| 摘要註: |
本實驗室自室外篩選出一放線菌菌株,編號為K-7,在先前的研究中顯示(劉,2008)此菌株可分泌一種以上之抗生物質,而由K-7所分泌之第一種抗生物質其生產純化及相關性質在先前亦已有透徹的研究。此菌株透過饋料培養後可產生第二種抗生物質,且此抗生物質為目前較罕見之水溶性抗生物質。經培養基檢討其最適的培養基組成為Peptone 0.8%、Yeast extract 0.4%及Casamino acid 0.4%,將K-7菌株培養於此培養基中,在30℃,振盪速率為150 rpm的條件下,培養24小時之後可得到最大的抗生物質產率(20µl的抑菌圈大小為0.6 cm)。粗發酵液經丙酮沉澱後,接著通過陰(AmberliteIRA 96)、陽離子(CM-650)交換樹脂,最後再以Bio-Gel P2及Sephadex G10管柱進行層析,經上述流程後所得之活性物質,以薄層層析進行初步純度鑑定,可得到單一且明亮的色帶,判定其為具一定純度之物質,且最終回收率為16%。此抗生物質具良好的耐熱性,以90℃水浴處理60分鐘後仍保有83%之活性,其對格蘭氏陽性菌(金黃色葡萄球菌、枯草桿菌)及格蘭氏陰性菌(大腸桿菌、綠膿桿菌)皆有抑制作用,且具有清除金黃色葡萄菌所生成的生物膜之能力,但對真菌(白色念珠菌)則無抑制效果。結構方面,經FTIR分析後顯示其可能具O-H與N-H結構之官能基,而經LC-Mass、1H-NMR與13C-NMR分析的結果顯示此抗生物質為一小分子化合物,且結構相當簡單或對稱,與目前已知的抗生物質相比,屬於相當特殊之現象,未來極具研究價值。 Our laboratory had screen an actinomycete strain from the field, named K-7, and revealed by previous researcher that the strain could produce at least two distinct classes of antibiotics (Liu, 2008), while the first class of antibiotic produced by k-7 strain had been discussed. It's worthwhile to say that the strain can secrete another antibiotic after feed-back culture, and the second antibiotic is water soluble, the rare property of general antibiotic. After the investigation of the components of culture, we found that the optimal culture medium contained Peptone 0.8%, Yeast extract 0.4% and Casamino acid 0.4%. When K-7 strain was incubated with the optimal culture medium for 24 hr at 30℃ and 150 rpm, the strain could produce the best yield of antibiotic (the clear zone size is 0.6 cm with 20µl antibiotic). The crude supernatant produced by K-7 strain was purified by combination of acetone precipitation, anion exchange chromatography (Amberlite IRA 96), cation exchange chromatography (CM-650), and final, the Bio-Gel P2 and Sephadex G10 column chromatography. After these purification steps we used above, we obtained one active component which could display single band on thin layer chromatography (TLC). We judged that the purified compound possessed with a degree of purity and final recovery was 16%. The antibiotic has good heat resistance, it still had 83% activity after water bath treatment at 90℃ for 60 minutes. The antibiotic against both Gram positive (Staphylococcus aureus, Bacillus spp.) and negative bacteria (Escherichia coli, Pseudomonas aeruginosa), and possessed with the abilities to reduce preformed biofilm of Staphylococcus aureus, but it couldn't against fungus (Candida albicans). In the structure, the antibiotic may contain the O-H and N-H functional groups by FT-IR analysis, and after LC-Mass, 1H-NMR and 13C-NMR analysis, the result revealed that the antibiotic is a compound with small molecular weight, and with very simple or Symmetrical structure. To compare with the known antibiotics, these structure characteristics are really rare. It's pretty valuable to be investigated in the future. |