請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39195
標題: | 大腸桿菌植酸酶於重組酵母菌Pichia pastoris中大量表現之研究 High level expression of E. coli phytase in recombinant yeast Pichia pastoris |
作者: | Chang-Chih Chen 陳長志 |
指導教授: | 黃慶璨 |
關鍵字: | ;乳糖操縱子,啟動子,表現,大腸桿菌,植酸酶,酵母菌, Escherichia coli,phytase,Pichia pastoris,expression,promoter,lac opreon, |
出版年 : | 2005 |
學位: | 博士 |
摘要: | 植酸酶可分解植酸為一常用之飼料添加酵素,本研究以AOX1啟動子系統於重組酵母菌Pichia pastoris大量生產Escherichia coli之植酸酶。除了以甲醇誘導調控之AOX1啟動子系統外,在P. pastoris表現系統中還有另一個持續表現模式之GAP啟動子系統。本研究中成功利用乳糖操縱子系統(lac operon)調控GAP啟動子之表現。利用AOX1啟動子表現E. coli植酸酶基因時,須以BMGY培養基培養至生長定常期後再以含甲醇之誘導培養基BMMY置換生產。若BMGY中不添加蛋白腖、酵母氮源對菌體生長影響不大。而移除誘導培養基BMMY中酵母氮源與蛋白腖並將酵母膏濃度降為原配方十分之一 (0.1%)更有助於植酸酶之生產。將P. pastoris以修飾培養基mBMGHY培養再以誘導培養基mBMMHY置換誘導P. pastoris生產植酸酶,可提高植酸酶產量並降低生產成本。甲醇濃度對P. pastoris在醱酵槽誘導植酸酶生產過程中非常重要,在醱酵槽中利用甲醇控制儀器監控控制培養基中甲醇濃度為0.5%,提供菌體穩定之碳源與誘導物可有效提高植酸酶產量。批次醱酵誘導生產後之菌體可離心回收再次進行誘導生產植酸酶,且以回收菌體進行第二與第三批次之誘導生產,由於回收利用之菌體在誘導過程中已經適應了甲醇之代謝,其植酸酶生產速率皆高於第一批次生產,降低生產成本與時間。P. pastoris以全合成培養基FBSH於5公升醱酵槽中培養,並經過甘油饋料批次培養後,培養液之OD600與生菌數分別為321與2.6 x 1010 CFU/mL。將菌體離心回收,懸浮於mBMMHY誘導培養192小時後,胞外蛋白質產量達6.4 g/L,植酸酶活性則為4,946 U/mL。
P. pastoris之GAP啟動子為持續表現之強力啟動子,可能會在培養過程中影響菌體之生長,故本研究利用E. coli之乳糖操縱子系統來調控GAP啟動子之表現。將乳糖阻遏物(lac repressor, LacI)基因轉形至P. pastoris宿主中,建構一可於胞內表現乳糖阻遏物之菌株P. pastoris KM71I。將乳糖操縱基因(lac operator, lacO)插入GAP啟動子不同位置,並將E. coli植酸酶基因接至GAP-lacO融合啟動子下游作為報導基因。在ATG轉譯起始點前214 bp位置插入lacO序列造成表現量下降50%,顯示此位置對GAP啟動子表現扮演著重要角色。若於GAP啟動子下游之ATG轉譯起始點前插入lacO序列,對GAP啟動子於P. pastoris KM71表現強度影響不大,但於乳糖阻遏物表現宿主P. pastoris KM71I中,則表現活性明顯受到抑制降為22%。培養基中添加IPTG可成功誘導lacO-GAP融合啟動子之表現,而在10 mM IPTG濃度下表現量可回復為88%。本篇為首篇利用乳糖操作子系統成功調控GAP啟動子之研究。 Phytase can degrade phytic acid and commonly be used as feed additive. The Escherichia coli phytase was highly expressed in methylotrophic yeast P. pastoris under control of alcohol dehydrogenase (AOX1) promoter. In addition to the inducible AOX1 promoter, the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was used for protein expression in P. pastoris. In this study we successfully regulate the GAP promoter by the lac operator-repressor system. After cultivation of P. pastoris in BMGY until stationary phase, cells were recovered and resuspended in fresh BMMY medium for induction can efficiently improve the protein expression. The formulas of BMGY and BMMY were modified by reducing the concentrations of peptone, yeast nitrogen base (YNB) and yeast extract (YE). Removing the peptone and YNB in BMGY did not affect cell growth significantly. In addition, phytase production in modified induction medium mBMMHY was better than that in BMMY or FBSH. In mBMMHY, peptone as well as YNB in BMMY was removed and YE concentration was reduced to 1/10 (0.1%). Methanol concentration is very important during induction period of P. pastoris fermentation. Using an on-line methanol monitor and controller to maintain the methanol concentration at steady state in fermentor significantly increased the phytase production. After one batch of phytase production, cells may be reused for another batch induction protein production. The phytase production of second and third induction batches were faster than the first batch which may due to the adaptation of methanol metabolism. High cell density was achieved with glycerol fed-batch in minimal medium in 5-L fermentor. The optical density at 600 nm and viable cells reached 321 and 2.6 x 1010 CFU/mL before induction. The cells were resuspended in mBMMHY for induction. After 192 hours induction 6.4 g/L protein was produced in culture supernatant and the phytase activity was 4,946 U/mL. The constitutive expression of foreign protein under control of GAP promoter in P. pastoris may affect the cell growth. In this study, the E. coli lac operator-repressor system was used to control the expression of GAP promoter. The LacI repressor gene was transformed into P. pastoris host strain and successfully expressed. The lacO operator sequence was integrated into GAP promoter of pGAPZ |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39195 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-94-1.pdf 目前未授權公開取用 | 1.14 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。