臺灣博碩士論文加值系統

摘 要

Iturin A是由七個胺基酸所組成的環狀胜�L結構物，為液化澱粉芽孢桿菌(Bacillus amyloliquefaciens)所分泌的二次代謝產物之一。 Iturin A為一強效抗菌活性之抗生物質，並擁有生物界面活性劑性質，但其應用上長期受限於產量偏低的瓶頸，因此提高iturin A產量的研究成為重要的課題。為提高iturin A的產量，本研究利用培養基中添加金屬鹽類及調控發酵液pH值之方法，探討對iturin A產能影響。其次，利用不同分離純化方法，以其提高iturin A之回收效果，再利用高效液相層析儀(HPLC)進行定性定量分
析，同時評估毛細管電泳(CE)應用於iturin A定性分析之可行性。
液態發酵生產iturin A的研究方面，實驗結果顯示，以台糖紅糖(brown sugar)最碳源培養6天，可得到iturin A之最佳產量為70.48 mg/L。此外，經由搖瓶的初步測試也發現，若發酵液之pH低於6.0，則會造成酸化而使iturin A產能降低；過鹼也會造成菌絲生長不良，而使iturin A產能降低，而當調pH值控制於pH6.64時有較佳之產能。其次，添加金屬離子實驗方面，結果發現，硫酸亞鐵、硫酸鎂二種金屬鹽類在生合成iturin A的能力有明顯促進作用，其中以添加濃度0.20 mM硫酸亞鐵時iturin A之產能最佳。以最適條件台糖紅糖為碳源，添加0.2mM FeSO4 調控pH值6.64
可得最佳產量為121.36 mg/L。
分離純化方面，發酵液先以甲醇萃取回收，再以Silica gel管柱做初步的分離，再經半製備型的HPLC管柱收集純化之iturin A，濃縮經冷凍乾燥得黃棕色iturin A粉末，經此步驟的iturin A可作為定性定量分析的標準品，此方法可應用於高純度抗生物質樣品的製備。
在建立高效液相層析儀(HPLC)與毛細管電泳(CE)進行抗生物質分析的策略中，藉由比較發酵液、超過濾濾液、甲醇萃取液的之HPLC與CE圖譜的建立，即可辨識出抗生物質之波峰。實驗結果，HPLC的最佳分離條件為：C18的層析管柱，以10.0 mM 醋酸氨緩衝液與acetonitrile (60 : 40, v/v)組成之移動相沖提，可得到良好的分離效果；CE的最佳分離條件為： 以50 mM sodium dodecyl sulfate, 20 mM Boric acid, pH 8.72 / ACN (90 : 10, v/v)組成之電泳液、融矽毛細管為50 µm I.D. × 57 cm、電壓 20 kV、檢測波長214 nm，於8分鐘可得到分析波峰。本研究發現，HPLC及CE的分析技術可直接應用於抗生物質iturin A之分析，並可互相輔助鑑定此抗生物質。

Abstract

Iturin A consists of a peptide ring of seven amino acid residues with the chiral sequence LDDLLDL closed by a b-amino acid bearing a hydrophobic tail, which includes 11–12 carbon atoms. It is included in a family of lipopeptides which can be extracted from the culture media of a number of strains of Bacillus amyloliquefaciens. The iturin A exhibits a strong antifungal, and antibacterial activity as well as biosurfactant property. The applications of this compound have been limited due to its poor production yield. Accordingly, the addition of metal salts in the fermentation medium along with the controlling the cultivation pH to improve the iturin A productivity were investigated. Furthermore, the improvement of isolation, and purification methods were also engaged. Analysis by high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE) were undertaken to evaluate the possibility of qualitative and quantitative this compound.
The best yield for the iturin A was found at level of 70.48 mg/L after 6 days cultivation with brown sugar as a carbon source. While the pH level dropped below 6.0 resulted in the acidification of broth caused the decreased in the iturin A production. Optimal pH for the production of iturin A was centered at neighborhood of 6.64. Moreover, the supplement of ferrous sulphate (0.2 mM) and magnesium sulphate in the culture medium improved the production of iturin A.
A separation and purification of iturin A from Bacillus amyloliquefaciens fermentation broth was proposed. The fermentation broth was extracted with methanol and then purified by silica gel chromatography, and semi-preparative HPLC chromatography. Quality of the semi-preparative products was unique. Consequently, the method can also be adapted for the preparation of antibiotic sample.
A general approach for the analysis of iturin A using HPLC and CE methods was also proposed. By comparing the chromatograms of fermentation broth, ultrafiltraction filtrate, and the methanol-antibiotic extract, the peaks corresponding to antibiotic can be identified. The optimal condition for HPLC was: 10 mM ammonia acetate /acetonitrile (60:40, v/v) as a mobile phase. On the other hand, the optimal conditions for CE assay were: 50 mM SDS containing 20 mM boric acid (pH 8.72)/acetonitrile (90:10, v/v) as a running buffer with the capillary size of 50 mm ID ´ 57 cm under the voltage of 20 kV. The sample can be detected at 214 nm within 8 mins.

