臺灣博碩士論文加值系統

利用基因重組技術 (recombinant DNA technology) 於大腸桿菌 (Escherichia coli；E. coli) 中生產 penicillin acylase (PAC)，過量生產常常被限制於細胞間質的處理中 (periplasmic processing) 中，而內涵體 (inclusion body) 在細胞間質 (periplasm) 中大量生成。對於蛋白之過量表達所需要增加的不只是轉錄 (transcription)、轉譯 (translation) 等效率，我們需要更進一步的考慮酵素過量表達時，在於基因表現步驟 (如：轉錄、轉譯、轉譯後修飾 (post-translational processing) 等步驟) 以及蛋白摺疊 (folding) 步驟，於宿主細胞中應保持整體蛋白合成通量 (flux) 的平衡，以避免聚胜肽 (polypeptide) 中間體的累積。在此篇研究中，我們進行 PAC 酵素的胞外生產，於生產 PAC 酵素時共同表現 brp 基因以生產 bacteriocin release protein (BRP) 蛋白和 pac基因，其釋放程度表現可以考慮到不同的條件，包括：宿主細胞 (host)、誘導劑 (inducer) 濃度以及誘導 (induction) 時機，以達一最佳化的操作值，另外釋放 PAC 酵素對內涵體生成的影響也被詳細的研究，內涵體會隨著 brp 基因的表現增加而上升，其形成並非具有活性的PAC蛋白過度累積所致。在第二部分的研究中，我們論證了生產 PAC 酵素時共表現細胞間質中的蛋白切割酵素 (protease) DegP 以增加 PAC 酵素生產之可行性，經由共表現 DegP酵素，細胞間質中的內涵體很明顯的減少，而 PAC 蛋白活性也明顯的提高了許多，在大腸桿菌中雖然 DegP並非生產 PAC 時之必要酵素，但由結果顯示 DegP 酵素可能參與了細胞間質中的處理程序，以生產具有活性的 PAC酵素，PAC酵素內涵體生成的首要原因應為細胞間質中先驅物被限制於切割處理效率。基於以上結果，限制 PAC 酵素生成的原因被鑑定出來。

To produce penicillin acylase (PAC) by recombinant DNA technology in Escherichia coli (E. coli), the overproduction was often limited by periplasmic processing and inclusion bodies were formed at a large amount in the periplasm. This raises an important issue that, for the overproduction of recombinant proteins, not only the transcriptional and/or translational efficiency has to be increased but also a ‘balanced’ protein synthesis flux throughout various gene expression (i.e., transcription, translation, and posttranslational processing) and folding steps should be properly maintained to avoid the accumulation of polypeptide intermediates. In this study, we demonstrated the extracellular production of penicillin acylase (PAC) by coexpression of the brp gene encoding bacteriocin release protein (BRP) and the pac gene. The performance for the production and release of PAC was optimized by taking several culture parameters, including host, inducer concentration, and induction timing for brp expression, into consideration. The effect of PAC release on inclusion body formation was also investigated. It was observed that the amount of inclusion bodies was significantly increased by brp expression. The formation of inclusion bodies was not caused by over-accumulation of active PAC. In the second part of this study, we demonstrated the enhancement of recombinant penicillin acylase (PAC) production in E. coli by increasing the intracellular concentration of the periplasmic protease DegP. The amount of these periplasmic inclusion bodies was significantly reduced and PAC activity was significantly increased upon coexpression of DegP. The results suggest that DegP could in vivo assist the periplasmic processing though the enzyme was shown to be not absolutely required for the formation of active PAC in E. coli. The formation of PAC inclusion bodies should be primarily caused by limitation of the proteolysis on periplasmic PAC precursors. In addition, the steps limiting the production of PAC were identified.

