臺灣博碩士論文加值系統

漆氧化酶 (laccase) 首於漆樹中被發現並分離，為含銅的多酚氧化酵素 (EC 1.10.3.2)，屬於多銅氧化酵素家族的成員，已知廣泛分布於真菌、高等植物和細菌當中。這類含銅酵素可參與催化多種基質的氧化反應，同時將氧分子還原成水。目前生物科技上被認為最具應用潛力的漆氧化酶來自於真菌，主要歸因於其具有較高的氧化還原能力，且作用時只需要空氣、副產物只有水的生成，為一理想的綠色酵素。已知有超過六十種真菌的品系證實具有漆氧化酶活性，包括子囊菌綱、半知菌綱以及擔子菌綱，尤以擔子菌綱中的白腐菌在生態上有助於垃圾分解並且具有外生菌根，為目前已知之漆氧化酶的最高生產者。本實驗成成功選殖樟芝 (Taiwanofungus camphoratus) 漆氧化酶讀譯區全長片段 (tclac)，其中包含1572個鹼基，推測可轉譯出523個胺基酸。經生物資訊軟體預測N端23個胺基酸為樟芝漆氧化酶之訊息胜肽，且樟芝漆氧化酶cDNA經GenBank資料庫比對結果與其他真菌物種之漆氧化酶有高度相似性。樟芝漆氧化酶酵素活性於第五天與第七天液態培養基中高達0.8 U/L，同時培養初期呈現高度基因表現，推測樟芝漆氧化酶的表現可能與真菌孢子的形成以及對環境的適應力有關。此外，本研究發現熱逆境處理會使固態培養菌絲體顏色轉變為乳白色或淡橘褐色，與自然狀態下日照區域的野生種顏色相似；且短時間熱逆境處理之固態與液態培養條件下皆能提升樟芝漆氧化酶基因的表現與酵素活性。實驗進一步將其轉譯區選殖入表現載體pET-22b(+)，以大腸桿菌Escherichia coli BL21(DE3) 做為表現宿主，加入最終濃度為0.25 mM的IPTG於20℃誘導24小時，經酵素活性檢測與原態電泳分析得知可成本冗犮X具活性的胞外重組蛋白 (47 U/L)。此研究將有助於闡明此酵素之特性，以及快速、低成本、大量生產，有利於未來開發綠色能源及生技產業之應用。

Laccase was discovered and isolated in Rhus verniciflua at first, it is a multi-copper containing oxidase (EC 1.10.3.2), belongs to the multicopper oxidase family. Its activity has been detected in fungi, higher plants and bacteria. These copper containing enzymes catalyze the oxidation of various substrates with the simultaneous reduction of oxygen molecules to water. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements: they only need air, and they produce water as the only by-product. Over 60 fungal strains belonging to Ascomycetes, Deuteromycetes and especially Basidiomycetes show laccase activities. Among the latter group, white-rot fungi participate in litter-decompose and are ectomycorrhizal fungi, as well as the highest producers of laccase. In this study, we interest in the brown-rot fungus, Taiwanofungus camphoratus, which is discovered especially in Taiwan, and we analyze the characteristics of laccase. We successfully isolated and cloned a laccase cDNA sequence, named tclac (Taiwanofungus camphoratus laccase) for the nonce. The open reading frame of tclac is 1572 base pairs in length and can translate 523 amino acids. By way of bioinformatics software, the 23 amino acids in N terminal is the deduced signal peptide of laccase from Taiwanofungus camphoratus. According to the alignment to GenBank database, the cDNA sequence of the tclac reveals high similarity with laccase genes in other fungal species. Laccase gene could be expressed in liquid-cultured mycelia and laccase activity was detected in the cultured medium under the primary condition. The highest laccase activity (0.8 U/L) was detected in 5- and 7-day liquid- cultured medium, as well as the high mRNA expression level in primary phase. It is possible that the laccase activity of brown-rot fungi may be enhanced by the adaptation of spores to environmental changes. Moreover, color of heat-stress mycelium is similar to that of wild type grows in light area, appears ivory or light-brown colors. In addition, the gene expression and enzyme activity of laccase were increased in both heat stress-treated solid and liquid cultures in short term treatment. Further, the coding region was cloned into a pET-22b(+) vector then the recombinant DNA was transformed into Escherichia coli BL21(DE3). The recombinant laccase activity (47 U/L) was detected under the 24-hour induction of 0.25 mM IPTG in LB medium at 20℃and native-PAGE analysis showed that this protein is in extra-soluble form. In future, heterologous expression of laccase in Escherichia coli would be better suited for efficient, low-cost and large-scale production and study of the properties of the enzyme may prove beneficial for future applications in environmental and biotechnology.

