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研究生:林志成
研究生(外文):Chih-Cheng Lin
論文名稱:探討通氣量對於樟芝發酵生產與純化脂解酵素之研究
論文名稱(外文):The study of production and purification of lipase fermented from Antrodia camphorata by aeration rate
指導教授:徐敬衡徐敬衡引用關係
指導教授(外文):Chin-Hang Shu
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程研究所
學門:工程學門
學類:化學工程學類
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:77
中文關鍵詞:樟芝發酵純化脂解酵素
外文關鍵詞:lipasepurificationAntrodia camphoratafermentation
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本研究以樟芝Antrodia camphorata BCRC 35396於攪拌式發酵槽培養下,探討不同通氣量對於樟芝發酵生產脂解酵素的影響,並將最適條件之發酵液進行酵素的分離純化。由實驗結果顯示,0.3 vvm的最大脂解酵素活性為1.151 U/ml,而1.0 vvm的最大酵素活性為2.588 U/ml,大約提升了2.25倍,所以通氣量的增加有利於脂解酵素活性的提升。而YL/P(specific activity)顯示,0.8 vvm有較高的比活性(29.493 U/mg)。在蛋白質的生成方面,0.3 vvm的最大蛋白質含量為0.061 mg/ml,而1.0 vvm的最大蛋白質含量為0.120 mg/ml,大約提升1.97倍,所以提升通氣量對於蛋白質含量有提高的效果。
脂解酵素的純化步驟是將發酵澄清液利用冷凍乾燥再經由DEAE Sepharose陰離子交換管柱層析分離純化。經過冷凍乾燥處理的脂解酵素活性及比活性分別為341.6 U及29.19 U/mg。而通過陰離子交換管柱層析後,純化倍率為4.50倍,回收率為8.28%。最後由SDS-PAGE電泳分析,發現最後純化的脂解酵素分子量是60.0 kDa。
This work studied the production and purification of lipase fermented from Antrodia camphorata BCRC 35396 by aeration rate in the stired tank. Shown by the experimental result, the more aeration rate can enhance the lipase activity. The biggest lipase activity of 1.0 vvm was 2.25 times higher than that of 0.3 vvm. The specific activity YL/P show the 0.8 vvm has the higher quality than other aeration rate. The biggest protein of 1.0 vvm was 1.97 times higher than one of 0.3 vvm.
Lipase fermented from Antrodia camphorata BCRC 35396 was purified by lyophilization as concentration, follow by anion exchange chromatography(DEAE-Sepharose). The specific activity of the purified enzyme was 4.5 times higher than that of the crude broth after lyophilization. The lipase had a molecular weight of 60.0 kDa when examined by SDS-polyacrylamide gel electrophoresis.
摘要……………………………………………………………………...I
目錄……………………………………………………………………...Ⅱ
表索引…………………………………………………………………...Ⅵ
圖索引…………………………………………………………………...Ⅶ

第一章 緒論………………………………………………………….....1
1-1研究動機…………………………………………………….. .....1
1-2研究目的…………………………………………………….. .....2
第二章 文獻回顧…………………………………………………….....3
2.1菇類…………….…………………………………………….. .....3
2.2 樟芝…………………………………………………………........3
2.2.1 分類…………….………………………………………. .....3
2.2.2 型態…………………………………..……………......4
2.2.3 成分……………..…………………………………......5
2.2.4 功用…………………………………………………......5
2.3 酵素………………………………………………………......6
2.3.1 脂解酵素………………………………………………....9
2.3.1.1 脂解酵素簡介……………………………………….9
2.3.1.2 脂解酵素應用…..……………………………….....14
2.4 酵素之分離與純化………………………………………….18
2.4.1 濃縮方法………………………………………………..18
2.4.1.1 離心………………………………………………...18
2.4.1.2超微薄膜過濾法…….……………………………...19
2.4.1.3 其他濃縮方法……………………………………...20
2.4.1.4 層析法……………………………………………...20
2.4.1.4.1 離子交換管柱層析…………………………....21
2.4.1.4.2疏水性作用管柱層析………………………….22
2.4.1.4.3親和性作用管柱層析………………………….22
2.4.1.4.4膠體過濾管柱層析…………………………….23
2.4.2 快速蛋白質液相層析儀(FPLC)……………………….23
第三章 實驗材料與方法……………………………………………..25
3.1 實驗材料…………………………………………………….25
3.1.1 實驗菌株………………………………………………..25
3.1.2 實驗藥品………………………………………………..25
3.1.3 實驗儀器及其他設備…………………………………..26
3.1.4 實驗裝置......................................27
3.2 實驗方法…………………………………………………...27
3.2.1 菌株保存………………………………………………27
3.2.2 培養基組成.…………………………………………….28
3.2.3 操作方法………………………………………………29
3.2.4分析方法…………………………………………………30
1. 發酵液處理流程……………………………………………30
2. 菌重分析……………………………………………………32
3. 葡萄糖殘量分析…………………………………………....32
4. 冷凍乾燥……………………………………………………33
5. 管柱層析…………………………………………………....33
陰離子交換管柱層析……………………………………...33
6. 酵素活性分析……………………………………………….35
7. 蛋白質含量測定...................................36
8. SDS膠體電泳(SDS-PAGE)……………………………...37
第四章 結果與討論………………………………………………….41
4.1樟芝菌絲型態…………………………………………………41
4.1.1固態培養………………………………………………….41
4.1.2 搖瓶培養…………………………………………………42
4.1.3 攪拌式發酵槽……………………………………………43
4.2 不同通氣量攪拌槽批次實驗………………………………...44
4.2.1發酵時間曲線比較……………………………………….44
4.2.2不同通氣量對於菌體生長的影響…………… ………....47
4.2.3不同通氣量對於生產脂解酵素的影響………………….49
4.2.4 不同通氣量對於生產蛋白質的影響……………………51
4.3 脂解酵素的純化……………………………………………...53
4.3.1 發酵澄清液………………………………………………53
4.3.2 陰離子交換管柱層析……………………………………54
4.3.3 SDS膠體電泳(SDS-PAGE)…………………………55
第五章 結論與未來展望…………………………………………58
5-1結論…………………………………………………………...58
5-2未來展望………………………………………………. .59
參考文獻………………………………………………………………..60
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