臺灣博碩士論文加值系統

白腐真菌能產生三種主要的胞外木質素分解酵素：漆氧化酵素，木質素過氧化酵素以及錳離子過氧化酵素。錳離子過氧化酵素為具有高氧化還原電位，是一種帶有亞鐵離子的的血紅素醣蛋白分子，能應用於許多頑強污染物的氧化與降解，例如多環芳香碳氫化合物、偶氮染料。
本篇研究從菌種中心、林業試驗所取得和本實驗室篩得一共八株白腐真菌 (BCRC 36319、TFRI 20、TFRI 31、TFRI 45、TFRI 554、TFRI 691、TFRI707、F5)。針對此八株菌的錳離子過氧化酵素 (MnP) 進行初步的酵素產量分析，以低氮源 (1.2 mM)的無機鹽培養基培養篩選出TFRI 20、TFRI 554及TFRI 691此三株具較高錳離子過氧化酵素之菌株。在高/低氮源的培養實驗中，高氮源培養基 (含有3 g tryptone peptone)對於錳離子過氧化酵素具有抑制的效果。另外針對在不同時間點各菌株的MnP活性進行測試，最高活性出現在培養後第4至6天，TFRI 20的 MnP活性為13631 U/L，TFRI 554為21931 U/L，TFRI 691為37619 U/L。TFRI 554與TFRI 691在後續不同氮源種類培養的實驗中，TFRI 554的MnP活性受到氯化銨以及硝酸鉀的抑制，而TFRI 691的MnP在酒石酸銨及硝酸鉀的環境下都有高量的活性表現(約為6,0000 U/L)，但仍會受到氯化銨些微的影響。更換不同pH值的酵素分析試劑實驗中TFRI 554及TFRI 691兩者的MnP活性皆在pH 6下有最佳的反應活性，隨著反應環境越趨酸性，酵素活性則逐漸減弱。而在不同溫度測試中TFRI 691的MnP在40℃及45℃下加熱一小時後酵素活性有些微的提高，但兩株菌的MnP對於50℃的高溫並沒有明顯的耐受性，處理三小時後僅剩餘40％~50%的活性。不論添加少量的碎稻桿(0.5% W/V)或veratryl alcohol (1mM、2mM)，這些誘導物都能顯著的提升TFRI 554及TFRI 691的MnP產量，但過量的veratryl alcohol (>2 mM)則反而會抑制MnP的生成。TFRI 554與TFRI 691兩株菌在半固體培養實驗中均有很好的MnP產量，甚至比一般的液體震盪培養還要來的高(74700 U/L; 45553 U/L)，而通氣培養則能維持MnP的活性穩定，持續到培養後第18天。TFRI 554與691的MnP經由SDS-PAGE及Native PAGE分析後發現具有不同的酵素圖譜。TFRI 554的錳離子過氧化酵素具有蛋白質條帶，大小分別為65 kDa、40 kDa、35 kDa；TFRI 691的錳離子過氧化酵素三個蛋白質條帶，大小分別為40kDa、38 kDa、35 kDa。

White-rot fungi is the most common degrader in lignin degradation. They produce three major extra cellular lignolytic enzymes-laccase, lignin peroxidase and manganese peroxidase. Manganese peroxidase (MnP) is a heme containing glycoprotein which has a high redox potential, could be applied in oxidizing and degrading many recalcitrant pollutants such as PAHs (polycyclic aromatic hydrocarbons), azo dyes.
There are eight isolates were screened for the production of MnP. TFRI 20, TFRI 554 and TFRU 691 cultivated in low nitrogen phosphate (1.2 mM) buffer medium has the highest MnP activities (13631 U/L, 21931 U/L, 37619 U/L, respectively). In the later experiment different nitrogen sources were applied in cultivation with TFRI 554 and TFRI 691. The MnP activity of TFRI 554 was suppressed by ammonium chloride and potassium nitrate; TFRI 691 under ammonium tartrate and potassium nitrate exhibited high amount of MnP activity (around 6,0000 U/L), but the activity was also reduced slightly while cultivating under ammonium chloride. In the analysis of MnP activities, different pH of analysis buffer were applied, the MnP of TFRI 554 and TFRI 691 both exhibited highest activity under pH 6, but the MnP activities decreased as the pH of analysis buffer decreased. When study the effect of temperature on MnP stability, MnP activity of TFRI 691 raised slightly after one hour treatment under 40℃ even 45℃. MnP of both TFRI 554 and TFRI 691 did not exhibit the stability to high temperature. Whether the addition of milled rice straw (0.5% W/V) or veratryl alcohol (1, 2 mM), these inducers could be suitable for inducing higher MnP production of TFRI 554 and TFRI 691, but an excess addition of veratryl alcohol (4 mM) could conversely inhibit the MnP production. In the semi-solid state study, both TFRI 554 and TFRI 691 produce high amounts of MnP (74700 U/L; 45553 U/L, respectively), even higher than which in liquid cultivation. MnP activity of TFRI 554 maintained stable amount in air pumping experiment until the eighteenth day of cultivation, much longer than that of non-air pumping cultivation. According the SDS-PAGE analysis, the MnP zymogram of TFRI 554 and TFRI 691 are different. There were three bands of MnP found in TFRI 554 (65 kDa, 40 kDa, 35 kDa respectively); and there were three bands found in TFRI 691 (40 kDa, 38 kDa, 35 kDa respectively).

表一：實驗使用菌株....................................................................41
圖一：各菌株培養六天後的MnP活性比較....................................................................42
圖二：TFRI 20, 554, 691於無機鹽低氮源培養基及GPCSL培養基中的MnP表現...43
圖三：TFRI 691,554,20 在不同培養天數下的MnP活性表現………………..…….....44
圖四：TFRI 20, 554於30℃下培養5天的菌絲生長情形比較…………………………45
圖五：TFRI 554培養於不同氮源下(1.2 mM)生產MnP產量............………………....46
圖六：TFRI 691培養於不同氮源下(1.2 mM)生產MnP產量............………………....47
圖七：TFRI 554在不同pH緩衝液下的MnP表現……………………………………...48
圖八：TFRI 691在不同pH緩衝液下的MnP表現……………………………………...49
圖九：TFRU 554、691在不同錳離子濃度下的MnP產量...........................................50
圖十：TFRI 554於各前處理溫度的MnP活性表現…………………………………….51
圖十一：TFRI 691於各前處理溫度的MnP活性表現………………………………….52
圖十二：誘導物veratryl alcohol的濃度對於TFRI 554、TFRI691 MnP產量的影響…...53
圖十三：TFRI 554於通氣培養基中MnP的活性表現…………………………………54
圖十四：TFRI 691於通氣培養基中MnP的活性表現…………………………………55
圖十五：TFRI 554、691 的MnP穩定性測試結果.........................................................56
圖十六：TFRI 554、691於半固態培養基下的MnP活性表現…………………………57
圖十七：TFRI 554、691的native-PAGE及經coomassie blue染色結果……………..58
圖十八：TFRI 554、691液體培養液的SDS-PAGE結果………………………………59

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