臺灣博碩士論文加值系統

在全球暖化及能源危機的大環境下，生質能源更加的受到重視，其中以生長時不與糧食作物爭奪土地、生產資源，及不具有轉化過程中難以被分解的木質纖維素的大型海藻做為原料生產生質能源最具有潛力。在大型海藻的褐藻個體中，含量最高的碳水化合物即是褐藻膠 (alginate)，若能夠研發降解褐藻膠的技術並搭配醱酵產程，以褐藻做為原料生產生質能源將會是解決能源問題的一個契機。本研究於基隆潮間帶分離到一株能夠分解褐藻膠並且能夠以其做為單一碳源生長的溶藻弧菌Vibrio alginolyticus KL-10。以全基因體已解序的Vibrio alginolyticus 40B的褐藻膠裂解酶 (alginate lyase) 基因之同源性序列設計引子對alg-F及alg-R對V. alginolyticus KL-10染色體DNA進行PCR，成功地從其中增幅出一段2 kb的DNA片段，將此PCR產物以TA cloning的方式接合至yT&A載體，重組質體yT&A-alg以熱轉型法送入E. coli DH5α保存，以核苷酸定序分析得知其中1566 bp為藻膠裂解酶基因，可轉譯出521個胺基酸，分子量約為60 kDa。重組質體yT&A-alg以限制酶Hind III去除褐藻膠裂解酶基因中的signal peptide序列後，以Hind III切位重組至表現載體pET30c，得到的重組質體pET30c-alg以熱休克轉型法送至E. coli BL21 (DE3)，轉型株E. coli BL21 (DE3) (pET30c-alg) 以1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) 於16℃下誘導表現18小時，於胞內粗酵素液可測得褐藻膠裂解酶的活性69.82 unit/mg。胞內粗酵素液經由nickel column純化後的洗提液，可測得褐藻膠裂解酶的活性135.82 unit/mg，經由SDS-PAGE及zymogram分析可知褐藻膠裂解酶融合蛋白的大小約為70 kDa，在20-37℃及pH 7-9中具有酵素活性，且在30℃及pH 8有最高酵素活性。在改造生質能源生產菌的部分已成功構築穿梭質體pKT230-alg及pIMP1-alg，但對於生物資源保存及研究中心 (BCRC) 購得的Zymomonas mobilis ATCC 10988及本實驗室前人篩選到一株能夠生產氫氣的Clostridium xylanolyticum Ter3皆無法以電穿孔轉形獲得帶有重組質體的轉形株，僅有Saccharomyces cerevisiae BY4741成功的以LiAc / PEG轉型獲得帶有重組質體pRS426-alg的轉型株，但轉形株S. cerevisiae BY4741 (pRS426-alg) 目前尚未測得褐藻膠裂解酶基因的表現活性。

Global warming and energy crisis are major drivers for a shift from the use of fossil fuels to renewable energy, and then, brown macroalgae exhibit several key features of an ideal feedstock for production of biofuels, such as requiring no arable land, fertilizer, or fresh water resources. Alginate is the most abundant sugars in brown macroalgae, therefore when microorganism fermentation combine alginate degradation technology, it will be a new strategy for bioethanol production. This study has isolated a gram-negative bacterium, which could degrade alginate and use as the sole carbon source for its growth, and it was taxonomically identified as Vibrio alginolyticus KL-10 by 16S rRNA sequencing. By referring to the homologus sequence of alginate lyase gene of Vibrio alginolyticus 40B which has been finished whole genome sequencing to design the PCR primers, the alginate lyase gene of Vibrio alginolyticus KL-10 was amplified from genomic DNA and sequenced. The alginate lyase gene was 1566 base pairs in length and could code for a protein product of 57.6 kDa including signal peptide. Cloning this gene with expression vector pET30c into the E.coli BL21 (DE3) and inducing with 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) at 16℃ for 18 hour, the alginate lyase activity of 69.82 Umg-1 was obtained from the cell curde extract, but the activity increased to 135.82 Umg-1 after purifing by nickel column. SDS-PAGE and zymogram analysis displayed that the fusion protein of alginate lyase and 6X His-tag had a molecular mass of 70 kDa, its optimum temperature and pH for enzyme activity were 30℃ and pH 8, respectively. In order to heterologously express the alginate lyase gene in ethanol-producing microorganisms, four shuttle vectors: pKT230 of Zymomonas mobilis/E.coli, pIMP1 of the Clostridium spp./E. coli, and pRS423 and pRS426 of Saccharomyces cerevisiae/E. coli were used to construct recombinant plasmids pKT230-alg, pIMP1-alg ,pRS423-alg, pRS426-alg. However, pKT230-alg and pIMP1-alg can not be transformed into Z. mobilis and C. xylanolyticum Ter3, respectively, by electroporation transformation. On the other hand, only pRS426-alg was successfully introduced into S. cerevisiae BY4741 by LiAc/PEG transformation, but for unknown reason the transformant did not express alginate lyase activity.

