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研究生:吳世揚
研究生(外文):Shih-Yang Wu
論文名稱:BacillussubtilisDB430饋料式發酵及基因重組蛋白表現之研究
論文名稱(外文):Development of fed-batch culture and recombinant proteins expression system in Bacillus subtilis DB430
指導教授:曾慶平
指導教授(外文):Ching-Ping Tseng
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:77
中文關鍵詞:枯草桿菌發酵
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過去的研究得知,Bacillus subtilis有很多的優點,並適合基因重組蛋白質產品生產用。本論文研究目的是為了提高Bacillus subtilis發酵產物的產量,在已知最佳的碳源(葡萄糖)及氮源(酵母抽出物)搭配下去調控其他發酵條件,使達到工業化生產水準。我們同時也進行菌株突變以獲得更適合之菌種,並改造質體使其在菌株內的穩定性上升。從搖瓶式發酵培養枯草桿菌的實驗中得知氮源(酵母抽出物)的消耗速率為每百分之一酵母抽出物使菌體O.D.600上升8、最佳的鹽類添加、及最佳的起始濃度為NaCl 0.3g/l、 K2HPO4 0.4g/l、 CaCl20.2g/l、 MgSO4 0.5g/l、 FeSO4 30mg/l、 MnSO4 20mg/l。進一步在發酵槽中用上述之條件進行大量培養,並以電腦控制葡萄糖、酵母抽出物及各種鹽類的進料速率。由於枯草桿菌為好氧菌,其對氧濃度的變化十分敏感,因此我們根據D.O.數值來調配進氣量及其進氣含氧的濃度。當D.O.值保持在10~20%之間,在精密的控制下件進行饋料培養,可得到最高的菌體濃度,其OD600可達154,菌體乾重(biomass)可達54 g/l。另一方面,為了保護重組DNA所表現的蛋白質不被破壞,因此構築recE的突變株。我們取得Kmr及Cmr基因並將兩基因分別插入正常recE基因中使其失活。再把失活recE送入DB430中行double crossover recombinant,將正常的recE基因換掉,得到含Cmr的recE突變株。此外本研究中外來表現蛋白質為口蹄疫病毒之表面蛋白VP1及VP3,分子量均為25kDa。從蛋白質電泳圖得知VP1及VP3均有表現,其中VP3在DB430中經饋料式發酵可得到每公升1.4克的蛋白表現。另外已知Restriction - Modification system有助質體穩定,現已從
E coli RY13取得EcoRI之R-M system,轉殖到質體的工作目前尚在進行中。

Bacillus subtilis has many advantages for biotechnology that including nonpathogenic, easy to cultivate, store and secrete protein. Therefore, it has been used to product the recombinant DNA proteins. The goal of this study is to improve the biomass as well as recombinant proteins production ability in Bacillus subtilis. In order to obtain the high cell density, the optimization of fed-batch culture was studied. The experiment was carried out by using glucose as carbon source and yeast extract as nitrogen source. The parameters of control fermentation have been examined which includes how to manipulate
Carbon and nitrogen sources feeding, agitation and aerate, as well as trace elements supply. The results showed that the optimal glucose concentration is 0.5% ( w/v ), and the consumption tare of yeast extract is 1%( w/v ) when O.D.600 value increased 8. When Bacillus subtilis DB430 grew in 2.5L fermenter at 37℃ and 700 rpm of stir rate and used 50% of oxygen, we can got the highest cell density ( O.D.600 value is 154 ) and the cell dry weight ( biomass is 54 g/l ). The highest expression of recombinant VP3 protein was1.4 g/l in fed-batch culture at 37℃, 700 rpm of stir rate. To protect the recombinant protein, a recE mutant was also construct in Bacillus subtilis DB430.

