輔酵素Q10 (Coenzyme Q10)是人體電子傳遞鏈中生成能量adenosine triphosphate (ATP)的必須成分，為一種抗氧化物，具有防止脂質過氧化和清除體內過氧化物的功能，如自由基之清除。
本論文研究目的主要探討如何將光合菌之ㄧ---球形紅桿菌(Rhodobacter sphaeroides BCRC 13100)菌株生成之輔酵素Q10從醱酵液中分離並且純化。分成三部分探討。(1)前處理破菌方法，如酵素法、冷熱法、化學法和高壓破菌法，結果顯示，以酵素法而言，其產量最高可達2.85 mg/g-DCW，但其成本花費較高。其次是高壓破菌法效果最好，其產量也可達2.11 mg/g-DCW。另外則使用乙醇破菌，並且直接萃取，立即可省掉破菌的成本，其產量也可達2.01 mg/g-DCW；(2)分離方法分成兩種，破菌後，利用一些有機溶劑從細胞中萃取出輔酵素Q10、另一方法即利用乙醇在40℃水浴下經過30分鐘，直接破菌並且兩次萃取，結果顯示產量可達到2.46 mg/g-DCW，其萃取率可達到酵素法的0.86倍；(3)純化方法分成兩種方法進行，利用矽膠管柱層析使粗萃取物達到純化的效果，另一方法即使用正己烷做液-液萃取，再經過結晶後，結果顯示，純度可達到96.9%。

Coenzyme Q10 acts as an essential component of adenosine triphosphate (ATP) generation in the oxidative phosphorylation chain and as an antioxidant preventing lipid peroxidation and scavenging superoxide.
The purpose of this study will focus on separation from the fermentation broth and purification of CoQ10 that produced by Rhodobacter sphaeroides BCRC 13100. Three topics were discussed as follows：(1) Disruption methods：the cells were disrupted by using enzyme, freezing and heating, chemicals or high press homogenizer. The results showed that the product yield could achieve 2.85 mg/g-DCW for enzyme method but its cost was higher. Secondly, the method of high press homogenizer was better that the product yield can achieve 2.11 mg/g-DCW. On the other hand, the ethanol could be used to disrupt and extract directly. The product yield could also achieve 2.01 mg/g-DCW. (2) Separation methods：CoQ10 was quantitatively extracted with various organic solvents. Using ethanol twice extraction could separate the products to release into the surrounding solution.CoQ10 was extracted by ethanol at 40℃ for 30min. The product yield could also achieve 2.46 mg/g-DCW. The product yield of ethanol disrupted method is 0.86 fold of the product yield using enzyme method. (3) Purification method：The crude extract was purified by silica gel column chromatography. Another method was liquid-liquid extraction with hexane. After crystallization, the purity determined by means HPLC could achieve 96.9%.