目錄
中文摘要……………………………………………….……………….Ⅰ
Abstract………...……………………………………………..…………Ⅱ
目錄…………………………………………………….……………….Ⅳ
表目錄………………………………………………………….……….Ⅷ
圖目錄…………………………………………..……………………....Ⅸ
第一章 緒言……………………………………………………………..1
1.1 前言…………………………………………………………….1
1.2盤尼西林醯胺分解酵素之用途….…………………………….2
1.3 PAC酵素的來源以及種類………………..…………………….3
1.4 PAC酵素成熟路徑………………….………………….…..…..4
1.5高量表達重組蛋白的策略……………….....…………..………6
1.6改善 pac 基因表現之策略……………………..………...……7
1.7研究方向…………...……………………………...…………….9
第二章 實驗方法………………………………………………………13
2.1 菌種的處理…………………………………………………...13
2.1.1 本研究所使用菌種種類……………………………….13
2.1.2 培養基的製備………………………………………….13
2.1.3 抗生素的配製………………………………………….13
2.1.4 菌種的儲存與活化…………………………………….13
2.1.5 接菌 (inoculum) 的方法與細胞濃度的測量………...14
2.2 處理DNA技術……………………………………….………15
2.2.1 本研究所使用質體DNA種類……………….…..……15
2.2.2 質體 DNA的萃取……………………….……..….…..15
2.2.3 可轉形性細胞 (competent cell) 的製備………...……16
2.2.4 轉形 (transformation)………………………………….16
2.3 菌種培養…………………………..…………………………...18
2.3.1 批次菌種醱酵培養…………………………….………18
2.3.2 生化反應器之醱酵培養……………………………….18
2.4酵素活性之定量分析…………..……………….……...……...19
2.4.1 PAC 酵素活性分析原理………………………….…...19
2.4.1.1 酵素樣品之製備……………………………………..19
2.4.1.2 酵素反應.……………….……………………………20
2.4.1.3 呈色反應…………………….……………………….20
2.4.2 b-半乳糖苷酶 (b-galactosidase；b-gal) 分析…….…...20
2.5 蛋白質SDS-PAGE分析法及免疫分析 (Immunological analysis)……………………………………………...….……21
2.6 實驗藥品……………………………………………………...22
2.6.1 培養基部分…………………………………….………22
2.6.2 轉形部分……………………………………….………23
2.6.3 酵素分析部分…………………………………….……23
2.6.4 b-半乳糖苷酶分析部分…....…………………………..24
2.6.5 蛋白質電泳部分…………………………………….…24
2.6.6 免疫分析部分………………………………….………24
2.7 實驗設備………………………………………………….…..25
第三章 結果與討論………………………………………….…….…..26
3.1在於生產 PAC酵素時共表現 BRP蛋白……….………..…26
3.3.1共表現 BRP 蛋白對於 PAC 酵素活性以及釋放之影響………………………………………………………26
3.1.2 延遲 brp 基因誘導時機對於細胞生理狀況、PAC 酵素活性以及釋放之影響…………………………………28
3.1.3共表現 BRP 蛋白對於內涵體生成之影響…..……….29
3.2 在於生產 PAC 酵素時共表現 DegP酵素……………….…30
3.2.1 共表現 DegP 酵素對不同宿主/質體組合於 PAC 酵素活性以及內涵體之影響……………..……………..30
3.2.2 DegP 酵素在於生產 PAC 酵素時可能佔有之地位…33
3.3 過量生產 PAC 酵素之限制步驟…..………………………..35
第四章 結論與展望……………………………………………………38
參考文獻………………………………………………………………..56
表目錄
表一、本研究使用菌種列表…………………………………………...14
表二、本研究使用培養基的組成……………………………………...15
表三、本研究使用質體 DNA 列表…………….…………………….17
表四、蛋白質 SDS-PAGE 凝膠的製備………….………………….. 22
表五、不具生產 DegP 酵素能力之宿主細胞生產 PAC 酵素之情形………………………………………………………………..34
圖目錄
圖一、利用基因重組技術大量生產蛋白質……………………………..1
圖二、酵素法生產6-APA……………………………………………….3
圖三、在大腸桿菌 ATCC11105 中生產 PAC 酵素的途徑……….…..5
圖四、以 HB101 為宿主細胞；pTrcKnPAC2092 為 PAC 表現質體；pSW1為 BRP 表現質體，利用生化反應器及 MAPC 培養基培養，探討生產 BRP對於 PAC 生產及釋放之影響。…………………………………………………………..….40
圖五、以 HB101 為宿主細胞；pTrcKnPAC2092 為PAC表現質體；pSW1為 BRP 表現質體，利用生化反應器及 MAPC 培養基培養，以西方墨漬法之不可溶蛋白分析，探討生產 BRP 對於 PAC內涵體之影響。…………………………………….……..42
圖六、以 MDDP7 為宿主細胞；pTrcKnPAC2092 為 PAC 表現質體；pSW1為 BRP 表現質體，利用生化反應器及 MAPC 培養基培養，探討生產 BRP 對於 PAC生產及釋放之影響。……………….…………………………...……………….43
