臺灣博碩士論文加值系統

肌紅蛋白(myoglobin)為一種血基質蛋白(heme-protein)，在脊椎動物體內具有儲存及攜帶氧氣的功能。本論文研究主要是將不具酵素活性的肌紅蛋白利用飽和定點突變成H93X；及將具有過氧化酵素 (peroxidase) 活性的突變H64D/V68L/I107M肌紅蛋白，再Leu29位置利用飽和突變技術改變L29X/H64D/V68L/I107M這個推測可改變活性的胺基酸，以進一步分析比較這些突變蛋白的過氧化酵素能力。將其與過氧化氫和2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid),ABTS,反應後利用UV-Vis分光光譜儀偵測並分析其一個電子傳遞的過氧化酵素活性與機制。發現四突變的過氧化氫催化能力不比三突變的活性差，但是對ABTS的催化活性不到三突變的一半，可得知Leu-29這位置對於ABTS進入反應中心有非常重要的調控機制。而His-93的突變幾乎沒有活性，證明過氧化催化能力，主要取決於反應的環境，與鐵金屬的原子軌域影響較小。

Myoglobin (Mb) is a heme-protein, functioning as oxygen carrier in vertebrates. In previous study, MbH64D/V68L/I107M had been engineered into an enzyme with peroxidase activity. We further investigated the L29X/H64D/V68L/I107M and site-saturated H93X mutational effects on peroxidase activity. A well established peroxidase activity with one-electron oxidation reacted with 2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was observed by the UV/Vis spectrophotometer and demonstrated its capability in electron transfer reaction. When ABTS was used as substrate, the results showed that the activity of quadruple mutants was less than triple mutant, indicating the important role of Leu-29 in maintaining peroxidase function, especially the catalysis of ABTS. The catalyses of H2¬¬O¬¬2 of MbL29I/H64D/V68L/I107M, MbL29F/H64D/V68L/I107M, and MbL29M/H64D/V68L/I107M were as good as that of triple mutants. The H93X mutants exhibited no activity, indicating that the peroxidase activity was related with the environment.

Contents
1. Introduction
1-1 Myoglobin 1
1-2 Heme proteins 2
1-3 Peroxidase 4
1-4 Mutated Myoglobin 6
1-5 Peroxidase Cycle of Mutated Mb 10
1-6 Enzyme Kinetics 11
1-7 Research Goal 14
2Materials and Methods 21
2-1 Materials 21
2-1-1 Chemicals 21
2-1-2 Buffers and Reagents 23
2-1-3 Equipments 25
2-1-4 Bacterial strains, vectors, animal, and cell 25
2-2 Method 26
2-2-1 Molecular modeling 26
2-2-2 Preparation of Myoglobin Variants 26
2-2-3 Preparation of Clones 31
2-2-4 Preparation of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 31
2-2-5 Protein detection 32
2-2-6 Crude Extract 34
2-2-7 Expression of Apo-Mb 35
2-2-8 Crude extract of inclusion body 35
2-2-9 Denature and refolding of inclusion body 36
2-2-10 Purification of Apo-Mb 36
2-2-11 Reconstitution of Apo-Mb with heme 37
2-2-12 Analysis of Peroxidase Activity 38
3 Results and Discussion 39
3-1 All clones 39
3-2 Protein detection 41
3-3 Activity Test of Crude Extract 42
3-4 Purification of mutated Mbs 46
3-5 Reconstitution of mutated Mbs 48
3-6 Enzyme kinetics measurements 50
4 Conclusion and Future Perspective 58
5 References 62
6 Appendix 64

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