目錄
中文摘要……………………………………………………………… ……..i
英文摘要…………………………………………………………………...…..ii
目錄……………………………………………………………………………iii
圖表目錄………………………………………………………………….…...vi
壹 緒論………………………………………………………………… …….1
1.1前言……………………………………………………………… ……..1
1.2 研究目的…………………………………………………… …………2
貳. 文獻回顧………………………………………………………… ………3
2.1. 枯草桿菌屬 (Bacillus) 簡介……………………………… ……….3
2.2. Bacillus 屬細菌在醱酵工業上之應用…………………… ………..4
2.2.1. 生產 Peptide antibiotic……………………………………… ……..4
2.2.2. 桿菌屬細菌生產之殺蟲劑 (insecficidal toxins) 胞外酵素
(extracellular enzyme) ………………………………………… ……..5
2.2.3. 利用枯草桿菌屬生產重組 DNA (recombinant DNA) 產物 ……..5
2.3. 宿主的選擇 (Bacillus subtilis DB 428 及 DB 430)………… …….6
2.4. recE 基因之功能……………………………………………… ……7
2.5. 批式培養、連續培養、饋料批式培養……………………………..8
2.5.1 批式培養……………………………………………………………..8
2.5.2 連續培養………………………………………………………….….8
2.5.3 饋料批式培養…………………………………………………….….9
2.6. 通氣、攪扮與溶氧……………………………………………….…12
2.7. 養料及電解質的濃度…………………………………………….…14
2.8. Bacillus spp. 質體選擇與質體穩定性研究………………………..14
2.8.1. Bacillus subtilis 之質體……………………………………………14
2.8.2. 質體穩定性 ( plasmid stability )……………………………..…….17
2.8.3. 生長環境對質體穩定性之影響……………………………………19
2.9. R-M system ( Restriction-Modification system )……………………20
參. 實驗材料與方法…………………………………………………………24
3.1 宿主及載體……………………………………………………………24
3.1.1菌株 …………………………………………..……………………24
3.1.2質體 ………………………………………..………………………24
3.2 藥品及儀器……………………………………………………………24
3.2.1藥品及試劑 …………………………………………………………24
3.2.2 培養基及培養液…………………………………………………….25
3.2.3 設備……………………………………………………………………….26
3.3 實驗方法……………………………………………………………….27
3.3.1搖瓶式發酵…………………………………………………………...27
3.3.2批次式發酵…………..……………………………………………….27
3.3.3饋料式發酵…………………………………………………….……..27
3.3.4 枯草桿菌質體 DNA 的純化………………………………………28
3.3.5 枯草桿菌染色體DNA的純化………………………………………29
3.3.6 洋菜膠體中DNA的分離……………………………………………29
3.3.7 轉形作用…………………………………………………………….30
3.3.8 載體穩定性偵測…………………………………………………….32
3.3.9 recE 突變株的構築………………………………………………….32
3.3.10 大腸桿菌質體DNA的純化、分離與轉形作用…………………..33
3.3.11 葡萄糖定量分析…………………………………………………36
3.3.12 R-M system之架構……………………………………………….38
3.3.13 菌體乾重(Biomass)測定………………………………………...39
3.3.14 蛋白質電泳分析……………………………………………..….39
肆. 實驗結果…………………………………………………………………41
4.1. 比較枯草桿菌DB428與DB430饋料式發酵之生長…….………...41
4.2. 枯草桿菌之最適化發酵條件探討…………………………..……….41
4.2.1. 不同的攪拌速率對菌體生長之影響………….…………..……….42
4.2.2. 氮源的饋料方式對DB430發酵之影響……………………………42
4.2.3. 鈉、鉀離子濃度對DB430發酵之影嚮…………………..……….44
4.2.4. 氧氣對DB430發酵之影嚮………………………….…….……….44
4.3. 構築 recE 突變株……………….……………………….….……….45
4.4. 葡萄糖濃度對DB430發酵之影嚮…………………….…….………46
4.5 不同發酵條件對DB430發酵之影嚮……………….……..…………47
4.6 R-M system……………………………………………….………….47
五. 討論………………………………………………………….…….……..49
5.1 菌種的選擇…………………………………………….….…………..49
5.2 攪拌速率對DB428生長之影響………………………….………….49
5.3 供氧量對枯草桿菌DB430高密度發酵之影響………….………….50
5.4 不同發酵方式對枯草桿菌DB430生長之影響………….………….50
5.5 饋料方式對枯草桿菌DB430發酵之影響………………….……….51
5.5.1酵母抽出物在饋料發酵方式中之探討………… …….……………51
5.5.2 葡萄糖濃度在饋料方式之探討…………………………………….52
5.7. VP3 及VP1蛋白質的表現………………………………………..53
參考文獻……………………………………………………………………...71
圖表目錄
圖一 以饋料式發酵比較DB428與DB430菌株之生長…………………………54
圖二. 不同攪拌速率對DB428批次發酵之影響……………………………….…55
圖三 添加氮源對DB430饋料發酵之影響………………………………………..56
圖四 不同濃度之酵母抽出物對DB430生長之影響……………………………..57
圖五 饋料式發酵中氮源添加方式不同對DB430生長之影響…………………..58
圖六.不同鈉及鉀離子濃度對搖瓶批次培養DB430生長之影響…………59
