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研究生:黃筵嘉
研究生(外文):Yan-Jia Huang
論文名稱:篩選特殊單體組成之聚羥基烷酸酯生產菌並分析其PHA合成酶基質專一性
論文名稱(外文):Screening polyhydroxyalkanoate producer with unique composition and analyzing substrate specificity of the PHA synthase
指導教授:李佳音李佳音引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:103
中文關鍵詞:聚羥基烷酸酯
外文關鍵詞:PHA
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本研究利用MSM培養基培養及Nile red染色法,從活性污泥菌株中初步篩選與分離,再經氣相層析儀分析後,可從PHA生產菌株中篩選出,TO7、PO6、PO21、TS18及TF1-1五株具高的PHA生產率。PO6及PO21菌體內的PHA包含短鏈及中鏈的PHA單體組成,且中鏈PHA單體組成的莫耳百分比(mol%)可經由培養基中不同的碳氮比(C/N ratio)調控,當C/N ratio越高則中鏈PHA單體組成的mol%則越高,然而經丙酮劃分及氣相層析儀分析後確定PO6及PO21菌體內所生產的PHA是屬於混合的PHA (blend PHA),而不是短鏈及中鏈單體隨機合成在同一條高分子鏈上的雜合的PHA (hybrid PHA)。分離株TS18及TF1-1的PHA單體組成為3-HB,可利用廉價的蔗糖做為碳源累積PHA,並且在使用果糖及propionic acid複合式碳源時,其累積的PHA單體有3-HB及3-HV兩種。菌株TS18在使用1.5%果糖為碳源時,其PHA產率更可高達75.7%的菌體乾重,且使用果糖及propionic acid複合式碳源時,其累積的PHA (3HB-co-3HV)產率高達60%以上。TF1-1的3-HV單體mol%可經由不同的碳源比值1.5%/0.025% ~ 1.5%/0.1% (果糖/propionic acid)可調控範圍為17 mol% ~ 42 mol%。篩選菌株TO7是中鏈
PHA生產菌,當提供0.5% octanoate為唯一碳源時,PHA產率為56.4%,而其中3-HO單體組成比率占PHA之93.7%。此5株PHA生產菌株經16S rDNA系統演化分析後,鑑定出TO7、PO6及PO21分別為Pseudomonas mosselii、Pseudomonas nitroreducens及Pseudomonas citronellolis,TS18及TF1-1為Duganella zoogloeoides。將中鏈PHA生產菌TO7之PHA合成酶PhaC1及PhaC2選殖,再分別送入PHA合成酶缺陷株Pseudomonas putida GPp104 PHA-中表現其基因產物。當使用0.5% octanoate為碳源時,TO7的PHA合成酶PhaC1及PhaC2在Pseudomonas putida GPp104 PHA-中表現分析後,其PHA的組成也是以3-HO單體為主的PHA。
We screened the poly(3-hydroxyalkanoates) (PHAs) producers by cultivated in MSM containing 0.5 µg/ml Nile red from environment. Nine PHA producers had been isolated and verified for their PHA accumulation ability by flask fermentation and gas chromatography analysis. Among the wild type strains, TO7, PO6, PO21, TS18, and TF1-1 possess high PHA accumulation ability. The monomer compositions of PHAs produced by PO6 and PO21 possessed short-chain-length (scl) and medium- chain-length (mcl) monomers. The mcl monomer mol% of PHA produced by PO6 and PO21 could be regulated by the C/N ratio in PHA accumulation medium. The PHAs produced by PO6 and PO21 were further identified as scl-mcl blend PHA not the scl-mcl hybrid PHA by hot-acetone fractionation and GC analysis. The monomer composition of PHA accumulated by strains TS18 and TF1-1 was only 3-hydroxybutyrate (3-HB). Strains TS18 and TF1-1 could use cheaper carbon source such as sucrose to produce PHA. The PHA yield of TS18 could reach up to 75.7% of cell dry weight while fructose was used as the sole carbon source. Strains TS18 and TF1-1 both could incorporate 3-hydroxyvalerate (3-HV) monomer into PHB polymer when propionic acid was supplemented in the medium. 3-HV monomer composition of PHA produced by TF1-1 could also be regulated from 17 mol% to 42 mol% by supplementing various concentration of propionic acid with constant fructose as
carbon sources, concentration ratio of fructose to propionic acid from 60 to 15. Strain TO7 was the mcl-PHA producer and 3-hydroxyoctanoate (3-HO) monomer composition of mcl-PHA was 93.7% of PHA content from octanoate as sole carbon sources. Strains TO7, PO6 and PO21 were considered to belong to Pseudomonas mosselii, Pseudomonas nitroreducens and Pseudomonas citronellolis. TS18 and TF1-1 were the genus Duganella zoogloeoides by 16S rDNA analysis. PHA synthase of Pseudomonas mosselii, TO7, was analyzed by recombinant P. putida GPp104 harboring PHA synthase gene. The 3-HO monomer was the major composition of PHA when P. putida GPp104 PHA- harboring phaC1 and phaC2 accumulated PHA.
目 錄
頁次
壹、前言
一、PHA之介紹
1. PHA之發現………………………………..………1
2. PHA在菌體內之定位……………………………..2
3. PHA之化學結構…………………………………..2
4. PHA之生物可分解性……………………………..3
5. PHA之合成模式…………………………………..3
6. 合成PHA 相關之環境因子……………………...4
二、短鏈PHA
1. PHA之合成路徑…………………………………..4
2. PHA之物理性質…………………………………..5
3. PHA之應用………………………………………..5
三、中鏈PHA
1. PHA之合成路徑…………………………………..6
2. PHA之物理性質…………………………………..6
3. PHA之應用……………………………………….7
四、合成PHA之相關酵素………………………………7
五、PHA合成酶之分類………...………………………..8
六、生產PHA的技術發展……………………………….9
七、研究的緣起與目的………………………………….10

