臺灣博碩士論文加值系統

利用枯草桿菌(Bacillus subtilis)當作蛋白質生產的宿主細胞具有多項優點；包括其為非病原菌，對於密碼子(codon)無特殊偏好，且具強而有力的分泌系統(secretory system)。特別是最後一項優點，是可將蛋白質分泌至培養基中，因而可便於下游蛋白質的純化與回收，這也是枯草桿菌在基因工程應用上最具吸引力的一項特色。然而枯草桿菌的轉型效率不若大腸桿菌一般便利，因此為增進基因操作的方便性，過去的研究即發展枯草桿菌/大腸桿菌穿梭質體。其中以結合金黃色葡萄球菌(Staphylococcus aureus)pUB110質體和大腸桿菌的pUC系列的質體所建構而成的穿梭載體最為廣泛使用。然而，此類穿梭質體在枯草桿菌中往往發生質體不穩定的現象，其中可區分為分離不穩定(segregational instability)與結構不穩定(structural instability)兩種。過去的研究顯示使用pUB110/pUC結合之穿梭載體在枯草桿菌中極易產生巨分子量DNA(high molecular weight DNA)，以致質體產生結構不穩定的現象。基於此，為了提升穿梭質體在枯草桿菌中的穩定性，本研究建構一種結合質體pUB110和大腸桿菌R6K質體的新型穿梭質體。結果發現該質體在枯草桿菌中可完整維持結構穩定性，並可以穩定且大量生產胞外納豆激酶。為了進一步達到量產高純度納豆激酶之目標，我們以系統分析的方法找尋限制納豆激酶生產的因子，隨後利用回應曲面實驗設計法(Experiment design of response surface methodology)設計最佳培養基配方。配合最適化發酵策略，在實驗室規模發酵槽培養下，重組枯草桿菌可生產500 mg/L的納豆激酶而純度可達98%以上。

Bacillus subtilis has many attractive traits serving as a producer cell for foreign protein production. These include the non-pathogenic nature, non-biased codon usage, fast growth, and an efficient secretory capacity. The latter trait allows the direct export of extracellular proteins into culture medium and thereby greatly simplifying the down stream processing of proteins. This is the most attractive feature of B. subtilis when applied for the industrial use. Owing to the low transformation efficiency of B. subtilis, the B. subtilis-Eschericha coli shuttle vectors have been developed in the past to facilitate its gene manipulation. Most of these shuttle vectors are constructed by the fusion of plasmid pUB110 from Staphylococcus aureus with the pUC series plasmids from E. coli. However, these hybrid plasmids are highly unstable in B. subtilis. Two types of plasmid instability are observed, including segregational and structural instability. In particular, the pUB110/pUC-based shuttle vector was previously shown to produce high molecular weight (HMW) DNAs which is responsible for the occurrence of its structural instability in B. subtilis. In this study, a new shuttle vector was constructed by fusion plasmid pUB110 with plasmid R6K of E. coli. This new vector was found to exhibit full stability and could be used to achieve the high-level production of extracellular nattokinase in B. subtilis. To obtain a large production of nattokinase, the factors limiting the nattokinase production was identified by the method of systematic analysis. With these limiting materials, the response surface methodology was then exploited for the formulation of medium optimized for nattokinase production. Finally, using the optimum medium, the recombinant B. subtilis strain was able to produce nattokinase with 500 mg/L and 98% purity on a lab-fermenter scale.

