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研究生:杜遠朋
研究生(外文):Yuan-Peng Du
論文名稱:牡蠣蛋白片段對碳酸鈣礦化過程之影響
論文名稱(外文):Effect of Pif80 Protein Fragments on the Mineralization Process of Calcium Carbonate
指導教授:陳振中陳振中引用關係
指導教授(外文):Jerry Chun-Chung Chan
口試委員:李度楊家銘
口試委員(外文):Tu-Lee
口試日期:2015-06-16
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:125
中文關鍵詞:生物礦化碳酸鈣晶體成長理論生物物理固態核磁共振光譜晶體成核
外文關鍵詞:biomineralizationcalcium carbonatecrystal growth theorybiophysicsPif80 proteinsolid-state nuclear magnetic resonance spectroscopynucleation
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生物礦物為一種由無機物與有機分子組成的複合性材料,這些有機物通常為蛋白質或多醣類等生物分子。有趣的是此類生物分子可以精準地控制礦物的晶相與晶體成長的位置。但是因為缺乏一個好的研究系統,至今我們還不是非常了解生物中的有機分子如何去調控並引導晶體的生長。
在本論文中我們建立了一個生物礦化的實驗系統,並由生物物理與晶體成長理論兩大角度切入進行研究。我們參考一種對於日本牡蠣珍珠層的成長相當重要的蛋白,依照其序列合成了一系列含有天門冬氨酸─天門冬氨酸─精氨酸─賴氨酸序列的胜肽。我們發現這些蛋白片段可以很專一地與文石這個碳酸鈣晶相進行結合,且胜肽的結合量與上述特殊序列的重複次數有著高度的相關性。而這些成功合成出來的胜肽─碳酸鈣仿生礦物可以使我們能夠更進一步地應用固態核磁共振光譜技術去研究有機與無機物的關係以及給予我們胜肽的結構訊息。此外我們也利用幾丁聚糖與牡蠣蛋白片段胜肽來模仿珍珠層的環境,使我們可以更進一步探討碳酸鈣在生物系統中可能的成長模式。我們發現片段胜肽的加入後,某些幾丁質模版的區域有較多數量的晶體形成,而這些晶體數目密度較高的區域中,文石的數量有著特別多的趨勢。以上結果使我們總結在胜肽分子與幾丁質的協同作用下,文石晶體成核的活化能可以被這些生物分子降低,因此引導了文石晶相的生成。本論文的研究與實驗系統對於碳酸鈣晶相的調控機制上有了更進一步的看法,我們相信後續的實驗將對於仿生材料的設計與開發會有重要的貢獻。


Biomineral is a composite material in organisms and it comprises an inorganic part as well as biomolecules like proteins and polysaccharides. These biomolecules can
precisely control the crystal polymorphism and crystallization sites. However, the relationship between the two diverse organic and inorganic phases is still obscure and waiting to be explored because of lacking a good model system for study.
In this thesis we have established a model system for investigating biomineralization process from the perspectives of biophysics and crystal-growth theory. Our model peptides were synthesized as motivated by the repeated sequence (DDRK) in Pif80, which is a crucial protein controlling the formation of the aragonitic nacreous layer in Pinctada fucata. We found that the Pif80 fragments can specifically associate with the aragonite phase of calcium carbonate and their binding propensity is highly dependent on the number of the repetition of DDRK sequence. These biomimetic peptide-mineral composites prepared here enable us to further utilize solid-state nuclear
magnetic resonance to address the conformation of Pif80 peptides. In the view of crystal-growth theory, we employed β-chitin as a substrate with various Pif80 fragments to mimic the nacreous environment in order to investigate how biomolecules affect the crystallization of CaCO3 in nacre. Scanning electron microscopic analysis has shown that Pif80 peptides can induce the nucleation of aragonite on the β-chitin, which is
distinct from the results without the substrate. Among the three peptides, Pif80-11 can increase the population densities of crystallites in the regions occupied by aragonite and we suspect this result is related to the higher binding affinity of Pif80-11. This binding effect probably reduces the energy barrier of nucleating aragonite and further induces the formation of aragonitic crystallites. We believe that the model system developed here can facilitate the future study in the mechanistic aspect of biomineralization that can be applied for material science.

