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研究生:李承軒
研究生(外文):Chen-hsuan Lee
論文名稱:嗜甲基酵母菌表現融合透明颤菌血紅素之葡萄糖氧化酶(VHb-GOX)之研究
論文名稱(外文):The Study of Glucose Oxidase Fuses with Vitreoscilla Hemoglobin(VHb-GOX) Expression in Methylotrophic Yeast Pichia pastoris
指導教授:李振綱李振綱引用關係
指導教授(外文):C. K. Lee
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:126
中文關鍵詞: 明颤菌血紅素嗜甲基酵母菌葡萄糖氧化酶
外文關鍵詞:Vitreoscilla HemoglobinPichia pastorisGlucose oxidase
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Aspergillus niger所產的葡萄糖氧化酶(Glucose oxidase, GOX)是屬於一種醣化蛋白質,在食品工業及醫療上皆有廣泛的應用。利用嗜甲基酵母菌Pichia pastoris可進行大量表現此酵素。在菌體中表現顫菌血紅蛋白(Vitreoscilla Hemoglobin, VHb ),已證明可來提升菌體之氧氣利用率,而提高菌體的生產速率,因此本研究將VHb與GOX融合在AOX1啟動子調控下,利用甲醇當誘導劑在P. pastoris GS115中表現可分泌至胞外之VGOX融合蛋白。在YPF(Yeast 1%, Peptone 2%, Fructose 2%)培養基中添加1% Casamino acid會大幅提升VGOX的表現量。在搖瓶培養時,添加了Casamino acid之VGOX活性可由1.5 U/ml增加至19.3U/ml提高約13倍。VGOX經過Immobilized Metal Affinity Chromatography (IMAC)純化過後,比活性可達到328.6 U/mg,相較於GOX 的237.9 U/mg提升約1.4倍,主要原因可能是融合之VHb可供氧給GOX而加速其反應速率。而Casamino acid添加對提升VGOX活性之效應,主要可能是防止菌體本身所分泌之蛋白分解酶分解VGOX及提供足夠的胺基酸用於VGOX之表現。
The glucose oxidase(GOX)from Aspergillus niger is a glycoprotein. it is widely use in food industry and diagnosis kit for glucose because it catalyzes the glucose oxidation with O2. The methylotrophic yeast Pichia pastoris can been used to extracellularly express GOX. Vitreoscilla Hemoglobin(VHb)from Vitreoscilla sp. has been demonstrated that it can enhance oxygen uptake and cell growth when expressed intracellularly in most microorganisms. In this study, VHb was fused with GOX(VGOX) in order to take advantage of O2 carrying ability of VHb so that GOX activity enhancement can be expected. The extracellular expression of fusion protein VGOX in P. pastoris GS115 was under to control of methanol induced AOX1 promoter. Casamino acid(CA)was found to be the crucial nutrition factor that determined the expressed GOX activity in the culture of VGOX expressing strain GS115 / καVG. The activity of GOX increased from 1.5 U/ml to 19.3 U/ml when 1 % CA was employed in YPF medium. Maintaining culture at neutral pH to prevent expressed VGOX from degradating by usually expressed acidic protease of GS115 was found to be one of the reasons that a higher GOX activity could be obtained in the culture supplemented with CA. The specific activity of IMAC purified VGOX is 328.6 U/mg. It is about 1.4 fold higher than that of GOX(237.9 U/mg).
