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標題: | 嗜熱菌屬生物膜中自誘導物與胞外多醣之性質鑑定 Characterization of Autoinducer and Exopolysaccharide in Thermus spp. Biofilms |
作者: | Miao-Hsia Lin 林妙霞 |
指導教授: | 吳世雄 |
關鍵字: | 嗜熱菌,生物膜, Biofilm,Exopolysaccharide, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 先前研究指出嗜熱菌屬在自然溫泉環境下,並非自由存在於外界,而是以生物膜的型式,緊密附著在週遭的石頭或礦物鹽上,以求取生存。粗萃取生物膜多醣是經由將Thermus aquaticusYT-1此株菌塗在覆蓋有滅菌後的cellophane膜的Thermus固體培養基上,於60 ℃培養48小時後取得。粗萃取的外多醣可經由TSK HW-65F膠體進一步純化,可收集到4個分離的部分,分別稱為231-1、231-2、231-3與231-4,其分子量範圍分佈從10 kDa至500 kDa。藉由酸水解與衍生化反應後可將這四個分離的部分以氣相層析質譜儀與HPAEC-PAD系統測得單醣組成。結果顯示T. aquaticus YT-1株菌的生物膜含有大量的galactose與N-acetyl galactosamine,兩者比例約是1比1。同時也利用氣相層析質譜儀分析主要單醣鍵結確定在231-1樣品中含有:1,3-linked Gal、1,4-linked Gal、1,3-linked GalNAc;在231-2中有:1,4-linked Gal;231-3含有1,4-linked Gal;231-4則有1,4-linked Gal與1,3-linked GalNAc。然而真正確切結構還有待進一步研究探討。
在培養後的T. thermophilus HB-8液體培養基中發現含有促進生物膜生成的因子,即自誘導物。培養後的液體培養基先經過分子量為10 kDa的centricon濾過後,再經由TSK HW-50F的膠體過濾層析系統分離純化,初步可將之分為七個部分進行生物活性分析。結果顯示有兩個分子量較小的部分具有誘導NTU407菌株形成生物膜的能力,之後將會需要更進一步的純化與實驗,進而將分子的結構鑑定出來 In previous studies, bacterial species of genus Thermus living in natural hot springs are firmly attached to rocks and sands by a complex structure called biofilm. The crude biofilm exopolysaccharides (EPS) are obtained from Thermus aquaticusYT-1 plated on sterile cellophane membrane on Thermus agar plate incubation at 60 ℃ for 48 h. The crude EPS passes through TSK HW-65 gel for progressive purification and four major fractions called 231-1, 231-2, 231-3 and 231-4 with molecular weight range from 10 kDa to 500 kDa are collected. After acidic hydrolysis and derivative, the monosaccharide compositions of these four fractions are analyzed by GC-MS and HPAEC-PAD system. The results indicated that the major components of T. aquaticus YT-1 biofilm are galactose and N-acetyl galactosamine with the molar ratio of 1:1. The main glycosidic linkages are also characterized by GC-MS, showing that there are 1,3-linked Gal, 1,4-linked Gal, 1,3-linked GalNAc in 231-1, 1,4-linked Gal in 231-2, 1,4-linked Gal in 231-3 and 1,4-linked Gal, 1,3-linked GalNAc in 231-4. However, the detail structure should be further investigated. The cultural medium of T. thermophilus HB-8 can induce to form biofilm and show to contain the autoinducer. The cultural medium is incubated overnight and collected by filtering through 10 kDa centricon. Then the filtered cultural medium is subjected to TSK-50F gel filtration chromatography for fractionation. Seven fractions are collected for bioassay. Two fractions with the smaller MW possess bioactivities to promote NTU407 biofilm formation. More studies are under preparation to elucidate their detailed structures. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36612 |
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顯示於系所單位: | 生化科學研究所 |
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