Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90081
標題: Characterization and directed evolution of polyphenol oxidase Tfu_1114 from the actinomycetes Thermobifida fusca
褐色嗜熱裂孢菌之多酚氧化酶T.fu_1114之特性分析及其定向進化
作者: 陳弘軒
Hung-Shuan Chen
關鍵字: 褐色嗜熱裂孢菌
多酚氧化酶
Thermobifida fusca
T.fu_1114
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摘要: 嗜高溫放線菌 Thermobifida fusca 為革蘭氏陽性菌,具分解木質纖維素 (Lignocellulose) 之能力。本實驗室自 T. fusca 10-1 中取得一多酚氧化酶Tfu_1114,該酵素屬於含銅離子的氧化還原酵素,可將許多苯酚類受質氧化。此外,該蛋白於大腸桿菌中表現時,利用穿透式電子顯微鏡以免疫金標定觀察發現該酵素分別存在於大腸桿菌外膜表面、膜間腔及細胞質中。本實驗利用不同方式分別取得大腸桿菌外膜表面、膜間腔及細胞質中之Tfu_1114蛋白,以 2,6-dimethylphenol (2,6-DMP) 為受質進行酵素活性的測定,發現分泌到大腸桿菌外膜表面及膜間腔的酵素具有漆酶受質活性,而存在於細胞質中的酵素則否。進行蛋白質結構模擬後發現,雙硫鍵的形成與正確鍵結與否可能與酵素活性有關,因此進一步探討雙硫鍵在蛋白質結構組成與銅離子鍵結中扮演的角色。此外,以甘蔗渣為反應物測定 Tfu_1114 幫助水解木質纖維素的能力發現,Tfu_1114 可促進數種不同來源的纖維水解酵素分解蔗渣,但效果不一。並以此分解液中所含之還原糖代替 R2 培養基中的葡萄糖做為碳源,進行大腸桿菌的培養,證明甘蔗渣分解所產生之還原糖的實用性。另外以易錯聚合酶連鎖反應 (Error prone PCR) 對 Tfu_1114 基因進行隨機突變,隨機突變基因體庫依序經過固態篩選、液態篩選及分泌量篩選後,得到三株有益突變株 MKH-48、MHD-20及MUC-16,其中 MHD-20及MUC-16 分別可以將比活性提高 1.6 倍及2.8 倍,而 MKH-48 則會使 Tfu_1114 被分泌到培養基中,且培養基中的酵素比活性與野生型相當。
Thermobifida fusca, a Gram-positive bacterium, can degrade lignocellulose. Tfu_1114 was cloned from T. fusca 10-1. It is a copper-containing oxidase that can catalyze the oxidation of various phenolic compounds, coupled to the reduction of molecular oxygen. Interestingly, Tfu_1114 was present on the outer membrane surface, periplasmic space, and cytoplasm when it was expressed in E. coli according to the immune-gold labeling and transmission electron microscopy (TEM). In this study, classic polyphenol oxidase substrates 2,6-dimethylphenol (2,6-DMP) was used to measure Tfu_1114 enzyme activity isolated from different places of E. coli. Only the protein from outer membrane surface and periplasm exhibited enzymatic activity, implying that correct fold of the enzyme occurs only after secretion. Correct disulfide bonds formation or copper binding by cysteine residues may be critical according to a protein model of Tfu_1114 using SWISS-MODEL Workspace. Therefore, this study aimed to explore the roles of cysteine residues in this regards. In addition, sugarcane bagasse was used to measure whether Tfu-1114 can help commercial cellulase to hydrolyze lignocellulose. Results showed that enzymes can help commercial cellulase to hydrolyze bagasse to produce reducing sugar. Hydrolyzate was used to substitute medium's carbon source to cultivate E. coli BL21 (DE3). In addition, error-prone PCR was used to select mutant enzymes with higher catalytic efficiency and thermostability. After activity and secretion screening, we found 3 mutant MKH-48, MHD-20 and MUC-16. The specific activity was enhanced 1.6-fold and 2.8-fold in the MHD-20 and MUC-16 mutant respectively. MKH-48 can be secreted into medium.
URI: http://hdl.handle.net/11455/90081
文章公開時間: 2017-08-31
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