目錄
封面內頁
授權書
誌謝
中文摘要………………………………………………………………………Ⅰ
英文摘要...........................................................................................................Ⅲ
目錄....................................................................................................................Ⅴ
圖目錄…………………………………………………………………………Ⅷ
表目錄………………………………………………………………………ⅩⅠ



第一章 緒論……………………………………………………………………1
1-1研究動機及目的……………………………………………………………1
1-2液化澱粉芽孢桿菌(Bacillus amyloliquefaciens)………………………......2
1-2-1芽孢桿菌屬(Bacillus spp.)簡介……….…………………………….2
1-2-2液化澱粉芽孢桿菌的演進…………….............................................3
1-2-3液化澱粉芽孢桿菌特性…………………………………………….4
1-2-4液化澱粉芽孢桿菌的研究和發展………………………………….4
1-2-5 B. subtilis和B. amyloliquefaciens之胜�L抗生物質..........................5
1-2-6 液化澱粉芽孢桿菌發酵環境及系統……………………………...6
1-3 Iturin A的結構性質與應用………………………………………………7
1-3-1 Iturin A 的介紹…………………………………………………...7
1-3-2 Iturin A的結構性質………………………………………………8
1-3-3. Iturin A的應用……………………………………………..…….9
1-4 毛細管電泳的應用………………………………...……………………10

第二章 材料及方法……………….………………………………...……......22
2-1 實驗材料及藥品………………………………………………………….22
2-1-1 實驗用菌種……………………………………………………….22
2-1-2 實驗用培養基種類及組成…………………………………….....22
2-1-3 實驗用藥品…………………………………………………….....22
2-2實驗用儀器設備………………………………………...………………...24
2-3實驗方法……………………………...…………………………………...24
2-3-1 菌種保存…………………………………………...……………24
2-3-2 前培養………………………………………………...…………..25
2-3-3 主培養…………………………………………………...………..25
2-3-4 Iturin A之合成發酵……………………………………...………..26
2-3-5 Iturin A之分離及純化…………………………………...………..26
2-3-6 Iturin A之定性定量分析…………………………………...……..26

第三章 Iturin A 培養基配方之探討
3-1 前言……………………………………………………………………….30
3-2 合成培養基與天然複合培養基之選擇……………………………….....31
3-3 酸鹼值對Iturin A產能之影響…………...………………………………32
3-4 金屬鹽類添加效應對Iturin A產能之影響...............................................33
3-4-1硫酸亞鐵添加效應…………………………………….…………..34
3-4-2硫酸鎂添加效應………………………………………….………..35
3-4-3硫酸亞鐵/硫酸鎂混合效應…………………………….………….35
3-4-4結果與討論……………….…………………………….………….37
3-5 最適化培養條件………………………………………………………….37

第四章 Iturin A的分離純化與分析之探討
4-1 前言……………………………………………………………………….63
4-2 分離純化……………………………………………………...…………..63
4-3 分析方法………………………………………………………...………..65
4-3-1 高效液相層析儀（HPLC）定性定量分析……………...…………65
4-3-2 毛細管電泳（CE） 定性分析……..………………………………65
4-3-3 質譜儀（Mass spectrometer FAB-MS）定性分析….……………67
4-4結果與討論……………………………………………………....………..67

第五章 結論與展望……. ……………………………………...…………….93
5-1 結論……………………………………………………………………….93
5-2 展望……………………………………………………………………….95

