跳到主要內容

臺灣博碩士論文加值系統

(34.236.192.4) 您好!臺灣時間:2022/08/17 18:32
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:謝雅雪
研究生(外文):Ya-Hsueh Hsieh
論文名稱:利用Streptomycesclavuligerus生產clavulanicacid之研究─在進料批式培養中檢討glycerol、ammoniumion的效應
指導教授:陳國誠陳國誠引用關係
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:70
中文關鍵詞:進料批式操作氨離子
外文關鍵詞:Clavulanic acidStreptomyces clavuligerusfed-batchammonium ion
相關次數:
  • 被引用被引用:1
  • 點閱點閱:141
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
Clavulanic acid為β-lactamase抑制劑,常與青黴素類(penicillins)抗生素合併使用來克服病原菌的抗藥性。clavulanic acid與amoxicillin的合併藥方是目前在醫療上被廣泛的使用。
本論文利用S. clavuligerus進行clavulanic acid的發酵生產,在批式和進料批式的操作,探討前培養的特性對clavulanic acid生產的影響,並在進料批式培養中,探討glycerol在培養液中最適維持濃度及檢討glycerol與ammonium ion的進料對clavulanic acid生化合成的效應。
前培養的菌體特性與生長形態會影響後續clavulanic acid的發酵生產。以不同的接種菌齡與接種比例進行clavulanic acid的批式與進料批式的發酵培養,得到最適的接種菌齡及接種比例分別為28小時與10%。以間歇進料的方式進料glycerol進行搖瓶進料批式培養,探討不同glycerol在培養液中最低維持濃度及其進料量,得知glycerol在培養液中的濃度變化維持在1∼5 g/l,其clavulanic acid產量最佳。不論是否有調控培養液中的pH或者初始添加ornithine與否,以glycerol與ammonium ion合併進料的結果皆顯示,微量的添加ammonium ion (5 mg)的clavulanic acid產量比單獨glycerol進料的培養的增加約15∼30%,之後隨著ammonium ion進料量的增加,產量會受到抑制。以初始含ornithine 5 mM,每次進料0.25 g glycerol和ammonium ion 5 mg,並控制pH的進料批式培養所得到的clavulanic acid產量最佳,為批式培養的2.74倍。
Clavulanic acid is a specific and irreversible inhibitor of a wide range of bacterial β-lactamases. Clavulanic acid has been used in human therapy for several years in combination with amoxicillin. The effect of inoculum quality on the production of clavulanic acid was first investigated in batch or fed-batch flask cultures with S. clavuligerus. A methodology to obtain an optimal concentration maintaining range for glycerol in the culture was developed to enhance the clavulanic acid production. Furthermore, the effect of intermittently feeding both glycerol and ammonium ion on the biosynthesis of clavulanic acid was also investigated.
The inoculum quality and the morphology did influence the clavulanic acid production. The optimal age and size of inoculum for the production of clavulanic acid in batch and fed-batch flask culture was 28 hrs and 10%, respectively. The optimal concentration range of glycerol could be maintained low in the culture, and the concentration range for clavulanic acid biosynthesis were 1~5 g/l. Whatever we try to control the pH of the cultures or initially add ornithine to the cultures, intermittently feeding glycerol of 0.25 g and ammonium ion of 5 mg in shake flask cultures resulted in the maximum production of clavulanic acid that was 15~30% higher than that with only feeding glycerol culture. Subsequently, the biosynthesis of clavulanic acid was inhibited due to the increasing amount of ammonium ion continuously added. The highest maximum production of clavulanic acid could be obtained under the conditions: initially concentration of ornithine of 5 mM, and simultaneously and intermittently feeding glycerol of 0.25 g and ammonium ion of 5 mg with the pH controlled in the culture. It was 2.74-fold higher than batch culture.
