嗜甲烷菌之甲烷單氧化酶 (pMMO) 及其重組 (PmoB) 次單元和甲醇脫氫酶 (MDH) 的純化，表徵和催化電化學研究

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研究生：	蔡奕堃 I-Kune Tsai
論文名稱：	嗜甲烷菌之甲烷單氧化酶 (pMMO) 及其重組 (PmoB) 次單元和甲醇脫氫酶 (MDH) 的純化，表徵和催化電化學研究 Purification, Characterization, and the Catalytic Electro-chemical Study of the Particulate Methane Monooxygenase (pMMO) with its Recombinant Subunit B Variants and Methanol Dehydrogenase (MDH) from Methylococcus capsulatus (Bath)
指導教授：	俞聖法 Sheng-Fa Yu 江志強 Jyh-Chiang Jiang
口試委員:	陳長謙 Sunney I. Chen 陳皇州 Huang-Chou Chen 章為皓 Wei-Hau Chang
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：	2020
畢業學年度：	108
語文別：	中文
論文頁數：	87
中文關鍵詞：	嗜甲烷菌、甲烷單氧化酶、甲醇脫氫酶、催化電化學
外文關鍵詞：	Catalytic Electro-chemical, Methylococcus capsulatus, Particulate Methane Monooxygenase, Methanol Dehydrogenase
相關次數：	點閱：231 下載：0
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摘要----------------------------------------------------------------------------------I
Abstract---------------------------------------------------------------------------III
誌謝--------------------------------------------------------------------------------V
Table of Contents---------------------------------------------------------------VII
Lists of Figures--------------------------------------------------------------------X
List of Tables--------------------------------------------------------------------XII
I.    Introduction-------------------------------------------------------------------1
1-1 Purpose--------------------------------------------------------------------1
1-2 Motivation----------------------------------------------------------------2

II.    Background and Significance----------------------------------------------3
2-1 Methylococcus capsulatus (Bath)-------------------------------------4
2-2 C1 proteins in Methylococcus capsulatus (Bath)-------------------7
2-2-1 soluble methane monooxygenase (sMMO)------------------7
2-2-2 particulate methane monooxygenase (pMMO)--------------7
2-2-3 Methanol dehydrogenases (MDH)---------------------------10
2-3 Construction of the expression plasmids pMAL-p2X (PmoB33-414 & PmoB55-414)---------------------------------------------------------------11
2-4 Electrocatalysis---------------------------------------------------------13
2-4-1 Cyclic voltammetry--------------------------------------------13
2-4-2 The protein concentration about electrocatalytic-----------15
2-4-3 Electron transfer rate-------------------------------------------15
2-5 Cryogenic Electron Microscopy-------------------------------------16
III.    Methods and Material------------------------------------------------------17
3-1 Chemicals---------------------------------------------------------------17
3-2 Instruments and equipment-------------------------------------------19
3-3 Cell culture of Methylococcus capsulatus (Bath)------------------20
3-3-1 Growth medium for culturing --------------------------------20
3-3-2 Cell culture of Methylococcus capsulatus (Bath)----------23
3-4 Cell culture of Escherichia coli (E. coli) ---------------------------24
3-5 Isolation of MDH and pMMO from M. capsulatus (Bath) cells
3-5-1 Purification of MDH-------------------------------------------25
3-5-2 Purification of pMMO-----------------------------------------26
3-6 Determination of the molecular mass of purified MDH----------27
3-7 Preparation and purification of the recombinant MBP-PmoB protein------------------------------------------------------------------------28
3-7-1 Preparation of membranes ------------------------------------28
3-7-2 Purification of membrane proteins---------------------------29
3-7-3 Western blotting------------------------------------------------29
3-8 Protein quantification--------------------------------------------------30
3-9 Quantitative of ICP-OES ---------------------------------------------31
3-9-1 Copper quantification------------------------------------------31
3-9-2 Calcium quantification ----------------------------------------32
3-10 The electrocatalytic reaction----------------------------------------32
3-11 Protein structure analysis (Cryo-EM)------------------------------33

IV.    Results and Discussion-----------------------------------------------------35
4-1 Protein purified of pMMO from M. capsulatus (Bath) cells-----35
4-2 Protein purified of MDH from M. capsulatus (Bath) cells-------37
4-3 Electrocatalytic pMMO, PmoB33-414 and PmoB55-414 in the
membranes-------------------------------------------------------------------40
4-4 Analysis of the purified pMMO structure--------------------------43
Discussion-------------------------------------------------------------------46

V.    Conclusion and Future work----------------------------------------------50
5-1 Conclusion--------------------------------------------------------------50
5-2 Future work-------------------------------------------------------------51

VI.    References-------------------------------------------------------------------53
Supplementary information ----------------------------------------------------57

                                

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全文公開日期 2025/07/09 (校內網路)
全文公開日期 2025/07/09 (校外網路)
全文公開日期 2025/07/09 (國家圖書館：臺灣博碩士論文系統)

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