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研究生:林惠敏
研究生(外文):Hui-Min Lin
論文名稱:嗜鹽菌HaloferaxmediterraneiPHA生合成之探討
論文名稱(外文):STUDY ON THE PHA BIOSYNTHESIS IN HALOPHILIC Haloferax mediterranei
指導教授:林銘澤
指導教授(外文):Ming-Tse Lin
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:141
中文關鍵詞:嗜鹽菌
外文關鍵詞:Haloferax mediterraneihalophilicPHA
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本實驗探討嗜鹽菌Haloferax mediterranei在醱酵各時期之PHA synthase以及PHA depolymerase之酵素活性情形及與PHA生合成之關係。以葡萄糖、擠壓米糠澱粉以及丁酸為碳源來培養Haloferax mediterranei,結果含0.1%以上之丁酸培養基會抑制菌生長,而以米糠澱粉生長最好,且其PHA synthase活性較高,PHA depolymerase活性較低。利用pH-stat方式進行重覆式批次饋料醱酵時,結果發現無論是PHA synthase或是PHA depolymerase,其活性分布與菌的生長曲線傾向相似,但是時間上會比菌生長較慢一點。利用液態培養為種菌進行連續式批次饋料培養,僅需50hr即可達菌體最大量,僅為以菌落懸浮液為種菌所需之時間之一半,且PHA synthase比PHA depolymerase之活性高,但是菌體與PHA之最大量及PHA的含量較低,顯示PHA之生合成與菌體生理狀態有關,約於對數生長期的末期,PHA synthase開始表現。
利用Q-TOF定序PHB顆粒上的蛋白質之序列,結果並無法得到一段較完整的序列,因此進一步將其純化。結果蛋白質雖被部分純化,但經超高速離心後酵素的回收率偏低,純化過程當酵素極易失活,因此應再試其他操作條件與調整保存方式。
This study is focus on the variation of activities for PHA synthase and PHA depolymerase as well as the PHA biosynthesis in the fermentation of haliphilic Haloferax mediterranei.
H. mediterranei was cultured in shacking culture with the glucose, extructed bran/starch, or n-butanic acid as carbon sources. Above 0.1 % n-butanic acid would inhibited the growth of H. mediterranei. The extructed bran/starch was the most favorable carbon source for this bacterial growth and it had higher activity of PHA synthase but lower of PHA depolymerase. In the fermentation of continuous feeding-batch controlled by pH-stat, the activity curves for PHA synthase and PHA depolymerase were similar to the growth curve but delayed for a few hours. Using liquid culture as starter for the fermentation of continuous feeding- batch, it took 50 hrs to the maximum of cell dry weight and was only the half of time by using the suspension of colony from plate. It also had higher activity of PHA synthase than activity of PHA depolymerase. However, the CDW, PHA amount, and PHA content were lower. It showed that the biosynthesis of PHA was related to the bacterial physiologic state and PHA-related enzymes were expressed at late exponential phase.
It was failure to merge the peptide sequences of PHB granule bound proteins obtained from the sequencing results by Q-TOF. The PHB relative enzymes had been partially purified, but the recovery of these enzymes activities were very low after ultracentrifugaion due to their unstability in purification procedure. It is necessary to study the enzyme stability in future.
中文摘要 I
ABSTRACT II
目錄 IV
表目錄 VIII
圖目錄 IX
第一章 前言 1
1.1 緒論 1
1.2 生物分解性塑膠 2
1.2.1定義 2
1.2.2 檢測標準 3
1.2.3 分類 5
1.2.4 分解塑膠發展之限制與解決方法 6
1.3 PHA之簡介 7
1.3.1 PHA研究之發展 8
1.3.2 PHB合成酶 12
1.3.3 PHA depolymerase 16
1.3.4 PHB的合成途徑 17
1.3.5 PHA之應用 20
1.4 嗜鹽菌Haloferax mediterranei之介紹 23
1.4.1 嗜鹽菌的分類 23
1.4.2 嗜鹽菌的特性 25
1.5 以Haloferax mediterranei生產PHA之研究 27
1.6 實驗目的 29
第二章 材料與方法 30
2.1 實驗儀器 30
2.2 實驗材料 31
2.2.1 菌種來源 31
2.2.2 培養基與藥品 31
2.3 實驗方法 32
2.3.1 菌種保存 32
2.3.2 菌種活化與種菌培養 33
2.3.3 含丁酸培養基之培養 34
2.3.3.1 以丁酸培養液培養 34
2.3.3.2 不同濃度丁酸培養 35
2.3.3.3 額外添加不同濃度丁酸 35
2.3.4 米糠澱粉培養 35
2.3.5 醱酵培養 35
2.3.6 菌體分析 38
2.3.6 超音波破菌 40
2.3.7 超高速離心分離純化PHB granules 40
2.3.8 酵素活性之測定 41
2.3.9 SDS-PAGE 43
2.3.10 蛋白質定序 45
2.3.11 蛋白質純化 47
第三章 結果與討論 50
3.1 以不同碳源搖瓶培養 50
3.2 探討進行饋料醱酵之各時期酵素活性分布 56
3.3 蛋白質序列定序 66
3.4 蛋白質純化 68
第四章 結論 77
第五章 未來研究之建議 78
參考文獻 79
附錄 84
附錄1 CDW And OD520 Relation Curve 84
附錄1.1 CDW vs. OD520nm Curve for the Low Cell Concentration (CDW < 20 g/L) 84
附錄1.2 CDW vs. OD520nm Curve for the High Cell Concentration (CDW > 20 g/L) 84
附錄2 The Standard Curve of PHA Detection 86
附錄3 The Modified Curve of PHA Concentration 87
附錄4 以Trypsin截切定序後比對之結果 88
附錄5 以ArgC截切定序後比對之結果 95
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