臺灣博碩士論文加值系統

利用聚合鏈鎖反應增幅含L-aspartate β-decarboxylase基因之DNA片段作為探針，與Alcaligenes faecalis CCRC 11585之基因庫進行雜交篩選得到六株選殖株，其中一株含重組質體pBK-asdAE1之選殖株具有L-aspartate β-decarboxylase活性。將pBK-asdAE1其中3,003 bp部份定序，分析定序所得之核酸序列發現一段由1,599 bp構成之開讀架構序（open reading frame, ORF），其所轉譯之蛋白質共533個胺基酸殘基。此段ORF與Pseudomonas dacunhae CCRC 12623之L-aspartate β-decarboxylase基因有84 %相同性，而胺基酸序列則有93 %相同性。Alcaligenes faecalis CCRC 11585之L-aspartate β-decarboxylase胺基酸序列與多種轉胺（aminotransferase）有51∼59 ％相似性，將L-aspartate β-decarboxylase之胺基酸序列與5種aspartate aminotransferases及2種tyrosine aminotransferases比對，發現共有17個保守性胺基酸，其中包含了轉胺之PLP binding site大部分的保守性胺基酸。分析Alcaligenes faecalis CCRC 11585之L-aspartate β-decarboxylase胺基酸序列發現一段序列符合aminotransferase class-I pyridoxal-phosphate attachment site motif，其中包括與pyridoxal 5'-phosphate共價結合之lysine殘基。另外L-aspartate β-decarboxylase也發現一段與ATP/GTP binding motif A（P-loop）相符之序列。利用PCR增幅Alcaligenes faecalis CCRC 11585之L-aspartate β-decarboxylase基因後選殖至表現載體pQE-30中，轉形至E. coli Nova-Blue表現，發現選殖株E. coli Nova-Blue / pQE-asdA1之胞內可溶性蛋白與內涵體（inclusion body）部份有L-aspartate β-decarboxylase活性，由SDS-PAGE電泳結果發現E. coli Nova-Blue/pQE-asdA1之內涵體中比E. coli Nova-Blue / pQE-30多了一約62 kDa之蛋白質產物，與由胺基酸序列預測之分子量相符。

The DNA fragment containing L-aspartate β-decarboxylase gene was amplified by polymerase chain reaction (PCR). Six clones were isolated from Alcaligenes faecalis CCRC 11585 genomic library that was hybridized by the PCR-amplified fragment. One of the six clones harvesting plasmid pBK-asdAE1 could express L-aspartate β-decarboxylase activity and 3,003 bp of the cloned DNA fragment was sequenced. Sequence analysis revealed an open reading frame (ORF) consisting of 1,599 bp that encodes a 533 amino-acids polypeptide. The nucleotide sequence of the L-aspartate β-decarboxylase gene from Alcaligenes faecalis CCRC 11585 showed 84 % identity with that from Pseudomonas dacunhae CCRC 12623, and the amino acid sequence showed 93 % identity. The amino acid sequence of the L-aspartate β-decarboxylase from Alcaligenes faecalis CCRC 11585 showed 51-59 % homology with various aminotransferases. Comparison of the L-aspartate β-decarboxylase with 5 aspartate aminotransferases and 2 tyrosine aminotransferases revealed 17 conserved amino acid that include most of the conserved amino acid residues within PLP binding domain of aminotransferases. L-aspartate β-decarboxylase contains the aminotransferases class-I pyridoxal-phosphate attachment site motif including the lysine residue covalently bound to pyridoxal 5'-phosphate. L-aspartate β-decarboxylase also contains the (A,G)XXXXGK(S,T) motif, characteristic of ATP/GTP -binding site motif A (P-loop). The L-aspartate β-decarboxylase gene of Alcaligenes faecalis CCRC 11585 was cloned into expression vector pQE-30 to express the L-aspartate β-decarboxylase in Escherichia coli Nova-Blue. Intracellular soluble form protein and inclusion body of E. coli Nova-Blue / pQE-asdA1 exhibit L-aspartate β-decarboxylase activity. A protein band sized at 62 kDa is present on the SDS-PAGE gel of the inclusion body of E. coli Nova-Blue / pQE-asdA1 that is absent from the gel of E. coli Nova-Blue / pQE-30.