目 錄
一、研究緣起 1
二、研究背景與目的 2
2-1β-lactam抗生素簡介 2
2-1-1β-lactam抗生素之分類 2
2-1-2β-lactamase的抗藥性機制 2
2-2 Clavulanic acid簡介 4
2-2-1 Clavulanic acid生產菌 4
2-2-2 Clavulanic acid生化特性及其用途 5
2-3 Clavulanic acid生化合成路徑 6
2-4 前培養活性對發酵生產的影響 9
2-5 碳源、氮源進料對發酵生產的影響 11
2-5-1基質進料對發酵生產的影響 11
2-5-2 Ammonium進料的影響 12
2-6 Fed-batch培養簡介 13
2-6-1 Fed-batch培養原理及其優缺點 13
2-6-2 Fed-batch培養在抗生素生產的應用 13
2-7 先前研究結果 14
2-8 研究目的 15
三、實驗材料與方法 16
3-1 實驗藥品 16
3-2 Clavulanic acid生產菌 16
3-2-1 菌種 16
3-2-1 菌種活化與菌種保存 16
3-3 接種及培養方法 17
3-4 分析方法 18
3-4-1菌體量測法 18
3-4-2 Clavulanic acid濃度分析 18
3-4-3 Glycerol濃度分析 20
3-4-4 Ammonium濃度分析 20
3-5 實驗設備 21
四、結果與討論 22
4-1 接種菌體活性對clavulanic acid生化合成的影響 22
4-1-1 前培養的生長曲線 22
4-1-2 不同前培養時間對批式生產培養之影響 22
4-1-3 不同接種比例對批式生產培養之影響 25
4-1-4 不同前培養時間對進料批式生產培養之影響 26
4-2 Glycerol進料對clavulanic acid生化合成的影響 27
4-2-1 Glycerol最低維持濃度及其進料量的影響 27
4-3 Glycerol與ammonium ion同時進料對clavulanic acid 生化合成的影響 30
4-3-1 Ammonium ion合併進料對clavulanic acid生化 合成的影響 30
4-3-2 控制培養液的pH之影響 33
4-3-3 初始添加ornithine之影響 34
五、結論 36
六、參考文獻 38
七、圖表 46
[1] 劉英俊,最新微生物應用工業,中央圖書出版社,1996。
[2] Betina V. Bioactive secondary metabolites of microorganisms. Elsevier Science, Netherlands, 1994.
[3] Brody TM., Larner J and Minneman KP. Human pharmacology, molecular to clinical. Mosby-Year Book, Inc., Missouri, 1998.
[4] Higgens CE and Kastner RE. Streptomyces clavuligerus sp. nov. a β-lactamases antibiotics producer. International Journal of Systematic Bacteriology 1971;21: 326-331.
[5] Buchanan R E and Gibbons N E. Group 25: Streptomycetes and related genera. In Bergey’s Manual of Determinative Bacteriology, eds R. E. Buchanan and N. E. Gibbons, Williams & Wilkins, Baltimore, 1974. p. 667-675..
[6] Hopwood DA. Genetic studies of antibiotics and other secondary metabolites. In Genetics as a Tool in Microbiology (Symposia of the Society for General Microbiology), eds S. W. Glover and D. A. Hopwood, Cambridge University Press, 1981. p. 187-218.
[7] Nagarajan R. β-Lactam antibiotics from Streptomyces. In Cephalosporin and penicillins: Chemistry and biology, ed E. H. Flynn, Academic Press, New York, 1972. p. 636.
[8] Brown AG, Butterworth D, Cole M, Hanscomb G, Hood JD, Reading C and Robinson GN. Naturally occurring β-lactamase inhibitors with antibacterial activity. Journal of Antibiotics 1976;29:668-669.
[9] Reading C and Cole M. Clavulanic acid: a beta-lactamase-inhibiting beta-lactam from Streptomyces clavuligerus. Antimicrobial Agents and Chemotherapy 1977;11:852-857.
[10] Box SJ. Preparation of clavulanic using Streptomyces jumonjinensis. Beecham Group Ltd. US patent 4072569, 1978.
[11] Kitano K. New species Streptomyces katsurahamanus. Takeda Yakuhin Kogyo. JP patent 58081778, 1983.
[12] Okamurak K. Preparation of clavulanic acid. Sanraku Inc. JP patent 55162993, 1980.
