臺灣博碩士論文加值系統

在植物的種子中含有大量的phytate (myo-inositol hexaphosphate，IP6) 來負責儲存磷酸 (Pi)，以利未來在發芽時所用。Phytase的作用為將phytate水解產生磷酸根及inositol polyphosphate衍生物。在畜牧業上的應用方面，也會將phytate及phytase作為飼料中的添加劑，以做為家禽或豬隻的磷酸來源。
一般而言，phytase可分為Histidine acid phytase family及Alkaline phytase family兩種。而我們所要討論的Selenomous ruminantium phytase的作用機制並不屬於之前所了解的Histidine acid phytase family或Alkaline phytase family。藉由結構比對我們發現到其活性位置的結構高度相似於dual specificity phosphatase family。並且也發現到相較於其他物種的phytase，S. ruminantium phytase有著較高的催化能力。所以我們希望經由S. ruminantium phytase的研究來增加在商業上的應用價值。
我們試著藉由點突變的方法，將主要負責催化的P-loop上Cys241改為Ser241、Ala241及有輔助弁鄋斡PD-loop上Asp212與Pro216分別改為Ala212與Gly216，來測量其在不同溫度下活性的改變，試著了解這些loop上這一些高度保留的胺基酸對催化活性的影響。發現到P216G的活性會隨著溫度的升高而下降。C241A、C241S、D212A則都會失去活性，這意味著這些位置跟催化弁鄏陬蛪奶j的關係。同時利用CD spectra，試圖由Tm值來檢視活性的改變是否是因為由於溫度的升高而使構型改變所造成。最後將C241A以E. coli大量表現，並以sodinm malonate為主沉澱劑來結晶，以X-ray的方式來了解phytase在結構上的特性及其與substrate的相互作用。由結構上卻意外的發現作為主沉澱劑的malonate會結合於活性位置。這個結構可以幫助我們了解polycarboxyl acid如何對phytase active site產生競爭抑制作用。

Phytase can hydrolyzes phytate（myo-inositol hexaphosphate）during plant germination to produce phosphate（Pi）and inositol polyphasphate derivatives. In poultry and pig farms, phytase and phytate are fed to livestock as additives to provide Pi. In this study, we analyzed the active site of Selenomous ruminantium phytase . It exhibits higher catalytic activity than many other phytases. We wish our analysis could improve its applicability in the livestock industry.
Most phytases can be classified into two big families: Histidine acid phytase family and Alkaline phytase family. S. ruminantium phytase, however, belongs to neither of them. Through structural alignment we have found that the active site of S. ruminantium phytase greatly resembles to members of the dual specificity phosphatase family in that it contains conserved Cys241 in the primary catalytic site, P-loop, and Asp212 and Pro216 in the auxiliary site, WPD-loop. To study the functions of these conserved amino acid residues, we employed site-directed mutagenesis to change Cys241 into Ser241 or Ala241, and Asp212 and Pro216 into Ala212 and Gly216, respectively, and measured their catalytic activity of these mutant forms. We found that the activity of P216G decreases as the temperatures increases, whereas mutations in Cys241 and Asp212 abrogate the activity of S. ruminantium phytase. These results suggest that these sites are very important for the function of S. ruminantium phytase. We also examined CD spectra of these mutant forms to test whether the increase in temperature causes conformational changes. To better understand the structural properties of phytase and its interaction with the substrates, C241A recombinant protein is over expressed in E. coli, and crystallized by using sodium malonate as precipitant, and subject to X-ray crystallography. We found that malonate binds into the active site of C214A. This observation has gain insight that how the competitive inhibition of polycarboxyl acid acts to the phytase active site.

