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研究生:彭昭蓉
研究生(外文):Chao-Jung Peng
論文名稱:利用農業廢棄物促進白腐真菌之錳過氧化酵素的生產
論文名稱(外文):Production of manganese peroxidase during semi-solid culture of agriculture wastes with the white rot fungi
指導教授:劉秀美劉秀美引用關係
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:海洋生物研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:110
中文關鍵詞:白腐真菌錳過氧化酵素農業廢棄物
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於35℃在半固態培養下找出白腐真菌之錳過氧化酵素的最佳生產條件,發現培養基中添加10 g/L的glucose、6 mM的MnSO4.H2O、0.5%(w/v)的Tween 80和5 g的玉米穗軸在pH 6中,可使白腐真菌MB-F26有最佳的錳過氧化酵素活性產量產生。以最適偵測條件偵測酵素活性產量,即調整分析試劑的含量,使之為0.5 mM的sodium tartrate buffer(pH 3)、30 mM的guaiacol、3 mM的MnSO4.H2O和0.5 mM的H2O2來偵測酵素活性產量時,農業廢棄物除了高梁酒糟之外,其它如稻稈、構樹內木、構樹樹皮和玉米穗軸可以產生活性高於前人研究2000倍以上的錳過氧化酵素,其酵素活性產量分別高達2295 U/mL、1797 U/mL、1972 U/mL和1735 U/mL,且在培養期間可使農業廢棄物中的酚類化合物濃度大幅減少。另外在添加稻稈的半固態培養基中測試不同白腐真菌之錳過氧化酵素活性產量,發現其中MB-F1、MB-F12和MB-F14,可達最高酵素活性產量分別為7.85 U/mL、114 U/mL和150 U/mL,此為前人研究的數十到數百倍。實驗中所生成的錳過氧化酵素極其穩定,在4℃下可維持90%以上的活性達50天之久,在室溫(25℃)下,可維持80%以上的活性達20天之久。
White rot fungi, a group of organisms that can oxidize lignin to CO2 and H2O, are able to produce a lignin-degrading enzymatic system, mainly composed of manganese peroxidase (MnP), lignin peroxidase (LiP) and laccase. These enzymes can degrade hazardous chemicals and wastes. MnP plays an importmant role in delignification of kraft pulp, decolorization of bleach plant effluents. Each year about 6.54 million tons of argriculture wastes were produced in Taiwan. Among them, a big portion belongs to lignincellulose wastes. Therefore, in this study different agriculture wastes were used to induce MnP production for industrial applications. High production of MnP activity was obtained by semi-solid culture of MB-F26, supplementing with 10 g/L glucose, 6 mM MnSO4.H2O, 0.5% (w/v) Tween 80 and 5 g corn cob. Under the optimum culture conditions, supplementing the agriculture waste material such as rice straw, bark of tapa cloth tree, core of tapa cloth tree or corn cob could induce the maximum MnP activity to 2295 U/mL, 1797 U/mL, 1972 U/mL and 1735 U/mL, respectively. Other fungi such as MB-F13, MB-F12 and MB-F14 also could produce MnP activity. The maximum MnP activity of these fungi were 114 U/mL, 150 U/mL and 7.85 U/mL, respectively, when rice straw was included in the medium. Moreover, concentrations of phenolic compounds in these agriculture wastes were decreased during incubation. More than 90% and 80% of its initial MnP activity were retained even after 50 days incubation at 4℃ and 20 days incubation at 25℃, respectively.
