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研究生:江易原
研究生(外文):I-Yuan Chiang
論文名稱:利用枯草桿菌突變株BacillussubtilisNTU710生產聚麩胺酸之研究
論文名稱(外文):Study on Production of Poly-γ-glutamic acid by a mutnat, Bacillus subtilis NTU 710
指導教授:潘子明
指導教授(外文):Tzu-Ming Pan
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:81
中文關鍵詞:聚麩胺酸枯草桿菌
外文關鍵詞:Poly-γ-glutamic acidBacillu subtilis
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本研究利用紫外線照射及EMS處理本研究室保存之聚麩胺酸生產菌株,以紫外線照射及EMS處理等方法進行誘導變異,得到高產量之菌株Bacillus subtilis NTU 710。
利用Bacillus subtilis NTU 710菌株以500 mL Hinton氏三角瓶探討生產聚麩胺酸之最適培養基組成份為40 g/L sodium L-glutamate, 40 g/L glycerol, 30 g/L citric acid,7 g/L (NH4)2SO4,0.5 g/L K2HPO4,0.5 g/L MgSO4‧H2O, 0.05 g/L FeCl3‧6H2O,0.2 g/L CaCl2‧2H2O,0.104 g/L MnSO4‧H2O及50μ/L biotin,培養基之起始pH值為7.0。以100 mL之裝液量,在30℃,200 rpm下振盪培養48小時,可得26 g/L 之聚麩胺酸。
利用5公升醱酵槽以批次方式進行聚麩胺酸醱酵生產試驗,其最適操作條件為:接種量5%,培養溫度30℃,通氣量1 vvm,攪拌速率300 rpm,裝液量3公升,培養56小時後,聚麩胺酸的產量可達16 g/L。在培養16小時後,調高攪拌速率及通氣量 (500 rpm及2vvm),在培養64小時之後可得聚麩胺酸18.8 g/L。
Poly-γ-glutamic acid (PGA) which is water-soluble and biodegradable, can be used as thickeners, humectants, biodegradable plastics and drug carrier. In this study, mutants were induced by UV irradiation and EMS treatment. Highly sticky colonies were selected after mutation treatment. The most productive strain is Bacillus subtilis NTU 710, and it produces about 26 g/L of PGA while the medium contains 30 g/L of citric acid, 7 g/L of ammonium sulfate and 40 g/L of sodium glutamate, 0.5 g/L of K2HPO4, 0.5 g/L of MgSO4‧H2O, 0.05 g/L of FeCl3‧6H2O, 0.2 g/L of CaCl2‧2H2O, 0.104 g/L of MnSO4‧H2O, 50μg/L biotin and shaking cultured at 30℃ for 48 hours (in 500 mL Hinton’s flask). The optimal cultural conditions in 5 liter jar fermentor were as following: inoculum size, 5 %; temperature, 30℃; aeration rate, 1 vvm; agitation speed 300 rpm; with 3 liter medium. After 56 hours’ cultivation with optimal condition, Bacillus subtilis NTU 710 produced 16 g/L polyglutamic acid in culture broth. After cultivating for 16 hours, the agitation speed is increasing from 300 rpm to 500 rpm and the aeration is from 1 vvm to 2 vvm, the production of polyglutamic acid could reach to 18.8 g/L. The cultivation and PGA purification methods had been set up, and it might be scale up in the future.
目 錄 -------------------------------------------------------------------- I
中文摘要 -------------------------------------------------------------------- IV
英文摘要 -------------------------------------------------------------------- V
圖 次 -------------------------------------------------------------------- VI
表 次 -------------------------------------------------------------------- VII
縮 寫 表 -------------------------------------------------------------------- VIII
第一章 前言-------------------------------------------------------------- 1
第一節 聚麩胺酸之發現及其應用----------------------------------- 1
一、聚麩胺酸之發現
二、聚麩胺酸之特性及應用
第二節 生產聚麩胺酸之研究─文獻回顧-------------------------- 5
一、Bacillus licheniformis ATCC 9945
二、Bacillus licheniformis A35及Bacillus subtilis TAM-4
三、Bacillus subtilis F-2-01
四、Bacillus subtilis IFO3335
五、Bacillus subtilis IFO3336
六、Bacillus subtilis IFO16449
七、Bacillus subtilis (natto) Asahikawa
第三節 研究動機及目的----------------------------------------------- 15
第二章 材料與方法---------------------------------------------------- 16
第一節 使用菌株-------------------------------------------------------- 16
第二節 培養方法-------------------------------------------------------- 16
一、培養基
二、種菌製備
第三節 突變方法-------------------------------------------------------- 17
一、紫外線照射
二、EMS處理法
三、NTG處理法
四、篩菌用平板培養基
第四節 三角瓶之培養-------------------------------------------------- 20
一、培養基組成及最適培養條件之探討
第五節 五公升醱酵槽之培養----------------------------------------- 21
一、醱酵槽之操作程序
二、醱酵槽之生產培養
三、醱酵槽之培養條件探討
第六節 分析方法------------------------------------------------------- 24
一、菌體生長量之測定 (OD660、生菌數、乾重)
二、聚麩胺酸之純化與定量
三、胺基酸組成分析 (TLC、amino acid analyzer)
四、醣類分析 (phenol-sulfuric acid method)
五、聚麩胺酸分子量測定 (SDS-PAGE)
六、蕃石榴果汁製備及黏度之測定
七、掃瞄式電子顯微鏡標本之製作
第三章 結果與討論----------------------------------------------------- 36
第一節 聚麩胺酸生產菌Bacillus subtilis之菌種改良----------- 36
一、紫外線照射法
二、EMS處理法
三、優良突變株之篩選
第二節 聚麩胺酸純化法之建立-------------------------------------- 44
一、純化法之建立
二、產物分析
第三節 聚麩胺酸生產條件之探討----------------------------------- 49
一、起始pH值對聚麩胺酸生產之影響
二、溫度對聚麩胺酸生產之影響
三、Biotin濃度對聚麩胺酸生產之影響
四、金屬陽離子種類對聚麩胺酸生產之影響
五、碳源對聚麩胺酸生產之影響
六、甘油及檸檬酸對聚麩胺酸生產之影響
七、氮源對聚麩胺酸生產之影響
八、有機氮源對聚麩胺酸生產之影響
九、麩胺酸對聚麩胺酸生產之影響
十、聚麩胺酸生產菌株之比較
第四節 五公升醱酵槽之聚麩胺酸生產條件探討----------------- 65
一、醱酵槽之操作
二、培養基之組成
三、攪拌速率對聚麩胺酸生產之影響
四、通氣量對聚麩胺酸生產之影響
五、培養中提高溶氧對聚麩胺酸生產之影響
六、討論
第五節 聚麩胺酸對果汁之增稠效果-------------------------------- 71
第四章 結論-------------------------------------------------------------- 74
參考文獻 ------------------------------------------------------------------ 75
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