現今環保議題受到重視，聚羥基鏈烷酸酯 (PHA) 作為可替代塑料的材料之一，其具有良好的生物相容性及生物可降解性，但受限於高的生產成本阻礙了PHA的商品化。PHA從細菌細胞中的萃取過程佔了總成本的30％至50％，是降低PHA生產成本進入商業化的關鍵之一。本研究將溶菌酶基因與Cupriavidus sp. L7L或Ralstonia eutropha H16之pha合成基因組於大腸桿菌中共同表現。在PHA累積條件下，溶菌酶基因的表現不會抑制細菌生長。累積結果顯示以葡萄糖酸 (gluconic acid) 為碳源，並在有溶菌酶表現時，其生物生質量可提高約1 dry wt g/L及約20 dry wt % PHA含量。溶菌酶基因與pha合成基因組共同表現可有效提升PHA的萃取效率，萃取過程不使用氯仿及二氯甲烷等有機溶劑。本研究建立的最佳純化方式包含添加EDTA及界面活性劑處理、20%甲醇清洗菌體、蛋白水解酶 (Alcalase) 消化蛋白質、最終以50%甲醇清洗後，其結果顯示萃取後可獲得高純度 (95 dry wt ％) 的PHA顆粒。進一步以1% SDS清洗PHA顆粒後其純度可達99 dry wt %。最終以SDS-PAGE與CBR染色分析PHA顆粒表面蛋白，幾乎偵測不到蛋白質的存在。本研究建立PHA最佳化純化方式在未來有望運用於工業化使用。

Environmental threats are considered. Polyhydroxyalkanoates (PHAs), as one of the materials that can replace the plastics, possess biocompatibility and biodegradability. The high production cost of PHA hampered its commercialization. The extraction process of PHA from bacterial cells accounts for 30% to 50% of the total cost, which is one of the keys to reducing the production cost of PHA into commercialization. In this study, the lysozyme gene and the pha operon derived from Cupriavidus sp. L7L or Ralstonia eutropha H16 were co-expressed in Escherichia coli. In the PHA accumulation experiments, the expression of lysozyme gene had no adverse effect on bacterial growth. The biomass and PHA content were increased about 1 dry wt g/L and 20 dry wt%, respectively, when gluconic acid was the carbon source. The co-expression of lysozyme gene with pha operon effectively improved the extraction efficiency of PHA. The extraction process does not use organic solvents such as chloroform and methylene chloride. The best purification method established in this study includes the treatment of bacterial cells with EDTA and surfactant Triton X-100, washing the cell with 20 methanol, digesting the cells with proteolytic enzyme (Alcalase), and finally washing the cells with 50% methanol. The purity of PHAs reached 95 dry wt % after purification precess. After further cleaning the PHA particles with 1% SDS, the purity can even reach 99 dry wt %. The protein on the PHA granules are not detectable by means of SDS-PAGE and CBR staining. The optimal purification method of PHA exhibited in this study is expected to be applied to industrial use in the future.

