(3.230.173.249) 您好!臺灣時間:2021/04/21 04:08
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:蔡俊杰
研究生(外文):Jiun-Jie Tsai
論文名稱:自Rhodobacter sphaeroides分離與純化輔酵素-Q10之研究
論文名稱(外文):Separation and Purification of Coenzyme Q10 from Rhodobacter sphaeroides
指導教授:吳和生吳和生引用關係
指導教授(外文):Ho-Shing Wu
學位類別:碩士
校院名稱:元智大學
系所名稱:化學工程與材料科學學系
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:79
中文關鍵詞:輔酵素Q10光合菌Rhodobacter sphaeroides醱酵萃取純度。
外文關鍵詞:Coenzyme Q10Rhodobacter sphaeroidesfermentationExtractionPurity
相關次數:
  • 被引用被引用:0
  • 點閱點閱:371
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:2
  • 收藏至我的研究室書目清單書目收藏:0
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
1.Abe, K., Y. Matsuo, J. Kadekawa, S. Inoue and T.
Yanagihara, Effect of Coenzyme Q10 in Patients with
Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis,
and Stroke-like Episodes (MELAS): Evaluation by
Noninvasive Tissue Oximetry, J Neurol Sci 162, 65-68.
(1999)
2.Alleva, R., M. Tomasetti, S. Bompadre and G.P. Littarru,
Oxidation of LDL and Their Subfractions: Kinetic Aspects
and CoQ10 Content, Mol. Aspects Med., 18, s105 (1997)
3.Battino, M., R. Fato, G. Parenti-Castelli and G. Lenaz,
Coenzyme Q10 can Control the Efficiency of Oxidative
Phosphorylation, Int J Tissue React, 12, 137-144(1990).
4.Beyer, R., J. Segura-Aguilar, S. Di Bernardo, M.
Cavazzoni, R. Fato, D. Fiorentini, M. Galli, M. Setti,
L. Landi and G. Lenaz, The Role of DT-diaphorase in the
Maintenance of the Reduced Antioxidant Form of Coenzyme
Q in Membrane Systems, Proc Natl Acad Sci USA, 93, 2528-
2532(1996).
5.Burke, B.E., R. Neuenschwander and R.D. Olson,
Randomized, Double-blind, Placebo-controlled Trial of
Coenzyme Q10 in Isolated Systolic Hypertension, South
Med. J., 94, 1112-1117(2001).
6.Cao, X.L., Y.T. Xu, G.M. Zhang, S.M. Xie, Y.M. Dong, Y.
Ito, Purification of Coenzyme Q10 from Fermentation
Extract: High-Speed Counter-Current Chromatography
versus Silica Gel Column Chromatography, Journal of
Chromatography A, 1127,92-96(2006).
7.Cadenas, E., P. Hochstein and L. Ernster, Pro- and
Antioxidant Functions of Quinones and Quinone Reductases
in Mammalian Cells, Adv Enzymol Relat Areas Mol Biol 65,
97-146(1992).
8.Crane, F.L., Y.Hatefi, R.L.Lester and C.Widmer,
Isolation of A Quinone From Beef Heat Mitochondria,
Biochim.Biophys.Acta, 25, 220-221 (1957).
9.Di Giovanni, S., M. Mirabella, A. Spinazzola, P.
Crociani, G. Silvestri, A. Broccolini, P. Tonali, S.
DiMauro and S. Servidei, Coenzyme Q10 Reverses
Pathological Phenotype and Reduces Apoptosis in Familial
CoQ10 Deficiency, Neurology 57, 515-518. (2001)
10.Ernster, L. and G. Dallner, Biochemical, Physiological
and Medical Aspects of Ubiquinone Function, Biophys
Acta ,1271(1), 195-204(1995).
11.Fink, M., Cytopathic Hypoxia. Mitochondrial Dysfunction
as Mechanism Contributing to Organ Dysfunction in
Sepsis, Crit Care Clin, 17, 219-237(2001).
12.Forsmark-Andrée, P., G. Dallner and L. Ernster,
Endogenous Ubiquinol Prevents Protein Modification
Accompanying Lipid Peroxidation in Beef Heart
Submitochondrial Particles, Free Radic Biol Med, 19, 749-
757 (1995).
13.Folkers, K. and R. Simonsen, Two Successful Double
blind Trials with Coenzyme Q10 (Vitamin Q10) on Muscular
Dystrophies and Neurogenic Atrophies, Biochim Biophys
Acta, 1271, 281-286(1995).
14.Forsmark-Andrée, P. and L. Ernster, Evidence for A
Protective Effect of Endogenous Ubiquinol Against
Oxidative Damage to Mitochondrial Protein and DNA During
Lipid Peroxidation, Mol Aspects Med, 15(Suppl), S73-81
(1994).
15.Gerald, K., Cell and Molecular Biology Concepts and
Experiments Fourth Edition, Wiley, New York (2005).
16.Harris, E. L. V. and S. Angal, Protein Purification
Methods:A Practical Approach. New York: IRL Press
(1989).