圖七、以 MDDP7 為宿主細胞；pTrcKnPAC2092 為 PAC 表現質體；pSW1為 BRP表現質體，利用生化反應器及 MAPC 培養基培養，以西方墨漬法之不可溶蛋白分析，探討生產 BRP 對於 PAC 內涵體之影響。……………………...…………….……..45
圖八、以 MDDP7 為宿主細胞；pTrcKnPAC2092 為 PAC 表現質體；pSW1為 BRP表現質體，利用生化反應器及 MAPC 培養基培養，探討延遲生產 BRP對於 PAC生產及釋放之影響。…………………….………………………………………..46
圖九、以 MDDP7 為宿主細胞；pTrcKnPAC2092 為PAC 表現質體；pSW1為 BRP 表現質體，利用生化反應器及 MAPC 培養基培養，以西方墨漬法不可溶蛋白分析，探討探討延遲生產 BRP對 PAC生成之影響。……………………..…………………..48
圖十、以 HB101 為宿主細胞；pTrcKnPAC2092 為 PAC 表現質體；pKS12為 DegP 表現質體，利用生化反應器及 MAPC 培養基培養，探討 DegP 對於 PAC之影響。.……………..……49
圖十一、以 MDDP7 為宿主細胞；pTrcKnPAC2092 為PAC表現質體；pKS12為 DegP 表現質體，利用生化反應器及 MAPC 培養基培養，探討 DegP對於 PAC生產之影響。…………..…50
圖十二、不同宿主細胞內含質體 pTrcKnPAC2092 共表現 DegP ，以西方墨漬法之不可溶蛋白分析。………………...……………51
圖十三、以 MDDP7 為宿主細胞；pCLL2092 為PAC表現質體；pKS12為 DegP 表現質體，利用生化反應器及 MAPC 培養基培養，探討 DegP 對於 PAC生產之影響。………………….…52
圖十四、以 MDDP7 為宿主細胞；pTrcKnPAC3221p 為PAC表現質體；pKS12為 DegP 表現質體，利用生化反應器及 MAPC 培養基培養，探討 DegP 對於 PAC生產之影響。……………53
圖十五、以 MDDP7 為宿主細胞；pTrcKnPAC2092 為PAC表現質體；pKS12為 DegP 表現質體，利用生化反應器及 MAPC 培養基培養，探討 DegP 對於過量 PAC生產之影響。……….54
圖十六、MDDP7內含質體 pTrcKnPAC3221p 共表現 DegP以及MDDP7內含質體將pTrcKnPAC2902 過度誘導，對於內涵體生成情形。……………………………………………...…...….55

1. Makrides SC., Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 1996, 60, 512-538.
2. Shewale, J.G. and Sivaraman, H., Penicillin acylase: Enzyme production and its application in the manufacture of 6-APA. Process Biochem. 1989, 24, 146-154.
3. Shewale, J.G., Deshpande, B.S., Sudhakaran, V.K. and Ambedkar, S.S., Penicillin acylases: Applications and potentials. Process Biochem. 1990, 25, 97-103.
4. Valle, F., Balbas, P., Merino, E. and Bolivar, F., The role of penicillin amidases in nature and in industry. Trends Biochem. Sci. 1991, 16, 36-40.
5. Mayer, H., Collins, J. and Wagner, F., Cloning of the penicillin G acylase gene of Escherichia coli ATCC 11105 on multicopy plasmids. In Plasmids of medical, environmental, and commercial importance, Timmis, K. N., Puhler, A., Ed., Elsevier/North-Holland Biomedical Press: Amsterdam 1979, pp 459-470.
6. Garcia, J. L. and Buesa, J. M., An improved method to clone penicillin acylase genes: Cloning and expression in Escherichia coli of penicillin G acylase from Kluyvera citrophila. J. Biotechnol. 1986, 3, 187-195.
7. Daumy, G.O., Williams, J.A., McColl, A.S., Zuzel, T.J. and Danley, D., Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli. J. Bacteriol. 1986, 168, 431-433.
8. Meevootisom, V. and Saunders, J.R., Cloning and expression of penicillin acylase genes from overproducing strains of Escherichia coli and Bacillus megaterium. Appl. Microbiol. Biotechnol. 1987, 25, 372-378.
9. Konstantinovic, M., Marjanovic, N., Ljubijankic, G. and Glisin, V., The penicillin amidase of Arthrobacter viscosus (ATCC 15294). Gene 1994, 143, 79-83.
