拮抗性鏈黴菌S1和S3菌株之抗生物質生產與特性分析

Abstract

本研究主要目標在探討鏈黴菌Streptomyces sp. S1菌株與Streptomyces griseobrunneus S3菌株所產生之抗生物質的分子與生物特性。此二菌株已知於液態培養下具優異產孢能力，且對多種植物病原真菌具高度之抗生活性並可行超寄生作用，有關其作為微生物殺菌劑的應用潛力已由系列相關試驗成果加以證實。既有研究結果顯示，此二菌株對病原真菌的超寄生作用為其達病害防治效用之重要因子，其中幾丁質水解酶以及葡聚醣水解酶皆已被證實於此超寄生作用中扮演關鍵角色。本論文相關研究主要目的是想瞭解此二鏈黴菌菌株所產生抗生物質於上述拮抗真菌活性中所扮演之角色。由對峙培養試驗結果證實，此二菌株可強力抑制荔枝露疫病菌(Peronophythora litchii)、番椒疫病菌(Phytophthora capsici)及草莓灰黴病菌(Botrytis cinerea)之菌絲生長。以玻璃環法試驗進而證實，二菌株皆可產生具抗生活性之代謝產物。在液態培養條件下，經篩檢添加不同碳、氮素源於培養基中對於S1與S3菌株所產生抗生物質活性之影響，試驗結果證實於八種供試碳素源中以市售純化自馬鈴薯之可溶性澱粉具最佳應用效果，可使此二菌株之抗生活性達到最高。然於氮素源試驗中，供試的硝酸態與氨態氮皆未見對抗生活性的提昇有明顯的促進效果。此些結果顯示相關抗生物質的生合成應為碳代謝作用攸關而非氮代謝關聯之特性。另者，此二菌株產生抗生物質之活性明顯有隨不同種類天然澱粉基質之應用而改變之現象，當以玉米粉及玉米澱粉作為碳素源時，對此二菌株產生抗生物質之活性表現明顯有加速及提昇之效果。在抗生物質萃取純化方面，以可溶性澱粉做為碳素源之供試菌培養液，經以乙酸乙酯萃取、及進而減壓濃縮蒸餾乾後，以適量甲醇回溶，並以70 %甲醇為沖提液進行Sep-Pak C18管柱的純化分離。所獲得具抗生活性樣品接著經由薄層色層分析及定性染色檢測，證實其結構中帶有糖基組成。經逆相式高效液體層析(HPLC)分析結果進而證實S1與S3菌株各自產生的兩種抗生物質(S1-1、S1-2；S3-1、S3-2)皆具有近乎一致的紫外光譜吸收特性。由HPLC分析純化所得到的抗生物質經以質譜儀分析，由50-800質荷比(m/z)全譜域掃描結果發現S1菌株的S1-1可見含分子量分別為390、454、538與556之四個離子碎片；S1-2則含分子量分別為390、556、588、749與767之五個離子碎片。雖然此純化抗生物質的結構仍待進一步釐清，但由既有質譜分析結果已可看出，此S1-1與S1-2應為結構極為近似、分子量大於700之新型抗生物質；本研究中另以核磁共振儀進行氫譜分析也證實此二物質確具極高相似性。此些純化之抗生物質另外經不同溫度及pH處理，證實其在100 ℃處理30分鐘或是置pH 4-9液體環境中處理5個小時，其仍可保有抗生活性。於生體外培養系統中，此抗生物質已證實可抑制露疫病菌孢囊的產生與菌絲的生長，並導致發芽管畸形；然由導電度測試則發現其對細胞膜功能的影響並非如預期明顯，其主要抗生機制顯非造成細胞膜通透性改變所致。在人工接種之荔枝果實的露疫病防治應用試驗方面，本研究發現施用去除菌體之培養濾液對標的病原之感染較之原培養液可有較佳的抑制效果。綜合本研究中所見理化特性、生物活性等試驗結果，所萃取純化抗生物質在抑制卵菌類病原感染為害上之應用性確具發展潛力。

