抗藥性的病原菌日形嚴重，威脅大眾健康，故公私機構的研究人員正努力開發新藥，以解決此一問題。由於海洋環境範圍廣及具有多樣性的生物種類，從海洋生物中篩出治療疾病的物質為新的且深具潛力的研發途徑，過去數年在醫藥應用的研究已逐漸增多。本研究旨在分離出海洋黴菌及放線菌，以其生產新抗生素，並瞭解微生物及所產之抗生物質對其周遭環境生物的影響和所扮演的角色。已篩得海洋黴菌Acremonium altematum T1-1及Aspergillus ochraceus N7和放線菌Nocardiopsis dassonvillei F5可以產生有效的抗生物質，經由層析法分離純化、NMR光譜、及結晶決定結構後得知A. altematum T1-1可生產5,6-dihydropenicillic acid (DHPA)，A. ochraceus N7可生產penicillic acid (PA)，N. dassonvillei F5生產iodinin。PA和DHPA具五元環lactone，iodinin是phenazine衍生物。A. altematum T1-1在30℃、6%鹽度下生長最佳，在25℃、鹽度0% 所產DHPA量最多。A. ochraceus N7在25℃、6%生長最好，在25℃、3% 時可生產最多PA。N. dassonvillei F5在30℃、6%生長最好，在25℃、1-3% 時可生產最多iodinin。在抑菌能力上只有PA較具活性，其對B. cereus MIC為75 mg/ml。PA對於P-388、HT-29、A549三種癌細胞株有毒殺活性。 Iodinin則對P-388、A549有毒殺活性。同時發現PA與DHPA在二十分鐘時抑制輪蟲游泳能力的ED50分別為1.03及2.5 mg/ml，顯示在常溫海水環境下，若A. altematum T1-1和A. ochraceus N7的滋長，則會產生毒殺輪蟲的物質。而N. dassonvillei F5及iodinin餵食白蝦則會造成白蝦死亡，並使白蝦更容易遭白斑病毒的侵害。

The antibiotic-resistant bacteria seriously threaten public heath. Scientists of both public and private sectors are devoted to develop new drugs to overcome this problem. Marine species are highly diversity and have been shown to be a potential sources to find efficient compounds. The medicine application of the natural products from ocean increases in last decade. The purpose of the project was to isolate antibiotic-producers from sea-bottom mud. Three filament microorganisms from marine environment, Acremonium altematum T1-1, Aspergillus ochraceus N7 and Nocardiopsis dassonvillei F5, can produce bioactivity compounds. All compounds purified using silica chromatographic separation from culture extraction and determination of their structure by NMR spectrum and X-ray diffraction pattern for crystals. A. altematum T1-1 produced 5,6-dihydropenicillic acid (DHPA), A. ochraceus N7 produced penicillic acid (PA), and N. dassonvillei F5 produced iodinin. The structures of DHPA and PA were identified as derivatives of five-member lactone, and iodinin is a derivative compound of phenazine. The optimal growth of DHPA -producer A. altematum T1-1 was in 6% salinity at 30℃ and best DHPA production was in 0% salinity at 25℃. The optimal growth of PA-producer A. ochraceus N7 was in 6% salinity at 25℃, and best PA production conditions was below 3% salinity at 25 ℃. The optimal growth of iodinin-producer N. dassonvillei F5 was in 6% salinity at 30℃ and best iodinin production condition was 1-3% salinity at 25℃. Only PA has highly inhibited of B. cereus at the MIC of 75 mg/ml. The PA had cytotoxicity against cancer cell lines of P-388, HT-29 and A549 while iodinin inhibited P-388 and A549. The ED50 on the swimming ability of rotifer was 1.03 and 2.5 mg/ml for PA and DHPA, respectively. It revealed that the growth of these two mold can occur in regular marine environment and produce toxin to kill rotifer. The growth of N. dassonvilleiand F5 and iodinin caused the death and virus-invasion of shrimp.

目 錄
謝辭 ------------------------------------------------------------------------------------------ I
中文摘要 ------------------------------------------------------------------------------------ II
Abstract -------------------------------------------------------------------------------------- III
目錄 ------------------------------------------------------------------------------------------ IV
表目錄 --------------------------------------------------------------------------------------- VI
圖目錄 --------------------------------------------------------------------------------------- VII
附件目錄 ------------------------------------------------------------------------------------ IX
壹、前言 ------------------------------------------------------------------------------------ 1
一、抗生素發展簡史 -------------------------------------------------------------- 1
二、目前常見陸地微生物所產生的醫用抗生素 ----------------------------- 2
三、抗生素的作用機制 ----------------------------------------------------------- 4
四、細菌的抗藥性產生 ----------------------------------------------------------- 6
五、克服抗藥性的研發 ------------------------------------------------------------9
六、抗生素於抗癌的應用 ---------------------------------------------------------11
七、抗癌天然物的發展 ------------------------------------------------------------11
八、海洋天然物開發的新趨勢 ---------------------------------------------------12
九、有關海洋黴菌的天然物相關研究 ------------------------------------------13
十、研究目的 ------------------------------------------------------------------------15
貳、材料與方法 --------------------------------------------------------------------------16
一、海洋絲狀菌的篩選及培養 ---------------------------------------------------16
二、海洋絲狀菌所產抗生素抑菌測試 ------------------------------------------17
三、海洋絲狀菌之培養 ------------------------------------------------------------18
四、抗生素定量 ---------------------------------------------------------------------18
五、抗生素純化與結構鑑定 ------------------------------------------------------19
六、純物質最低抑制濃度(MIC)測定 ------------------------------------------- 20
七、癌細胞毒殺活性之測定 ------------------------------------------------------ 21
八、抗生素對原生動物毒性測試 ------------------------------------------------ 21
九、抗生素生物毒性對白蝦的影響 ----------------------------------------------22
十、海洋黴菌的鑑定 --------------------------------------------------------------- 23
十一、海洋放線菌之鑑定 --------------------------------------------------------- 26
參、結果 ------------------------------------------------------------------------------------- 27
一、菌種篩選 ------------------------------------------------------------------------ 27
二、菌種鑑定 ------------------------------------------------------------------------ 27
三、Acremonium altematum之生長與抗生素之純化 ------------------------- 27
四、A. altematum所產抗生物質之結構解析及生物活性探討 ------------- 29
五、Aspergillus ochraceus之生長與抗生素之純化 --------------------------- 30
六、A. ochraceus所產抗生物質之結構解析及生物活性探討--------------- 31
七、Nocardiopsis dassonvillei生長情形及所產抗生物質分析 ------------- 33
八、N. dassonvillei所產抗生物質之結構解析及生物活性 ----------------- 34
九、DHPA與PA對輪蟲的游動影響 --------------------------------------------36
十、海洋絲狀菌與所產抗生素及白斑病蝦對白蝦生存的影響 -------------36
肆、討論 ------------------------------------------------------------------------------------- 38
一、海洋黴菌ITS片段比對-------------------------------------------------------- 38
二、青黴酸 Penicillic acid (PA)的生物特性 ------------------------------------ 38
三、PA的物理和化學特質 --------------------------------------------------------- 40
四、PA的生合成及轉換 ------------------------------------------------------------ 40
五、PA的生物毒性 ------------------------------------------------------------------ 41
六、Iodinin的生產與生合成 ------------------------------------------------------- 42
七、Iodinin產生菌的病原性 ------------------------------------------------------- 43
伍、參考文獻 -------------------------------------------------------------------------------- 45

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