Chapter 1 Introduction 1
1.1 A brief introduction of nattokinase 1
1.2 The expression system of B. subtilis 2
1.2.1 The secretory system of B. subtilis 2
1.2.2. Signal peptides designate secretory proteins 3
1.2.3. The development of the recombinant B. subtilis 4
1.2.4. Plasmids instability in B. subtilis 4
1.2.5. E. coli-B. subtilis shuttle vectors 5
1.3. Experimental design for improvement of fermentation 6
1.4 Reference 11
Chapter 2 High-level secretory production of nattokinase by B. subtilis with a novel shuttle vector exhibiting full stability 24
2.1 Materials and methods 25
2.1.1 Bacterial strains and culture condition 25
2.1.2 Plasmid construction 25
2.1.3 Analytical methods 27
2.2 Results 28
2.2.1 The pUB110/pUC18-based plasmid exhibiting structural instability 28
2.2.2 Stable production of nattokinase in B. subtilis with the pUB110/R6K-based plasmid 29
2.3 Discussion 31
2.4 Reference 34
Chaper 3 Enhanced production of recombinant nattokinase in B. subtilis by theelimination of limiting factors 43
3.1 Methods 44
3.1.1 Culture condition 44
3.1.2 Plasmid construction 44
3.1.3 Analytical methods 45
3.2 Results 45
3.2.1 Extracellular production of recombinant nattokinase by B. subtilis 45
3.2.2 Identification of amino acids limiting nattokinase production 46
3.2.3 Increased production of nattokinase by metal ions in combination with Glu 47
3.3 Discussion 47
3.4 Reference 49
Chaper 4 Medium optimization for recombinant nattokinase in B. subtilis usingresponse surface methodology 54
4.1 Materials and methods 55
4.1.1 Bacteria and conditions 55
4.1.2 Analytical methods 55
4.1.3 Screening design of nutrients using a Placket-Burman design 55
4.1.4 Medium optimization of nutrients using Box-Behnken design 55
4.1.5 Batch fermentation 56
4.2 Results and discussion 56
4.2.1 Screening medium components using a Plackett-Burman design 56
4.2.2 Optimization of medium composition using a Box-Behnken design 57
4.2.3 Effect of factors response 57
4.2.4 Effect of initial pH during fermentation 58
4.2.5 fermentation of scale-up 58
4.3 Conclusion 58
4.4 Reference 60

Viles-Gonzalez, J. F., Fuster, V., and Badimon, J. J., 2004. Atherothrombosis: a widespread disease with unpredictable and life-threatening consequences. Eur Heart J., 25(14):1197-1207.
2.Sumi, H, Hamada, H, Tsushima, H, Mihara, H, and Muraki, H, 1987. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia, 43(10):1110-1111.
3.Nakamura, T, Yamagata, Y, and Ichishima, E, 1992. Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto). Biosci. Biotechnol. Biochem., 56(11):1869-1871.
4.Sumi, H, Hamada, H, Nakanishi, K, and Hiratani, H, 1990. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematologica, 84(3):139-143.
5.Sumi, H., Nakajima, N., and Mihara, H., 1992. In vitro and in vivo fibrinolytic properties of nattokinase. Thromb Haemost, 89:1267.
6.Fujita, M, Hong, K, Ito, Y, Fujii, R, Kariya, K, and Nishimuro, S, 1995. Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat. Biol. Pharm. Bull., 18(10):1387-1391.
7.Behnke, D., 1992. Protein export and the development of secretion vectors. Biotechnology., 22:143-188.
8.Harwood, C.R., 1992. Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol., 10:247-256.
9.Wong, S.-L., 1995. Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Current Opinion in Biotechnology, 6(5):517-522.
10.Bron, Sierd, Bolhuis, Albert, Tjalsma, Harold, Holsappel, Siger, Venema, Gerard, and van Dijl, Jan Maarten, 1998. Protein secretion and possible roles for multiple signal peptidases for precursor processing in Bacilli. Journal of Biotechnology, 64(1):3-13.
11.Manting, E.H., van der Does, C., Remigy, H., Engel, A, and Driessen, A.J.M., 2000. Sec YEG assembles into a tetramer to form the active protein translocase channel. EMBO J., 19:852-861.
12.Ogura, A., Kakeshita, H., Honda, K., H.Takamatsu, Nakamura, K., and Yamane., K., 1995. srb: a Bacillus subtilis gene encoding a homologue of the alpha-subunit of the mammalian signal recognition particle receptor. DNA Res., 2:95-100.
13.Deuerling, E., Paeslack, B., and Schumann, W., 1995. The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift. J. Bacteriol., 177:4105-4112.
14.van der Does, C., E. H. Manting, A. , Kaufmann, M. Lutz, and Driessen., A. J. M., 1998. Interaction between SecA and SecYEG in micellar solution and formation of the membrane-inserted state. . Biochemistry, 37:201-210.
15.Hirose, I., Sano, K., Shioda, I., Kumano, M., Nakamura, K., and Yamane, K., 2000. Troteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional protein electrophoretic study. Microbiology, 146:65-75.
16.Swaving, J., K.H.M., van Wely, and A.J.M., Driessen, 1999. Host specific functions of preprotein translocase subunits. J. Bacteriol., 181:7021-7027.
17.Schatz, G. and Dobberstein, B., 1996. Common principles of protein translocation across membranes. . Science, 271:1519-1526.
