臺灣博碩士論文加值系統

靈芝屬(Ganoderma spp.)是一種白腐型真菌，具有各種可分解木材的外泌酵素，包括纖維素水解酵素(cellulase)和半纖維素水解酵素(hemicellulase),、果膠質水解酵素(pectinase)、漆氧化酶(laccase)、木質素過氧化酶(lignin peroxidase)和錳過氧化酶(manganese peroxidase)。其中錳過氧化酶是一種含有血基質(heme)的酵素，在二價錳離子以及過氧化氫存在的環境中能將酚類多環物質氧化分解，白腐型真菌利用此酵素分解木質素結構使其菌絲能有效深入木質纖維中分解纖維素等多醣以獲得養分。本實驗以含有ABTS的培養皿測試十株靈芝屬菌株的胞外酵素，發現所測試靈芝屬真菌都有可分解ABTS(2,2''-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid))的外泌酵素。並利用Genome Walking、五端RACE和PCR增幅技術自台灣靈芝(G. formosanum 0109, BZ)、新日本靈芝(G. neo-japonicum 0813, VZ)和南方靈芝(G. australe 0705)中選殖出六段新的錳過氧化酶基因序列。以RT-PCR增幅基因首尾端具有限制酶切位的台灣靈芝0109錳過氧化酶的cDNA，並將此基因轉殖入大腸桿菌(Escherichia coli)以及嗜甲醇畢赤氏酵母(Pichia methanolica)進行異源表達，得到在C端接有六個組胺酸(histidine)的錳過氧化酶以利後續純化的重組蛋白。

Lignin, component of plant secondary cell wall, binds to hemicellulose by covalent bonds and defends bacteria or fungi invading into the core of the wood. Lignin is a complicated polymer composed of aromatic compounds, which are ferulic acid, sinapic acid and p-coumaric acid etc. In paper pulping industry, lignin is hard-degradable and influences the brightness of paper. Generally, people use concentrated acid or alkali to precipitate or dissolve lignin. In recent study, the paper would be brighter and whiter by using manganese peroxidase in pulping procedure.
Manganese peroxidase, which was first isolated from white rot fungi Phanerochaete chrysosporium by Glenn and Gold in 1985, belongs to heme-containing peroxidase family. This extracellular enzyme is a glycoprotein with five disulfide bonds in general and contains two calcium ions in the structure. When it catalyses the substrates, hydrogen peroxide which produced by glyoxal oxidase or aryl-alcohol oxidase is necessary as electron donor. Mn(II) would be oxidized to Mn(III) by heme-oxygen radical complex. Mn(III) cations would be stabilized by alfa-hydroxyl acids, such as succinic acid or lactic acid, and oxidize lignin or lignin-like compounds.
Manganese peroxidase has been found in many of white rot fungi, including Trametes versicolor, Pleurotus ostreatus, Armillaria mellea, etc, however, the studies of manganese peroxidase from Ganoderma spp. are few and most of them were focus on preotein level. The only published cDNA sequence was from G. applanatum. In this study, we cloned six new sequences of manganese peroxidase from G. formosanum, G. neo-japonicum and G. australe. Furthermore, we expressed the manganese peroxidase of G. formosanum in Escherichia coli and Pichia methanolica and produced recombinant enzyme.

