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研究生: 吳明哲
Ming-Zhe Wu
論文名稱: 以枯草芽孢桿菌外泌生產之自組裝生物催化劑解聚合聚對苯二甲酸乙二酯
Depolymerization of Polyethylene Terephthalate by Secretory Production of Self-assembly Biocatalysts in Bacillus subtilis
指導教授: 陳秀美
Hsiu-Mei Chen
蔡伸隆
Shen-Long Tsai
口試委員: 李振綱
Cheng-Kang Lee
王勝仕
Steven S.-S. Wang
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2023
畢業學年度: 111
語文別: 中文
論文頁數: 104
中文關鍵詞: 枯草芽孢桿菌分泌生產生物分解PET水解酶疏水蛋白
外文關鍵詞: B. subtilis, Secretion, Biodegradation, PET hydrolase, Hydrophobin
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碩士學位論文指導教授推薦書 I 碩士學位考試委員審定書 II 摘要 III ABSTRACT IV 致謝 VI 目錄 VII 圖目錄 X 表目錄 XII LIST OF ABBREVIATIONS XIII 第一章 緒論 1 1.1 研究背景 1 1.2 研究動機與目的 2 1.3 研究內容 3 第二章 文獻回顧 4 2.1 聚對苯二甲酸乙二酯 (Polyethylene terephthalate, PET) 之生物分解 4 2.2 枯草芽孢桿菌 (Bacillus subtillis) 7 2.3 細菌分泌系統 (Bacterial secretion system) 8 2.4 疏水蛋白 (Hydrophobin) 13 2.5 SpyTag-SpyCatcher接合系統 14 第三章 實驗材料與方法 16 3.1 實驗藥品與儀器 16 3.1.1 儀器設備 16 3.1.2 實驗藥品 17 3.1.3 菌種與質體 18 3.2 實驗方法 19 3.2.1 基因轉殖技術 19 3.2.2 蛋白質表達與純化 31 3.2.3 蛋白質分析方法 34 3.2.4 外泌重組蛋白質之功能性與活性測試 40 第四章 結果與討論 46 4.1 質體建構 46 4.1.1 pMK-P43m-PETase-Gln-SpyCat之質體建構 46 4.1.2 pMK-P43m-LipB-PETase-Gln-SpyCat之質體建構 49 4.1.3 pBE-S-aprE-PETase-Gln-SpyCat之質體建構 51 4.1.4 pBE-S-aprE-MHETase-SpyTag之質體建構 54 4.1.5 pBE-S-aprE-SpyCat-(G4S)3-HGFI之質體建構 56 4.1.6 pBE-S-aprE-SpyCat-(G4S)3-HGFI-(G4S)3-SpyCat之質體建構 58 4.1.7 pBE-S-aprE-SpyTag-(G4S)3-HGFI之質體建構 60 4.1.8 pBE-S-SpyTag-(G4S)3-HGFI-(G4S)3-SpyTag之質體建構 62 4.2 外泌重組蛋白質之純化與表達 64 4.2.1 外泌重組蛋白質之超濾膜濃縮純化處理與表達結果 64 4.2.2 外泌重組蛋白質之硫酸銨沉澱純化處理與表達結果 66 4.3 SpyTag-SpyCatcher系統之接合測試 68 4.3.1 外泌重組蛋白質之超濾膜濃縮純化處理與接合測試結果 68 4.3.2 外泌重組蛋白質之硫酸銨沉澱純化處理與接合測試結果 70 4.4 外泌重組蛋白質之活性測試 71 4.4.1 PETase-SpyCatcher之酵素活性測試 71 4.4.2 MHETase-SpyTag之酵素活性測試 73 4.4.3 外泌重組蛋白質之比例對於PET之生物分解的影響 75 第五章 結論 80 參考文獻 81 附錄 86

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(10) Müller, R.-J.; Kleeberg, I.; Deckwer, W.-D. Biodegradation of polyesters containing aromatic constituents. Journal of Biotechnology 2001, 86 (2), 87-95. DOI: https://doi.org/10.1016/S0168-1656(00)00407-7.
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(12) Maurya, A.; Bhattacharya, A.; Khare, S. K. Enzymatic Remediation of Polyethylene Terephthalate (PET)–Based Polymers for Effective Management of Plastic Wastes: An Overview. Frontiers in Bioengineering and Biotechnology 2020, 8, Review. DOI: 10.3389/fbioe.2020.602325.
(13) Yoshida, S.; Hiraga, K.; Takehana, T.; Taniguchi, I.; Yamaji, H.; Maeda, Y.; Toyohara, K.; Miyamoto, K.; Kimura, Y.; Oda, K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 2016, 351 (6278), 1196-1199. DOI: doi:10.1126/science.aad6359.
