臺灣博碩士論文加值系統

漆氧化酶為白腐真菌（white rot fungi）之特有胞外木質素分解酵素系統 (extracellular ligninolytic enzyme system)，主要的組成酵素之一。其為一多功能酵素，可催化多樣多種的反應，被廣泛的應用於紙漿造紙中的紙漿漂白、環境污染物去毒性作用、處理工業工廠之廢水及紡織和染料工業之染料脫色等。本實驗之目的便是將前研究人員從土壤中所分離出，能產生極佳的漆氧化酶活性之白腐真菌分離株A8，進行分離株A8之菌種鑑定，以傳統鏡檢方法及分子生物學方法－18S rDNA及ITS序列定序，分析分離株A8之類緣關係；並以蛋白質電泳膠體活性染色（Native-PAGE）分析此分離株所產之漆氧化酶，發現分離株A8於液態培養下能產生四種漆氧化酶（laccase isoforms，標示為L1、L2、L3及L4）；使用硫酸胺沈澱、透析、陰離子交換樹脂等方法分離純化出此分離株之多種漆氧化酶，將純化後之漆氧化酶經由二維蛋白質電泳（Two-dimensional SDS polyacrylamide gel electrophoresis）分離後，結合液相層析串聯質譜儀 ( LC/MS/MS ) 對蛋白質peptide進行序列分析，再進行database比對鑑定；同時亦探討有無人工介質存在下，此分離株之漆氧化酶對偶氮染料褪色之能力。結果顯示，分離株A8之18S rDNA序列與Spongipellis unicolor及Cerrena unicolor最為靠近；液體培養下之分離株A8能產生高量蛋白質（5 mg/mL）；laccase L1（本實驗標示）與Spongipellis sp. FERM P-18171之laccase 1 precursor與laccase 2 precursor皆有100 %相符合之peptide序列（matched peptides）。此外，分離株A8之漆氧化酶能夠降解偶氮染料，於50 mM ABTS人工介質存在下，酵素對染料之退染效果更加明顯，一天內可褪色剛果紅55 %以上，而經純化後之laccase L1三天的時間可以褪色橙G（orange G）93 %以上。

Laccase is one of the ligninolytic enzymes produced by white rot fungi which is a very versatile enzyme. It has been used extensively in pulp and paper industry, detoxifying the environmental pollutants, and food and cosmetic industries. The purposes of this study are to (1) provide identification of isolate A8, a wood rot fungus which excrete high amount of laccase, using both traditional and molecular biology technique; (2) purify and characterize laccase(s) produced by isolate A8; (3) evaluate the efficiency of decoloration activities of laccase produced by isolate A8. Based on ITS (internal transcribed spacer) and 18S rDNA (small-subunit) sequences information, isolate A8 was shown to be a member that related to Polyporaceae and can be grouped with Spongipellis unicolor and Cerrena unicolor in the same cluster. When cultural supernatant of isolate A8 was analyzed using Native-PAGE (polyacrylamide gel electrophoresis) with ABTS (2, 2’-azinobis 3-ethylbenzathiozone-6-sulfonic acid) as the substrate, four laccase isoforms (L1, L2, L3, and L4) were observed. Strain A8 produced high amount of protein in the culture liquid, about 0.5 milligram per milliliter. Purification of laccase was done by following the procedure described below. Proteins in the cultural supernatant were first precipitated by adding ammonium sulfate to the supernatant. After the salt in the precipitate was removed through dialysis against 0.01 M phosphate buffer (pH 7.0), they were concentrated by ultrafiltration using a membrane with molecular weight cutoff at 10K. The concentrated sample was applied to a DEAE-cellulose anion – exchange column that was pre-equilibrated with 0.01 M phosphate buffer (pH 7.0). The column was then eluted with a linear gradient of 0 to 1 M NaCl in the same buffer and a major protein peak was observed between fraction 35 (with 5 ml a fraction). Activity assay revealed that samples in this peak also showing high laccase activity, about 94591 units per milligram of protein. The purified strain A8 laccases were then analyzed with two-dimension SDS polyacrylamide gel electrophoresis and LC/MS/MS. Comparison of amino acid sequences of the four fragments of purified laccase L1 with the NCBI enzyme database showed 91~100% sequence similarity with laccase 1 precursor fragments encoded by Spongipellis sp. FERM P-1817. For the decolorization studies, crude and purified laccase was added to the dye (congo red and orange G) solution to a final concentration of 5 U/ml. For orange G, more than 93% of the dye added was decolorized in three days. By adding low concentration (50 μM ABTS) of mediator in the treatment process, we can effectively increased the range and rate of decolorization. For example, the original recalcitrant congo red can then be decolorized up to 55% by crude laccase preparation in 24 hours.

摘要………………………………………………………………………I
Abstract……………………………………………………………..III
目錄……………………………………………………………………..V
表目錄……………………………………………………………...VIII
圖目錄………………………………………………………………….IX
前言……………………………………………………………………..1
一、 白腐真菌之胞外木質素分解酵素系統…………………...1
二、 漆氧化酶(laccase)之結構及去酚作用機制………………4
三、 漆氧化酶(laccase)之應用…………………………………7
四、 本實驗之目的………………………………………………11
材料方法……………………………………………………………….12
一、 菌株來源……………………………………………………12
二、 藥品與器材設備……………………………………………12
三、 絲狀真菌之保存……………………………………………13
四、 分離株A8外觀型態之鑑定………………………………..15
五、 分子生物學rDNA與ITS序列分析之鑑定………………….16
六、 分離株A8產漆氧化酶之培養………………………………19
七、 漆氧化酶活性測定…………………………………………20
八、 蛋白質濃度測定……………………………………………22
九、 分離株A8漆氧化酶之純化…………………………………23
十、 蛋白質電泳膠體分析………………………………………26
十一、二維蛋白質電泳(2-D)…………………………………………27
十二、液相層析串聯質普儀（LC/MC/MC）定序分析……………….35
十三、酵素之染料褪色實驗………………………………………….36
結果…………………………………………………………………….39
一、 白腐真菌分離株A8外觀型態之鑑定………………………39
二、 分子生物學rDNA與ITS序列分析之鑑定………………….39
三、 蛋白質電泳膠體分析與分離株A8 漆氧化酶之純化…….44
四、 二維蛋白質膠體電泳分析與蛋白質氨基酸序列分析....46
五、 褪色染料實驗....................................48
討論…………………………………………………………………….52
一、 白腐真菌分離株A8之鏡檢與分子生物學方法之鑑定……52
二、 蛋白質電泳膠體分析……………………………………..56
三、 分離株A8 漆氧化酶之純化……………………………….57
四、 漆氧化酶序列分析…………………………………………59
五、 褪色染料實驗....................................61
結論…………………………………………………………….......63
參考文獻……………………………………………………………….65
圖表…………………………………………………………………….75
附錄一、………………………………………………………………112
附錄二、…………………………………………………………....113
附錄三、……………..………………………………………………114
附錄四、…………………………..…………………………......115
附錄五、.…………………………………………………………...116
附錄六、................................................117

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