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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 李昆達(Kung-Ta Lee) | |
dc.contributor.author | Hsien-Li Lin | en |
dc.contributor.author | 林軒立 | zh_TW |
dc.date.accessioned | 2021-06-15T03:51:05Z | - |
dc.date.available | 2010-07-16 | |
dc.date.copyright | 2010-07-16 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-14 | |
dc.identifier.citation | Alexandre, G., and Zhulin, I.B. (2000). Laccases are widespread in bacteria. Trends Biotechnol 18, 41-42.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44556 | - |
dc.description.abstract | 本研究使用選殖自Bacillus subtilis natto NTU18漆脢基因,分別轉殖到大腸桿菌的誘導型表現質體pQE-30 Xa和酵母菌Pichia pastoris X33的持續型表現質體pGAPZαA進行異源表現。在大腸桿菌表現系統方面,漆脢活性在Hinton氏搖瓶培養和7-L生物反應器批次培養中,分別達到0.084 U mL-1和0.87 U mL-1,菌體濃度分別為0.37 gDCW-1和4.40 gDCW L-1;但在7-L生物反應器中進行批次饋料培養,菌體濃度雖可達批次培養的13.3倍 (58.5 gDCW L-1),但活性卻下降了30%,只達到0.61 U mL-1,推測應為大腸桿菌在快速且大量的增殖時,表現質體無法穩定存在於細胞內,造成活性無法如預期般的提升。而在P. pastoris X33表現系統方面,漆脢活性在Hinton氏搖瓶培養和7-L生物反應器饋料批次培養中,則分別可達到0.13 U mL-1和0.62 U mL-1,菌體濃度分別為55.7 gWCW L-1和392.2 gWCW L-1。而重組納豆菌漆脢的最適反應溫度為85℃,相對於雲芝漆脢之最適反應溫度為65℃,重組納豆菌漆脢的最適反應溫度較高;且重組納豆菌漆脢經過55℃處理24小時之後,活性沒有下降,顯示其具有良好的熱穩定性。酵素動力學分析結果顯示,兩酵素在對於ABTS的親和性與最大反應速率並沒有很大的差異。 | zh_TW |
dc.description.abstract | The laccase gene of Bacillus subtilis natto NTU18, which was isolated form commercial natto by previous graduated student, was cloned into pQE-30 Xa and pGAPZαA. In Escherichia coli expression system, the laccase activity reached 0.084 U mL-1 in Hinton’s flask, and 0.87 U mL-1 in 7-L bioreactor batch culture. And the biomass was reached to 0.37 gDCW L-1 and 4.40 gDCW L-1. However, in 7-L bioreactor fed-batch culture, the biomass was 13.3 times to which in batch culture, up to 58.5 gDCW L-1. But the laccase activity was reduced by 30%, only 0.61 U mL-1 was obtained. The low activity may due to the genetic instability of plasmid replication during E. coli reproduction. In Pichia pastoris expression system, the laccase activity reached 0.13 U mL-1 in Hinton’s flask, and 0.62 U mL-1 in 7-L bioreactor fed-batch culture. And the biomass was 55.7 gWCW L-1 and 392.2 gWCW L-1. The optimum reaction temperature of recombinant laccase was 85℃, higher than the optimum reaction temperature of T. versicolor laccase, which was 65℃. The recombinant laccase also showed high thermostability under the treatment of 55℃ for 24 h. And there is no significant difference of the enzyme kinetic constant Km and Vmax between recombinant and T. versicolor laccase. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T03:51:05Z (GMT). No. of bitstreams: 1 ntu-99-R95b47105-1.pdf: 2424421 bytes, checksum: b9298bfab73c3fe72e3eefd515ca03e9 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 口試委員審定書 I
Abstract I 中文摘要 IV Abbreviation V Index VI Contents VII Contents of tables and figures IX Chapter 1 Introduction 1 1.1 Biofuel and lignocellulose 1 1.2 Laccases 3 1.3 Endospore binding laccase 3 1.4 Bacillus subtilis natto 6 1.5 The objective of this research 7 Chapter 2 Materials and Methods 9 2.1 Microorganisms and vectors 9 2.2 Molecular cloning of B. subtilis natto NTU18 laccase gene 9 2.2.1 Construction of E. coli JM109 / yT&A-pQE-30 Xa-laccase and E. coli JM109 / yT&A-pGAPZαA-laccase 9 2.2.2 Construction of E. coli JM109 / pQE-30 Xa-laccase and E. coli JM109 / pGAPZαA-laccase 11 2.2.3 Construction of E. coli M15 / pQE-30 Xa-laccase 13 2.2.4 Construction of P. pastoris X33 / pGAPZαA-laccase 13 2.3 Expression of recombinant laccase using E. coli M15 / pQE-30 Xa-laccase in Hinton’s flasks 15 2.3.1 Batch cultures of recombinant E. coli in a flask 15 2.3.2 Preparation of cell lysate of recombinant E. coli 16 2.4 Expression of recombinant laccase using E. coli M15 / pQE-30 Xa-laccase in a 7 L bioreactor 17 2.4.1 Batch cultures of recombinant E. coli in a 7 L bioreactor 17 2.4.2 Fed-batch cultures of recombinant E. coli in a 7 L bioreactor 18 2.5 Expression of recombinant laccase using P. pastoris X33 / pGAPZαA-laccase in Hinton’s flasks 19 2.6 Expression of recombinant laccase using P. pastoris X33 / pGAPZαA-laccase in a 7 L bioreactor 19 2.7 Laccase activity assay and enzyme kinetics coefficient determination 20 2.8 SDS-PAGE and activity staining 22 2.9 Analytical methods 23 Chapter 3 Results 24 3.1 Gene cloning of laccase 24 3.2 Expression of recombinant laccase in E. coli M15 24 3.2.1 Expression of the recombinant laccase using E. coli M15 / pQE-30 Xa-laccase in Hinton’s flasks 24 3.2.2 Scale-up expression of recombinant laccase in a 7-L bioreactor using E. coli M15 / pQE-30 Xa-laccase 25 3.3 Expression of recombinant laccase in P. pastoris X33 26 3.3.1 Expression of the recombinant laccase using P. pastoris X33 / pGAPZαA-laccase in Hinton’s flasks 26 3.3.2 Scale-up expression of recombinant laccase in a 7-L bioreactor using P. pastoris X33 / pGAPZαA-laccase 27 3.4 Evaluation of Enzyme properties 28 3.4.1 Thermostability, optimum temperature and pH 28 | |
dc.language.iso | en | |
dc.title | 納豆菌漆脢之異源表現 | zh_TW |
dc.title | Heterologous expression of laccase gene from Bacillus subtilis natto | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇遠志,黃鵬林,呂誌翼 | |
dc.subject.keyword | 納豆菌,漆脢,饋料批次培養, | zh_TW |
dc.subject.keyword | Laccase,Bacillus subtilis natto,fed-batch culture, | en |
dc.relation.page | 69 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-07-14 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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