臺灣博碩士論文加值系統

由戶外所得之土壤尋獲一可產生抗真菌性抗生素之革蘭氏陰性菌，編號為AF-714的菌種。AF-714在以3％蔗糖(sucrose)與0.5％黃豆粉(soy bean)之液態培養條件下培養20小時，於菌體外液中測得抗真菌物質活性。
粗培養液經由Acetone precipitation除去大部份蛋白質，續經Fractogel DMAE-650M column離子交換樹脂、Charcoal activated filtration活性碳抽濾、Biogel P-2 column膠體過濾管柱、Charcoal activated column活性碳管柱、Sephadex G-15膠體過濾管柱及Silica gel 60 PLC矽膠展開等純化處理後，獲得一白色粉末狀之抗真菌性物質，回收率為17%。
所獲得之抗真菌性抗生素在經由不同有機溶劑的溶解測試後，推斷為一親水性物質。該物質在水溶液中以0℃到100℃水浴或不同有機溶劑處理，活性皆不受影響。而且、此抗真菌性抗生素可抑制Candida albicans的生長，亦能有效控制植物病原菌如Rhizoctonia solani與Athelia rolfsil的生長。

A antifungal antibiotic producing bacterium strain AF-714, isolated from out door soil was identified as Gram negative. AF-714 strain produced the antifungal antibiotic extracellularly when incubated in liquid medium with 3% sucrose and 0.5% soybean for 20 hours. The antifungal antibiotic was purified from the crude extract of producing broth by acetone precipitation, Fractogel DMAE-650M column, Charcoal activated filtration, Biogel P-2 column, Charcoal activated column, Sephadex G-15 column and Silica gel 60 PLC. The purified antifungal antibiotic was obtained, with a recovery of 17%, and shows as a white color powder. The antifungal antibiotic showed extreme hydrophilicity and low molecular weight ( about 1000 ). The antifungal activity is no loss in the temperature range of 0℃to 100℃for 30 minutes. The purified antifungal antibiotic was activated even after incubation at 30℃ in organic solvent for 2 hours. It is active against Candida albicans and filamentous fungi such as Athelia rolfsil and Rhizoctonia solani.

ACKNOWLEDGEMENTS..........................................................................iii
ABSTRACT ( ENGLISH ) ..........................................................................iv
ABSTRACT ( CHINESE ) ...........................................................................v
TABLE OF CONTENTS..............................................................................vi
LIST OF TABLE.........................................................................................viii
LIST OF FIGURES.......................................................................................ix
CHAPTER 1. INTRODUCTION
1. 1 The peril of fungi..................................................................................... 1
1. 2 Antifungal antibiotics............................................................................... 3
1. 3 Purpose...................................................................................................13
CHAPTER 2. MATERIALS AND METHODS
2. 1 Screening and isolation of bacteria..........................................................15
2. 2 Assay methods.......................................................................................15
2. 2. 1 Cell number........................................................................................15
2. 2. 2 Antifungal assay used to monitor isolation.........................................15
2. 2. 3 TLC assay system...............................................................................16
2. 3 Investigation of media.............................................................................16
2. 3. 1 Effect of carbon sources on antifungal product production.................16
2. 3. 2 Effect of nitrogen sources on antifungal product production...............16
2. 3. 3 Effect of inorganic salts on antifungal product production..................17
2. 3. 4 Time course of antifungal production from AF-714...........................17
2. 4 Production and purification of antifungal product....................................17
2. 4. 1 Production of antifungal product.........................................................17
2. 4. 2 Purification of antifungal product........................................................18
2. 5 The recover rate computation of isolation................................................19
2. 6 The purity characterization of antifungal product.....................................20
2. 7 Properties of the antifungal product.........................................................20
2. 7. 1 Thermal stability of antifungal activity.................................................20
2. 7. 2 Solvent stability of antifungal activity..................................................20
2. 7. 3 Solvent solubility of antifungal product...............................................20
2. 7. 4 Minimum inhibit concentration (MIC) ...............................................21
2. 7. 5 Activity of antifungal spectrum...........................................................21
2. 7. 6 Antifungal mechanism of antifungal product......................................21
2. 8 Materials................................................ .................................................21
2. 8. 1 Chemicals...................................... .....................................................21
2. 8. 2 Gels.................................................... ................................................22
2. 8. 3 Strains.................................................... ............................................23
2. 8. 4 Medium.................................................... .........................................23
2. 9 Equipments.................................................... ........................................23
CHAPTER 3. RESULTS AND DISCUSSION
3. 1 Screening and characterization of the bacterium strain AF-714..............25
3. 2 Antifungal activity assay.........................................................................25
3. 3 Investigation of media............................................................................25
3. 3. 1 Effect of carbon sources on antifungal product production................30
3. 3. 2 Effect of nitrogen sources on antifungal product production..............30
3. 3. 3 Effect of inorganic salts on antifungal product production.................30
3. 3. 4 Optimal composition and concentration. ...........................................36
3. 3. 5 The growth curve...............................................................................36
3. 4 Production and purification of antifungal product..................................36
3. 4. 1 Production of antifungal product........................................................36
3. 4. 2 Purification of antifungal product.......................................................38
3. 5 The purity of antifungal product.............................................................40
3. 6 Properties of the antifungal product........................................................43
3. 6. 1 Structure of antifungal product...........................................................43
3. 6. 2 Thermal stability of antifungal activity...............................................43
3. 6. 3 Solvent stability of antifungal activity................................................43
3. 6. 4 Solvent solubility of antifungal product.............................................43
3. 6. 5 Minimum inhibit concentration (MIC) .............................................48
3. 6. 6 Antifungal mechanism of antifungal product.....................................48
3. 6. 7 Activity of antifungal spectrum..........................................................48
CHAPTER 4. CONCLUSIONS..................................................................55
REFERENCES.............................................................................................57

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