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標題: | 以重組大腸桿菌外泌生產耐熱型漆酶 Secretory production of thermostable laccase in recombinant Escherichia coli |
作者: | Bo-Kai Chiu 邱柏凱 |
指導教授: | 李昆達(Kung-Ta Lee) |
關鍵字: | 漆?,納豆菌,大腸桿菌,外泌生產, Laccase,Bacillus subtilis natto,Escherichia coli,Extracellular production, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 漆酶 (EC 1.10.3.2) 是自然界廣泛存在的一種藍色多銅氧化酵素,可氧化分解木質素及包括雙酚A (bisphenol A)、鄰苯二酚 (ortho-diphenol)、對苯二酚 (para-diphenol)、胺基苯酚 (aminophenol) 等多種酚類化合物物質,同時將氧氣還原為水,常應用於紙漿工業、工業廢水處理及生質能源開發等。本實驗室先前自納豆菌Bacillus subtilis natto NTU18中選殖漆酶基因cotA,並轉殖至大腸桿菌表現,發現重組納豆菌漆酶的熱穩定性較市售雲芝漆酶為佳。為進一步優化重組大腸桿菌生產納豆菌漆酶之效能與可調控性,減低工業化生產及純化程序成本,本研究利用訊號序列修飾目標基因,及調整胞膜間區 (periplasmic space) 釋放條件等方式,探討重組大腸桿菌外泌生產納豆菌漆酶的可行性。經實驗發現,納豆菌漆酶毋需修飾訊號序列,即能自然轉移至胞膜間區。此外,藉由菌體培養過程中添加 2% glycine及Triton X-100,並採「誘導」與「釋放」分開處理的方式,可有效提升納豆菌漆酶釋放至培養基的效率,使用Hinton’s flask振盪培養可生產納豆菌漆酶活性達1.06±0.007 U/mL ,再配合細胞高密度培養技術,經20小時培養後於胞外釋放之納豆菌漆酶活性可達5.72±0.38 U/mL,較原先胞內表現並破菌的方式所得活性提高近170倍,有利於工業化生產效能之提升。 Laccase (EC 1.10.3.2) is one of the blue multicopper oxidases that catalyze the oxidation of lignin and many aromatic substrates including bisphenol A, ortho-diphenol, para-diphenol, and aminophenol with reduction of oxygen to water. It has many applications such as dye removal in textile, industrial wastewater treatment, and biofuel development. The laccase gene cotA was cloned from Bacillus subtilis natto NTU18, and laccase CotA was produced in Escherichia coli expression system by our lab. In previous study, the higher optimum reaction temperature indicated that recombinant CotA was more themostable than commercial fungal (Trametes versicolor) laccase. In this study, 5 kinds of N-terminal and C-terminal signal peptides were added to cotA gene sequence to enhance the translocation of target protein, and the periplasmic protein was released with chemical method to upgrade the efficiency and controllability of recombinant CotA laccase production in E. coli. The results showed that CotA laccase can be transferred to periplasmic space without any signal peptide addition. And using 2% glycine and Triton X-100 after induction would increase CotA laccase release to the culture medium. The released laccase activity reached 1.06±0.007 U/mL in Hinton’s flask, and 5.72±0.38 U/mL in bioreactor with high cell-density culture for 20 hours, which was 170-fold to intracellular production. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78134 |
DOI: | 10.6342/NTU201600857 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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