18.Pohlschro¨der, M., Prinz, W. A., Hartmann, E., and Beckwith, J., 1997. Proteintranslocation in the three domains of life: variations on a theme. Cell, 91:563-566.
19.Bolhuis, A., Broekhuizen, C. P., Sorokin, A., Roosmalen, M. L. van, Venema, G., Bron, S., Quax, W. J., and Dijl, J. M. van, 1998. SecDF of Bacillus subtilis, a molecular Siamese twin required for the efficient secretion of proteins. J. Biol. Chem., 273:21217-21224.
20.Dalbey, R.E., Lively, M.O., Bron, S., and van Dijl, J.M., 1997. The chemistry and enzymology of the type I signal peptidases. Protein Sci., 6:1129-1138.
21.Dalbey, R. E. and Heijne., G. von, 1992. Signal peptidases in prokaryotes and eukaryotes: a new protease family. Trends Biochem. Sci., 17:474-478.
22.Bolhuis, A., Sorokin, A., Azevedo, V., Ehrlich, S. D., Braun, P. G., Jong, A. de, Venema, G., Bron, S., and Dijl., J. M. van, 1996 Bacillus subtilis can modulate its capacity and specificity for protein secretion by temporally controlled expression of the sipS gene for signal peptidase I. Mol. Microbiol., 22:605-618.
23.Tjalsma, H., M. A. Noback, S. Bron, G. Venema, K. Yamane, and J. M. van and Dijl., 1997. . Bacillus subtilis contains four closely related type I signal peptidases with overlapping substrate specificities: constitutive and temporally controlled expression of different sip genes. . J. Biol. Chem., 272:25983-25992.
24.Tjalsma, H., Bolhuis, A., Roosmalen, M. L. van, Wiegert, T., Schumann, W., Broekhuizen, C. P., Quax, W., Venema, G., Bron, S., and Dijl., J. M. van, 1998. Functional analysis of the secretory precursor processing machinery of Bacillus subtilis: identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases. Genes Dev., 12:2318-2331.
25.van Dijl, J. M., Jong, A. de, Vehmaanpera¨, J., Venema, G., and Bron, S., 1992. Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases. EMBO J. EMBO J., 11:2819-2828.
26.van Dijl, J. M., Bolhuis, A., Tjalsma, H., Venema, G., and Bron, S., 1998, The signal peptidase SipS. The handbook of proteolytic enzymes. , ed. A.J. Barret, et al. London, England.: Academic Press, Ltd. 451-452.
27.Pra´gai, Z., Tjalsma, H., Bolhuis, A., Dijl, J. M. van, Venema, G., and Bron, S., 1997. The signal peptidase II (lsp) gene of Bacillus subtilis. Microbiology, 143:1327-1333.
28.Tokunaga, M., Tokunaga, H., and Wu, H.C., 1982. Post-translational modification and processing of Escherichia coli prolipoprotein in vitro. Proc. Natl. Acad. Sci. USA, 258:12102-12105.
29.LF, Wang, WT, Hum, NK, Kalyan, SG, Lee, PP, Hung, and RH, Doi, 1989. Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis. Gene, 84:127-133.
30.Pero, J. and Sloma, A., 1993. Proteases. In: Sonenshein, A.L., Bacillus subtilis and other gram-positive bacteria, Biochemistry, physiology, and molecular genetics. Washington, D.C. American Society for Microbiology:939-952.
31.Wu, S.C., Lee, W., Tran, L., and Wong, S.-L., 1991. Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J. Bacteriol., 173:4952-4958.
32.Ye, R, Yang, L. P., and Wong, S.-L., 1996. Construction of protease-deficient Bacillus subtilis strains for expression studies: inactivation of seven extracellular proteases and the intracellular LonA protease. Proc. Internat. Symp Recent Adv. Bioindustry:160-169.
33.Antelmann, Haike, Yamamoto, Hiroki, Sekiguchi, Junichi, and Hecker, Michael, 2002. Stabilization of cell wall proteins inBacillus subtilis: A proteomic approach. Proteomics, 2(5):591-602.
34.Wu, S.-C., Ye, R., Wu, X.-C., Ng, S.-C., and Wong, S.-L., 1998. Enhanced secretory production of a single-chain antibody fragment from Bacillus subtilis by coproduction of molecular chaperones. J. Bacteriol., 180:2830-2835.
