臺灣博碩士論文加值系統

中文摘要
Bacillus kaustophilus CCRC11223白胺酸胺基胜肽酶(LAP)之完整胺基酸序列與原核及真核生物之LAPs有超過30﹪以上相似性。遺傳樹分析顯示B. kaustophilus LAP是與來自Bacillus subtilis 之酵素親源相近，而同歸類於M17 family。His6-tagged LAP 是經由轉殖白胺酸胺基胜肽酶基因至pQE-30產生，此重組酵素可利用鎳螯合層析管柱進行純化。純化酵素之最適pH值和溫度分別為9和65℃，而在60℃下其半衰期為32分鐘。
藉由利用選位突變可探討B. kaustophilus LAP Lys-265、Asp-270、Lys277、Asp-288、Asp-347、Glu-349和Arg-351等殘基之功能重要性。將其一級結構序列與十三種酵素比對，發現上述胺基酸殘基完全被保留。分子量約為54 kDa之變異株酵素可在Escherichia coli M15細胞中表現，並純化出均一蛋白質。野生型酵素之比活性為103.7 U/mg蛋白質，而其催化效率(kcat/Km）在重碳酸鹽參與下增加2倍。然而，純化之突變酵素沒有LAP活性，顯示這些殘基為酵素催化活性所必需。
B.kaustophilus LAP對於過氧化氫傷害敏感。欲改良其之氧化穩定性，利用定點突變方式將氧化敏感之甲硫胺酸以白胺酸置換。分子量為54 kDa之變異株酵素在重組E. coli M15細胞中表現，並純化至均一狀態。與野生型酵素比較，M282L、M285L、M289L和M321L的比活性下降超過43%，至於M400L、M426L、M445L和M485L之比活性分別上升191%、79%、313% 和103%。縱使突變並不對Km值造成顯著改變，但突變酵素M400L、M426L、M445L及 M485L之kcat/Km值上升超過67.8%。在50 mM過氧化氫存在下，Met-68、Met-224、Met-229、Met-400、Met-426、Met-445和Met-485變異酵素分別比野生型酵素穩定，顯示此酵素可藉由基因工程置換較關鍵甲硫胺酸殘基來改善其氧化穩定性。

Abstract
The complete amino acid sequence of Bacillus kaustophilus CCRC 11223 leucine aminopeptidase (LAP) showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped to M17 family. His6-tagged LAP was generated in E. coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65℃, respectively, and 50% of its activity remained after incubation at 60℃ for 32 min.
The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of B. kaustophilus LAP was explored by the approach of site-directed mutagenesis. Alignment of known primary sequences of thirteen enzymes revealed that the above-mentioned residues are completely conserved. The variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity. The specific activity for wild-type enzyme was 103.7 U/mg protein and its catalytic efficiency (kcat/Km) was activated about 2-fold by 1 mM bicarbonate. However, the purified variants had no LAP activity, implying these residues are essential for the catalytic reaction of the enzyme.
B. kaustophilus LAP was sensitive to oxidative damage by hydrogen peroxide. To improve its oxidative stability, the oxidation-sensitive methionine residues in the enzyme were replaced with leucine. The variants with an apparent molecular mass of approximately 54 kDa were overexpressed in the recombinant E. coli M15 cells and purified to homogeneity. The specific activity for M282L and M285L, M289L, and M321L decreased by more than 43%, while M400L, M426L, M445L, and M485L showed 191, 79, 313, and 103%, respectively, higher activity than the wild-type enzyme. Although the mutations did not cause a significant change in the Km value, more than 67.8% increase in the value of kcat/Km was observed in the M400L, M426L, M445L, and M485L. In the presence of 50 mM H2O2, Met-68, Met-224, Met-229, Met-282, Met-299, Met-400, Met-426, Met-445 and Met-485 variants were more stable with respect to the wild-type enzyme, indicating that the oxidative stability of the enzyme can be improved by engineering the critical methionine residues.