10. Kang, J.H., Hwang, Y. and Yoo, O.J., Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis. J. Biotechnol. 1991, 17, 99-108.
11. Ohashi, H., Katsuta, Y., Nagashima, M., Kamei, T. and Yano, M., Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis. Appl. Environ. Microbiol. 1989, 55, 1351-1356.
12. Alvaro, G., Fernandez-Lafuente, R., Rosell, C.M., Blanco, R.M., Garcia-Lopez, J.L. and Guisan, J.M., Penicillin G acylase from Kluyvera citrophila: New choice as industrial enzyme. Biotechnol. Lett. 1992, 14, 285-290.
13. Daumy, G.O., Danley, D. and McColl, A.S., Role of protein subunits in Proteus rettgeri penicillin G acylase. J. Bacteriol. 1985, 163, 1279-1281.
14. del Rio, G., Rodriguez, M.-E., Munguia, M.-E., Lopez-Munguia, A. and Soberon, X.., Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH. Biotechnol. Bioeng. 1995, 48, 141-148.
15. Erarslan, A., Terzi, I. Guray, A., and Bermek, E., Purification and kinetics of penicillin G acylase from a mutant strain of Escherichia coli ATCC 11105. J. Chem. Tech. Biotechnol. 1991, 51, 27-40.
16. Forney, L.J., Wong, D.C. L. and Ferber, D.M., Selection of amidases with novel substrate specificities from penicillin amidase of Escherichia coli. Appl. Environ. Microbiol. 1989, 55, 2550-2555.
17. Niersbach, H., Tischer, W., Weber, M., Wedekind, F. and Plapp, R., Isolation and mapping of a mutant penicillin G acylase with altered substrate specificity from Escherichia coli. Biotechnol. Lett. 1995, 17, 19-24.
18. Williams, J.A. and Zuzel, T.J., Penicillin acylase (E.C.3.4.11) substrate specificity. Modification by in vitro mutagenesis. J. Cell. Biochem. 1985, (Suppl.) 9B, 99.
19. Sobotkova, L., Stepanek, V., Plhackova, K. and Kyslik, P., Cloning of penicillin G acylase-encoding gene from Escherichia coli high-producing strain RE3. Biotechnol. Lett. 1995, 17, 723-728.
20. Sobotkova, L., Stepanek, V., Plhackova, K. and Kyslik, P., Development of a high-expression system for penicillin G acylase based on the recombinant Escherichia coli strain RE3(pKA18). Enzyme Microb. Technol. 1996, 19, 389-397.
21. Yee, L. and Blanch, H.W., Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli. Biotechnol. Bioeng. 1993, 41, 781-790.
22. Kane, J.F., Hartley, D.L., Formation of recombinant protein inclusion bodies in Escherichia coli. Trends Biotechnol. 1988, 6, 95-101.
23. Buchner, J., Pastan, I., Brinkmann, U., A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal. Biochem. 1992, 205, 263-270.
24. Hsih, M.H., Kuo, J.C., Tsai, H.J., Optimization of the solubilization and renaturation of fish growth hormone produced by Escherichia coli. Appl. Microbiol. Biotechnol. 1997, 48, 66-72.
25. Sizmann, D., Keilmann, C. and Bock, A., Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105. Eur. J. Biochem. 1990, 192, 143-151.
26. Keilmann, C., Wanner, G. and Bock, A., Molecular basis of the exclusive low-temperature synthesis of an enzyme in E. coli: Penicillin acylase. Biol. Chem. Hoppe-Seyler 1993, 374, 983-992.
27. Merino, E., Balbas, P., Recillas, F., Becerril, B., Valle, F. and Bolivar, F., Carbon regulation and the role in nature of the Escherichia coli penicillin acylase (pac) gene. Mol. Microbiol. 1992, 6, 2175-2182.
28. Chou, C. P., Yu, C.-C., Tseng, J.-H., Lin, M.-I. and Lin, H.-K., Genetic manipulation to identify limiting steps and develop strategies for high-level expression of penicillin acylase in Escherichia coli. Biotechnol. Bioeng. 1999, 63, 263-272.
29. Chou, C. P., Yu, C.-C., Lin, W.-J., Kuo, B.-Y. and Wang, W.-C., Novel strategy for efficient screening and construction of host/vector systems to overproduce penicillin acylase in Escherichia coli. Biotechnol. Bioeng. 1999, 65, 219-226.
