利用篩選之菌株Bacillus subtilis DYU1生產聚麩胺酸之研究呂媚君、吳建一

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利用篩選之菌株Bacillus subtilis DYU1生產聚麩胺酸之研究呂媚君、吳建一

E-mail: 9607697@mail.dyu.edu.tw

摘要

Bacillus subtilis DYU1於含有70 g/L L-glutamic acid、30 g/L maltose和5 g/L peptone的培養基中有最大poly-glutamic acid (PGA)產量61 g/L。C/N比會顯著地影響PGA產量及產率。當固定maltose濃度(30 g/L)時，最適C/N比為0.6-7.5；固定peptone濃度(5 g/L)時，最適C/N比為6-10。另外，本研究亦探討其他影響因子，如pH、振盪速度、金屬離子、NaCl 和Biotin對PGA生產之影響。另外，nuclear magnetic resonance (NMR) spectrometry的結果證實所純化之產物為PGA。而胺基酸分析結果顯示PGA之純度高達99%。以gel permeation chromatography (GPC)的結果顯示，PGA之分子量超過1,000 kDa；

光學異構物分析之結果顯示PGA是以98% D-glutamic acid所組成，證明在B. subtilis DYU1代謝路徑中，可將L-glutamic acid 轉化為D-glutamic acid。另外，本研究亦以動力學模式來解析B. subtilis DYU1生長、PGA生長和maltose消耗。Monod 和Michaelis-Meten models的結果顯示，當glutamic acid和maltose濃度分別高於70和50 g/L時，有基質抑制PG生產之情形產生。此外，logistic model除了可合理且精確地模擬B. subtilis DYU1生長、PGA生產及maltose消耗之情形，並證實PGA生產行為屬於混合相關模式。另外，本研究探討不同pH值和溫度之PGA溶液流變學行為，亦發展一套與溫度和PGA濃度相關

，且對PGA溶液外觀黏度影響之數學模式。本研究亦以活性碳來去除糖蜜廢液之色度，並探討稀釋倍數、吸附劑濃度

、pH值和溫度對色度去除之影響。最後，比較處理前後之廢液作為基質來生產PGA之可行性，以達到減廢之效果。

關鍵詞 : Bacillus subtilis ; 聚麩胺酸 ; 動力學模式 ; 流變學特性 ; 糖蜜廢液目錄

封面內頁簽名頁授權書... iii 中文摘要... iv 英文摘要... vi 誌謝... viii 目錄... ix 圖目錄... xiv 表目

錄... xx 符號說明... xxii 1.前言... 1 2.文獻回

顧... 4 2.1麩胺酸鈉鹽製程概述... 4 2.1.1發酵廢液分析... 4 2.1.2發酵廢液處理概述... 5 2.2聚醯胺(polyamide)之簡介... 12 2.3聚麩胺酸(PGA)之特性、生合成與降解... 15 2.3.1聚麩胺酸之特性... 15 2.3.2聚麩胺酸之生合成... 16 2.3.3合成聚麩胺酸有關之基因... 19 2.3.4 生化代謝上與合成聚麩胺酸有關之酵素... 21 2.3.5聚麩胺酸之降解作用... 24 2.4聚麩胺酸之生產菌株及其特性... 26 2.4.1需額外補充L-glutamic acid之菌株... 31 2.4.2不需額外補充L-glutamic acid之菌株... 40 2.5聚麩胺酸生產之環境影響因子... 44 2.5.1碳源... 44 2.5.2氮源... 48 2.5.3氧

氣... 49 2.5.4金屬離子... 52 2.6聚麩胺酸之應用... 53 2.6.1食品工業上之應用... 55 2.6.2生物醫藥/醫學材料上之應用... 56 2.6.3環境上之應用... 60 2.6.4其他之應用... 62 3.材料與方法... 64 3.1實驗材料... 64 3.1.1藥

品... 64 3.1.2儀器設備... 66 3.2菌株培養... 67 3.2.1菌株來源... 67 3.2.2菌株活化... 68 3.2.3聚麩胺酸生產培養... 68 3.3分析方

法... 68 3.3.1Maltose分析... 68 3.3.2多醣分析－酚-硫酸法... 69 3.3.3NH4+-N濃度之測定... 70 3.3.4黏度... 71 3.4聚麩胺酸之分析... 71 3.4.1膠體滲透層析... 71 3.4.2高效能液相層析儀(HPLC)分析... 72 3.4.3胺基酸分析... 73 3.4.4核磁共振(NMR)分析... 74 3.4.5PGA中D/L-光學異構物之比率分析... 74 3.5聚麩胺酸之回收與純化...

74 3.6糖蜜廢液之批次吸附試驗... 77 3.7色度分析... 78 3.8COD分析... 78 3.9BET (Brunauer-Emmett-Teller)表面積測定... 79 4.動力學模式解析... 80 4.1 Monod and Michaelis-Menten models... 80 4.2 Logistic and Luedeking-Piret model... 82 5.結果與討論... 88 5.1探討B. subtilis DYU1生產PGA之最適培養基及環境因子... 88 5.1.1不同L-glutamic acid濃度對B. subtilis DYU1 生產PGA之影響... 88 5.1.2不同碳源及濃度對B. subtilis DYU1生產PGA之影響... 92 5.1.3不同氮源及濃度對B. subtilis DYU1生產PGA之影響... 97 5.1.4 C/N比對B. subtilis DYU1生產PGA之影響.... 101 5.1.5 pH對B. subtilis DYU1生產PGA之影響... 103 5.1.6振盪速度對B. subtilis DYU1生產PGA之影響.. 105 5.1.7金屬離子對B.

subtilis DYU1生產PGA之產量及 D/L glutamic acid組成之影響... 107 5.1.8 NaCl和biotin對B. subtilis DYU1生產PGA之影響... 111 5.2PGA之基本特性... 115 5.3動力學模式... 120 5.3.1 Monod and Michaelis-Menten models... 120 5.3.2 Logistic model... 133 5.4 PGA溶液之流變學特性... 146

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5.4.1 pH對黏度的影響... 148 5.4.2溫度和PGA濃度對PGA溶液黏度的影響... 149 5.4.3溫度和PGA濃度與PGA溶液外觀黏度之關係... 150 5.5以活性碳去除糖蜜廢液色度之研究... 166 5.5.1不同稀釋度對糖蜜廢液色度去除之影響... 166 5.5.2不同活性碳吸附劑量對糖蜜廢液色度去除之影響166 5.5.3不同pH對糖蜜廢液色度去除之影響...

167 5.5.4不同溫度對糖蜜廢液色度去除之影響... 168 5.5.5活性碳之BET分析... 169 5.6利用糖蜜廢液生產PGA之可行性... 177 6.結論... 180 參考文獻... 182

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利用篩選之菌株Bacillus subtilis DYU1生產聚麩胺酸之研究 呂媚君、吳建一