臺灣博碩士論文加值系統

為了研究Bacillus stearothermophilus leucine aminopeptidase II (LAPII) 中結構與功能之關係，四個被高度保留的麸胺酸及天門冬醯胺酸分別利用定點突變的方式置換為白胺酸、天門冬胺酸及麸胺酸以進行分析。經由大量表現所得之野生型酵素與突變株酵素之分子量大約為 45 kDa，且可進一步以固相金屬親合性層析管柱進行純化。經由酵素活性分析可知 E250L、E316L、E316D、E316K、E340L、E340D 及 E340K之酵素比活性約下降了90% 之多，而E249L、E249D 及 E249K 則具有和野生型酵素相似之酵素活性；而動力學參數之分析結果則顯示，E250L、E316L、E316D、E316K、E340L、E340D 及 E340K這些突變株酵素之催化效率 (kcat/Km) 比野生型酵素下降95% 之多，然而 Glu-249 突變點之突變株酵素之Km 及kcat 值和野生型酵素相比卻無任何重大的改變。這些研究結果顯示，Glu-250、Glu-316 及 Glu-340 這三個殘基，對於 B. stearothermophilus LAPII 之催化特性可能扮演了重要的角色。而將Asn-245、Asn-335及 Asn-341置換為離胺酸後，會造成酵素活性嚴重的喪失；而以p-leucine-nitroanalide作為受質進行動力學參數分析之實驗結果顯示，Asn-373 突變株酵素由於 kcat 值上升，而 Km 值沒有重大的改變，因此使整體的催化效率增加了 2 倍之多；而熱穩定性實驗之分析結果則顯示，將Asn-335、Asn-341 及 Asn-373 置換為天門冬胺酸後，會造成突變株酵素於 70℃ 之半衰期延長，因此也顯示天門冬醯胺酸之脫胺反應 (deamination) 對於 LAPII 而言是具有傷害性的。而為了探討在B. stearothermophilus leucine aminopeptidase II 中參與聚集的關鍵性區域為何，本研究構築了許多由 N- 及 C-端刪除不同區域之變異株酵素以進行生化特性分析，由初步的實驗結果顯示 C-端的區域不論是對於酵素活性或是聚集反應皆較具重要性，但是確切的實驗結果仍需作進一步的分析，未來將嘗試運用 enterokinase 處理以去除融合蛋白 (Trx) ，並利用金屬固相親和性層吸管柱來做純化，以期獲得各個純粹的 LAPII片段，以進一步進行酵素活性分析，以及利用原子力顯微鏡來觀察野生型酵素與刪除不同區域之變異株酵素於形態上之差異。

To understand the structure-function relationships of Bacillus stearothermophilus leucine aminopeptidase II (LAPII), each of four conserved glutamic acid residues and asparagine residues was replaced with leucine, aspartate, and lysine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes with an apparent molecular mass of approximately 44.5 kDa were purified to homogeneity by nikel-chelate chromatography. The specific activity for E250L, E316L, E316D, E316K, E340L, E340D and E340K were decreased by more than 90%, while E249L, E249D and E249K showed similar activity to that of the wild-type enzyme. As compared with the wild-type enzyme, E250L, E316L, E316D, E316K, E340L, E340D and E340K exhibited a 95% decrease in the value of kcat/Km. Alterations in Glu-249 did not cause a signification change in the Km and kcat values. These results indicated that Glu-250, Glu-316 and Glu-340 of B. stearothermophilus LAP II were important for proper catalytic activity of the enzyme. Substitution of Asn-245, Asn-335, and Asn-341 with Lys generated the variants with a dramatic loss of LAP activity. Kinetic analysis of Asn-373 variants with p-leucine-nitroanalide as the substrate revealed an increase in kcat with no significant change in Km leading to more than 2-fold increase in the catalytic efficiency. Thermostability assays showed that replacement of Asn-335, Asn-341 and Asn-373 by aspartic acid markedly increased the half-life of the enzyme at 70°C, indicating that the deamination of these residues may have a deleterious effect on LAPII. To identify the region involved in the dimerization of B. sterothermophilus LAP II, several N- and C-deletion mutants were constructed and characterized. The preliminary result showed that the area of C-terminus may be more important, but it is still needed further work to prove that. In the future, we will utilize enterokinsae to remove Trx part in the fusion protein and use nikel-chelate chromatography to purify that which expect to get the passage of each deletion of LAPII. In addition to activity analysis, we will also use atomic force microscope to observe the difference between wild-type enzyme and deletion mutants.