中文摘要 I
英文摘要 III
本文目錄 VI
表目錄 XI
圖目錄 XII
英文縮寫對照表 XIV
第一章 緒論 1
1.1 樟芝 1
1.2 木質素 3
1.3 木頭腐生真菌 (wood rot fungi) 與木質素可分解酵素 6
1.4 漆氧化酶 7
1.4.1 漆氧化酶的蛋白質結構 8
1.4.2 漆氧化酶的基因表現 9
1.4.3 漆氧化酶的的應用 10
1.5 真菌漆氧化酶異源表達之研究現況 11
1.6 研究動機 12
第二章 材料與方法 13
2.1 藥品與儀器 13
2.1.1 藥品 13
2.1.2 儀器 16
2.2 菌種來源與培養條件 16
2.2.1 樟芝固態培養 16
2.2.2 樟芝液態培養 17
2.3 熱逆境處理條件 17
2.3.1 固態培養樟芝菌絲體之熱逆境處理 17
2.3.2 液態培養樟芝菌絲體之熱逆境處理 17
2.4 樟芝菌絲體之漆氧化酶活性分析 18
2.4.1 固態培養樟芝菌絲體之漆氧化酶活性分析 18
2.4.2 液態培養樟芝菌絲體之胞外活性分析 19
2.5 樟芝漆氧化酶基因cDNA (tclac) 之分離與選殖 19
2.5.1 液態培養樟芝菌絲體的樣品收集與分析 19
2.5.2 Total RNA之萃取 20
2.5.3 cDNA之合成 20
2.5.4 退化性引子設計與部分tclac cDNA之擴增 21
2.5.5 全長tclac cDNA之擴增 22
2.5.6 DNA片段之純化回收 22
2.5.7 接合反應 23
2.5.8 轉型作用 24
2.5.9 質體純化 25
2.6 漆氧化酶基因表現之分析 26
2.7 樟芝漆氧化酶於轉型大腸菌中之異源表達 28
2.7.1 異源表達系統中大腸桿菌轉型株的製備 28
2.7.1.1製備tclac讀譯區全長 (暫命名為total tclac，簡稱 ttclac) 片段 28
2.7.1.2重組載體pET22b-ttclac之構築 28
2.7.1.3轉型作用 29
2.7.2 誘導目標蛋白生產之策略 30
2.7.3 重組樟芝漆氧化酶蛋白的純化 31
2.8 SDS聚乙烯醯胺膠體電泳 32
2.9 蛋白質原態電泳與漆氧化酶活性染色 33
第三章 結果 35
3.1 樟芝漆氧化酶活性檢測 35
3.1.1 固態培養菌絲體之漆氧化酶活性分析 35
3.1.2 液態培養菌絲體之發酵液中漆氧化酶活性分析 36
3.2 熱逆境處理條件與樟芝漆氧化酶之研究 36
3.2.1 固態培養菌絲體之熱逆境處理誘導漆氧化酶活性 36
3.2.2 液態培養菌絲體於熱逆境處理後之變化 37
3.2.2.1菌絲體型態變化 37
3.2.2.2培養基pH值變化、漆氧化酶活性分析 38
3.3 樟芝漆氧化酶基因 (tclac) 之選殖與分析 38
3.3.1 total RNA之萃取與部分tclac cDNA產物之擴增 38
3.3.2 tclac cDNA之選殖 39
3.4 樟芝漆氧化酶之基因表現 40
3.5 大腸桿菌中樟芝漆氧化酶之異源表達 40
3.5.1 樟芝漆氧化酶基因完整讀譯區(total tclac, ttclac) 之cDNA選殖 40
3.5.2 重組載體pET22b- ttclac之建構 41
3.5.3 漆氧化酶異源表達最佳誘導條件測試 41
第四章 討論 44
4.1 樟芝漆氧化酶活性與基因表現之誘導條件 44
4.2 樟芝漆氧化酶cDNA與胺基酸序列分析 47
4.3 樟芝漆氧化酶之異源表達 49
第五章 結論 52
參考文獻 54
圖表 62

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