臺灣博碩士論文加值系統

自林業試驗所取得28株白腐真菌，以及從環境篩選出具降解染料(剛果紅)的11株真菌和實驗室驗室6株真菌，先用PDA含100 ppm剛果紅和低氮磷酸鹽培養基 (1.2 mM)含100 ppm剛果紅測試其對染料退色能力，選擇能完全將染料降解的菌株共12株真菌，將其培養在PDB中測試第2、4、6、8天時的漆氧化酵素產量，選擇酵素產量高於10000 U的菌株共5株。改變磷酸鹽培養基中的氮源：soytone、yeast extract、peptone、ammonium tartrate、NH4Cl、KNO3 、 NH4NO3，找出最適合漆氧化酵素生產的氮源，另外將這5株白腐真菌培養在30-70 ℃溫度範圍，測試其菌絲生長情形，發現A 8在30 ℃生長最快12.1 mm/day，但在40 ℃時則無法生長，而TFRI 707在40 ℃生長最快12.7 mm/day，超過50 ℃所有測試菌株都不生長。挑選氮源測試中漆氧化酵素生產量最高的兩株TFRI 707和A 8，培養在pH 3.5-5.5下測試酵素產量，發現在pH 5.5酵素產量最好。金屬離子則以不添加的方式測試其影響，其中以銅離子的影響最大。添加誘導物實驗中，veratryl alcohol可以同時誘導兩株菌的漆氧化酵素產量。初步分析TFRI 707和A 8分泌的漆氧化酵素特性，隨著pH值越接近中性酵素活性越低，在pH 3時對ABTS有最大的酵素活性，50 ℃時3小時內酵素活性還有70 %，隨著溫度上升酵素活性喪失越快，TFRI 707和A 8的酵素適應70 ℃超1小時後，酵素活性分別低於34 %和8 %。在活性染的結果發現，半固體培養下A 8有較多的同功異構酶，TFRI 707則剛好相反，分子量分別為50-90 kDa和40-50 kDa；在SDS-PAGE中A 8的漆氧化酵素分子量約為50-60 kDa，TFRI 707分子量則約30-40 kDa。

White rot fungi produce three main extracellular enzymes involved in ligninolysis, including laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP). These enzymes are capable of initiating the oxidation of lignin by a free-radical mechanism which also led to the degradation of a wide variety of normally very recalcitrant environmental pollutants. Laccase is a glycosylated polyphenol oxidase which contains four copper ions per molecule and has shown good potential to be used in bioremediation, biopulping, biobleaching, medicine, and textile industry. Forty-five isolates of white rot fungi were screened for their ability to degrade congo red. Based on the decoloration ability, twelve isolates were selected for further study. They were grown in PDB and phosphate buffer culture medium (PBCM) for laccase activity assay. Five isolates were chosen for their high level of laccase production (＞10000 U/L). In order to find out the best nitrogen source for laccase production, we have tested 7 different nitrogen sources in PBCM. With yeast extract and NH4Cl as nitrogen source, isolate TFRI 707 and A 8 have shown the highest laccase production, respectively. When study the effect of temperature on their growth, the data indicated that TFRI 707 and A 8 had higher growth rats on 40 ℃ and 30 ℃respectively. Various factors, including pH (3.5-5.5), metal ions (Mg, Mn, Ca, Cu, Fe, Zn) and inducer (ethanol, veratryl alcohol, ferulic acid, 2,5-xylidine) on laccase production were also studied. The optimal pH for laccase production was 5.5 for both isolates. When Cu supplement was deleted from the medium, decreases in laccase production were observed for both of them. As for the inducers, only veratryl alcohol had a substantial effect on laccase production. With ABTS as the substrate, laccase has an optimum activity at pH 3, and an optimum temperature at 30 ℃. In liquid and semi-solid medium, isolate A8 produce 3 and 4 isoenzymes of laccase, respectively, while 4 and 3 isoenzymes were detected for isolate TFRI 707. Molecular weight was determined using Native PAGE, and laccases secreted by isolate A 8 were between 50-90 kDa, and those excreted by TFRI 707 were between 40-50 kDa. When assaying with SDS-PAGD, the molecular weight of laccases secreted by A 8 were between 50-60 kDa and by TFRI 707 were between 30-40 kDa.

中文摘要………………………………………………………………..Ⅰ
英文摘要………………………………………………………………..Ⅲ
表目錄…………………………………………………………………..Ⅴ
圖目錄…………………………………………………………………..Ⅵ
前言………………………………………………………………………1
材料與方法………………………………………………………………9
結果……………………………………………………………………..16
討論……………………………………………………………………..23
參考文獻………………………………………………………………..31
圖表……………………………………………………………………..45

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