[13] Liras P and Rodriguez-Garcia A. Clavulanic acid, a β-lactamase inhibitor: biosynthesis and molecular genetics. Applied Microbiology and Biotechnology 2000;54:467-475.
[14] Hsu LY. Antibacterial activities of amoxicillin alone and in combination with clavulanic acid correlated with β-lactamase production. Chinese Journal of Microbiology Immunology 1991;24:272-280.
[15] Hsueh PR, Chang SC, Chen YC, Hsu LY, Luh KT and Hsieh WC. In vitro antibacterial activities of ticarcillin alone and ticarcillin plus clavulanic acid against β-lactamase producing and non-producing microorganisms. Chinese Journal of Microbiology Immunology 1992;25:149-159.
[16] Butterworth D. Clavulanic acid: properties, biosynthesis, and fermentation. In Biotechnology of Industrial Antibiotics, Ed. E. J. Vandamme, Marcel Dekker, New York, Chap 6, 1984. p. 225-235.
[17] Baggaley KH, Brown AG and Schofield CJ. Chemistry and biosynthesis of clavulanic acid and other clavams. Natural Product Reports 1997;14:309-333.
[18] Hodgson JE, Fosberry AP, Rawlinson NS, Ross HNM, Neal RJ, Arnell JC, Earl AJ and Lawlor EJ. Clavulanic acid biosynthesis in Streptomyces clavuligerus: gene cloning and characterization. Gene 1995;166:49-55.
[19] Thirkettle JE, Baldwin JE, Edwards J, Griffin JP and Schofield CJ. The origin of theβ-lactam carbons of clavulanic acid. Journal of the Chemical Society. Chemical Communications 1997:1025-1026.
[20] Khaleeli N, Li R and Townsend CA. Origin of the β-lactam carbons in clavulanic acid from an unusual thiamine pyrophosphate-mediated reaction. Journal of the American Chemical Society 1999;121:9223-9224.
[21] Jensen SE and Paradkar AS. Biosynthesis and molecular genetics of clavulanic acid. Antonie van Leeuwenhoek 1999;75:122-133.
[22] Paradkar AS, Aidoo KA and Jensen SE. A pathway-specific transcriptional activator regulates late steps of clavulanic acid biosynthesis in Streptomyces clavuligerus. Molecular Microbiology 1998;27:831-843.
[23] Egan LA, Busby RW, Iwata-Reuyl D and Townsend CA. Probable role of clavaminic acid as the terminal intermediate in the common pathway to clavulanic acid and the antipodal clavam metabolites. Journal of the American Chemical Society 1997;119:2348-2355.
[24] Nicholson NH, Baggaley KH, Cassels R, Davison M, Elson SW, Fulston M, Tyler JW and Wononieck SR. Evidence that the intermediate biosynthetic precursor of clavulanic acid is its N-aldehyde analogue. Journal of the Chemical Society. Chemical Communications 1994:1281-1282.
[25] McGowan SJ, Bycroft BW and Salmond GPC. Bacterial production of carbapenems and clavams: evolution of β-lactam antibiotic pathways. Trends in Microbiology 1998;6:203-208.
[26] Calam CT. Starting investigational and production culture, Process Biochemistry 1976;4:7-12.
[27] Buckland BC. The translation of scale in fermentation processes: the impact of computer process control. Bio/Technology 1984;2:875-883.
[28] Freeman A and Aharonowitz Y. Immobilization of microbial cell in crosslinked, prepolymerized, linear polyacrylamide gel: antibiotic production by immobilized Streptomyces clavuligerus cells. Biotechnology and Bioengineering 1981;23:2747-2759.
[29] Warr SRC, Gershater CJL and Box SJ. Seed stage development for improved fermentation performance: increased milbemycin production by Streptomyces hygroscopicus. Journal of Industrial Microbiology 1996;16:295-300.
[30] Kishimoto K and Akiyama SI. Stimulatory effect of ferrous ion on mildiomycin production by Streptoverticillium rimofaciens. Biotechnology Letters 1997;19(7):699-702.