35.Bron, S. and Luxen, E., 1985. Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis. Plasmid, 18:235-244.
36.Bron, S., Haima, P., van Belkum, M., and Luxen, E., 1988. Segretgational plasmid instability in Bacillus subtilis. In Genetics and Biotechnology of Bacilli, 2:305-309.
37.Bron, S., Luxen, E., and Swart, P., 1988. Instability of recombiant pUB110 plasmids in Bacillus subtilis: plasmid-encoded stability function and effects of DNA inserts. Plasmid, 19:231-241.
38.Bron, S, W., M., Holsappel, S, and Haima, P. , 1991. Plasmid instability and molecular cloning in Bacillus subtilis. Res. Microbiol., 142:875-833.
39.Gruss, A. and Ehrlich, S. D., 1989. The family of highly interrelated single-strand deoxyribonucleic acid plasmids. Microbiol. Rev., 53:231-241.
40.Gruss, A. and Ehrlich, S. D., 1988. Insertion of foreign DNA into plasmids from Gram-positive bacteria induces formation of high-molecular weight plasmids multimers. J. Bacteriol., 170:1183-1190.
41.Viret, J-F. and Alonso, J. C., 1988. A DNA sequence outside the pUB110 mimimal replicon is required for normal replication in Bacillus subtilis. Nucleic Acids Res., 16:4389-4406.
42.Ehrlich, S. D., Noirot, P., Petit, M. A., Janniere, L., B., Michel, and te Riele, H., 1986, Structural instability of Bacillus subtilis plasmids. In Genetic Engineering, ed. J.K. Setlow and A. Hollaender. Vol. 8: Plenum Publishing Corp.: New York.
43.Ehrlich, S. D., 1989, Illegitimate recombination in bacteria. In mobile DNA, ed. D. Berg and M. Howe: American Society for microbiology: Washington DC. 797-829.
44.Cordes, C., Meima, R., Twiest, B., Kazemier, B., Venema, G., van Dijl, J. M., and Bron, S., 1996. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis. J Bacteriol.
45.Meima, R., Haan, G. J., Venema, G., Bron, S., and de Jong, S., 1998. Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I. Nucleic Acids Res., 26:2366-2373.
46.Janniere, L, Bruand, C, and Ehrlich, S. D., 1990. Structurally stable Bacillus subtilis cloning vectors. Gene, 87:53-61.
47.Leonhardt, Heinrich and Alonso, Juan C., 1991. Parameters affecting plasmid stability in Bacillus subtilis. Gene, 103:107-111.
48.Vos, Willem M. and Venema, Gerard, 1981. Fate of plasmid DNA in transformation of Bacillus subtilis protoplasts. Molecular Genetics and Genomics, 182(1):39-43.
49.Smith, H., Bron, S., J., Van Ee., and G., Venema, 1987. Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis. J Bacteriol., 169:3321-3328.
50.Smith, Hilde, de Jong, Anne, Bron, Sierd, and Venema, Gerard, 1988. Characterization of signal-sequence-coding regions selected from the Bacillus subtilis chromosome. Gene, 70(2):351-361.
51.Smith, H., De Jong, Anne, can Dijl, J. M., Bron, S., and Venema, G., 1989, Protein secretion in Bacillus subtilis characterization of randeomly selected signal-sequence-coding regions. In Genetic Transormation and Expression, ed. L.O. Butler, C.R. Harwood, and B.E.B. Moseley: Intercept Ltd: Andover, UK. 519-527.
52.Janniere, L. and Ehrlich, S. D., 1987. Recombination between short repeated sequences is more frequent in plasmids than in the chromosome of Bacillus subtilis. Molecular Genetics and Genomics, 210(1):116-121.
53.Peeters, Ben P. H., Boer, Jan H., Bron, Sierd, and Venema, Gerard, 1988. Structural plasmid instability in Bacillus subtilis: Effect of direct and inverted repeats. Molecular Genetics and Genomics, 212(3):450-458.
54.Kennedv, M. J., Reader, S. L., and Davies, R. J., 1994. The kinetics of developing fermentation media. Process biochem., 29:529-534.
55.Weuster-Botz, Dirk, 2000. Experimental design for fermentation media development: Statistical design or global random search? Journal of Bioscience and Bioengineering, 90(5):473-483.
56.Plackett, R.L., and Burman, J. P., 1946. The design of optimum multifactorial. Biometrika, 33:305-325.
