嗜鹽菌Haloferax mediterranei醱酵生產胞外多醣之成分與結構分析

Composition and chemical structures analysis of exopolysaccharides produced by Haloferax mediterranei

林谷鴻(Gu-Hong Lin)

指導教授 ： 陳志成

大同大學/工程學院/生物工程學系所/碩士(2010年)

摘要

胞外多醣(exopolysaccharides, EPS)已廣泛應用於不同的領域，諸如食品、醫藥、化妝品及生物科技等產業。已知嗜鹽菌Haloferax mediterranei所生產胞外多醣中含有硫酸根與甘露糖。本研究乃利用廉價的飼料玉米粉作為H. mediterranei所生產胞外多醣之碳源，於添加濃度為15.5 g/L，溫度42℃，轉速180 rpm下進行胞外多醣之生產。經120小時，所測得吸光值OD520達到最高約為19.9。EPS經萃取後，於DEAE-Sepharose column之陰離子交換層析中以0 ~2 M NaCl鹽梯度進行洗脫程序，可分離出4個明顯的波峰(CP1、CP2、CP3、CP4)。其中以CP1回收率為最高達29.6 %，其次是CP3為8.7 %，CP2與CP4回收率均低，分別為4.3 %和4.8 %，總括所有fractions，約有50 %的回收率。經傅立葉轉換紅外線光譜(FTIR)之分析結果，此H. medierranei EPS CP1 ~ CP4分別於波長1039 ~ 1233 cm－1、1374 ~ 1418 cm－1、1540 ~ 1652 cm－1有波峰出現。其對應的官能基分別為S=O、CH bend與－CO－NH－R等。由元素組成分析可得知，CP1 ~ CP4其S含量各約為3.6 %、6.3 %、2.2 %及2.4 %。CP1 ~ CP4之1H NMR光譜分析之化學位移集中在1.0 ~ 5.2 ppm之範圍，其中CP1的anomeric protons分別為5.22 ppm、4.95 ppm與4.86 ppm；而CP2的anomeric protons於5.22 ppm、4.99 ppm與4.90 ppm。表示單元結構間之鍵結形式為1個α- 和2個β-linkgae，故推測CP1和CP2之化學結構可能為重複性的三醣所組成。而CP3和CP4 anomeric protons二者均有4個化學位移為5.02 ppm、4.56 ppm、4.42 ppm、4.35 ppm，表示含有1個α- 和3個β-linked sugars，推測CP3和CP4其化學結構可能由重複性的四醣單元構成。本研究提出H. mediterranei胞外多醣CP1與CP2之重複三醣單元以及CP3與 CP4重複四醣之單元之間的鍵結情形。

關鍵字

嗜鹽菌 ； 胞外多醣 ； 成分 ； 結構分析 ； 核磁共振

並列摘要

Extracellular polysaccharides (EPS) has been widely applied to different areas, such as food, medicine, cosmetics and biotechnology industries. The halophilic bacteria Haloferax mediterranei produces EPS with functional group of sulfate and mannose. This study use corn flour as the cheap carbon source at concentration of 15.5 g/L, under shaker flasks culture of temperature 42 ℃ and rotational speed of 180 rpm, to produce EPS. After 120 hours, the measured absorbance OD520 of cell concentration reached a maximum value of 19.9. EPS had been extracted and purified by ion-exchange chromatography on DEAE-Sepharose using a linear(0 ~ 2 M) NaCl gradient, and separated as four significant peaks (CP1, CP2, CP3, CP4) of different compositions. The overall recovery of EPS was 50 % with CP1, CP2, CP3 and CP4 of 29.6 %, 8.7 %, 4.3 % and 4.8 % recovery, respectively. Through the Fourier transform infrared spectroscopy (FTIR) analysis, CP1 ~ CP4, shown the absorption of wavelength at 1039 ~ 1233 cm-1, 1374 ~ 1418 cm-1 and 1540 ~ 1652 cm-1 with corresponding functional groups of S=O, CH bend, and－CO－NH－R, respectively. From the elemental composition analysis, CP1 ~ CP4 had sulfur content of 3.6 %, 6.3 %, 2.2 % and 2.4 % respectively. From the 1H NMR analysis, the chemical shifts of CP1 ~ CP4 focused on the range of 1.0 ~ 5.2 ppm. CP1 and CP2 shown the anomeric protons at 5.22 ppm, 4.95 ppm, 4.86 ppm , and at 5.22 ppm, 4.99 ppm, 4.90 ppm respectively. The anomeric protons of CP1 and CP2 indicated that the bonding between the monosaccharide units are in the form of one α- and two β-linkeage, therefore it is speculated that the chemical structure of CP1 and CP2 are tri-saccharides repeated units. The anomeric protons at CP3 and CP4 show four chemical shifts of 5.02 ppm, 4.56 ppm, 4.42 ppm, and 4.35 ppm, which represent one α- and three β-linkeage units, therefore it is speculated that CP3 and CP4 have chemical structure of tetra-saccharides. . This study proposes the chemical structure of tri-saccharides at CP1 and CP2, and also the structure of tetra-saccharides at CP3 and CP4.

並列關鍵字

exopolysaccharides(EPS) ； Haloferax mediterranei ； Composition ； chemical structures analysis ； NMR

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