Abstract (Chinese) ii
Abstract iii
Contents 1
List of Tables 4
List of Figures 5
Chapter 1 9
Introduction 9
1.1 Concept of Biomineralization 9
1.2 Diversity of Biomineral 10
1.3 Calcium Carbonate Based Biomineral 11
1.4 Crystal Growth Theory 13
1.4.1 Classical Nucleation Theory 13
1.4.2 Non-Classical Nucleation Theory 15
1.5 Organic Component in Biomineral 19
1.5.1 Disordered Characteristic of Biomineralization Related Protein 20
1.5.2 Functions of Organic Matrices in Biomineral 23
1.6 Motivation 26
Chapter 2 28
Materials and Experimental Section 28
2.1 Chemicals 28
2.2 Introduction of Solid Phase Peptide Synthesis 29
2.2.1 Deprotection 30
2.2.2 Activation 31
2.2.3 Coupling 31
2.2.4 Cleavage 32
2.2.5 Summary of SPPS 33
2.3 Preparation of Pif80 Fragmental Peptides 34
2.3.1 Method of Synthesis 34
2.3.2 Purification 35
2.3.3 Characterization 36
2.4 Effect of Pif80 Peptides on the Growth of CaCO3 36
2.5 Preparation of CaCO3 Bound with Peptides 38
2.6 Extraction of Beta-Chitin Substrate 39
2.7 Nucleation of CaCO3 on Beta-Chitin Substrate 39
Chapter 3 40
Characterization Method 40
3.1 Solid State Nuclear Magnetic Resonance 40
3.1.1 Basic Concept 40
3.1.2 Magic Angle Spinning 42
3.1.3 Cross Polarization 43
3.1.4 Experimental Conditions 45
3.2 Circular Dichroism 47
3.2.1 Introduction 47
3.2.2 Experimental Conditions 49
3.3 X-Ray Diffraction 49
3.3.1 Introduction 49
3.3.2 Experimental Conditions 50
3.4 Infrared Spectroscopy 51
3.5 Thermogravimetric Analysis 52
3.6 Scanning Electron Microscopy 53
Chapter 4 54
Results and Discussions 54
4.1 Effect of Pif80 Peptides on the Growth of CaCO3 54
4.1.1 Experiment of Pif80-7 54
4.1.2 Experiment of Pif80-11 56
4.1.3 Experiment of Pif80-22 57
4.1.4 Discussion 59
4.2 Characterization of Aragonite Bound with Peptides 62
4.2.1 Pif80-7 Aragonite 62
4.2.2 Pif80-11 Aragonite 66
4.2.3 Pif80-22 Aragonite 70
4.2.4 Discussion 73
4.3 CD and SSNMR Characterization of Pif80 Peptides 79
4.3.1 Circular Dichroism 79
4.3.2 Solid State Nuclear Magnetic Resonance 81
4.3.3 Discussion 84
4.4 Nucleation of CaCO3 on Beta-Chitin Substrate 86
4.4.1 Control Experiment 86
4.4.2 Experiment of Pif80-7 86
4.4.3 Experiment of Pif80-11 89
4.4.4 Experiment of Pif80-22 92
4.4.5 Discussion 95
Chapter 5 101
Conclusions and Final Remarks 101
References 103
Appendix I HPLC Profiles of Pif80 Peptides 112
Appendix II Mass Spectra of Pif80 Peptides 113
Appendix III IR Spectra of Pif80 Aragonite in Full Range 117
Appendix IV Spin Counting of D5G9Pif80-11 Aragonite 119
Appendix V Supporting SEM Micrographs 120
Appendix VI Derivation of Nucleation Barrier 124


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