57.S. K. Ahuja, Ferreira, G. M., and Moreira, A. R., 2004. Application of Plackett-Burman design and response surface methodology to achieve exponential growth for aggregated shipworm bacterium. Biotechnol. Bioeng., 85(6):666-675.
58.Naveena, B. J., Altaf, Md, Bhadriah, K., and Reddy, G., 2005. Selection of medium components by Plackett-Burman design for production of (+) lactic acid by Lactobacillus amylophilus GV6 in SSF using wheat bran. Bioresour. Technol., 96(4):485-490.
59.Box, GEP and Behnken, D. W., 1960. Some new three level designs for the study of quantitative variables. Technometrics, 2:455-475.
60.Francis, F, Sabu, A, Nampoothiri, K. M., Ramachandran, S., Ghosh, S., Szakacs, G., and Pandey, A., 2003. Use of response surface methodology for optimizing process parameters for the production of a-amylase by Aspergillus oryzae. Biochem. Eng. J., 15:107-115.
61.Li, W, Zhou, X, and Lu, P., 2004. Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Res Microbiol., 155(8):605-610.
62.Harwood, C. R. and Cutting, S. M., 1990, Molecular biological methods for Bacillus Plasmids, ed. S. Bron: John Wiley & Sons Ltd 118.
63.Keggins, K. M., Lovett, P. S., and Duvall, E. J., 1978. Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB110. Proc Natl Acad Sci U S A., 75(3):1423-1427.
64.Ye, R., Kim, J. H., Kim, B.G., Szarka, S., Sihota, E., and Wong, S. L., 1999. High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis. Biotechnol Bioeng., 62(1):87-96.
65.Gemma, Serrano Heras, Salas, Margarita, and Bravo, Alicia, 2005. A new plasmid vector for regulated gene expression in Bacillus subtilis. Plasmid, 54(3):278-282.
66.Wu, S. C. and Wong, S. L., 1999. Development of improved pUB110-based vectors for expression and secretion studies in Bacillus subtilis. J Biotechnol., 72(1-2):185-195.
67.Bron, S., Luxen, E. , and Swart, P., 1988. Instability of recombinant pUB110 plasmids in Bacillus subtilis: plasmid-encoded stability function and effects of DNA inserts. Plasmid, 19(3):231-241.
68.Kurland, C. G. and Dong, H, 1996. Bacterial growth inhibition by overproduction of protein. Mol Microbiol., 21(1):1-4.
69.Gill, R. T., Valdes, J. J., and Bentley, W. E., 2000. A Comparative Study of Global Stress Gene Regulation in Response to Overexpression of Recombinant Proteins in Escherichia coli. Metabolic Engineering, 2(3):178-189.
70.Wang, Zei Wen, Chen, Yuhsin, and Chao, Yun-Peng, 2006. Enhancement of recombinant protein production in Escherichia coli by coproduction of aspartase. Journal of Biotechnology, In Press, Corrected Proof.
71.Ramirez, D. M. and Bentley, W. E., 1993. Enhancement of recombinant protein synthesis and stability via coordinated amino acid addition. Biotechnol. Bioeng., 41:557-565.
72.Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Oh, H., Kwon, I., and Lee, S., 1996. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Appl. Environ. Microbiol., 62(7):2482-2488.
73.KIM, Seung-Ho and CHOI, Nack-Shick 2000. Purification and Characterization of Subtilisin DJ-4 Secreted by Bacillus sp. Strain DJ-4 Screened from Doen-Jang. Biosci. Biotechnol. Biochem., 64(8):1722-1725.
74.Jeong, Yong Kee, Park, Jeong Uck, Baek, Hyun, Park, Sung Hoon, Kong, In Soo, Kim, Dong Wan, and Joo, Woo Hong, 2001. Purification and biochemical characterization of a fibrinolytic enzymefrom Bacillus subtilis BK-17. World J. Microbiol. Biotechnol., 17:89-92.
75.Chang, C. T., Fan, M. H., Kuo, F. C., and Sung, H. Y., 2000. Potent Fibrinolytic Enzyme from a Mutant of Bacillus subtilis IMR-NK1. J. Agric. Food Chem., 48(8):3210-3216.
76.Ko, Ju Ho, Yan, Jun Peng, Zhu, Lei, and Qi, Yi Peng, 2004. Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 137(1):65-74.
77.van Wely, K.H.M., 2000, The general protein secretion pathway of Bacilius subtilis. Vol. 1. 27.