Chinese abstract I English abstract II
Acknowledgement III
Contents IV
List of Captions of Figures VIII
List of Captions of Tables X
Chapter 1 INTRODUCTION...................................1
1.1 Prolegomenon.........................................1
1.2 Coenzyme Q10.........................................2
1.2.1 Discovery of Coenzyme Q10........................3
1.2.2 Biosynthesis of Coenzyme Q10.....................3
1.2.3 Production of Coenzyme Q10 by Microbial
Fermentation ....................................4
1.2.4 Role of Coenzyme Q10 in Electron-Transport Chain.6
1.2.5 Antioxidant Role of Coenzyme Q10.................9
1.2.6 Application of Coenzyme Q10 in the Medicine.....10
1.3 Separation and Purification.........................11
1.3.1 Strategy for Purification of Nonprotein.........13
1.3.2 Solid/Liquid Separation---cell separation.......15
1.3.3 Pretreatment....................................16
1.3.3.1 Theory of Cell Disruption and Release.......17
1.3.3.2 Methods of Cell Disruption..................20
1.3.4 Concentration---Main Separation Process.........22
1.3.4.1 Extraction............ .....................22
1.3.4.2 Precipitation...............................22
1.3.5 Purification....................................23
1.4 Patent Search.......................................23
1.4.1 Published Patent in Taiwan......................24
1.5 Motivations and Purpose of this Study...............24
Chapter 2 EXPERIMENTAL SECTION..........................27
2.1 Materials...........................................27
2.2 Appatrtus...........................................27
2.3 Microorganism.......................................28
2.3.1 Preservation of Bacteria........................28
2.3.2 Medium..........................................28
2.3.3 Flask Culture...................................29
2.3.4 Fermentor Culture...............................29
2.4 Determination of Coenzyme Q10 by HPLC...............30
2.4.1 Analytic Apparatus..............................30
2.4.2 Calibration Curve...............................30
2.4.3 Precision and Accuracy of Calibration Curve.....31
2.5 EI-Mass.............................................32
2.6 Nuclear Magnetic Resonance Spectroscopy.............32
2.7 Disruption of Crude cell............................32
2.7.1 Enzyme Pretreatment.............................33
2.7.2 Freeze and Heat Pretreatment....................33
2.7.3 Chemical Pretreatment...........................33
2.7.3.1 Hydrochloric acid...........................33
2.7.3.2 Sodium hydroxide............................33
2.7.3.3 Ethanol.....................................34
2.7.3.4 n-propanol:hexane(volume ratio=3:5) mixture
solvent.....................................34
2.7.4 Combine Freeze, Heat and Hydrochloric acid......34
2.7.5 High Press Homogenizer..........................34
2.8 Extraction..........................................34
2.9 Evaporation.........................................35
2.10 Purification.......................................35
2.10.1 Purification with Silica Gel Chromatography....35
2.10.2 Liquid-Liquid Extraction.......................36
2.11 Crystallization....................................36
Chapter 3 RESULTS AND DISCUSSION........................37
3.1 Effect of pH in the Flask Culture...................37
3.2 Fermentation........................................38
3.3 Characterization of analytical CoQ10................42
3.3.1 Effect of Cell Concentration....................42
3.3.2 Effect of Temperature...........................43
3.3.3 Effect of Extraction Time.......................43
3.4 Pretreatment........................................44
3.4.1 Enzyme..........................................44
3.4.2 Freeze and Heat Pretreatment....................45
3.4.3 Chemical Pretreatment...........................45
3.4.3.1 Hydrochloric acid...........................45
3.4.3.2 Sodium hydroxide............................46
3.4.3.3 Ethanol.....................................46
3.4.3.4 n-propanol:hexane(volume ratio=3:5) mixture
solvent.....................................47
3.4.4 Combine Freeze, Heat and Hydrochloric acid......47
3.4.5 High Press Homogenizer..........................47
3.5 Separation..........................................48
3.5.1 Effect of Solvent for Extraction with
Disruption......................................48
3.5.2 Effect of Solvent for Extraction without
Disruption......................................49
3.5.3 Disruption and Separation by Ethanol Directly...51
3.5.3.1 Effect of Temperature for Ethanol Extraction
............................................51
3.5.3.2 Effect of Extraction Time for Ethanol
Extraction..................................51
3.5.3.3 Effect of the Volume Ratio of Ethanol and DCW
for Ethanol Extraction......................52
3.5.3.4 Twice Extraction with Ethanol without
Pretreatment................................53
3.5.3.5 Twice Extraction with Ethanol with Homogenizer
Pretreatment................................54
3.6 Purification........................................55
3.6.1 Silica Gel Chromatography.......................55
3.6.2 Liquid-Liquid Extraction........................56
3.6.2.1 Effect of Extraction Time for Liquid-Liquid
Extraction..................................56
3.6.2.2 Effect of Proportion of Hexane and Diethyl
Ether for Extraction Solvent................57
3.6.2.3 Effect of Volume Ratio of Ethanol Extract
Solution and Hexane.........................58
3.6.2.4 Effect of Concentration of Ethanol Extract
Solution....................................59
3.6.2.5 Washing Impurity by pH 6-8 of Water Solution
............................................60
3.7 Crystallization.....................................60
3.7.1 Effect of Ethanol Purity........................60
3.7.2 Effect of Crystallization Concentration.........61
3.7.3 Comparison of Ethanol Purity and Temperature for
Crystallization.................................62
3.8 Scale Up............................................63
3.9 Comparison of Separation Efficiency of CoQ10 with
Reference...........................................65
Chapter 4 Cost Evaluation...............................66
4.1 Fermentation Culture................................66
4.2 Pretreatment and Separation.........................66
4.3 Produce Cost of Overall Procedure for Scale Up......68
Chapter 5 Conclusions and Future outlook................69
5.1 Conclusions.........................................69
5.2 Future Outlook......................................70
Appendix................................................71
References..............................................74
Curriculum Vitae........................................79

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