目錄
致謝
中文摘要……………………………………………………………………..I
英文摘要…………………………………………………………………… II
目錄………………………………………………………………………….III
圖目錄……………………………………………………………………….VIII
表目錄……………………………………………………………………….XII
第一章 緒論…………………………………………………………………1
1.1 前言……………………………………………………………………..1
1.2 研究內容………………………………………………………………..4
第二章 文獻回顧……………………………………………………………5
2.1 嗜甲基酵母菌Pichia patoris 表現系統……………………………….5
2.2 葡萄糖氧化酶(Glucose oxidase , GOX)…………………………….8
2.3 透明顫菌血紅蛋白(Vitreoscilla Hemoglobin , VHb)………………..10
2.4 酪蛋白水解物(Casamino acid , CA)…………………………………12
2.5 氧氣質傳係數與氧氣吸收速率(Oxygen transfer coefficient , KLa & Oxygen uptake rate , OUR)……………………………………………………...14
2.5.1 Dynamic Method………………………………………………...17
第三章 實驗方法……………………………………………………………...18
3.1 實驗材料…………………………………………………………………. 18
3.1.1 菌株及來源……………………………………………………………18
3.1.2 質體……………………………………………………………………18
3.1.3 GS115 / καVG / VGOX基因建構示意圖………………………….....19
3.1.4 X33 / GαG / GOX基因建構示意圖…………………………………..19
3.1.5 X33 / PADH2 / VHb重組菌株…………………………………………..20
3.1.6 酵素…………………………………………………………………….20
3.1.7 抗體…………………………………………………………………….20
3.1.8 標準分子量溶液………………………………………………………..21
3.1.9 培養基…………………………………………………………………..21
3.1.10 各種緩衝液配置…………………………………………………...23
3.1.11 GOX活性反應液…………………………………………………..25
3.2 實驗方法…………………………………………………………………….26
3.2.1 蛋白質濃度測定(Bradford's method)…………………………..........26
3.2.2 蛋白質純化……………………………………………………………...26
3.2.2.1 固定化金屬親合層析(Immobilized metal affinity
chromatography , IMAC)…………………………...26
3.2.2.2 BioCAD蛋白質層析儀………………………………………..27
3.2.3 聚丙烯醯胺凝膠體蛋白電泳(SDS-PAGE)…………………………28
3.2.4 Western blot……………………………………………………………...29
3.2.5 P. pastoris重組菌株培養………………………………………….……30
3.2.5.1 GS115 / καVG菌株……………………………………………30
3.2.5.2 X-33 / GαG菌株…………………………………………….....31
3.2.5.3 VHb之X-33菌株……………………………………………...32
3.2.6 葡萄糖氧化酶(GOX)蛋白活性分析……………………………..….33
3.2.7 顫菌血紅蛋白葡萄糖氧化酶(VGOX)蛋白活性分析…………….... 34
3.2.8 發酵槽批次發酵P.pastoris……………………………………….………35
3.2.9 VGOX動力學參數測定…………………………………………………..36
3.2.10 氧氣吸收速率與氧氣傳送係數測定(Oxygen uptake rate ,
OUR ;Oxygen transfer coefficient , KLa)……………………36
3.2.11 VGOX穩定性…………………………………………………………38
3.2.11.1 VGOX之熱穩定性……………………………………………….38
3.2.11.2 VGOX 之pH穩定性…………………………………………….38
3.2.12 VGOX與GOX限氧活性分析……………………………………….39
3.3 儀器操作………………………………………………………………….……40
3.3.1 BioCAD蛋白質層析儀………………………………………………..…..40
3.3.2 發酵槽操作……………………………………………………………..….42
3.3.2.1 pH電極之校正與保存…………………………………………....42
3.3.2.2 溶氧(DO)電極之校正與保存…………………………………….43
第四章 結果與討論…………………………………………………………….…..45
4.1 醣及酪蛋白水解物(CA)對P. pastoris 生長影響…………………………45
4.2 GOX與VGOX蛋白質表現……………………………………………….….47
4.2.1 X33 / GαG表現GOX…………………………………………………...…47
4.2.2 GS115 / καVG表現VGOX…………………………………………….....50
4.3 活性分析……………………………………………………………………….54
4.3.1 GOX活性表現分析……………………………………………………….54
4.3.2 VGOX活性表現分析……………………………………………………..56
4.4 影響GS115 / καVG表現VGOX之因子………………………………..……59
4.4.1 胞外蛋白分解酶之影響…………………………………………………...59
4.4.2 酸鹼值對VGOX表現之影響……………………………………………..62
4.4.3 甲醇對X33 / GαG 及GS115 / καVG表現GOX活性之影響……….....69
4.4.4 最適甲醇誘導濃度………………………………………………………...71
4.4.5 最適CA培養濃度………………………………………………………...73
4.5 GOX及VGOX保存性………………………………………………………..75
4.6 VGOX之熱穩定性與pH穩定性……………………………………………..76
4.6.1 熱穩定性分析……………………………………………………………...76
4.6.2 pH穩定性分析…………………………………………………………….78
4.7 VHb特性對GOX影響探討…………………………………………………..79
4.7.1 VGOX動力學參數分析…………………………………………………...79
4.7.2 限氧下之氧化反應………………………………………………………...81
4.8 VHb對嗜甲基酵母菌(P. pastoris)生長之影響…………………………….82
4.8.1 發酵槽批次培養P. pastoris X33 host…………………………………..….83
4.8.2 批次發酵P. pastoris X33 / PADH2 / VHb…………………………….……..85
4.9 發酵槽批次培養P. pastoris GS115 / καVG……………………………….…..88
第五章 結論………………………………………………………………………...91
附錄一 圖…………………………………………………………………….……..93
附錄二 式……………………………………………………………………….…102
附錄三 表……………………………………………………………………….…103
參考文獻……………………………………………………………………….…..107
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