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標題: 表現吳郭魚金屬硫蛋白於KY3 酵母菌中以提升清除自由基能力及酒精耐受性之研究
Enhancement of Free Radical Scavenging Ability and Ethanol Tolerance in KY3 Yeast by Expression Tilapia Metallothionein
作者: 劉彥君
Liu, Yen-Chun
關鍵字: 金屬硫蛋白
Reactive oxygen species(ROS)
Ethanol productivity
出版社: 生命科學系所
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摘要: 金屬硫蛋白(metallothionein, MT)為一約6000 ~ 7000 Dalton大小的蛋白質,廣泛存在於各生物中,因其富含半胱胺酸(cysteine),故可利用cysteine的硫醇基(thiol group)和重金屬結合(例如:鎘、鋅、銅),進而降低重金屬於細胞內累積所造成的傷害。另外,有文獻指出當酵母菌暴露於乙醇(ethanol)壓力環境下時,粒線體是主要產生活性氧屬(reactive oxygen species, ROS)的位置,當自由基於粒腺體內無法完全清除時會漸漸擴散至細胞質,進而造成脂質、蛋白質和核酸受到傷害。由過去研究發現MT 亦具有清除自由基能力,故本研究希望於酵母菌 Kluyveromyces marxianus KY3 中大量表現(overexpression)MT 蛋白,以提升酵母菌在發酵過程中因酒精逐漸累積所造成氧化壓力環境的抗性。本實驗室先前研究發現,相較於人類及老鼠的MT 蛋白,吳郭魚(Tilapia)的MT 蛋白(簡稱TMT)具有更好的清除自由基能力,故本研究使用TMT 以及另外於TMT 蛋白的前端(即N端)融合(fusion)革蘭氏陽性菌之汞離子結合蛋白MerP的訊號序列(signal peptide),期望藉由此signal peptide 將TMT(簡稱spTMT)表現於粒線體內膜或是細胞膜處,以達到更好的清除ROS效果。除此之外,將TMT 及spTMT 的DNA 序列進行codon usage 的優化(optimization),期望目標基因在KY3 中有較好的蛋白表現量。TMT、TMTO、spTMT和spTMTO 四種轉植株經H2DCFDA assay 發現在誘導21個小時有最佳清除自由基效果,且相較於控制組皆有較好清除自由基能力。在酒精耐受度測試得知轉植株TMT和TMTO在含有5% 酒精濃度時生長情形較控制組好。接著利用ABTS assay 分別探討細胞質及細胞膜中的蛋白質清除自由基能力,實驗結果發現融合signal peptide 的TMT 蛋白應有表現於細胞中帶有「膜」的位置。利用氣相層析儀(gas chromatography, GC)分析四種轉植株發現TMT 及TMTO 在發酵前期約22.5~34.5 小時間具有較佳酒精生產力,故在未來可能將其應用在發酵前期以提升酒精產量。
Metallothioneins(MTs) are widely found in organisms, cysteine–rich and 6000 – 7000 Dalton molecular weight of proteins. It can utilize the thiol group of cysteine to bind heavy metals(ex: Zn, Cd and Cu)and to decrease the cytotoxic effects. Moreover, when yeast cells are exposed to the environment having ethanol will lead to generation of the reactive oxygen species(ROS). It was found that MT has the ability to scavenge free radicals. Therefore, we hope to increase the tolerance of ethanol by overexpressing MT protein under ethanol stress conditions. In addition, our laboratory has found that tilapia MT (designated as TMT) protein had better free radical scavenging ability when compared with human and mouse MT. This study used TMT protein and N-terminal of TMT protein fusion signal peptide of mercury-ion-binding protein MerP from Gram positive bacterial (designated as spTMT) to allow TMT protein be located in the cytosol membrane or inner membrane of mitochondrial to enhance the free radical scavenging ability. In addition, the DNA sequences of TMT and spTMT were optimized by codon usage to increase the TMT protein productivity in KY3 yeast. TMT、TMTO、spTMT and spTMTO four types of transformants have better free radical scavenging ability when protein was induced for 21 hours, revealing better free radical scavenging ability than the control group by H2DCFDA assay. Transformants had better growth abilities with medium containing 5% ethanol by ethanol tolerance test. ABTS assay was used to investigate the protein free radical scavenging ability The results suggest that the TMT protein was located in the membrane by the signal peptide. TMT and TMTO had better ethanol productivity in pre-fermentation approximately at 22.5 to 34.5 hours by gas chromatography analysis of the four transformants. In the future, the recombinant strains coulde be applied in pre-fermentation of alcohol to enhance ethanol production.
其他識別: U0005-3001201321305200
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