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標題: | 以畢赤氏酵母菌表現拱狀靈芝漆氧化酶及醣基化修飾對於漆氧化酶穩定性之影響 Expression and characterization of Ganoderma fornicatum laccase in Pichia pastoris and the role of glycosylation for laccase stability |
作者: | Wan-Ting Huang 黃琬婷 |
指導教授: | 黃慶璨(Ching-Tsan Huang) |
關鍵字: | 漆氧化酶,拱狀靈芝,畢赤氏酵母菌,醣基化修飾, Ganoderma fornicatum,Pichia pastoris,laccase,glycosylation, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 漆氧化酶 (laccase) 是一種具有醣基化修飾的多酚氧化酶,以銅離子為酵素催化中心,透過自由基的產生可氧化多種酚類及非酚類化合物,水是為唯一反應副產物。自然界中木質素分解需要漆氧化酶的參與,白腐型真菌其木質素分解能力較其他微生物高,因此成為漆氧化酶主要來源。漆氧化酶應用範圍相當廣泛,包含紙漿漂白、色素脫色、生物感測器、生物復育、有機合成及在食品工業上作為澄清劑。本研究由白腐型真菌拱狀靈芝 (Ganoderma fornicatum 0814) 中選殖出漆氧化酶基因lac1,基因全長為2019 bp具有9個內含子 (intron),其蛋白質由521個胺基酸組成,其中前21個胺基酸為訊息胜肽,具有11個N-醣基化 (N-linked glycosylation) 序列,在保守性胱胺酸Cys475之後第十個胺基酸為苯丙胺酸 (phenylalanine),屬於高還原電位之第三類漆氧化酶。以嗜甲醇酵母Pichia pastoris表現lac1基因,轉形株胞外上清液經蛋白質電泳與醣蛋白染色,確認此重組蛋白質有醣基化修飾,純化後以光譜分析確認為黃色漆氧化酶,有別於常見的藍色漆氧化酶。以2,2’-azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) (Km = 103.9 uM) 為基質其最適pH 2.5,而以2,6-dimethoxyphenol (DMP) (Km = 276.7 uM) 及guaiacol (Km = 1263.4 uM) 為基質其最適pH皆為3.0,三種基質最適反應溫度皆為55oC。在酵素穩定性方面,重組漆氧化酶於25 oC pH 2.5-10.0環境中24小時,可保有85%以上的活性;熱穩定性方面,於pH 5.0環境下60 oC 3小時,酵素仍保有70%以上活性; 50% (v/v) 甲醇、乙醇、DMF與DMSO於25 oC下3小時,仍可保有80%活性。拱狀靈芝來源的漆氧化酶具有良好的pH、熱穩定性及有機溶劑耐受性,成功在P. pastoris中表現並外泌到培養基中,可使用全合成培養基以醱酵槽大量生產,極具工業應用潛力。在促進表現量方面,以AOX1為啟動子其表現量是GAP啟動子的4.5倍;在P. pastoris KM71H表現量是營養缺陷株 (P. pastoris KM71) 的2.4倍;以P. pastoris慣用密碼子修改lac1序列,利用overlap PCR進行密碼子最適化合成,成功合成出mlac基因,轉形至P. pastoris KM71H表現,以微孔盤篩選284轉形株平均表現量提高30%。挑選活性最佳轉形株以試管或搖瓶培養,表現量可提升1倍,使用全合成培養基以醱酵槽大量生產,以甲醇誘導密碼子最適化之轉形株,其漆氧化酶表現量提升80%。漆氧化酶活性隨著醣基化抑制劑tunicamycin濃度增加而減少,添加30 ug/mL tunicamycin活性下降81.2%。經序列比對,拱狀靈芝漆氧化酶相較於其他物種具有較多醣基化位點,利用點突變方式將特定醣基化序列中天冬醯胺酸 (asparagine) 修改成天冬胺酸 (asparatate) (N7D、N168D、N190D、N302D及N334D),醣基化位點及數目會影響漆氧化酶的活性與穩定性,且影響對ABTS基質親合性。去除N4醣基化位點 (N190D) 漆氧化酶活性只有原本的32%,若除了N4外再去除1-4個位點則活性僅存20-27%,顯示N4醣基化位點對於漆氧化酶活性具有關鍵性影響。此外,去除N7醣基化位點 (N334D) 會影響漆氧化酶熱穩定性及對乙腈與乙醇的耐受性,然而個別醣位點去除對於酸鹼穩定性無顯著相關性,但去除越多個醣基化位點在較為極端酸鹼環境下酵素不穩定,對有機溶劑較為敏感且熱穩定性也有下降趨勢。多個或特定醣基化位點去除會使活性與酵素穩定性顯著下降,推測特定位點醣基化在拱狀靈芝漆氧化酶扮演重要角色。 Laccases, a group of copper-containing oxidizing enzymes, catalyze the oxidation of various aromatic compounds, particularly phenolic substrates with the concomitant reduction of molecular oxygen to water. This characteristic makes laccases of great potential for industrial applications, including bioremediation, lignocelluloses processings, organic syntheses, etc. White-rot fungi have been deemed as a good resource of laccase genes due to their high ligninolytic activity. In this study, the newly defined laccase gene, lac1, from the medicinal white-rot fungus Ganoderma fornicatum 0814 was cloned and expressed in Pichia pastoris. The corresponding open reading frame has 1563 nucleotides and encodes a protein