(14) Chen, C.-C.; Han, X.; Ko, T.-P.; Liu, W.; Guo, R.-T. Structural studies reveal the molecular mechanism of PETase. The FEBS Journal 2018, 285 (20), 3717-3723. DOI: https://doi.org/10.1111/febs.14612.
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(16) Son, H. F.; Joo, S.; Seo, H.; Sagong, H.-Y.; Lee, S. H.; Hong, H.; Kim, K.-J. Structural bioinformatics-based protein engineering of thermo-stable PETase from Ideonella sakaiensis. Enzyme and Microbial Technology 2020, 141, 109656. DOI: https://doi.org/10.1016/j.enzmictec.2020.109656.
(17) Seo, H.; Kim, S.; Son, H. F.; Sagong, H.-Y.; Joo, S.; Kim, K.-J. Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli. Biochemical and Biophysical Research Communications 2019, 508 (1), 250-255. DOI: https://doi.org/10.1016/j.bbrc.2018.11.087.
(18) Chen, Z.; Wang, Y.; Cheng, Y.; Wang, X.; Tong, S.; Yang, H.; Wang, Z. Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase. Science of The Total Environment 2020, 709, 136138. DOI: https://doi.org/10.1016/j.scitotenv.2019.136138.
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(20) Yafeng, S.; Jonas, M. N.; Dawei, Z.; *. Improving Protein Production on the Level of Regulation of both Expression and Secretion Pathways in Bacillus subtilis. J. Microbiol. Biotechnol. 2015, 25 (7), 963-977. DOI: 10.4014/jmb.1501.01028.
(21) Fu, G.; Liu, J.; Li, J.; Zhu, B.; Zhang, D. Systematic Screening of Optimal Signal Peptides for Secretory Production of Heterologous Proteins in Bacillus subtilis. Journal of Agricultural and Food Chemistry 2018, 66 (50), 13141-13151. DOI: 10.1021/acs.jafc.8b04183.
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(30) Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy, V. T.; Howarth, M. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 2012, 109 (12), E690-E697. DOI: doi:10.1073/pnas.1115485109.
(31) Han, X.; Liu, W.; Huang, J.-W.; Ma, J.; Zheng, Y.; Ko, T.-P.; Xu, L.; Cheng, Y.-S.; Chen, C.-C.; Guo, R.-T. Structural insight into catalytic mechanism of PET hydrolase. Nature Communications 2017, 8 (1), 2106. DOI: 10.1038/s41467-017-02255-z.
(32) Hirooka, K.; Tamano, A. Bacillus subtilis highly efficient protein expression systems that are chromosomally integrated and controllable by glucose and rhamnose. Bioscience, Biotechnology, and Biochemistry 2018, 82 (11), 1942-1954.
(33) Cui, W.; Han, L.; Suo, F.; Liu, Z.; Zhou, L.; Zhou, Z. Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond. World Journal of Microbiology and Biotechnology 2018, 34, 1-19.
(34) Choi, J.; Lee, S. Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied microbiology and biotechnology 2004, 64, 625-635.
(35) Jeong, H.; Jeong, D.-E.; Park, S.-H.; Kim, S. J.; Choi, S.-K. Complete genome sequence of Bacillus subtilis strain WB800N, an extracellular protease-deficient derivative of strain 168. Microbiology Resource Announcements 2018, 7 (18), e01380-01318.
(36) Jan, J.; Valle, F.; Bolivar, F.; Merino, E. Characterization of the 5′ subtilisin (aprE) regulatory region from Bacillus subtilis. FEMS Microbiology Letters 2000, 183 (1), 9-14. DOI: 10.1111/j.1574-6968.2000.tb08926.x (acccessed 6/19/2023).(1) Koshti, R.; Mehta, L.; Samarth, N. Biological Recycling of Polyethylene Terephthalate: A Mini-Review. Journal of Polymers and the Environment 2018, 26 (8), 3520-3529. DOI: 10.1007/s10924-018-1214-7.
(2) Webb, H. K.; Arnott, J.; Crawford, R. J.; Ivanova, E. P. Plastic Degradation and Its Environmental Implications with Special Reference to Poly(ethylene terephthalate). Polymers 2013, 5 (1), 1-18.
(3) Thachnatharen, N.; Shahabuddin, S.; Sridewi, N. The Waste Management of Polyethylene Terephthalate (PET) Plastic Waste: A Review. IOP Conference Series: Materials Science and Engineering 2021, 1127 (1), 012002. DOI: 10.1088/1757-899X/1127/1/012002.
(4) Moharir, R. V.; Kumar, S. Challenges associated with plastic waste disposal and allied microbial routes for its effective degradation: A comprehensive review. Journal of Cleaner Production 2019, 208, 65-76. DOI: https://doi.org/10.1016/j.jclepro.2018.10.059.