圖七 不同溶氧值對DB430饋料式發酵影響……………………………………..60
圖八 不同供氧方式對DB430饋料式發酵之影響………………………………..61
圖九 構築DB430 recE 之突變株…………………………………………62
圖十 不同供氧濃度對DB430使用葡萄糖之比較………………………………..63
圖十一 不同葡萄糖濃度下DB430饋料式發酵…………………………………..64
圖十二 比較DB430在不同發酵方式下之生長…………………………………..65
圖十三 EcoRI R-M system之取得…………………………………………66
圖十四 DB430與不同質體在不同培養方式下得之VP1蛋白質表現之電
泳圖………………………………………………………………….67
圖十五 不同培養方式與培養基下測得之VP3蛋白質表現量之電泳圖….68
表一 不同發酵方式所得之菌體乾重( biomass )與孢子形成量…………..69
表二 不同葡萄糖條濃度對菌體乾重、菌數、及O.D.600值之比較……..70
表三 不同培養方式下測得之VP3蛋白質表現量………………………..71

1. Arbige, M., and W. R. Chesbro,” Industrial enzymology: alook towards
the future.”, Trends Biotechnol, 7:330-335, 1989.
2. Priest, F. Handbooks of Biotechnology, vol.2, p.293-315, Bacilli., 1989.
3. Demain, A. L. Economic Microbiology. vol. 2. p178-208, 1987.
4. Leifert, C., S. Chidburee, S. Hampson, Workman, D. Sigee, H. A.S.
Epton and A. Marbour., J. Appl. Bacteriol. 45:97-108, 1995.
5. Feitelson, J. S., J. Pyane and L. Kim. Bio/technology., 10:271-275, 1992.
6. Kleinkauf, H., and H. von Dohren, Trends. Biochem. Sci, 8:281-283, 1983.
7. Bella, L.A., R. M. Faust, R. Andrews, and N. Goodman. Molecular
Biology of the Bacillus, vol. 2. p.186-210, 1985.
8. Burges H.D., Parasitol. 84:79-114, 1982.
9. Rowe, G. E. and A. Margaritis, Reviews in Biotechnology., 6:87-127, 1987.
10. Van R. J., W. H. McGaugheyt, D. E, Johson, B. D. barnett, and Van
Mellaert , H., Science 247:72-74, 1990.
1. 吳文希。「植物病害之生物防治」。永續農業研討會專集,237-245頁,台灣,民國82年.
12. Kulakauskas,S.,A. Lubys, and S.D.Ehtlich., J.Bacteriol, 177(12) :3451- 3454, 1995.
13. Lubys, A., A. Janulaitis, Gene. 157:25-29, 1995.
14. Naito, T., K. Kusano, and I. Kobayashi, Science, 267(10):897-899, 1995
15. Yarmolinsky, M.B.,.Science, 267(10):836-837, 1995.
16. Feitelson, J. S., J. Pyane and L. Kim, “Bacillus thuringiensis:insects and
beyond”, Bio/technology. 10:271-275, 1992.
17. Leifert, C., S. Chidburee, S. Hampson, Workman, D. Sigee, H. A.S. Epton and A. Marbour, J. Appl. Bacteriol, 45:97-108, 1995.
18. Arbige, M., and W. R. Chesbro. “Industrial enzymology: a look towards the future”, Trends Biotechnol, 7:330-335, 1989.
19. Demain, A. L., “Production of nucleiotides by microorganism”, Economic Microbiology, vol. 2. pp178-208, 1987.
20. Prist, F. G., “Products and applications. Handbooks of Biotechnology”, vol.2. Bacilli, pp.293-315, 1989.
21. Kleinkauf, H., and H. von Dohren, “Non-ribosomal peptide formation on multifuctional proteins”, Trends. Biochem. Sci, 8:281-283, 1983.
22. Bella, L. A., R. M. Faust, R. Andrews, and N. Goodman, Molecular Biology of the Bacillus, vol. 2. pp.186-210, 1985.
23. Anonymous , Regul. Toxicol. Pharmacol, 12 (Part 2):1-196, 1990.
24. Koga, T. Matsuse,I. Teramoto, Y. Ueda, S., “Synthesis and antimycotic activity of
cinnamy benzoate” Journal of Frementation & Gioengineering, V 76 n6. P524-
26, 1993
25. Thibault, J.F. De Dreu, R. Geraeds, C. Rombouts, F. M., “Studies on extraction of
pectin from citrus peel, apple mark and sugar-beet pulps with arabinanase and
galactanase” Carbohydrate Polymer, V 9 n 2, p 119-131, 1988.