[31] Narang S, Sahai V and Bisaria VS. Optimization of xylanase production by Melanocarpus albomycesg ⅡS68in solid state fermentation using response surface methodology. Journal of Bioscience and Bioengineering 2001;91(4):425-427.
[32] Martinkova L, Machek F, Ujcova E, Kolin F and Zajicek J. Effect of age, amount of inoculum and inoculation medium composition on lactic acid production from glucose by Lactobacillus casei subsp. Rhamnosus. Folia Microbiology 1991;36(3):246-248.
[33] Sunitha K, Kim YO, Lee JK and Oh TK. Statistical optimization of seed and induction conditions to enhance phytase production by recombinant Escherichia coli. Journal of Biochemical Engineering 2000;5:51-56.
[34] Neves AA, Vieira LM and Menezes JC. Effect of preculture variability on clavulanic acid fermentation. Biotechnology and Bioengineering 2001;72(6):628-633.
[35] Court, JR and Pirt SJ. The application of fed-batch culture to the penicillin fermentation. Abstract papers, 5th International Fermentation Symposium, ed. H. Dellweg. Verlag Versuchs, Berlin, 1976.
[36] Lounes A, Lebrihi A, Benslimane C, Lefebvre G and Dermain P. Effect of nitrogen/carbon ratio on the specific production rate of spiramycin by Streptomyces ambofaciens. Process Biochemistry 1996;31(1):13-20.
[37] Suzuki T, Yamane T and Shimizu S. Mass production of thiostrepton by fed-batch culture of Streptomyces laurentii with pH-stat modal feeding of multi-substrate. Applied Microbiology and Biotechnology 1987;25:526-531.
[38] Pual RL and Michael EB. Manipulation of the physiology of clavulanic acid production in Streptomyces clavuligerus. Microbiology 1997;143:3573-3579.
[39] Mayer AF and Deckwer WD. Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations. Applied Microbiology and Biotechnology 1996;45:41-46.
[40] Suzuki T, Yamane T and Shimizu S. Effect of ammonium feeding on production of thiostrepton by fed-batch culture. Applied Microbiology and Biotechnology 1988;28:188-192.
[41] Jeremy RC and Pirt SJ. Carbon-and nitrogen-limited growth of Penicillium chrysogenum in fed-batch culture: the optimal ammonium ion concentration for penicillin production. Journal of Chemical Technology and Biotechnology 1981;31:235-240.
[42] Trong KV and Gray PP. Influence of ammonium on the biosynthesis of the macrolide antibiotic tylosin. Enzyme and Microbial Technology 1987;9:590-593.
[43] Whitaker A. Fed-batch culture. Process Biochemistry 1980;15:10-18.
[44] 陳國誠,微生物酵素工程學,藝軒圖書出版社,1992。
[45] Yamane T and Shimizu S. Fed-batch techniques in Microbial Processes. Advances in Biochemical Engineering/Biotechnology 1984;30:147-194.
[46] Modak JM, Lim HC and Tayeb YL. General characteristics of optimal feed rate profiles for various fed-batch processes. Biotechnology and Bioengineering 1986;28:1396-1406.
[47] Pirt SJ. Principles of Microbes and Cell Cultivation. Wiley, New York, 1975.
[48] Gray PP and Trong Kvu. Production of the Macrolide Antibiotic Tylosin in Cyclic Fed-batch Culture. Biotechnology and Bioengineering 1987;29:33-40.
[49] Ates S, Elibol M and Mavituna F. Production of actinorhodin by Streptomyces coelicolor in batch and fed-batch cultures. Process Biochemistry 1997;32:273-278.
[50] Traugott CS, Fiedler HP, Zahner H. Optimized nikkomycin production by fed-batch and continuous fermentation. Applied Microbiology and Biotechnology 1993;39:433-437.
[51] Kempf M, Theobald U and Fiedler HP. Correlation between the consumption of amino acid and the production of the antibiotic gallidermin by Staphylococcus gallinarum. Biotechnology Letters 1999;21:959-963.
