臺灣博碩士論文加值系統

木質纖維素 (lignocellulose) 其主要組成中：纖維素 (cellulose) 與半纖維素 (hemicellulose) 能被降解轉換為簡單的醣類，之後透過醱酵用以生產生物燃料 (biofuel)。但是其纖維素被木質素 (lignin) 包裹並與半纖維素形成共價結合，以及其複雜的結構使得木質纖維素不易被降解利用。本實驗室從腐爛木頭上，以系列稀釋並以鹼木素作為唯一碳源之培養基篩選到之菌種：Serratia sp.。將其接種於基本固態生長培養基上，發現Serratia sp.具有顯著之鹼木素利用性。將0.5 g/L鹼木素以及0.125 g/L甘蔗渣作為誘導物誘導相關氧化蛋白表現。三天之後破壞菌體結構，離心收取上清液並以2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) 進行活性分析，其活性值為69 U/L。分析其對源於Trichoderma reesei之市售纖維水解酶 (Tre cellulase) 水解甘蔗渣時的幫助性。在pH 6.0的環境下，加入50 mU Serratia酵素液與1.8 U Tre cellulase於5 ml緩衝液作用時，還原糖含量為0.92 mg/ml，比未添加酵素液時提高了2.7倍。藉由硫酸銨沉澱、凝膠過濾層析以及陰離子交換層析純化之酵素液，由聚丙醯胺膠體分析，仍舊具有許多雜蛋白。Serratia sp.具有一多酚氧化酶CueO，推斷其為可能之相關酵素。將此段基因選殖至pETDuet-1載體中，於大腸桿菌E. coli BL21 (DE3) 中表現。發現純化之CueO，在相同條件下對Tre cellulose水解甘蔗渣時不具有幫助性。多酚氧化酶亦具有染劑脫色的能力。在pH 6.0的環境下，aniline blue與methyl blue於隔夜後，其脫色的比例達到91%與92%。在pH 8.0的環境下，remazol brilliant blue R於隔夜後，其脫色的比例達到62%。顯示其CueO具有染劑脫色之潛力。

Lignocelluloses contain cellulose and hemicellulose which can be hydrolyzed into simple sugars suitable for the production of biofuels by fermentation. However, cellulose is wrapped by lignin, in turn bound covalently to hemicelluloses; this complex structure makes lignocelluloses hard to be degraded. A strain of Serratia sp. from a piece of rotting wood was isolated using agar plates with alkaline lignin as the sole carbon source in this study. This strain grows fast in alkaline lignin-containing minimal medium. 0.5 g/L alkaline lignin and 0.125 g/L bagasse were used as inducers to induce the oxidative enzyme activity of Serratia sp.. After three day culture, the cell was disrupted, the supernatant was collected by centrifugation and the activity was analyzed by using ABTS as the substrate. The activity was 69 U/L. The hydrolysis of sugarcane bagasse by Trichoderma reesei cellulases (Tre cellulases) in the presence or absence of the Serratia crude enzyme was analyzed. At pH 6.0, the addition of 50 mU Serratia enzyme into 1.8 U Tre cellulases increase the content of reducing sugar by 2.7 times compared with the reaction with Tre cellulases alone. The enzyme preparation after purification steps of ammonium sulfate precipitation, gel filtration chromatography and anion exchange chromatography still contain many proteins bands on SDS-PAGE. Serratia sp. has a polyphenol oxidase CueO that was thought to be the enzyme promoting the hydrolysis activity of Tre cellulases. The CueO gene was cloned into pETDuet-1 vector and the recombinant protein was expressed in E. coli BL21 (DE3). Nonetheless, the purified recombinant Serratia CueO was not able to promote the hydrolysis of bagasse catalyzed by Tre cellulases as did the crude Sarratia enzyme preparation. On the other hand, the recombinant CueO had the ability to decolorize several dyes. At pH 6.0, aniline blue and methyl blue could be decolorized 91% and 92% after an overnight reaction. At pH 8.0, remazol brilliant blue R was decolorized 62% after an overnight reaction, showing that Serratia CueO has the potential for bleaching of the dyes.

中文摘要 i
Abstract ii
目錄 iii
圖目錄 v
表目錄 vi

第一章 緒言 1
一、 生質能源 (biomass energy) 1
二、 木質纖維素 (lignocellulose) 2
三、 木質素的降解 3
實驗目的 6

第二章 材料與方法 7
一、 使用之菌株與載體 7
二、 培養基組成 7
三、 菌株篩選 9
四、 菌種身份鑑定 10
五、 菌種對鹼木素之利用性 12
六、 活性蛋白之表達 12
七、 蛋白氧化活性之測定 12
八、 蛋白質定量 13
九、 不同比例之誘導物對蛋白活性之影響 13
十、 測定與纖維酶水解甘蔗渣時之幫助性 13
十一、 DNS法測定還原糖含量 14
十二、 與協助纖維酶水解相關之活性蛋白的純化 14
十三、 聚丙醯胺膠體電泳 17
十四、 蛋白之活性染色 17
十五、 純化蛋白性質之分析 18
十六、 Serratia sp.多酚氧化酶基因之選殖 19
十七、 多酚氧化酶之表達 22
十八、 多酚氧化酶之純化 22
十九、 多酚氧化酶對染劑脫色之影響 23

第三章 結果 27
一、 菌株身份鑑定 27
二、 蛋白活性分析 27
三、 與協助纖維酶水解相關之活性蛋白的純化 28
四、 純化蛋白性質之分析 29
五、 多酚氧化酶之表達與純化 30
六、 多酚氧化酶對染劑漂白之影響 31

第四章 討論 32
一、 菌株篩選 32
二、 蛋白之活性分析以及測定與纖維水解酶共同作用時的幫助性 32
三、 Serratia sp.之CueO對於染劑漂白之應用性 34

第五章 參考文獻 61

第六章 附錄 66
附錄一，FavorPrepTM GEL/PCR Purification Kit 66
附錄二，FavorPrepTM Plasmid Extraction Mini Kit 67

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