17.Harrison, R.G., P. Todd, S.R. Rudge and D.P. Petrides,
Bioseparations Science and Engineering, Oxford, New York
(2003).
18.Hatefi, Y., The Mitochondrial Electron Transport and
Oxidative Phosphorylation System, Annu Rev Biochem, 54,
1015-1069 (1985).
19.Karlsson, J., Heart and Skeletal Muscle Ubiquinone or
CoQ10 as Protective Agent Against Radical Formation in
Man, Adv Myochem, 1, 305-308(1987).
20.Kito, Y., K. Ohara, Y. Kosakai, K. Kawazoe, K. Hayashi,
Y. Ego, N. Fujii, H. Takano, Y. Naito, T. Fujita and H.
Manabe, Clinical Studies of the Effect of Coenzyme Q10
on the Myocardial Protection in Open Heart Surgery,
Nippon Kyobu Geka Gakkai Zasshi, 30, 1491-1495(1982).
21.Kaplan, P., I. Kucera and V. Dadak, Effect of Oxygen on
Ubiquinone-10 Production by Paracoccus denitrificans,
Biotech. Lett. , 15, 10, 1001-1002(1993)
22.Kuratsu, Y., M. Sakurai and H. Hagino, Aeration-
agitation Effect on Coenzyme Q10 Production by
Agrobacterium species, J. Ferment. Technol., 62, 3, 305-
308(1984)
23.Koroshetz, W., B. Jenkins, B. Rosen and M. Beal, Energy
Metabolism Defects in Huntington''s Disease and Effects
of Coenzyme Q10, Ann Neurol 41, 160-165. (1997)
24.Kazuyoshi, Y., K. Takahisa, K. Akihisa, U. Yasuyoshi,
Processes for Producing Coenzyme Q10, U.S Patent,
10,500,249(2005)
25.Lass, A., L. Kwong and R.S. Sohal, Mitochondrial
Coenzyme Q Content and Aging, BioFactors, 9, 199-205
(1999).
26.Lenaz, G., A Critical Appraisal of the Mitochondrial
Coenzyme Q Pool, FEBS Lett 509, 151-155. (2001)
27.Lockwood, K., S. Moesgaard, T. Yamamoto and K. Folker,
Progress on Therapy of Breast Cancer with Vitamin Q10
and the Regression of Metastases, Biochem. Biophys. Res.
Commun, 212, 172-177(1995).
28.Morton, R.A., G.M.Wilson, J.S. Lowe, and W.M.F. Leat,
Chemical Industry, 1649 (1957).
29.Mitchell, P., Protonmotive Redox Mechanism of the
Cytochrome b-c1 Complex in the Respiratory Chain:
Protonmotive Ubiquinone Cycle FEBS Lett, 56,1-6(1975)
30.Mitchell, P., Possible Molecular Mechanisms of the
Protonmotive Function of Cytochrome Systems,
J.Theor.Biol, 62,327-367(1976)
31.Olsson, J., L. Xia, L. Eriksson and M. Björnstedt.
Ubiquinone is Reduced by Lipoamide Dehydrogenase and
this Reaction is Potently Stimulated by Zinc. FEBS Lett
448, 190-192. (1999)
32.Ondarroa, M. and P.J. Quinn, Proton magnetic resonance
spectroscopic studies of the interaction of ubiquinone-
10 with phospholipids model membranes,
Eur.J.Biochem.,155, 353-361.(1986)
33.Papa, S., Mitochondrial Oxidative Phosphorylation
Changes in the Life Span. Molecular Aspects and
Physiopathological Implications, Biochim Biophys Acta,
1276, 87-105(1996).
34.Saraste, M., Oxidative Phosphorylation at the Fin De
Siecle, Science, 283, 1488-1493(1999).
35.Sharma, S., M. Kheradpezhou, S. Shavali, H. Refaey, J.
Eken, C. Hagen and M. Ebadi, Neuroprotective Actions of
Coenzyme Q10 in Parkinson’s Disease, Methods Enzymol.
382, 488-509(2004).
36.Schapira, A.H., J.M. Cooper, D. Dexter, J.B. Clark, P.
Jenner and C.D. Marsden, Mitochondrial Complex
Deficiency in Parkinson’s disease, J Neurochem, 54(3),
823-827(1990).
37.Sasikala, C. and C.V. Ramana, Biotechnological
Potentials of Anoxygenic Phototrophic Bacteria. I.
Production of Single-cell Protein, Vitamins,
Ubiquinones, Hormones and Enzymes and Use in Waste
Treatment, Adv.Appl.Microbiol, 41, 173-226(1995).
38.Sakato, K., H. Tanka, S. Shibata and Y. Kuratsu,
Agitation-aeration Studies on Coenzyme Q10 Production
Using Rhodopseudomonas sphaeroides, Biotech. Appl.