謝辭 i
摘要 iv
Abstract v
表目錄 viii
圖目錄 ix
附錄 xiii
前言 1
文獻回顧 3
材料與方法 21
結果 34
討論 47
參考文獻 58
表目錄
表一、六株白腐真菌在PDA上培養於35℃中的菌絲生長速率 71
表二、不同受質下偵測錳過氧化酵素 72
表三、白腐真菌在不同培養及酵素偵測條件下產生的最大錳過氧化酵素活性產量。 73











圖目錄
圖一、不同受質下偵測錳過氧化酵素活性產量其吸光值與反應時間的關係:a.以guaiacol為受質, b.以2,6-dimethoxyphenol為受質。 75
圖二、以ABTS為受質偵測laccase酵素活性產量其吸光值與反應時間的關係。 76
圖三、PVA檢量線:a.BP05, b.BF08, c.BF17。 77
圖四、Phenolic compounds濃度檢量線。 78
圖五、以不同氮源培養時培養液中錳過氧化酵素活性產量變化:a.有機氮源, b.無機氮源。 79
圖六、以不同NH4Cl濃度培養時培養液錳過氧化酵素活性產量變化。 80
圖七、添加不同濃度MnSO4.H2O於培養基時錳過氧化酵素活性產量變化:a.添加0.5∼10 mM的MnSO4.H2O, b.添加3∼10 mM的MnSO4.H2O。 81
圖八、添加不同種類PVA培養基中錳過氧化酵素活性產量變化。 82
圖九、添加0∼0.5%(w/v)的BP05於培養基中(a)錳過氧化酵素活性產量變化和(b)BP05之濃度變化。 83
圖十、添加不同添加物於培養基中錳過氧化酵素活性產量變化:a.添加0.5%的Tween 20、Tween 80及不添加任何Tween, b.添加0〜4 mM的 veratryl alcohol。 84
圖十一、以不同pH值之培養基培養時錳過氧化酵素活性產量變化:a.於sodium tartrate buffer(pH 3∼5), b.於sodium phosphate buffer(pH 5∼8)。 85
圖十二、以不同pH值之培養基培養時錳過氧化酵素活性產量。 86
圖十三、以不同農業廢棄物之培養時培養液中錳過氧化酵素活性產量變化。 87
圖十四、於含稻稈半固態培養液培養時添加不同glucose濃度對錳過氧化酵素活性產量變化。 88
圖十五、以不同白腐真菌培養於含稻草桿的培養液所產錳過氧化酵素活性產量變化。 89
圖十六、不同白腐真菌於最適酵素生產條件下錳過氧化酵素活性產量變化:a.除MB-F26以外的六株白腐真菌, b.除MB-F13和MB-F12以外的白腐真菌。 90
圖十七、不同稻稈含量下培養時錳過氧化酵素活性產量變化。 91
圖十八、在室溫(25℃)和4℃下保存時其錳過氧化酵素活性產量變化。 92
圖十九、不同pH值下偵測錳過氧化酵素活性產量。 93
圖二十、於不同sodium tartrate buffer濃度下偵測錳過氧化酵素活性產量。 94
圖二十一、不同MnSO4.H2O濃度下偵測錳過氧化酵素活性產量。 95
圖二十二、不同guaiacol濃度下偵測到的錳過氧化酵素活性產量。 96
圖二十三、不同過氧化氫濃度下偵測錳過氧化酵素活性產量。 97
圖二十四、MB-F26培養於不同農業廢棄物下其第13天時的phenolic compounds含量。control表示培養時不植入MB-F26共同培養。 98
圖二十五、培養時植入MB-F26與不植入MB-F26之農業廢棄物結構:(a),(c), core of tapa cloth tree和rice straw不植入MB-F26時的結構;(b),(d), core of tapa cloth tree和rice straw植入MB-F26後的結構。 99
圖二十六、培養時植入白腐真菌MB-F26與不植入MB-F26之農業廢棄物結構:(a),(c), bark of tapa cloth tree和corn cob不植入MB-F26時的結構;(b),(d), bark of tapa cloth tree和corn cob植入MB-F26後的結構。 100
圖二十七、以不同方法偵測錳過氧化酵素活性產量:1:分析試劑中含0.05 M的sodium phosphate buffer(pH 3)、30 mM的guaiacol和0.5 mM的H2O2;2:分析試劑中含0.05 M的sodium phosphate buffer(pH 3)和30 mM的guaiacol;3:分析試劑中含0.05 M的sodium phosphate buffer (pH 3)、30 mM的guaiacol和3 mM的MnSO4.H2O;4:分析試劑中含0.05 M的sodium phosphate buffer(pH 3)、30 mM的guaiacol、3 mM的MnSO4.H2O和0.5 mM的H2O2;5:laccase酵素活性產量。 101
圖二十八、培養於PDA及SSM中的MB-F26菌絲結構。(a),(b):於PDA上的菌絲結構,放大1000倍。(c):於SSM中的菌絲結構,放大1000倍。+ chlamydospore-like cells;- 釦子體結構。 102
圖二十九、添加不同濃度MnSO4.H2O時錳過氧化酵素活性產量變化。 103





附錄
附錄一、自然界中植物纖維素、半纖維素與木質素之分解循環之碳循環(Tuomela et al. 2000) 104
附錄二、 苯基丙烷(丁 1990) 105
附錄三、木質素之前趨物質(Crawford 1981) 105
附錄四、木質素之結構(Crawford 1981) 106
附錄五、木質素過氧化酵素之催化循環(Wariishi and Gold 1989;Barr and Aust 1994) 107
附錄六、Laccase的催化循環(Reinhammar 1997) 108
附錄七、錳過氧化酵素的催化循環(Hofrichter 2002) 109
附錄八、錳過氧化酵素與不同基質作用後形成的自由基(Hofrichter 2002) 110
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