參考文獻……………………………………………………………………....97
圖目錄

圖1-1. Bacillus屬之樹狀分類圖…… ……….…………………………….15
圖1-2.液化澱粉芽孢桿菌的菌體與孢子…….…………….…........………16
圖1-3. Iturin A 之結構式…………………………………………………..18
圖1-4. Bacillus spp. 對病原菌和植物之間的關係...………………...….…19
圖3-1.不同培養基對菌體生長之影響...........................................................39
圖3-2.不同培養基對Iturin A產量之影響………………………………….40
圖3-3. Iturin A生合成中，不同起始pH值時間變化之改變 ……………41
圖3-4.不同起始酸鹼值對Iturin A產量之影響…………………………………..42
圖3-5.不同金屬鹽類對菌體生長之影響……………………………………43
圖3-6.不同金屬鹽類對Iturin A產量之影響………………………………..44
圖3-7.三種碳源添加0.2mM FeSO4對菌體生長之影響……………………45
圖3-8.三種碳源添加2mM FeSO4對菌體生長之影響……………………..46
圖3-9.三種碳源添加10 FeSO4對菌體生長之影響…………………………47
圖3-10.以Glucos為碳源，添加不同濃度FeSO4對菌體生長之影響………48
圖3-11.以Glucos為碳源，添加不同濃度FeSO4對Iturin A產量之影響…..49
圖3-12.以Fructose為碳源，添加不同濃度FeSO4對菌體生長之影響……50
圖3-13.以Fructose為碳源，添加不同濃度FeSO4對Iturin A產量之影響.51
圖3-14.台糖紅糖為碳源，添加不同濃度FeSO4對菌體生長之影響………52
圖3-15.台糖紅糖為碳源，添加不同濃度FeSO4對Iturin A產量之影響…53
圖3-16.以Glucose為碳源，添加不同濃度MgSO4對菌體生長之影響…54
圖3-17.以Glucose為碳源，添加不同濃度MgSO4對Iturin A產量之影響...55
圖3-18.以Fructose為碳源，添加不同濃度MgSO4對菌體生長之影響….56
圖3-19.以Fructose為碳源，添加不同濃度MgSO4對Iturin A產量之影響..57
圖3-20.以台糖紅糖為碳源，添加不同濃度MgSO4對菌體生長之影響….58
圖3-21.以台糖紅糖為碳源，添加不同濃度MgSO4對Iturin A產量之影響.59
圖3-22.以台糖紅糖為碳源，不同濃度FeSO4與不同濃度MgSO4對菌體生
長之影響………………………………………………………….60
圖3-23.以台糖紅糖為碳源，不同濃度FeSO4與不同濃度MgSO4對Iturin A產量之影響……………………………………………………….61
圖3-24.以最適培養條件：台糖紅糖為碳源，比較起始pH值6.64、添加
0.2mM FeSO4及添加0.2mM FeSO4 調控pH值6.64對Iturin A產
量之影響………………………………………………………….62
圖4-1. Iturin A 製備純化流程圖……………………………………………70
圖4-2. Iturin A 之標準曲線…………………………………………………71
圖4-3. Iturin A標準品之 HPLC層析圖……………………………………72
圖4-4.液化澱粉芽孢桿菌發酵液之HPLC層析圖………………………..73
圖4-5.溫度對遷移時間之影響…………………………………………….74
圖4-6.電壓對遷移時間之影響…………………………………………….75
圖4-7.酸鹼pH值對遷移時間之影響………………………………………76
圖4-8. cetonitrle體積濃度對遷移時間之影響…………………………..77
圖4-9. SDS濃度對遷移時間之影響………………………………………78
圖4-10.Buffer濃度對遷移時間之影響……………………………………79
圖4-11.Iturin A標準品之CE圖譜…………………………………………..80
圖4-12.液化澱粉芽孢桿菌發酵液之CE圖譜…………………………...81
圖4-13.經離心酸化後上清液之HPLC圖譜………………………………82
圖4-14.發酵液、經管柱層析及純化後Iturin A之HPLC 圖譜…………..83
圖4-15.經半製備管柱收集之Iturin A的HPLC 圖譜……………………..84
圖4-16.經純化製倍之Iturin A的HPLC 圖譜……………………………85
圖4-17.經純化分離之Iturin A2、Iturin A3的HPLC 圖譜………………….86
圖4-18.經純化分離之Iturin A4、Iturin A5的HPLC 圖譜…………………87
圖4-19.經純化分離之Iturin A6、Iturin A7的HPLC 圖譜…………………88
圖4-2.0經純化分離之Iturin A8的HPLC 圖譜………………………..........89
圖4-21.經半製備管柱純化的Iturin A 之Mass 圖譜……………………89
圖4-22.經矽膠管柱純化的Iturin A 之Mass 圖譜………………………..90
圖4-23. Iturin A標準品之 Mass 圖譜……………………………………91



表目錄

表1-1.枯草桿菌(Bacillus subtilis) 商品化之生物製劑…………………….13
表1-2.枯草桿菌(Bacillus subtilis)產生之胜�L抗生物質……………………17
表1-3 . turins 家族之結構表………………………………………………..20
表1-4 . turin A 的7種不同的衍生物……………………………………….21

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