臺灣博碩士論文加值系統

纖維素、半纖維素、木質素是自然界中存量最多的大分子物質，其中木質素由於結構複雜最難以分解。已知有三種酵素具有分解木質素之功能分別為漆氧化酶（laccase, 1.10.3.2）、含錳過氧化酶(manganese peroxidase, 1.11.1.13)和木質素氧化酶( lignin peroxidase, 1.11.1.14)。
靈芝屬真菌屬於白腐型真菌，具有能夠分解木質素的能力。本文將探討靈芝屬真菌中漆氧化酶基因之種類、特性與異源表現之結果。以漆氧化酶基因之保守性序列設計引子，對十一株靈芝屬真菌進行聚合酶鍊鎖反應，將產物定序比對後顯示，靈芝屬真菌皆具有兩條以上的漆氧化酶基因序列，並自靈芝屬真菌G. lucidum RZ、G. tsuage 1109、G. fornicatum 0814中，分別選殖出漆氧化酶基因RZ.lac4、0814.lac1、1109.lac1，其蛋白質各具有520、521、521個胺基酸，其中前21個胺基酸為signal peptide，在保守性Cysteine 之後第十個氨基酸為Phenylalanine，顯示三者皆屬具有高還原電位之第三類漆氧化酶。而RZ.lac4基因上游Promoter位置有TATA、CAAT序列，以及MRE、STRE序列，顯示此基因之表現可能受到金屬離子之調控。
使用AOX1起動子，將所選殖之三條基因轉殖入嗜甲醇酵母Pichia pastoris KM71進行異源表現，所得之重組蛋白質皆具有漆氧化酶活性，重組蛋白reRZ.lac4、re0814.lac1、re1109.lac1之最適反應pH值為3.0，最適反應溫度分別為55、60、65℃。

Cellulose, hemicellulose, and lignin are most abundant macromolecules in nature. Lignin is hardest to be degraded for its complex constitutions. There are three enzymes have the ability to degrade lignin: laccase (1.10.3.2), manganese peroxidase (1.11.1.13), and lignin peroxidase (1.11.1.14).

Whitr-rot fungi, such as Ganoderma spp., can degrade lignin. In this study, gene family, characteristics, and heterologous expression of laccase genes from Ganoderma spp. are discussed. The specific primers according to laccase conserved copper-binding regions used to amplify the laccase genes in the eleven strains of Ganoderma spp. The result shown that there are at least two laccase genes in each strain. Laccase cDNA of RZ.lac4、0814.lac1、1109.lac1 from G.. lucidum RZ、G. tsuage 1109、G. fornicatum 0814 were cloned and encodes for proteins with 520, 521, and 521 amino acids, including a 21-residue secrection signal peptide for each protein. Phenylalnine in additional residue 10 amino acids downstream of the conserved cysteine of each encoding protein shows that these proteins belong class 3 laccase and may have high redox potential of the cupric ion.
The Mental-responsive elements (MREs) and stress-responsive promoter element (STRE) found in the promoter of RZ.lac4 suggest that RZ.lac4 might be regulated by mentals.(Abadulla et al. 2000)

The cloned cDNA were expressed in Pichia pastoris KM71 under the control of the AOX1 promoter. The transformants were found to secrete active recombinant enzymes after induction with methanol. The optimal temperature of the recombinant proteins, reRZ.lac4, re0814.lac1, re1109, are 55, 60, and 65℃. The optimal pH for the recombinant proteins is 3.0.