摘要………………………………………………………..……………………………..i
Abstract……………………………………………………………….…………………ii
目錄……………………………………………………………………..………….…iv
表目錄………………………………………...………………………...………………vi
圖目錄……………………………………………….……………………….………vii
附圖目錄……………………………………………….……………………………viii
第一章 前言………………………………………………………………….…………1
一、石化塑膠之危害………………………………………………..……...…..…1
二、聚羥基烷酸酯 (polyhydroxyalkanoates, PHAs) ……………..……….……1
1.聚羥基烷酸酯……………………………..………………………………..1
2.PHA之化學結構與物理性質……………….……………..…………….…2
3.生合成路徑……………………………..……………………….………….3
4.PHA合成酶的分類………………………….……………………...………4
5.聚羥基烷酸酯的純化………………………………………………………5
6.聚羥基烷酸酯的應用………………………………………..………..……7
三、葡萄糖酸 (gluconic acid, GA) …………………………………..…..………8
四、溶菌酶 (lysozyme) …………………………………………..……..………...9
1.蛋清溶菌酶 (egg white lysozyme) ……………………..….……..………..9
2.噬菌體T7溶菌酶 (T7 bacteriophage lysozyme) …………………..……..9
五、研究目的與策略………………………………………………………..……10
1.研究目的……………………………………………………………..……10
2.研究策略………………………………………………………..…………10
第二章 材料與方法……………………………………………….………..…………11
一、實驗菌株及培養基……………………………………………………..……11
二、藥品與儀器………………………………………………………..…………11
三、質體建構…………………………………………………………..…………11
1.基因建構………………………………………………………..…………11
2.接合作用………………………………………………………..…………12
3.轉形作用………………………………………………………..…………12
四、PHA累積……………………………………………..…………..…………13
五、PHA純化方法測試………………………………………………....………13
1.滲透壓差破菌……………………………..………………………....……13
2.超音波震盪……………………………..…………………………………13
3.醇類清洗對PHA萃取之影響………………………….………....………13
4.添加界面活性劑對於PHA萃取之影響………………...…………..……13
5.以蛋白酶Alcalase處理菌體對PHA純度的影響………..….……..……14
6.有機溶劑純化PHA……………………………………………………..…14
六、PHA含量分析……………………………………………………………..…14
七、十二烷基硫酸鈉聚丙烯醯胺凝膠電泳…………………………………..…15
1.電泳膠體配製…………………………………………………..…..…..…15
2.樣品處理及蛋白質電泳………………………..…………………………15
第三章 結果…………………………………………………………………….…..…16
一、建構溶菌酶基因與pha基因組共表現載體…………………………..…16
二、溶菌酶基因與pha基因組共同表現對PHA累積的影響…………….…16
三、以滲透壓破菌法萃取細菌PHA………………………………………..…17
四、超音波震盪提高PHA萃取效率…………………………….……….…..…17
五、以醇類或酮類清洗菌體對PHA純度的影響……………...…………….…17
六、添加界面活性劑對純化PHA之影響…………………………………...…17
七、蛋白酶處理菌體對PHA純度的影響…………………………………….18
八、測試蛋白酶反應時間………………………..…………………………….18
九、純化PHA的最佳化流程………………………………………………..…18
十、PHA顆粒表面蛋白質分析…………………………….………………19
第四章 討論………………………..………………………………………….…20
結論………………………..………………………………...…………………….…23
參考文獻………………………..……………………………………………..….....…24

表目錄
表一、本研究使用之引子………………………………………...…………………..28
表二、本研究與pha基因組共同表現對PHA累積之結果…..…………………..29
 
圖目錄
圖一、本研究建構之基因圖譜………………………………………………………..30
圖二、超音波震盪萃取PHA之效率分析……………………………………….......31
圖三、醇類或酮類清洗菌體之結果……………………………................................32
圖四、不同濃度之Trotion X-100對PHA萃取效率的影響………..………………33
圖五、不同清潔劑對PHA萃取效率的影響……………………………..…….……34
圖六、不同濃度之SDS對PHA萃取效率的影響………………………..…………35
圖七、滲透壓試驗經介面活性劑後以甲醇清洗等條件之PHA純度變的結果…...36
圖八、蛋白水解酶Alcalase萃取方法前處理之甲醇濃度的影響結果…………….37
圖九、T7溶菌酶共表現的試驗組 (pT7C16AB16) 測試蛋白水解酶Alcalaes反應
時間對萃取影響之結果……………………………………………….……38
圖十、最佳化純化流程………………………………………………………………..39
圖十一、測試最佳化純化流程於各階段PHA變化的結果…………………...40
圖十二、PHA顆粒表面蛋白質分析…………………………....……………….41


 
附圖目錄
附圖一、PHA之化學結構……………….………………...…………………….……..2
附圖二、PHA之合成代謝路徑……………….………..………………………..……..3
附圖三、PHA合成酶分類……………………….…………………………………..…5
附圖四、PHA應用領域………………………….……………………………………..7
附圖五、溶菌酶作用位置示意圖………………………………..………………….…10
附圖六、基因建構示意圖…………………………………..………………………….12
附圖七、Triton X-100及EDTA作用位置示意圖……………….…….……...………21

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