of 521 amino acids, including secretion signal with a 21-residue peptide. The ten amino acids downstream of a conserved Cys residue existing within the laccases encoded by the lac1 are phenylalanine, suggesting that the laccases from G. fornicatum 0814 may demonstrate high redox potential and good ligninolytic activity. The absorption spectrum of the purified laccase rLac1 indicated that it is a yellow laccase rather than the common blue laccase. The optimum pH of rLac1 was 2.5, 3.0 and 3.0 for ABTS, DMP and guaiacol, respectively. The kinetic parameter Km of rLac1 was 103.9, 276.7 and 1263.4 uM for ABTS, DMP and guaiacol, respectively. The optimal temperature for rLac1 was 55°C for all of the substrates. rLac1 is stable up to 60°C for 3 h and in the pH range 2.5 to 10.0 for 24 h. Sodium azide and thioglycolic acid strongly inhibited rLac1 activity, but it was not affected by EDTA. rLac1 retained more than 80% activity in 50% (v/v) ethanol, methanol, DMF and DMSO after incubation at 25°C for three-hour. Less than 20% of activity was found in 50% (v/v) acetone and acetonitrile after incubation at 25°C for three-hour. The results show that rLac1 is thermostable, pH-stable and tolerant to organic solvents. It has also been produced in fermentations of P. pastoris with synthetic media. These properties suggest that rLac1 has potential uses in industrial applications. To enhance laccase expression in P. pastoris, lac1 was modified to the optimal codon usage of P. pastoris. The expression level was thus increased up to 30%. Glycosylation retards protein overexpression, although it helps stabilize enzymes and avoids protein degradation. Through sequence alignment, it is shown that laccase from G. fonicatum has more glycosylation sites than that from other species. Enzyme activity decreases drastically when the asparagine of some glycosylation sites were replaced by aspartic acid (N7D, N168D, N190D, N302D and N334D) by site-directed mutagensis. With the deletion of the glycosylation site N4 (N190D), only 32% of the laccase activity remains. Only 20-27% activity remains if 1-4 more glycosylation site is deleted, in addition to the deletion of the N4 site. Thus, N4 glycosylation site had a critical effect on the activity of the laccase. Moreover, the thermal stability and actonitrile- and ethanol-tolerance were affected when the N7 glycosylation site was deleted (N334D). Any individual site deletion had little effect on the pH-stability. However, multiple site deletiond had significant effection the pH-stability. It was deduced that glycosylation at these specific sites are important for the activity and stability of laccase (Lac1) from G. fonicatum. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10478 |
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