目錄

目錄……………….………………………………………….……………I
縮寫－全名對照表…………….…………………………………………IV
表目錄…………….………………………………………….…………V
圖目錄…………….………………………………………….…………VI
中文摘要…….………………………………….………………………IX
英文摘要…….……………….…………………………….……………X
第一章、緒論……………….…………………….………………………1
一、木質素……………….…………………….……………………2
二、木質素分解酵素……………….………….……………………5
三、漆氧化酶……………….………….……………………………7
1. 漆氧化酶之來源……………….………….…………………7
2. 漆氧化酶之生理功能……………….………….………………8
3. 漆氧化酶之誘導……………….………….…………………10
4. 漆氧化酶之多型性……………….………….………………12
5. 漆氧化酶之演化關係……………….………….……………16
6. 漆氧化酶之EC number……………….………….…………17
7. 漆氧化酶蛋白質結構……………….………….……………18
8. 漆氧化酶之反應機制……………….………….……………22
9. 漆氧化酶之應用……………….………….…………………26
10. 漆氧化酶之異源表達……………….………….……………28
四、靈芝屬真菌……………….………….…………………………32
1. 靈芝屬真菌之分類……………….………….………………32
2. 靈芝屬真菌之漆氧化酶……………….………….…………33
五、研究動機與研究架構……………….………….………………35
1. 研究動機……………….………….…………………………35
2. 研究架構……………….………….…………………………35
3. 預期目標……………….………….…………………………36
第二章、材料方法……………….………….……………………………38
一、實驗材料……………….………….……………………………38
1. 基因來源……………….……….……….……………………38
2. 基因保存……………….………….……….…………………38
3. 異源表達系統……………….……….….……………………38
二、實驗方法……………….……….………….……………………40
1. 靈芝屬漆氧化酶基因確認……….……….………….………40
1.1 靈芝DNA之抽取………….……….………….…………40
1.2 引子設計與聚合酶鍊鎖反應（PCR）……………….……41
1.3 演化計算……………….….……….……………………41
2. 以Genome walking選殖靈芝屬漆氧化酶全長基因…………44
2.1 Genome walking原理……………….………….…………44
2.2 靈芝DNA之萃取……………….………….……………44
2.3 以限制酶截切靈芝genomic DNA………………………45
2.4 Linkers製備……………….………….……………………45
2.5 Ligation……………….………….…….…………………46
2.6 聚合酶鍊鎖反應……………….………….………………46
3. 靈芝屬漆氧化酶cDNA全長之選殖……………….….……52
3.1 養菌條件測試……………….………….…………………52
3.1.1誘導條件……………….………….…………………52
3.1.2漆氧化酶素活性測試………………….……………52
3.1.3活性染色……………….……….……………………53
3.1.4 SDS-PAGE………….………….……………………54
3.2 cDNA選殖……………….………….……………………56
3.2.1靈芝誘導培養……………….………….……………56
3.2.2 Total RNA之萃取……………….……………………56
3.2.3 RT-PCR……………….………….……………………57
3.2.4 PCR…….…………….………….……………………57
4. 以Pichia Pastoris 表達靈芝屬漆氧化酶基因………………59
4.1 質體建構………………….………….……………………59
4.2.1轉形DNA之製備……………….………….…………59
4.2.2勝任細胞(competent cell)之製備……………….……60
4.2.3電穿孔……………….………….……………………60
4.3 轉形株之培養……………….………….…………………61
5. 酵素特性分析……………….………….……………………61
5.1 最適pH值之測定……………….………………………61
5.2 最適反應溫度……………….………….…………………61
5.3 比活性…………………….………….……………………62
5.4 蛋白質電泳與活性染色圖……………….………………62
第三章、結果與討論……………….……………….……………………63
一、靈芝屬漆氧化酶基因確認……………….………….…………63
二、演化分析……………….………….……………………………72
三、靈芝屬漆氧化酶誘導測試……………….………….…………74
四、Genome walking結果……………….………….………………77
五、RT-PCR結果……………….……………….……………………82
六、基因序列分析……………….…………….……………………83
七、氨基酸序列分析……………….………….……………………93
八、表現載體之建構………………………….………….…………97
九、電穿孔轉型結果………………………………………………100
十、轉型株誘導培養誘導結果與酵素特性分析…………………101
1. 轉型株誘導後胞外上清液活性比較………………………101
2. 轉型株胞外液活性染色圖……………….…………………103
3. 最適pH值………………. …………….……………………104
4. 轉型株胞外漆氧化酶之最適反應溫度……………………104
5. 重組蛋白之胞外上清液之比活性評估……………………105
第四章、結論……………….…………………….……………………114
參考文獻……………….………….……………………………………115
附錄一、靈芝漆氧化酶部分基因序列………………………………123

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