目 錄


英文摘要 1
中文摘要 3
前言
一、蛋白酶之分類 5
二、蛋白分解酶之生理功能及應用 6
三、胜肽胺基分解酶之分類及結構 7
四、白胺酸胺基胜肽酶 (LAPs) 之生理功能及應用 9
五、牛眼LAP之結構及催化活性中心 12
六、鈷-蛋白質 (cobalt proteins)
七、Bacillus stearothermophilus 之分類及相關研究 15
17
八、目前所要進行之研究 19
材料與方法
一、藥品 25
二、菌株質體及培養基 26
三、方法 28
1. 重組DNA技術 28
2. 選位突變 31
3. 特定區域之刪除變異 39
4. 利用IMAC技術純化His6-tagged bsLAPII 45
5. 蛋白質電泳分析與活性染色 49
6. 酵素之生化特性分析
7. bsLAPII 突變酵素之拉曼光譜測定
8. HPLC 膠體過濾層析法
9. enterokinase 處理 54
61
63
66
結果
一、B. stearothermophilus白胺酸胜肽酶II (LAPII) 中假定活性中心殘基之選位突變 67

1. 重組酵素之表現與純化 67
2. 野生型 bsLAPII 與 Glu 突變株酵素之動力學分析
3. 拉曼光譜 68
68
二、B. stearothermophilus白胺酸胜肽酶II (LAPII) 中與熱穩定性相關殘基 (Asparagine) 之選位突變 78

1. Asparagine 突變株之誘導及表現
2. 野生型 bsLAPII 與 Asn 突變株酵素之動力學分析
3. 野生型 bsLAPII 與 Asn 突變株酵素之熱穩定性分析
4. 拉曼光譜 78
79
79
80
三、B. stearothermophilus白胺酸胜肽酶II (LAPII) 刪除特定區域變異株之產生 88

1. 刪除特定區域突變株之誘導及表現
2. 野生型 bsLAPII 與刪除特定區域突變株酵素之酵素比活性分析 88
90

3. HPLC 膠體過濾層析 90
4. enterokinase 處理 91
5. 拉曼光譜 91
討論
一、B. stearothermophilus白胺酸胜肽酶II (LAPII) 中假定活性中心殘基之選位突變 101

1. 重組酵素之表現與純化 101
2. 野生型 bsLAPII 與 Glu 突變株酵素之酵素比活性與動力學分析 102

3. 拉曼光譜 103
二、B. stearothermophilus白胺酸胜肽酶II (LAPII) 中與熱穩定性相關殘基 (Asparagine) 之選位突變 105

1. 重組酵素之表現與純化 105
2. 野生型 bsLAPII 與 Asn 突變株酵素之酵素比活性與動力學分析 106

3. 野生型 bsLAPII 與 Asn 突變株酵素之熱穩定性分析
4. 拉曼光譜 106
107
三、B. stearothermophilus白胺酸胜肽酶II (LAPII) 刪除特定區域變異株之產生 108

1. 刪除特定區域突變株之誘導及表現
2. 野生型 bsLAPII 與刪除特定區域突變株酵素之酵素比活性分析 108
110

3. HPLC 膠體過濾層析
4. enterokinase 處理 111
111
5. 拉曼光譜 111
四、結論 113
參考文獻 114
附錄一 124
附錄二 126
附錄三 127











圖 目 錄


圖一、蛋白酶之分類。 6
圖二、牛眼睛LAP之單體結構。 13
圖三、牛眼睛LAP之三倍及四倍體結構。 13
圖四、推論之blLAP催化機制。 15
圖五、E. coli methionine aminopeptidase (eMetAP) 金屬離子催化活性中心之立體結構圖。 16