貳、實驗方法及步驟
I. 實驗材料
一、實驗菌株及質體…………………………………….13
二、篩選樣品…………………………………………….13
三、培養基……………………………………………….13
四、藥品與試劑………………………………………….14
五、實驗中使用之套組(kit) ……………….…………….15
六、儀器……………………………………..…………..16
七、引子………………………………………………….17
II. 實驗方法
一、由環境中篩選出PHA生產菌之方法
1. 廢水樣品採樣與菌株分離…………………….18
2. PHA產率與單體組成分析……….…..………...18
二、PHA生產菌醱酵生產與單體組成調控
1. 提供不同碳源調控PHA組成...……………….19
2. 利用不同C/N比例調控PHA組成……………19
三、利用熱丙酮劃分分析PHA polymer………..……20
四、利用16SrDNA序列演化樹分析野生菌株之菌種..20
五、篩選菌株TO7的phaC基因的選殖及分析
1. 篩選菌株TO7染色體DNA的製備……….….....21
2. 引子之設計……….……..……………….…….…22
3. 利用PCR選殖TO7的phaC基因……….…….….22
4. 接合反應……….……..……………………..……23
5. 勝任細胞之製備……….……..………..…………23
6. 電衝法轉形作用……….……..……………….….23
7. 基因定序與序列分析……….……..………….….24
8. 表現載體之建構……….……..…………….….…24
9. 兩段培養方式累積PHA………………….……..25
10. 分析TO7 PHA合成酶PhaC1及PhaC2…………25
11. PHA合成酶的胺基酸序列比對………………….25

參、實驗結果
一、由廢水樣品中篩選出具特殊單體組成之PHA高產率菌株.…………………….……………………….26
二、同時生產以3-HB為主之短鏈PHA及含少量中鏈PHA之生產菌株PO6及PO21的PHA組成分析
1. 不同碳源對PHA組成的影響………………....27
2. 不同C/N比例對PHA組成及產率的影響……27
3. 利用熱丙酮劃分分析PO6及PO21 PHA聚分子…………………………………………………28
三、對PHA高產率菌株TS18及TF1-1的PHA組成分析
1. 不同碳源對PHA組成的影響……….………..29
2. 使用兩段培養方式在不易生長的碳源做PHA的組成測試……………………………………..29
3. 不同濃度的propionic acid對PHA組成的影響..29
四、不同碳源對中鏈PHA高產率菌株TO7的PHA組成影響…………………………………………...…30
五、對目標篩選菌株的鑑定………..………………….31
六、篩選菌株TO7的PHA合成酶基因序列的選殖…31
七、篩選菌株TO7的中長鏈PHA合成酶之分析……32