目 錄
英文摘要 1
中文摘要 3
前言
一、蛋白酶之分類 5
二、LAP之生理功能及應用 6
三、LAP之結構 8
四、LAP之催化活性中心 11
五、過氧化氫對酵素穩定性之影響 13
六、Bacillus kaustophilus之分類及相關研究 14
七、目前所要進行之研究 17
材料與方法
一、藥品 22
二、菌株質體及培養基 22
三、方法 24
1. 重組DNA技術 24
2. 選位突變 27
3. 轉型至E.coli M15 33
4. 利用IMAC技術純化His6-tagged bkLAP 34
5. 蛋白質電泳分析與活性染色 36
6. 酵素之生化特性分析 41
結果
一、B. kaustophilus LAP之特性 51
1. 胺基酸序列比對 51
2. 重組酵素之表現和純化 51
3. bkLAP之酵素特性 51
二、將Bacillus kaustophilus白胺酸胜肽酶中假定活性中心殘基之選位突變 62
1. 重組酵素之表現與純化 62
三、B. kaustophilus白胺酸胜肽酶的抗氧化變異株之產生 66
1. bkLAP之過氧化氫的氧化 66
2. 聚丙烯膠體電泳 66
3. 拉曼光譜 66
4. bkLAP抗氧化突變株之誘導及表現 67
5. bkLAP之野生型與抗氧化突變株酵素之動力學分析 67
6. 野生型和突變酵素之氧化穩定性 68
討論
一、B. kaustophilus LAP之特性 79
1. 胺基酸序列比對 79
2. 演化樹狀圖 80
3. 重組酵素之表現和純化 81
4. bkLAP之酵素特性 82
二、將B. kaustophilus白胺酸胜肽酶中假定活性中心殘基之選位突變 83
1. 重組酵素之表現與純化 83
2. 鋅配位殘基之突變 84
3. 活性殘基之突變 86
三、B. kaustophilus白胺酸胜肽酶之抗氧化變異株之產生 88
1. bkLAP之過氧化氫氧化 88
2. 野生型與突變酵素之純化 89
3. 野生型和突變酵素之氧化穩定性 90
參考文獻 93
附錄一 104
附錄二 105
附錄三 107
附錄四 108
附錄五 109
圖 目 錄
圖一、蛋白酶之分類。 5
圖二、牛眼睛LAP之單體結構。 10
圖三、牛眼睛LAP之三倍及四倍體結構。 11
圖四、推論之blLAP催化機制。 12
圖五、Methionine殘基之氧化過程。 14
圖六、bILAP之活性中心結構。 19
圖七、LAPs與bILAP和PepA環繞於活性中心位置之胺基酸序列比對。 20
圖八、一些生物體來源白胺酸胜肽酶胺基酸序列之比對。 53
圖九、由序列相似性來建立演化樹狀圖。 55
圖十、以SDS-PAGE分析大腸桿菌NovaBlue (pQE-LAP)總細胞蛋白質以及自鎳金屬螯合層析管柱進行分離萃取所純化之酵素。 58
圖十一、溫度對純化bkLAP活性（A）及穩定性（B）之影響。 60
圖十二、B. kaustophilus LAP與鋅配位有關胺基酸殘基經定點突變之相對位置。 63
圖十三、B. kaustophilus LAP與催化活性有關胺基酸殘基經定點突變之相對位置。 64
圖十四、野生型及突變株酵素在大腸桿菌中大量表現後，以SDS-PAGE分析回收純化酵素。 65
圖十五、純化之bkLAP之過氧化氫不活化。 69
圖十六、bkLAP於自然狀態及氧化狀態下未變性之膠體電泳分
析。 70
圖十七、bkLAP於自然狀態及氧化狀態下之紅外光-拉曼光譜圖
譜。 72
圖十八、B. kaustophilus LAP序列中甲硫胺酸殘基經定點突變之相對位置。 73
圖十九、純化之bkLAP甲硫胺酸突變株酵素之SDS/PAGE分析。 74
圖二十、M17 家族酵素圍繞保留假硫胺酸殘基之序列比對。 76
圖二十一、純化bkLAP野生型及甲硫胺酸突變株之氧化穩定性。 78
表 目 錄
表一、一些微生物來源白胺酸胺基胜肽酶之特性 7
表二、純化rLAP之受質專一性 17
表三、本實驗所使用之菌株及質體 23
表四、B. kaustophilus LAP與鋅配位相關及催化相關殘基之置換所
用的寡核甘酸 29
表五、B. kaustophilus LAP與甲硫胺酸殘基之置換所用的寡核甘酸 30
表六、二價金屬離子對純化的rLAP之活性影響 61
表七、野生型和bkLAP甲硫胺酸突變株之比活性及動力學參數 75
圖 目 錄
圖一、蛋白酶之分類。 5
圖二、牛眼睛LAP之單體結構。 10
圖三、牛眼睛LAP之三倍及四倍體結構。 11
圖四、推論之blLAP催化機制。 12
圖五、Methionine殘基之氧化過程。 14
圖六、bILAP之活性中心結構。 19
圖七、LAPs與bILAP和PepA環繞於活性中心位置之胺基酸序列比對。 20
圖八、一些生物體來源白胺酸胜肽酶胺基酸序列之比對。 53
圖九、由序列相似性來建立演化樹狀圖。 55
圖十、以SDS-PAGE分析大腸桿菌NovaBlue (pQE-LAP)總細胞蛋白質以及自鎳金屬螯合層析管柱進行分離萃取所純化之酵素。 58
圖十一、溫度對純化bkLAP活性（A）及穩定性（B）之影響。 60
圖十二、B. kaustophilus LAP與鋅配位有關胺基酸殘基經定點突變之相對位置。 63
圖十三、B. kaustophilus LAP與催化活性有關胺基酸殘基經定點突變之相對位置。 64
圖十四、野生型及突變株酵素在大腸桿菌中大量表現後，以SDS-PAGE分析回收純化酵素。 65
圖十五、純化之bkLAP之過氧化氫不活化。 69
圖十六、bkLAP於自然狀態及氧化狀態下未變性之膠體電泳分
析。 70
圖十七、bkLAP於自然狀態及氧化狀態下之紅外光-拉曼光譜圖
譜。 72
圖十八、B. kaustophilus LAP序列中甲硫胺酸殘基經定點突變之相對位置。 73
圖十九、純化之bkLAP甲硫胺酸突變株酵素之SDS/PAGE分析。 74
圖二十、M17 家族酵素圍繞保留假硫胺酸殘基之序列比對。 76
圖二十一、純化bkLAP野生型及甲硫胺酸突變株之氧化穩定性。 78
表 目 錄
表一、一些微生物來源白胺酸胺基胜肽酶之特性 7
表二、純化rLAP之受質專一性 17
表三、本實驗所使用之菌株及質體 23
表四、B. kaustophilus LAP與鋅配位相關及催化相關殘基之置換所
用的寡核甘酸 29
表五、B. kaustophilus LAP與甲硫胺酸殘基之置換所用的寡核甘酸 30
表六、二價金屬離子對純化的rLAP之活性影響 61
表七、野生型和bkLAP甲硫胺酸突變株之比活性及動力學參數 75

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