中文摘要 i
Abstract ii
目錄 iv
表目錄 vii
圖目錄 viii
壹. 前言 1
一、 生質能源 1
（一） 生質能源概況 1
（二） 以海藻研發生質能源 1
（三） 褐藻 2
二、 褐藻膠介紹 2
（一） 來源 2
（二） 組成 3
（三） 褐藻膠的應用 4
三、 褐藻膠裂解酶 6
（一） 褐藻膠裂解酶的來源 6
（二） 褐藻膠裂解酶的作用方式 9
（三） 褐藻膠裂解酶的特性及測定方式 10
（四） 褐藻膠裂解酶的應用 10
貳. 研究動機 12
參. 材料與方法 13
一、 實驗材料 13
（一） 菌株 13
（二） 質體 14
（三） Primer 15
（四） 儀器/軟體 15
（五） 藥品 15
（六） 培養基 16
（七） 試劑及緩衝溶液 18
二、 實驗方法 21
（一） 褐藻膠分解菌的分離純化與菌種鑑定 21
1. 分離褐藻膠分解菌 21
2. 菌種保存 21
3. 培養基添加不同含量褐藻膠之生長曲線 21
4. 菌種鑑定 21
5. 褐藻膠裂解酶活性測定方法 22
（二） 褐藻膠裂解酶基因的選殖及定序分析 23
1. V. alginolyticus KL-10的chromosomal DNA製備 23
2. 質體DNA的萃取 24
3. 聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 24
4. PCR產物純化 24
5. 瓊脂膠體電泳 (agarose gel electrophoresis) 24
6. 從電泳膠體萃取DNA (gel extraction) 25
7. 限制酶的切割 25
8. DNA片段之接合 25
9. E. coli勝任細胞的製備及熱休克法轉型作用 26
10. 轉型株的快速篩選 26
11. DNA片段定序分析 27
（三） 重組褐藻膠裂解酶特性分析 27
1. 重組褐藻膠裂解酶基因以IPTG誘導表現 27
2. 重組褐藻膠裂解酶的純化 (nickel-column purification) 27
3. 重組褐藻膠裂解酶的SDS-PAGE分析 28
4. 蛋白質標準曲線之制定與定量 28
5. 褐藻膠裂解酶最適反應溫度測定 29
6. 褐藻膠裂解酶最適反應pH值測定 29
（四） 褐藻膠裂解酶基因的異源表現表現 29
1. S. cerevisiae穿梭質體構築及LiAc/PEG轉型 29
2. Z. mobilis穿梭質體的構築及電穿孔轉型 30
3. Clostridium屬穿梭質體的構築及電穿孔轉型 31
肆. 結果 33
一、 褐藻膠裂解酶生產菌的分離及菌種分析 33
（一） 褐藻膠分解酶生產菌的分離 33
（二） 菌種鑑定分析 33
（三） 以褐藻膠為單一碳源之生長曲線 33
（四） V. alginolyticus KL-10 褐藻膠裂解酶分析 34
二、 褐藻膠裂解酶基因的選殖 35
（一） 褐藻膠裂解酶基因選殖 35
（二） 褐藻膠裂解酶基因定序分析 35
三、 褐藻膠裂解酶特性分析 37
（一） 以表現載體pET30c選殖褐藻膠裂解酶基因 37
（二） 褐藻膠裂解酶基因在E.coli BL21 (DE3) 的誘導表現 37
（三） 活性染色分析 (zymogram) 38
（四） 重組褐藻膠裂解酶之最適溫度及最適pH 38
四、 褐藻膠裂解酶基因的異源表現 40
（一） S. cerevisiae BY4741 40
（二） Z. mobilis ATCC 10988 40
（三） C. xylanolyticum Ter3 41
伍. 討論 42
參考文獻 45
圖表 53
附錄 84

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