肆、討論
一、由廢水樣品中篩選出可生產PHA的菌株…..…..33
二、對目標篩選菌株的PHA組成分析…………….…33
三、篩選菌株P. mosselii (TO7)的PHA合成酶基因序列的選殖……………………………………………...35
四、篩選菌株TO7的PHA合成酶酵素之分析………36

伍、結論…………………..…………………………...38

陸、參考文獻………………………………………….39
表次
頁次
表一、本研究所使用之菌株與質體……………………...45
表二、選殖TO7的PHA合成酶基因所使用之引子…...46
表三、從台南的廢水樣品中篩選出PHA生產菌株…47
表四、不同碳源對篩選菌株PO6的PHA組成…..….48
表五、不同碳源對篩選菌株PO21的PHA組成….….49
表六、不同的C/N比對篩選菌株PO6的PHA組.......50
表七、不同的C/N比對篩選菌株PO21的PHA組.....51
表八、使用熱丙酮劃分分析篩選菌株PO6的PHA聚分子…………………………………………......…52
表九、使用熱丙酮劃分分析篩選菌株PO21的PHA聚分子.………………………………………….…53
表十、不同碳源對篩選菌株TS18的PHA組成…….54
表十一、不同碳源對篩選菌TF1-1的PHA組成…....55
表十二、使用兩段培養方式在不同碳源時對篩選菌株TS18的PHA組成進行測試….……………...56
表十三、使用兩段培養方式在不同碳源時對篩選菌株TF1-1的PHA組成進行測試………………...57
表十四、果糖及propionic acid複合式碳源中,添加不同濃度的propionic acid對篩選菌株TS18的PHA組成分析..………………………………58
表十五、在果糖及propionic acid的複合式碳源中,添加不同濃度的propionic acid對篩選菌株TF1-1的PHA組成分析……………………………..59
表十六、不同碳源對篩選菌株TO7的PHA組成分析…..…………………………………………..60
表十七、將TO7的PHA合成酶基因轉殖到Pseudomonas putida GPp104中表現分析…..61
表十八、TO7菌株的PHA合成酶胺基酸序列利用BLASTP與其他PHA合成酶之比較………..62



圖次
頁次
圖一、五株Pseudomonas菌株的phaC1、phaZ、phaC2及phaD基因序列比對結果……………………...64
圖二、TO7的phaC基因選殖載體建構示意圖……...84
圖三、TO7的PHA合成酶表現載體建構示意圖…....85
圖四、利用16S rDNA序列對目標篩選菌株PO6、PO21、TF1-1、TS18及TO7做演化樹分析…………...86
圖五、PCR TO7的phaC1及phaC2 DNA片段之電泳圖………………………………………………...87
圖六、T pB2SK-TO7phaC1與 pB2SK- TO7phaC2經限制酶作用後之電泳圖……………………………..88
圖七、TO7的PHA合成酶PhaC1之核酸序列及胺基酸序列............................................................................89
圖八、TO7的PHA合成酶PhaC2之核酸序列及胺基酸序列............................................................................91
圖九、pBHR2-TO7phaC1及pBHR2- TO7phaC1經限制酶作用後之電泳圖……………………………..93





附錄表次
頁次
附錄表一、不同PHA組成之物理性質…………..……94




附錄圖次
頁次
附錄圖一、PHA的一般化學結構通式及命名……….....95
附錄圖二、PHA依據單體碳數可分為三大類………….96
附錄圖三、合成PHA的兩種模式………………………97
附錄圖四、短鏈PHA合成路徑………………………....98
附錄圖五、P(3HB-3HH)的合成路徑………………...…..99
附錄圖六、經由fatty acid biosynthesis pathway合成中鏈PHA的路徑………………………………...100
附錄圖七、合成PHA的各種路徑……………………..101
附錄圖八、一般Pseudomonas菌種合成中鏈PHA的路徑…………………………………………...102
附錄圖三、3-HHx單體經由fatty acidβ-oxidation代謝循環產生後與acetyl-CoA直接接合延長PHA單體的路徑示意圖…………………………...103
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