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論文中文名稱:鑑定鏈黴菌YX44分泌的抗微生物因子 [以論文名稱查詢館藏系統]
論文英文名稱:Identification of antimicrobial factors secreted by a new Streptomyces spp. YX44 [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:化學工程與生物科技系生化與生醫工程碩士班
畢業學年度:103
畢業學期:第二學期
中文姓名:王螢萱
英文姓名:Ying- Xuan Wang
研究生學號:102688004
學位類別:碩士
指導教授中文名:黃志宏
口試委員中文名:楊千金;陳月茸;黃姿雯;黃志宏
中文關鍵詞:鏈黴菌、抗菌因子、金黃色葡萄球菌
英文關鍵詞:Streptomyces, antibiotics, S. aureus
論文中文摘要:鏈黴菌是一種土壤中的微生物,在其生長過程中可分泌一些水解酶及二次代謝分子到環境中,其中部分的二次代謝分子具有抗菌活性可抑制其他微生物生長,而目前市面上的抗生素七成以上生產自鏈黴菌。本實驗原為找尋其他微生物分泌可誘發鏈黴菌大量產生抗生素的小分子,篩選過程中發現編號44分離菌株能夠抑制S. coelicolor M145的生長能力,我們對該菌株進行16S rRNA親源比對,發現NCBI資料庫中沒有序列與目前菌種完全相同,但與鏈黴菌的親源性最高,而它的外部型態也與鏈黴菌相似,因此將此菌暫時命名為Streptomyces spp. YX44。本論文分兩部分研究,第一部分為YX44在固態培養基上與其它微生物共同培養,發現對六種測試微生物[金黃色葡萄球菌(簡稱MSSA)、大腸桿菌、綠膿桿菌、白色念珠球菌、黑麴菌、酵母菌]有不同的抑菌效果,其中對MSSA的抑菌效果最好,因此第二部分為針對抑制MSSA的抗生物質做物理性分析。首先找到最佳化的培養基與培養條件,發現其抗MSSA因子極為親水性,與市面常見為親油性的抗生素不同。另外,此物質對proteinase K、E不敏感,用分子篩分離,發現它的分子量小於1000 Da;而且不耐熱,在100℃ 20分鐘便失去活性,但可耐酸鹼範圍在pH 1~13,能夠抑制多種抗藥性序列大腸桿菌與MRSA。初步的細胞毒性測試,48小時內不會抑制老鼠纖維母細胞生長;由LC-MS/MS偵測為461.15 Da,經比對資料庫並無相同化合物,目前推測為一種全新的抗生物質。
論文英文摘要:Streptomyces, a gram positive bacteriuml is normally found in soil. It secretes catabolic enzymes and secondary metabolites to its surroundings, many of the secondary metabolites are antibiotics show anti-microbe activity which inhibit the growth of the other microorganisms. More than 70% of known antibiotics are found in Streptomyces. In the beginning, we want to screen microorganism that can secrete small molecules to induce the production of antibiotics in S. coelicolor. We found that one of the screened microbes inhibited the growth of S. ceolicolor. Comparing the 16S rRNA sequence in the NCBI database, it was found that streptomycetes is the closest one, therefore, the screened stain was named Streptomyces spp. YX44. For testing the anti-microbe ability of YX44, we grew it closely to other microorganisms on an agar plate and found it could inhibit the growth of a variety of pathogens such as Staphylococcus aureus(MSSA), Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, Saccharomyces cerevisiae. And MSSA is the most sensitive to YX44. Therefore, my study was shifted to identify the anti-Staphylococcus aureus factors produced by YX44. We first found the anti-microbe factor is very hydrophilic, this charateristis is different from the most of known antibiotics shown hydrophobic. This factor is small since it passed 1 kDa cutoff dialysis membrane and lost its activity after incubated at 100℃ for 20 minutes. It resists to proteinase E and K and is stable in wide range of acid and base (pH 1~13). It showed the growth inhibition of variety of drug resistance E. coli and Methicillin-resistant Staphylococcus aureus. The preliminary result of cell toxicity test showed no effect after 48 hours co-incubation. After a series of purification, one molecule was identified with 461.15 dalton by LC-MS/MS analysis. There is no molecule matched this molecular weight in the database of Maybidge, Chembrige, and Zinc. This anti-microbe factors is a novel antibiotic.
論文目次:摘 要 i
ABSTRACT ii
致 謝 iii
表目錄 vii
圖目錄 viii
第一章 緒論 1
1-1鏈黴菌的簡介 1
1-2 誘導鏈黴菌產生二級代謝物的小分子 1
1-3 抗生素的簡介 2
1-4 本實驗中測試的微生物介紹 3
1-5本論文的內容 5
第二章 材料與方法 7
2-1菌種與細胞: 7
2-2 Primer: 8
2-3 Plasmid: 8
2-4藥品及培養基: 8
2-5 儀器與材料: 8
2-6 固態培養的抑菌測試 9
2-7菌種鑑別 9
2-8 抑菌能力測試 10
2-9 YX44分泌抗生物質的狀態 10
2-10 分泌抗MSSA因子的時間點 10
2-11 抗MSSA因子對MRSA的抑制測試 10
2-12 YX44對具抗藥性E. coli的抑制測試 11
2-13抗藥性能力測試 11
2-14 抗MSSA因子親水性測試 11
2-15 抗MSSA因子是否耐酸鹼度 11
2-16 不同pH值環境是否改變抗MSSA因子親油性 12
2-17 pH值中性下是否改變抗MSSA因子電荷性 12
2-18 抗MSSA因子是否為蛋白質水解酶的敏感性測試 12
2-19 抗MSSA因子的溫度敏感度 13
2-20 抗MSSA因子的純化方法與分析分子量 13
2-21 抗MSSA因子是否有相同分子量化合物 14
2-22 抗MSSA因子是否對真核細胞有毒性 14
第三章 實驗結果 16
3-1 篩檢的YX44對鏈黴菌有抑菌效果 16
3-2 YX44對多種致病菌抑菌能力 17
3-3 外部型態與序列分析顯示YX44為鏈黴菌 19
3-4在YM培養液所分泌抗MSSA因子為最佳效果 21
3-5 培養72小時為分泌抗MSSA因子的最佳效果 22
3-6 抗MSSA因子對帶有不同抗藥性序列的MRSA具有抑制作用 22
3-7 YX44對帶有不同抗藥性序列的E.coli具有抑制作用 24
3-8 YX44可抗多種抗生素 25
3-9 抗MSSA因子可穿透分子量1000的透析膜 25
3-10 抗MSSA因子物性分析-親水性 26
3-11 抗MSSA因子物性分析-耐強酸強鹼 28
3-12 抗MSSA因子物性分析-不同pH值不會改變抗MSSA因子的親水性 28
3-13 抗MSSA因子物性分析-在pH值中性環境下不帶電荷 29
3-14抗MSSA因子物性分析-對proteinase E、 K不敏感 30
3-15抗MSSA因子物性分析-高溫下不穩定 32
3-16 抗MSSA因子分子量應該為461.15 33
3-17目前資料庫中無461.15 Da的化合物 38
3-18 抗MSSA因子物性分析-不會抑制老鼠纖維母細胞生長 38
第四章 討論 41
參考文獻 45
附錄 47
Appendix A.Media 47
Appendix B. 抑制其他微生物的抗生物質物性分析 48

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論文全文使用權限:同意授權於2015-07-29起公開