字(中)

★ 高溫菌
★ 蛋白體
★ 熱穩定酵素

關鍵字(英)

★ geobacillus kaustophilus
★ thermophile
★ proteome
★ thermal stable enzymes

論文目次

中文摘要…………………...…………………………………………….. I
英文摘要…………………...…………………………………………….. III
誌謝…………………...………………………………………………….. V
目錄……………………...……………………………………………….. VI
圖目錄………………………………………………………………......... IX
表目錄…………………………………………………………………..... XI
附錄目錄……………………………………….………………………… XII
縮寫與全名對照………………………………………………………..... XIII
壹、緒論………………………………………………………………….. 1
1-1 嗜熱微生物…………..…………………………………………… 1
1-2 熱穩定酵素.……………………………………...……………….. 3
1-2-1 熱穩定酵素的來源和特性.……………………………….. 3
1-2-2 熱穩定酵素的應用性……………..….....................……… 4
1-3 Geobacillus 的介紹……………..….....................………………… 5
1-3-1 Geobacillus 的源起……………..………………………….. 5
1-3-2 Geobacillus kaustophilus……….………………………….. 6
1-4 蛋白質體學於Bacillus/Geobacillus 之研究……........................... 7
1-5 研究背景與目的……………………………….…………………. 8
1-5-1 研究背景…………………………………………………... 8
1-5-2 前人的研究成果…………………………………………... 9
1-5-3 本研究之目的……………………………………………... 10
貳、材料與方法………………………………………….………………. 12
2-1 菌株與培養基………...................................................................... 12
2-2 蛋白體製備………………………………….................................. 13
2-3 蛋白質定量………………………………….................................. 14
2-4 蛋白質一維電泳………………….................................................. 14
2-5 蛋白質二維電泳 ………………………………............................ 16
2-6 蛋白質消化…….............................................................................. 19
2-7 蛋白質鑑定………………….......................................................... 20
2-8 基因選殖.......................................................................................... 21
2-9 表現載體之構築.............................................................................. 23
2-10 酵素表現與純化............................................................................ 24
2-11 酵素活性測定................................................................................ 25
2-12 實驗儀器與化學藥品.................................................................... 26
?、結果……………………………………………………….……….…. 30
3-1 G. kaustophilus 之生長..………………………….……………….. 30
3-2 蛋白質體一維電泳分析………………………………………….. 30
3-3 蛋白質體二維電泳……………………………………………….. 31
3-4 蛋白質鑑定……….......................................................................... 33
3-5 蛋白質之功能分類……………………………………………….. 35
3-6基因構築與表達…….…………………………………….……….. 36
3-7重組熱穩定酵素之純化.................................................................... 38
3-8重組熱穩定酵素的活性測定與熱穩定性分析……….….……….. 40
肆、討論………………………………………………………...………… 43
4-1 胞外蛋白體之探討…….…………………………………………. 43
4-2 本研究三種方法探討胞內蛋白體之優缺點比較……………….. 45
4-3 蛋白質體學方法研究基因體已解序的原核生物之比較……….. 45
4-4 探討本研究G. kaustophilus ATCC8005 所鑑定之蛋白質與G.
kaustophilus HTA426 基因註譯之蛋白質...……………………………... 47
4-5 比較胞內蛋白體與55oC 熱處理30 分鐘之胞內次蛋白體……… 48
4-6 本研究熱穩定酵素之活性和熱穩定性探討.................................. 48
伍、結論……….………………………………………………………….. 52
陸、參考文獻…………………………………………………………….. 53
柒、圖…………………………………………………………………….. 58
捌、表…………………………………………………………………….. 85
玖、附錄…………………………………………………………………..103

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指導教授

黃雪莉(Shir-Ly, Huang)

審核日期

2008-12-3

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