Biochem. , 16, 19-28 (1992)
39.Sasaki, K. and S. Nagai, The Optimum pH and Temperature
for the Aerobic Growth of Rhodopseudomonas Gelatinosa
and Vitamin B12 and Ubiquinone Formation on a Starch
Medium, J. Ferment. Technol., 57, 383-386(1979)
40.Stocker, R., V. Bowry and B. Frei, Ubiquinol-10
Protects Human Low-Density Lipoprotein More Efficiently
Against Lipid Peroxidation than does α-tocopherol, Proc
Natl Acad Sci USA, 88, 1646-1650(1991).
41.Tomasetti, M., G. Littarru, R. Stocker and R. Alleva,
Coenzyme Q10 Enrichment Decreases Oxidative DNA Damage
in Human Lymphocytes, Free Radic Biol Med, 27, 1027-1032
(1999).
42.Urakami, T. and T. Yoshida, Production of Ubiquinone
and Bacteriochlorophyll a by Rhodobacter sphaeroides and
Rhodobacter sulfidophilus, J. Ferment. Bioeng. , 76, 191- 194(1993)
43.Wolf, D.E., C.H. Hoffman, N.R. Trenner, B.H. Arison,
C.H. Shunk, B.O. Linn, J.F. McPherson and K. Folker,
Coenzyme Q Structural Studies Coenzyme Q Group,
J.Am.Chem.Soc, 80, 4752 (1958).
44.Winkler, K., FR. Wiedemann, CW. Wallesch and WS. Kunz,
Effect of Coenzyme Q10 on the Mitochondrial Function of
Skin Fibroblasts from Parkinson Patients, Journal of
Neurological Sciences, 220, 41-48(2004).
45.朱勝忠,非蛋白質產品之回收純化,田蔚城主編,生物技術,九
州圖書文物有限公司出版,183-202(1996)
46.松永尚夫,君塚房夫,落合一頼,大林晃,田邊脩,補酵素Q10
的精製方法,JP 公開特許公報,531-534(1982)
47.林藹寧,蛋白質產品之回收純化,田蔚城主編,生物技術,九州
圖書文物有限公司出版,167-182(1996)
48.胡淼琳,Coenzyme Q10 的生化營養性質以及它的醫療功效,自
由基生物學與醫學,第二卷,第一期,46-53(1994)
49.孫彥,生物分離工程,化學工業出版社,中國北京(2005)
50.郭秋媚,Coenzyme Q10 ,BCRC News, vol 18, No 2 (2005)
51.廖敏宏,磁性奈米載體在生物觸媒和生化分離之應用,國立成功
大學化學工程研究所博士論文,台南(2002)
52.劉昌峰,探討不同培養基組成對光合菌Rhodobacter
sphaeroides 生產Coenzyme Q10 之研究,國立中央大學化學工
程研究所碩士論文,桃園(2001)
53.劉昌豪,以光合菌Rhodobacter Sphaeroides BCRC 13100 生產
Coenzyme Q10 醱酵製程之開發,私立元智大學生物技術暨生物
資訊研究所碩士論文,桃園(2007)
54.賴明良,木聚糖水解酵素之純化與特性探討,國立成功大學化學
工程研究所碩士論文,台南(2001)
55.蘇遠志編著,應用微生物學,華香園出版社(1997)
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 宋國城、陳力、陳彥傑(2004)。有關旗山斷層的一些新觀察。地質,23(3),31-40。
2. 汪靜明(2000)。學校環境教育的理念與原理。環境教育季刊,43,11-27。
3. 汪靜明、燕琍婷(1996):自然攝影及其環境教育角色。環境教育季刊,30,31-40。
4. 吳清基、蘇進棻(2001)。新世紀的教育展望。研習資訊,18(2),1-5。
5. 王鑫(1994)。環境保護教育。環境教育季刊,23,5-9。
6. 王鑫(1999)。地球環境教育與永續發展教育。環境教育季刊,37, 87-103。
7. 王俊秀(2000b)。通識教育與永續發展教育的連結:議題與展望。環境教育季刊,43,8-17。
8. 許世璋(2003)。大學環境教育課程對於環境行動與其它環境素養變項之成效分析。科學教育學刊,11(1),97-119。
9. 郭實渝(1999)。以生態文化教育的觀點看環境教育。環境教育季刊,40,15-23。
10. 楊冠政(1989)。環境教育概述(上)。環境教育季刊,1,6-17。
11. 楊冠政(1991)。學校課程環境化──學校實施環境教育的首要工作。環境教育季刊,11,5-20。
12. 葉欣誠、陳永昌、莊育禎、呂文銘(2003)。綠色大學評量指標系統之建構研究。載於國立高雄師範大學環境教育研究所舉辦之綠色大學理論與實務研習會(頁4-1~4-10),高雄。
13. 詹聖惠(2004)。六年級學生參與「校園學習步道手冊」製作之心理歷程研究。南投文教,20,70-74。
14. 靳知勤(1994)。從環境知識、態度與行為間的關係-論環境教育目標之達成。環境教育季刊,23,31-39。
15. 廖麗貞、林寶英、洪振方(2000)。將達爾文演化論發展史融入大學生命科學通識課程之研究。科學教育學刊,8(2),179-198。
 
系統版面圖檔 系統版面圖檔