壹、前言一、 序言................................................................................................................................................................................ 1二、 L-aspartate β-decarboxylase酵素特性1. L-aspartate β-decarboxylase之發現....................................................................................................................................32. L-aspartate β-decarboxylase之結構................................................................................................................................... 43. L-aspartate β-decarboxylase之aminotransferase活 性及添加PLP與α-keto acid對活性之影響.............................. 54. L-aspartate衍生物對L-aspartate β-decarboxylase 之影響.............................................................................................. 75. 哺乳類動物細胞與真菌中之L-aspartate β-decarboxylase............................................................................................ 8三、 L-aspartate β-decarboxylase基因之選殖.................................................................................................................... 10四、 L-aspartate β-decarboxylase之工業應用.................................................................................................................... 11五、 研究緣起與目的.......................................................................................................................................................... 11貳、材料與方法I. 實驗材料一、 使用菌株、噬菌體、質體與引子（primer）......................................................................................................... 13二、 培養基............................................................................................................................................................................ 13三、 藥品與儀器................................................................................................................................................................... 14四、 緩衝溶液與試劑 ......................................................................................................................................................... 16五、 其他................................................................................................................................................................................ 19II. 實驗方法一、 L-aspartate β-decarboxylase活性測試1. 胞內粗酵素液製備............................................................................................................................................................ 202. 薄層色層分析（TLC）..................................................................................................................................................... 20二、 A. faecalis CCRC 11585之L-aspartate β- decarboxylase基因選殖1. 質體DNA之小量抽取........................................................................................................................................................ 212. 染色體DNA之大量抽取....................................................................................................................................................223. A. faecalis CCRC 11585基因庫之建立1) 載體（vector）製備............................................................................................................................................................232) 選殖基因片段製備.............................................................................................................................................................233) 接合作用（ligation）..........................................................................................................................................................234) 宿主細胞（host cell）處理................................................................................................................................................245) Packaging.............................................................................................................................................................................. 246) Titering test............................................................................................................................................................................ 244. 溶菌斑漬片之製備.............................................................................................................................................................255. 南方氏漬片（Southern blot）製備................................................................................................................................... 256. 探針（probe）製備.......................................................................................................................................................... ..267. 探針標幟法（probe labeling）........................................................................................................................................... 278. 雜交反應（hybridization）之條件................................................................................................................................... 279. 單一選殖株質體釋出法（single clone excision）...........................................................................................................2810. 菌落漬片（colony blot）之製備.................................................................................................................................... 3011. 電衝法（electroporation）勝任細胞（competent cell） 之製備...............................................................................2912. 以電衝法（electroporation）進行轉形作用（transformation）..................................................................................30三、 A. faecalis CCRC 11585之L-aspartate β-decarboxylase基因定序及序列分析........................................................ 31四、 A. faecalis CCRC 11585之L-aspartate β-decarboxylase基因於Escherichia coli的表現1. 細胞劃分............................................................................................................................................................................. 312. SDS-PAGE蛋白質電泳分析............................................................................................................................................. 32參、結果一、 A. faecalis CCRC 11585之L-aspartate β-decarboxylase活性測試................................................................................33二、 A. faecalis CCRC 11585之L-aspartate β-decarboxylase基因的選殖1. L-aspartate β-decarboxylase基因探針之設計.................................................................................................................. 