26. Roy, S. Derochers, M. Jurasek, L., “Protein as potential retention acids in the pulp
and paper”, Journal of Wood Chemistry & Technology, V 9 n p 407-420, 1989.
27. Rubio, M.C. Molina, O.E., “Treatment of potato waste effluents with bavterial
protein production”, Biological Wastes, V 29 n 3 p 221-228, 1989.
28. Sneath, P. H. A., “Endospore-forming Gram-positive rods and coocci”, Bergey's Manual of Systemstic Bacteriology. Vol. 2. Section 13.
29. Prist, F. G., “Systematics and ecology of Bacillus. Bacillus subtilis and
other Gram-positive bacteria biochemistry”, Physiology and molecular
genetics, pp.3-12, 1993.
30. Ferrari, E., A. S. Jarnagin, and B. F. Schmidt, “Commercial Production of Extracellular Enzymes. Bacillus subtilis and other Gram-positive bacteria biochemistry”, Physiology and molecular genetics, pp.917-920, 1993.
31. Aronson, A. T., “Insecticidal Toxins. Bacillus subtilis and other Gram-positive bacteria biochemistry”, physiology and molecular genetics, pp.953, 1993.
32. Saunders, C. W., B. J. Schmidt, R. L. Mallonee, and M. S. Guyer,
“Secretion of human serum albumin from Bacillus subtilis”, J. Bacteriol,
v169:2917-2925, 1987.
33. Novikov, S., I. Borukhov, and A. Strongin, “Bacillus amyloliquefaciens α
- amylase signal sequence fused in frame with human proinsulin is properly
processed by Bacillus subtilis cell. Biochem. Biophys”, Res. Commun,
169:297-301, 1990.
34. Schein, C. H., K. Kashiwagi, A. Fujijawa, and C. Weissmann, “Secretion of muture interferon alpha-2 and accumulation with a hybrid alpha amylase signal sequence interferon alpha-2 gene”, Bio/Technol, 4:719-725, 1986.
35. He, X. S., Y. T. Shyu, S. Nathoo, S. L Wong, and R. H. Doi, “Construction and use of a Bacillus subtilis mutant deficient in multiple protease genes for the expression of eukaryotic genes”, Ann. N. Y. Acad. Sci, 646:69-77, 1991.
36. Gruss, A., and S. D. Ehrilich, “Insertion of foreign DNA into plasmid from Gram-positive bacteria induce formation high molecular weight plasmid multimers”, J. Bacteriol, 170:1188-1190, 1988.
37. Lovett, L. M., JR., P. E. Love, and R. E. Yasbin, “Competence-specific induction of the Bacillus subtilis recA protein analog: evidence for dual regulation of a recombination protein”, J. Bacteriol. 171:2318-2322, 1989.
38. Stranathan, M. C., K. W. bayles, and R. E. Yasbin. “The nucleotide sequence of the rec E+ gene of Bacillus subtilis”, Nucleic. Acids. Research, 18:4249, 1990.
39. Ceglowski, P., G. Luder, and J. C. Alonso. “Genetic analysis of rec E activities in Bacillus subtilis”, Mol. Gen. Genet, 222:411-445, 1990.
40. 王三郎,應用微生物學,高立圖書公司,台北,民國83年
41. 田蔚城主編,生物技術的發展與應用,九州圖書文物公司,台北,
民國86年
42. 劉國詮主編,生物工程下游技術,曉園出版社,台北,民國85年
43. Yoshida, F., T. Yamane, and K. Nakamoto, “Fed-batch hydrocarbon fermentation with collosidal feed”, Biotech. Bioeng, 15:257-270, 1973.
44. Prit, S, J., Principles of Microbe and Cell Cultivation. Blackwell, Oxford, 1975.
45. Saunders, C. W., B. J. Schmidt, R. L. Mallonee, and M. S. Guyer,
“Secretion of human serum albumin from Bacillus subtilis”, J. Bacteriol,
169:2917-2925, 1987.