圖六、blLAP之活性中心結構。 21
圖七、E. coli methionine aminopeptidase (eMetAP) 之活性中心結構。 21

圖八、不同微生物來源之胺基胜肽酶活性區域比對圖。 22
圖九、保留的asparagine 殘基之周圍序列比對圖。 23
圖十、B. stearothermophilus LAPII 與鈷配位有關胺基酸殘基經定點突變週遭位置之DNA序列。 70

圖十一、以 SDS-PAGE 分析野生型及麩胺酸突變株酵素在大腸桿菌中之表現情形。 71

圖十二、以 SDS-PAGE (A) 及活性染色 (B) 分析經純化之野生型及麩胺酸突變株酵素。 72

圖十三、以紅外光－拉曼光譜分析野生型及麩胺酸-249 突變株酵素之二級結構。 74

圖十四、以紅外光－拉曼光譜分析野生型及麩胺酸-250 突變株酵素之二級結構。 75

圖十五、以紅外光－拉曼光譜分析野生型及麩胺酸-316 突變株酵素之二級結構。 76

圖十六、以紅外光－拉曼光譜分析野生型及麩胺酸-340 突變株酵素之二級結構。 77

圖十七、B. stearothermophilus LAPII 中 asparagines 殘基經定點突變週遭位置之DNA序列。 81

圖十八、以 SDS-PAGE 分析野生型及天門冬醯胺酸突變株酵素在大腸桿菌中之表現情形。 82

圖十九、以 SDS-PAGE (A) 及活性染色 (B) 分析經純化之野生型及天門冬醯胺酸突變株酵素。 83

圖二十、野生型與置換 Asp變異株酵素之熱穩定性。
圖二十一、以紅外光－拉曼光譜分析野生型及天門冬醯胺酸突變株酵素之二級結構。
圖二十二、以紅外光－拉曼光譜分析野生型及天門冬醯胺酸-373 突變株酵素之二級結構。
圖二十三、以 SDS-PAGE 分析野生型及刪除不同區域之突變株酵素在大腸桿菌中之表現情形。
圖二十四、以 SDS-PAGE 分析經純化之野生型及刪除不同區域之突變株酵素。
圖二十五、以 non-SDS-PAGE (A) 及活性染色 (B) 分析經純化之野生型及刪除不同區域之突變株酵素。
圖二十六、野生型及刪除不同區域突變株酵素之酵素比活性參數。
圖二十七、膠層析之標準曲線。
圖二十八、以 HPLC 膠層析分析野生型及刪除不同區域之突變株酵素之層析圖譜。 85
86

87

93

94

95

96

97
98

圖二十九、以 SDS-PAGE 分析經純化之 bsLAPII△N100 於20℃以 enterokinase 作用不同時間之結果。
圖三十、以紅外光－拉曼光譜分析野生型及刪除不同區域之突變株酵素之二級結構。 99

100



表 目 錄


表一、一些微生物來源白胺酸胺基胜肽酶之特性 11
表二、已知之鈷-蛋白質。 16
表三、本實驗所使用之菌株及質體。 27
表四、B. stearothermophilus LAPII 於鈷配位之相關殘基置換時所用寡核苷酸。 33

表五、B. stearothermophilus LAPII 於天門冬醯胺酸殘基置換時所用之寡核苷酸。 34

表六、B. stearothermophilus LAPII 於刪除特定區域變異所用之寡核苷酸。 40

表七、野生型及麩胺酸突變株酵素之酵素比活性及酵素動力學參數。 73

表八、野生型及天門冬醯胺酸突變株酵素之酵素比活性及酵素動力學參數。 84

郭莉瑛。2004。重組 Bacillus stearothermophilus 白胺酸胜肽胺基切割酶II之生化特性及其突變分析。碩士論文，生物學研究所，東海大學，台中。
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