30. Chou, C.P. Kuo, B.-Y. and Lin., W.-J., Optimization of the Host/Vector System and Culture Conditions for Production of Penicillin Acylase in Escherichia coli. Journal of Bioscience and Bioengineering 1999, 88, 160-167.
31. Castanié, M.-P., Bergès, H., Oreglia, J., Prère, M.-F. and Fayet, O., A Set of pBR322-Compatible Plasmids Allowing the Testing of Chaperone-Assisted Folding of Proteins Overexpressed in Escherichia coli. Analytical Biochemistry 1997, 254, 150-152.
32. Hayhurst, A. and Harris, W.J., Escherichia coli Skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments. Protein Express. Purif. 1999, 15, 336-343.
33. Chou, C.P., Tseng, J.-H., Kuo, B.-Y., Lai, K.-M., Lin, M.-I. and Lin, H.-K. Effect of SecB chaperone on production of periplasmic penicillin acylase in Escherichia coli. Biotechnology Progress 1999, 15, 439-445.
34. van der Wal, F.J., ten Hagen-Jongman, C.M., Oudega, B. and Luirink, J., Optimization of bacteriocin-release-protein-induced protein release by Escherichia coli: Extracellular production of the periplasmic molecular chaperone FaeE. Appl. Microbiol. Biotechnol. 1995, 44, 459-465.34.
35. Kasche, V., Lummer, K., Nurk, A., Piotraschke, E., Rieks, A., Stoeva, S. and Voelter, W., Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli. Biochim. Biophys. Acta 1999, 1433, 76-86.
36. van der Wal, F.J., Luirink, J. and Oudega, B., Bacteriocin release protein: mode of action, structure, and biotechnological application. FEMS Microbiol. Rev. 1995, 17, 381-399.
37. Strauch, K.L., Johnson, K. and Beckwith J., Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature. J. Bacteriol. 1989, 171, 2689-2696.
38. Stader, J.A. and Silhavy, T.J., Engineering Escherichia coli to secrete heterologous gene products. Methods Enzymol. 1990, 185, 166-187.
39. Boyer, H.W., Roulland-Dussoix, D., A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 1969, 41, 459-472.
40. Chung, C.T. and Miller, R.H., Preparation and storage of competent Escherichia coli cells. Methods Enzymol. 1993, 218, 621-627.
41. Gang, D.M. and Shaikh, K., Regulation of penicillin acylase in Escherichia coli by cyclic AMP. Biochim. Biophys. Acta 1976, 425, 110-114.
42. Balasingham, K., Warburton, D., Dunnill, P. and Lilly, M.D., The isolation and kinetics of penicillin amidase from Escherichia coli. Biochim. Biophys. Acta 1972, 276, 250-256.
43. Miller, J. H., Experiments in molecular genetics; Cold Spring Harbor Laboratory: Cold Spring Harbor 1972.
44. Towbin, H., Staehelin, T. and Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nat. Acad. Sci. USA 1979, 76, 4350-4354.
45. Chou, C.P., Tseng, J.-H., Lin, M.-I., Lin, H.-K. and Yu, C.-C., Manipulation of carbon assimilation with respect to expression of the pac gene for improving production of penicillin acylase in Escherichia coli. J. Biotechnol. 1999, 69, 27-38.
46. Lindsay, C.D., Pain, R.H., The folding and solution conformation of penicillin G acylase. Eur. J. Biochem. 1990, 192, 133-141.
47. Lindsay, C.D. and Pain, R.H., Refolding and assembly of penicillin acylase, an enzyme composed of two polypeptide chains that result from proteolytic activation. Biochemistry-USA 1991, 30, 9034-9040.
48. Scherrer, S., Robas, N., Zouheiry, H., Branlant, G. and Branlant C., Periplasmic aggregation limits the proteolytic maturation of the Escherichia coli Penicillin G amidase Precursor polypeptide. Appl. Microbiol. Biotechnol. 1994, 42, 85-91.
49. Sriubolmas, N., Panbangred, W., Sriurairatana, S. and Meevootisom, V., Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase. Appl. Microbiol. Biotechnol. 1997, 47, 373-378.
50. Enfors S.-O., Control of in vivo proteolsis in the production of recombinant proteins. Trends Biotechnol. 1992, 10, 310-315.
51. Gottesman S., Minimizing proteolysis in Escherichia coli: Genetic solutions. Methods Enzymol. 1990, 185, 119-129.
52. Meerman, H.J. and Georgoiu, G., Construction and characterization of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins. Bio/Technology 1994, 12, 1107-1110.