[52] Chen KC, Lin YH, Tsai CM, Hsieh CH, Houng JY. Optimization of glycerol feeding for clavulanic acid production by Streptomyces clavuligerus with glycerol feeding. Biotechnology Letters 2002;24:455-458.
[53] Foulstone M and Reading C. Assay of amoxicillin and clavulanic acid, the components of Augmentin, in biological fluids with HPLC. Antimicrobial Agents and Chemotherapy 1982;22:753-762.
[54] Mccullough H. The determination of ammonia in whole blood by a direct colorimetric method. Clinica Chimica Acta 1967;17:297-304.
[55] Sebastine IM, Stocks SM, Cox PW, and Thomas CR. Characterisation of percentage viability of Streptomyces clavuligerus using image analysis. Biotechnology Techniques 1999;13:419-423.
[56] Gouveia ER, Baptista-Neto A, Azevedo AG, Badino-Jr AC, Hokka CO. Improvement of clavulanic acid production by Streptomyces clavuligerus in medium containing soybean derivatives. World Journal of Microbiology and Biotechnology 1999;15:623-627.
[57] Romero J, Liras P, Martin JF. Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus. Applied Microbiology and Biotechnology 1984;20:318-325.
[58] Lee MS, Lim JS, Oh KK, Yang DR, and Kim SW. Enhancement of cephalosporin C production by cultivation of Cephalosporium acremonium M25 using a mixture of inocula. Letters in Applied Microbiology 2001;32:402-406.
[59] Ellis H, Hundt HKL, Swart KJ, Hundt AF, Joubert AL, Essen GH and Plessis JB. A fast and simple method for the determination of clavulanic acid in human plasma using derivatisation reaction kinetics. Journal of Pharmaceutical and Biomedical Analysis 2000;22:933-937.
[60] Park SY, Momose I, Tsunoda K and Okabe M. Enhancement of cephamycin C using soybean oil as the sole carbon source. Applied Microbiology and Biotechnology 1994;40:773-779.
[61] Sarkar S, Sreekanth B, Kant S, Banerjee R, Bhattacharyya BC. Production and optimization of microbial lipase. Bioprocess Engineering 1998;19:29-32.
[62] 謝禎皓,利用Streptomyces clavuligerus生產clavulanic acid之研究,國立清華大學化學工程系碩士論文,2000。
[63] 蔡政閔,利用進料批次操作以Streptomyces clavuligerus生產clavulanic acid之研究,國立清華大學化學工程系碩士論文,2001。
[64] Prave P, Faust U, Sittig W, Sukatsch DA. Fundamentals of Biotechnology. Weinheim; Deefield Beach, FL: VCH, 1987:68-104.
[65] Suzuki T, Yamane T, Shimizu S. Kinetics and effect of nitrogen source feeding on production of poly-β-hydroxybutyric acid by fed-batch culture. Applied Microbiology and Biotechnology 1986;24:366-369.
[66] Lee SD, Park SW, Oh KK, Hong SI, Kim SW. Improvement for the production of clavulanic acid by mutant Streptomyces clavuligerus. Letters in Applied Microbiology 2002;34:370-375.
[67] Romero J, Liras P, Martin JF. Utilization of ornithine and arginine as specific precursors of clavulanic acid. Applied and Environmental Microbiology 1986;52:892-897.
[68] Mendz GL, Hazell SL. The urea cycle of Helicobacter pylori. Microbiology 1996;142:2959-2967.
[69] Ives PR, Bushell ME. Manipulation of the physiology of clavulanic acid production in Streptomyces clavuligerus. Microbiology 1997;143:3573-3579.
[70] Romero J, Liras P, Martin JF. Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus. Applied Microbiology and Biotechnology 1984;20:318-325.
[71] Lebrihi A, Lamsaif D, Lefebvre G, Germain P. Effect of ammonium ions on spiramycin biosynthesis in Streptomyces ambofaciens. Applied Microbiology and Biotechnology 1992;37:382-387.
[72] Romero J, Liras P, Martin JF. Utilization of ornithine and arginine as specific precursors of clavulanic acid. Applied and Environmental Microbiology 1986;52: 892-897.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top