46. Whitaker, A., “Fed-batch culture”, Proc. Biochem, 15(4):10-15, 1980.
47. Queener, S. and R. Swarts., Penicillins; Biosynthetic and semisynthetic. In Economic Microbiology, 3. Secondry Products of Metabolism, pp. 35-123. Academic Press, London. 1979.
48. Waki, T., K. Luga, and K. Ichikawa, Production of cellulase in fed-batch culture. In Advances in Biotechnology, 1. Scientific and Engineering Principles. pp. 359-364. Pergamon Press, Toronto, 1982.
49. Rushton, J. H> and Oldahue, J. Y., Chem. Eng. Progress, 49, 161, 1953,
50. Karow, H. O., Bartholomew, W. H. And Sfat, M. R., J.Agric. Fd. Chem, 1, 302, 1953.
51. Yoshida, F., Ind. Eng. Chem, 52, 435, 1960.
52. Nagai, S. and Aiba, S.,“Reassessment of maintenace and energy uncoupling
in the growth of Azotobacter vinelandii.” J. Gen. Microbiol, 73. 531, (1972
53. Nagai,S. and Aiba, S., “ Reassessment of maintenace and energy
uncoupling in the growth of Azotobacter vinelandii “, J. Gen Microbiol, 73,
531,1972.
54. Michael J., et al., Microbiology concepts abd applications, McGRAW-
HILL, New York, 1993.
55. Jianbo C., Arthur A.G., Terry A. K., “A two-gene ABC-type transport
system that extrudes Na+ in Bacillus subtilis is induced by ethanol or
protonophore”, Molecular Microbiology , 23(6), 1107-1120, 1997
56. 熊定宇,「具網狀導流管氣舉式反應器之設計及其在蘇力菌發酵上之應用」
國立青華大學,博士論文,民國84年.
57. Nilsson, J., and S. G. Skognam., “Stabilization of Escherichia coli tryptophane production vectors in continuous culture: A comparison of three different system”, Bio/Technology. 4:901-903, 1986.
58. Bron, S., and E. Luxen, “Segregational instability of pUB110-derived
recombinant plasmids in Bacillus subtilis”, Plasmid, 14:235-239, 1985.
59. Jones, I., S. B. Primrose., A. Robinson, and D. C., “Ellwood. Maintenance of some Col E1-type plasmids in chemostat culture”, Molec. Gen. Genet, 180:579-581, 1980.
60. Noack, D., M. Roth, R. Geuther, and S. Gaspar, “Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 grown in a chemostat”, Mol. Gen. Genet, 184:121-125, 1981.
61. Aiba, S., and J. koizumi., “Effect of temperature on plasmid stability and penicillinase productivity in a transformant of Bacillus stearothermophillus. Biotechnol”, Bioeng, 26:1026-1030, 1984.
62. Pinches, A., M. E. Louw, and T. G. Watson, “Growth, plasmid stability, and α-amylase production in batch fermentations using a recombinant B. subtilis strain”, Biotechnol. Lett, 7(9):621-626, 1985.
63. Margaritis, A., and A. S. Bassi, “Plasmid stability of recombinant DNA microorganisms”, Recombinant DNA Technology and Applications. pp.316-321, 1991.
64. Benjamin J., GENES VI, Oxfurd, New York, 1997.
65. Taku N., Kohji K., Ichizo K., “Selfish behavior of restriction-modification
systems”, Science, vol. 267, 897-9, 1995.
66. Lovett, P. S., and N. P. Ambulos, “Genetic manipulation of Bacillus subtilis”, Handbook of Biotechnology, Vol. 2. Bacilli, pp.115-126, 1989.
67. Schein, C. H., K. Kashiwagi, A. Fujijawa, and C. Weissmann, “Secretion of muture interferon alpha-2 and accumulation with a hybrid alpha amylase signal sequence interferon alpha-2 gene”, Bio/Technol, 4:719-725, 1986.
68. Lee, Y. L., and H. N. Chang, “High cell density culture of a recombinant Escherichia coli producing penicillin in a membrane cell recycle fermentor”, Biotech. Bioeng, 36:330-337, 1990.
69. Nishizawa, Y., Nagai, S. and Aiba, S., “Effect of dissolved oxygen on
electron transport system of Azotobacter vinelandi in glucose-limited and
oxygen limited chemostat cultures.”, J. Gen. Microbiol. 17, 131, 1971.
70. Patricia J., Madhu G., Herbert W. B., “Sequence analysis of the DNA encoding
the Eco RI endonucleasa and methylase”, J. of Biological Chemistry, 2143-51,
1981.

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