目錄
目錄 i
圖目錄 vi
表目錄 viii
英文摘要 ix
中文摘要 x

目次
第一章 序論 1
1. 引言 1
2. 植酸（Phytate） 1
3. Phytase 2
(1) Alkaline phytase family 3
(2) Histidine acid phytase family 5
(3) Selenomonas ruminantium phytase 5
第二章 材料與方法 7
材料 7
儀器 10
實驗方法 11
1. 點突變（site-directed mutagenesis）之設計 11
(1) 點突變（site-directed mutagenesis）標的物之選擇 11
(2) 引子（primer）的設計 11
2. 點突變菌株製備 12
(1) 目標蛋白之點突變製備 12
(2) 限制酶處理（Enzyme cutting） 12
(3) 去除雜質（clean up） 13
(4) 轉型（Transformation）至E. coli XL-1 Blue 13
(5) 確認轉型成功 13
(6) 轉型至表現用宿主細胞E. coli BL21（DE3） 13
(7) 確認轉型成功 14
3. 表現測定 14
4. 純化方法 14
(1) 粗抽液製備 14
(2) 鎳親和性管柱（Ni-NTA column）層析法 15
(3) 蛋白質電泳 16
(4) 去鹽管柱（desalting column）層析法 16
(5) 真空乾燥 16
5. 蛋白質分子量鑑定 16
6. Phytase活性測試 17
(1) 原理 17
(2) 方法 17
7. 以圓二色（Circular Dichroism, CD）光譜分析Tm值 18
(1) 原理 18
(2) 方法 19
(3) Phytase與phytase mutant光譜 20
(4) Tm值的計算 20
8. 共晶體（co-crystal）培養 20
(1) 晶體培養原理 20
(2) 共結晶樣品準備 21
(3) 共結晶 21
9. X光繞射數據收集與處理（data collection and processing） 22
10. 繞射數據初步分析 22
11. 相位問題（phasing problem） 22
12. 分子模型建立與最佳化（model building and refinement） 23
13. 結構確認（validation） 24
14. 分子結構圖形繪製 24
15. 分子間作用力計算 24
第三章 實驗結果 25
1. 點突變之設計 25
2. 點突變菌株製備 26
3. 表現測定 26
4. Phytase mutant之純化 26
(1) 鎳親和性管柱層析 26
(2) 蛋白質電泳 27
(3) 去鹽管柱層析法 27
5. 蛋白質分子量鑑定 27
6. Phytase活性測試 27
7. Tm值的計算 28
8. 共結晶條件 29
9. X光繞射數據收集與處理 29
10. 繞射數據初步分析 30
11. 相位問題 31
12. 分子模型建立與最佳化 31
13. 結構確認 31
14. Phytase（C241A）晶體之空間結構 31
15. Phytase（C241A）分子之立體結構 32
16. Phytase（C241A）與malonate之間作用力 32
第四章 討論 34
1. D212A 34
2. P216G 35
3. C241S 36
4. C241A 37
第五章 結論與未來展望 39
附圖 41
附表 75
參考文獻 81

圖目錄
圖一 點突變在結構上之位置 42
圖二 點突變在WPD-loop與p-loop上的位置 43
圖三 E. coli BL21（DE3）轉型成功確認 44
圖四 可溶性蛋白質表現確認 45
圖五 鎳親和性管柱層析純化 46
圖六 Phytase mutant 純度鑒定 48
圖七 去鹽管柱層析法純化 50
圖八 ESI-MS觀測結果 52
圖九 不同mutant活性測試之ΔOD700nm 54
圖十 Phytase活性之標準曲線 55
圖十一 不同mutant活性測試之相對活性 56
圖十二 Wild type與不同mutants的CD光譜 57
圖十三 利用不同波長CD所計算之Tm值 58
圖十四 以wild type結晶條件對C241A結晶 59
圖十五 不同mutants的co-crystals 60
圖十六 Ramachandran plot 61
圖十七 C241A之單位晶格 62
圖十八 單位晶格中的C241A 63
圖十九 C241A單體之立體結構 64
圖廿 C241A單體之表面與活性位置 65
圖廿一 C241A與phytase-apo及phytase-ihs的結構重疊 66
圖廿二 C241A單體與phytase-apo的結構重疊與活性位置 67
圖廿三 C241A單體與phytase-ihs的結構重疊與活性位置 68
圖廿四 D212在C241A單體與phytase-ihs活性中心的位置 69
圖廿五 P216在C241A單體與phytase-ihs活性中心的位置 70
圖廿六 Malonate在C241A與phytase-ihs活性中心的位置 71
圖廿七 Ihs與malonat與活性中心的作用 72
圖廿八 Malonate會阻擋ihs的活性位置 73
圖廿九 Malonate與Ala241及Cys241的距離 74


表目錄
表一 點突變所設計之引子（primer） 76
表二 ESI-MS所得觀測值與理論值 77
表三 以CDPro分析wild type與C241S的CD光譜 77
表四 Phytase活性測試所得之ΔOD700nm值 78
表五 Phytase活性測試所得之活性 78
表六 Phytase活性測試所得之相對活性 79
表七 Crystal data collection statistics 80
表八 Refinement and Ramachandran Plot statistics 80

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