(5) Huang, X.; Cao, L.; Qin, Z.; Li, S.; Kong, W.; Liu, Y. Tat-Independent Secretion of Polyethylene Terephthalate Hydrolase PETase in Bacillus subtilis 168 Mediated by Its Native Signal Peptide. Journal of Agricultural and Food Chemistry 2018, 66 (50), 13217-13227. DOI: 10.1021/acs.jafc.8b05038.
(6) Qi, X.; Ma, Y.; Chang, H.; Li, B.; Ding, M.; Yuan, Y. Evaluation of PET Degradation Using Artificial Microbial Consortia. Frontiers in Microbiology 2021, 12, Original Research. DOI: 10.3389/fmicb.2021.778828.
(7) PlasticsEurope. Plastics-the facts 2022: an analysis of global plastics production, demand and waste data; PlasricsEurope, Brussels, Belgium, 2022.
(8) INSIGHTS, F. B. Polyethylene Terephthalate (PET) Market Size, Share & Covid-19 Impact Analysis, By Type, Application, and Regional Forecast, 2023-2030. 2023. (accessed 2023.
(9) Barnard, E.; Rubio Arias, J. J.; Thielemans, W. Chemolytic depolymerisation of PET: a review. Green Chemistry 2021, 23 (11), 3765-3789, 10.1039/D1GC00887K. DOI: 10.1039/D1GC00887K.
(10) Müller, R.-J.; Kleeberg, I.; Deckwer, W.-D. Biodegradation of polyesters containing aromatic constituents. Journal of Biotechnology 2001, 86 (2), 87-95. DOI: https://doi.org/10.1016/S0168-1656(00)00407-7.
(11) Hiraga, K.; Taniguchi, I.; Yoshida, S.; Kimura, Y.; Oda, K. Biodegradation of waste PET. EMBO reports 2020, 21 (2), e49826.
(12) Maurya, A.; Bhattacharya, A.; Khare, S. K. Enzymatic Remediation of Polyethylene Terephthalate (PET)–Based Polymers for Effective Management of Plastic Wastes: An Overview. Frontiers in Bioengineering and Biotechnology 2020, 8, Review. DOI: 10.3389/fbioe.2020.602325.
(13) Yoshida, S.; Hiraga, K.; Takehana, T.; Taniguchi, I.; Yamaji, H.; Maeda, Y.; Toyohara, K.; Miyamoto, K.; Kimura, Y.; Oda, K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 2016, 351 (6278), 1196-1199. DOI: doi:10.1126/science.aad6359.
(14) Chen, C.-C.; Han, X.; Ko, T.-P.; Liu, W.; Guo, R.-T. Structural studies reveal the molecular mechanism of PETase. The FEBS Journal 2018, 285 (20), 3717-3723. DOI: https://doi.org/10.1111/febs.14612.
(15) Son, H. F.; Cho, I. J.; Joo, S.; Seo, H.; Sagong, H.-Y.; Choi, S. Y.; Lee, S. Y.; Kim, K.-J. Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. ACS Catalysis 2019, 9 (4), 3519-3526. DOI: 10.1021/acscatal.9b00568.
(16) Son, H. F.; Joo, S.; Seo, H.; Sagong, H.-Y.; Lee, S. H.; Hong, H.; Kim, K.-J. Structural bioinformatics-based protein engineering of thermo-stable PETase from Ideonella sakaiensis. Enzyme and Microbial Technology 2020, 141, 109656. DOI: https://doi.org/10.1016/j.enzmictec.2020.109656.
(17) Seo, H.; Kim, S.; Son, H. F.; Sagong, H.-Y.; Joo, S.; Kim, K.-J. Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli. Biochemical and Biophysical Research Communications 2019, 508 (1), 250-255. DOI: https://doi.org/10.1016/j.bbrc.2018.11.087.
(18) Chen, Z.; Wang, Y.; Cheng, Y.; Wang, X.; Tong, S.; Yang, H.; Wang, Z. Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase. Science of The Total Environment 2020, 709, 136138. DOI: https://doi.org/10.1016/j.scitotenv.2019.136138.
(19) Moog, D.; Schmitt, J.; Senger, J.; Zarzycki, J.; Rexer, K.-H.; Linne, U.; Erb, T.; Maier, U. G. Using a marine microalga as a chassis for polyethylene terephthalate (PET) degradation. Microbial Cell Factories 2019, 18 (1), 171. DOI: 10.1186/s12934-019-1220-z.
(20) Yafeng, S.; Jonas, M. N.; Dawei, Z.; *. Improving Protein Production on the Level of Regulation of both Expression and Secretion Pathways in Bacillus subtilis. J. Microbiol. Biotechnol. 2015, 25 (7), 963-977. DOI: 10.4014/jmb.1501.01028.
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