The molecular and biological characteristics of antibiotics produced by Streptomyces sp. S1 and Streptomyces griseobrunneus S3 were investigated. The two streptomyces strains were known to be superior in sporulation in liquid culture system and were strongly antagonistic against and mycoparasitic on wide spectrum of fungal pathogen; their potential application as microbial bio-fungicide has been addressed in a serial researches. The importance of myco-parasitism in the control of fungal diseases has been explored where that the explicit role of hydrolytic chitinases and glucanases had been demonstrated. In the presented works, efforts were devoted to learn how the antibiotic production by the two bacteria might also contribute to the antifungal activity observed. By dual culture assay, the antagonistic effectiveness of the 2 tested strains were screened against Peronophythora litchii, Phytophthora capsici, and Botrytis cinerea, the mycelial extention of all three target fungal pathogens were strongly inhibited. A followed glass-ring assay further indicated the observed inhibitory effect a function of antifungal metabolites produced by the antagonistic bacteria. In a broth culture system, the effect of various carbon and nitrogen sources on the antifungal metabolite production was screened. Soluble starch manufactured from potato tubers was found among 8 tested carbon sources the best for the antibiotic production by both streptomyces strains. The screening test for nitrogen sources, however, appeared to be a failure since none of the tested nitrate- or ammonia-nitrogen sources showed stimulating effects on antibiotic production. The lack of effectiveness seemed to implicate the link of the biosynthetic activity to carbohydrate rather than to nitrogen metabolism. It was also noted that the antibiotic production changed greatly with the change of natural starch containing substrates applied. With the use of corn powder or corn starch as major carbon sources, an earlier increase and greater productivity of the antibiotics were observed. The antibiotics produced were extracted by addition of equal volume of ethyl acetate from the broth culture where in soluble starch of potato origin was added as the sole carbon source. The crude extracts obtained were concentrated to dryness by flash evaporator, re-dissolved with appropriate amount of methanol, cleared-up and enriched by Sep-Pak C18 cartridge filtration using 70% methanol as eluting solvent. By thin layer chromatography (TLC), the partial purified antibiotic preparations of both tested strains were shown to contain sugar moiety. A continued reverse phase high performance liquid chromatography (HPLC) further resolved the preparations of each tested strain into 2 components (S1-1 and S1-2 for S1 strain, S3-1 and S3-2 for S3 strain) with nearly identical UV absorption spectrum. The molecular characterization of the HPLC purified antibiotics by a triple-quad mass detector equipped HPLC system (LC/MS/MS) by whole span scanning revealed the detection of the major product ions at m/z=390, 454, 538, and 556, respectively for S1-1, and m/z=390, 556, 588, 749 and 767, respectively for S1-2. Although the molecular structure of the antibiotic purified awaited to be elucidated, the close similarity of the detected product ions implicated the identity of S1-1 and S1-2 and their uniqueness as a novel molecular weight (above 700) antibiotic. The close identity of these two components was further demonstrated by nuclear magnetic resonance (NMR) spectroscopy. By bioassay, it was demonstrated that the antibiotic purified remained stable in its antifungal activity after subjecting to 100 C treatment for 30 minutes or been adjusted to pHs ranging from 4 to 9 for 3 hours. In an in vitro culture system, the application of the antibiotic was found leading to inhibited sporangial germination, malformed germ tube development and inhibited mycelial extension of Peronophythora litchii. However, the antibiotic treatment led to only limited extent of membrane leakages of the test fungus indicating that membrane damage might not be the primed cause of the antifungal activity observed. On litchi fruits, the application of the purified antibiotics was shown to be effective in reducing significantly the infection of the artificially inoculated Peronophythora litchii. Their potential application as a therapeutic biochemical fungicide for the control of diseases caused by members of Oomycetes has worth great attention.