目錄

中文摘要……………………………………………..………………….V
英文摘要…………………………………………..…………...……….VI
表目錄…………………………………………..………………….......VII
圖目錄………………………………………..………...…………......VIII
第一章 前言…………………………………………………..…..……1
1.木質素………………………………………………………..…..…….1
1.1.木質素的來源及構造………………………………………..………1
1.2木質素在構型上的相似物…………………………………..………5
1.3木質素及其相似物對工業環境或生命之負面影響………..………5
1.4分解木質素的方法…………………………………………..…..…...7
2.錳過氧化酶…………………………………………………..…..…….7
2.1錳過氧化酶的研究歷史……………………………………..…..…...7
2.2與錳過氧化酶功能相似的酵素……………………………..…..…...9
2.3錳過氧化酶的特性及適用條件……………………………..……….9
2.4錳過氧化酶的基質和應用…………………………………..…..….14
2.5已開發之錳過氧化酶重組表現系統………………………..…..….18
2.5.1同源表達系統..………………………………………………..…..18
2.5.2異源表達系統..………………………………………………..…..19
2.5.2.1原核表達系統..………….…………………………………..…..19
2.5.2.2真核表達系統.…………….……………………..…………..….20
3.靈芝屬真菌..…………………………………………….………..…..22
3.1靈芝屬真菌的特性…………………………………….………..…..22
3.2靈芝屬真菌對植物的病害………………..…………….………..…24
3.3靈芝屬真菌木質分解酵素的研究………..…………….………..…24
4.異源表達系統……………………………..……………….……..…..26
4.1原核表達系統…………………………………………….……..…..26
4.2真核表達系統…………………………………………….……..…..26
4.2.1嗜甲醇畢赤氏酵母表達系統…………………….…….……..…..26
5.研究動機與目的…………………………………………….……..…27
第二章 材料與方法……………………………………….……..…...28
1.實驗材料…………………………………………………….……..…28
1.1實驗菌株………………………………………………….……..…..28
1.2實驗質體………………………………………………….……..…..30
1.3菌種保存………………………………………………….……..…..30
2.實驗流程及方法…………………………………………...……..…..30
2.1靈芝屬真菌ABTS分解能力測定..........………………...……..…..30
2.1.1培養皿測試…………………………………………......……..…..30
2.1.2胞外液酵素活性測試………………………………......……..….32
2.2錳過氧化酶基因選殖.........................................................................33
2.2.1真菌DNA製備……………………………………….......…..…..33
2.2.2錳過氧化酶片段聚合酶連鎖反應增幅………………......…..…..34
2.2.3 PCR產物純化及定序…………………………………….......…..34
2.2.4真菌RNA製備……….………………………………….......……34
2.2.5各菌株錳過氧化酶選殖方法…..…………….......………..…..….35
2.2.5.1台灣靈芝(G. formosanum 0109, BZ)錳過氧化酶基因選殖……35
2.2.5.1.1 Rapid Genome Walking……………………………………….35
2.2.5.1.2片段cDNA合成………………………………………...……36
2.2.5.1.3五端RACE ( Rapid Amplification of cDNA Ends)……..……36
2.2.5.1.4 台灣靈芝(G. formosanum BZ)錳過氧化酶基因選殖……….37
2.2.5.2 南方靈芝(G. australe 0705)錳過氧化酶基因選殖……………37
2.2.5.3新日本靈芝(G. neo-japonicum 0813, VZ)錳過氧化酶基因選殖………………………………………………………………………..37
2.2.6胺基酸序列分析…………………………………………………..38
2.3錳過氧化酶基因之異源表達系統建構及表達......……..………….42
2.3.1.1大腸桿菌表達系統建構………………….......….…..………….42
2.3.1.2以大腸桿菌表達錳過氧化酶…………….......…...…………….42
2.3.2.1嗜甲醇畢赤氏酵母表達系統建構……….....….…..…..……….43
2.3.2.2以嗜甲醇畢赤氏酵母表達錳過氧化酶….......…...…..…..…….43
2.3.2.2.1培養皿測試………………………..…….......……..………….44
2.3.3蛋白質分析……………………………….......……..……...…….44
2.3.3.1活性測試……………………………..........……..………..…….44
2.3.3.2 蛋白質定量…………….………………………………………44
2.3.3.3十二烷基磺酸鈉聚丙烯醯胺膠體電泳...…...…….……...…….44
2.3.3.4.西方雜合分析………………………….….......……..…...…….45
2.3.3.5重組蛋白之純化……………………………...…………………45
第三章 結果…………………………………….......……..…....…….46
1.靈芝屬真菌分解ABTS能力之初步測定………….......……....…….46
1.1培養皿測試………………………….......……..…...……………….46
1.2 菌絲體胞外酵素活性測定………….......……..…......……………50
2. 靈芝屬真菌錳過氧化酶基因選殖…….......……..…...…….………50
2.1 錳過氧化酶基因片段選殖………….......……..…...……...………51
2.2 錳過氧化酶轉譯區間選殖………….......……..…...……...………51
2.2.1 台灣靈芝0109基因選殖…………………...……………………51
2.2.1.1Genome Walking…………..…………….………………………51
2.2.1.2 RT-PCR和五端RACE…..…………….………………………..54
2.2.1.3台灣靈芝BZ錳過氧化酶基因選殖………….…………………54
2.3.2 南方靈芝0705錳過氧化酶基因選殖…………….…..…………57
2.3.3 新日本靈芝0813, VZ錳過氧化酶基因選殖….…...……………57
2.3.4 錳過氧化酶片段基因分析………….………………..…….……60
2.3.5 南方靈芝0705和台灣靈芝0109錳過氧化酶轉譯區間比較..…60
2.3.6 靈芝屬物種錳過氧化酶胺基酸序列和其他物種之比較…....…69
3.靈芝錳過氧化酶的異源表達………….………………..……..……..72
3.1大腸桿菌表達系統………….………………..……..…………..…..72
3.1.1表達載體建構………….………………..……..…………..……...72
3.1.2 目標蛋白表達………….………………..……..…………..…….72
3.2嗜甲醇畢赤氏酵母菌表達系統……………..……..……………….77
3.2.1表達載體建構………….………………..……..…………..……...77
3.2.2目標蛋白表達………….………………..……..…………..……...77
第四章 綜合討論與結果…………..……..……….…………..……...80
第五章 未來展望……………………………………………………..84
參考文獻………….……………………..……..………………..……...85

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