332. A. faecalis染色體DNA之南方氏雜交.............................................................................................................................. 353. A. faecalis CCRC 11585基因庫之建立及L-aspartate β-decarboxylase基因之篩選....................................................353-1. 以溶菌斑漬片雜交篩選L-aspartate β-decarboxylase基因.......................................................................................... 363-2. 以菌落漬片雜交篩選L-aspartate β-decarboxylase基因............................................................................................... 374. 質體pBK-asdAE1之限制圖譜........................................................................................................................................385. 質體pBK-asdAE1之次選殖.................................................................................................................................................38三、 A. faecalis CCRC 11585之L-aspartateβ-decarboxylase基因之定序與分析.................................................................39四、 A. faecalis CCRC 11585之L-aspartate β-decarboxylase基因於E. coli中之表現1. 質體pQE-asdA1之構築......................................................................................................................................................... 412. L-aspartate β-decarboxylase於E. coli Nova-Blue/pQE-asdA1之活性分布......................................................................... 42肆、 討論一、 A. faecalis CCRC 11585之L-aspartate β- decarboxylase基因的選殖.............................................................................44二、 A. faecalis CCRC 11585之L-aspartate β- decarboxylase基因序列分析........................................................................ 46三、 A. faecalis CCRC 11585之L-aspartate β- decarboxylase胺基酸序列資料庫比對分析.............................................. 47四、 A. faecalis CCRC 11585之L-aspartate β- decarboxylase 基因於E. coli中之表現......................................................... 51五、 未來展望............................................................................................................................................................................ 52伍、 結論..................................................................................................................................................................................... 53陸、 參考資料............................................................................................................................................................................. 54 表次 頁次表一、 本研究所使用之菌株與噬菌體.....................................................................................................................................60表二、 本研究所使用之質體..................................................................................................................................................... 61表三、 本研究所使用之引子（primers）.................................................................................................................................62表四、 A. faecalis CCRC 11585與P. dacunhae CCRC 12623之L-aspartate β-decarboxylase胺基酸組成比較表................. 63表五、A. faecalis CCRC 11585之L-aspartate β- decarboxylase胺基酸序列於Swiss-Prot資料 庫比對之結果................... 64 圖次 頁次圖一. aminotransferase活性與添加PLP或α-ketoacid對 L-aspartate β-decarboxylase活性之影響......................................... 65圖二. 利用L-aspartate β-decarboxylase合成L-alanine與 D-aspartate之反應.............................................................................. 66圖三. 以薄層色層分析測試P. dacunhae與A. faecalis CCRC 11585之L-aspartate β-decarboxylase活性.............................. 67圖四. 以PCR增幅含P. dacunhae之L-aspartate β-decarboxylase基因的探針............................................................................. 68圖五. 以ADr1、ADr2為引子，不同濃度P. dacunhae 與A. faecalis染色體為模板之PCR電泳圖....................................... 69圖六. A. faecalis CCRC 11585染色體DNA經不同限制截切後以asdA0.5探針與asdA探針雜交之結果.........................70圖七. 以asdAF-1、asdAR-1為引子，P. dacunhae與A. faecalis CCRC 11585染色體DNA為模板之PCR產物.................. 71圖八. 三株A. faecalis染色體DNA以asdA探針雜交之結果....................................................................................................... 72圖九. A. faecalis CCRC 11585染色體經限制截切後以asdA探針雜交之結果.....................................................................73圖十. A. faecalis CCRC 11585之L-aspartate β- decarboxylase基因選殖流程..............................................................................74圖十一. 含A. faecalis CCRC 11585之L-aspartate β-decarboxylase基因的噬菌體基因庫放大後以 asdA0.4探針雜交之結果..............................................................................................................................................................................................................75圖十二. 以asdAF-1、asdAR-1及ADr1、asdAR-1為引子進行PCR確認選殖株質體..............................................................76圖十三. A. faecalis CCRC 11585之L-aspartate β- decarboxylase基因可能選殖株質體經EcoRI截切 後之電泳圖並以asdA0.4探針雜交之結果.................................................................................................................................................................. 77圖十四. 含A. faecalis CCRC 11585染色體基因庫之選殖株質體以EcoRI截切後之電泳圖................................................... 78圖十五. A. faecalis CCRC 11585染色體基因庫之菌落漬片以asdA0.4探針雜交之結果........................................................ 79圖十六. 以pBK-asdAE1質體為模板，ADr1，ADr2；asdAF-1，asdAR-1為引子之PCR產物電泳圖................................ 80圖十七. 質體pBK-asdAE1經限制截切後之電泳圖並以asdA探針於60℃雜交之南方氏漬片結果............................... 81圖十八. pBK-asdAE1中選殖基因片段asdAE1經限制 截切後之電泳圖並以asdA探針於60℃雜交之南方氏漬片結果.............................................................................................................................................................................................................. 82圖十九. 質體pBK-asdAE1之限制圖譜....................................................................................................................................... 83圖二十. 次選殖株質體經限制截切後以1%電泳片分析之結果........................................................................................... 84圖二十一. 以薄層色層分析測試次選殖株之L-aspartate β-decarboxylase活性........................................................................ 85圖二十二. pBK-asdAE1之次選殖與L-aspartate β-decarboxylase活性......................................................................................... 86圖二十三. A. faecalis CCRC 11585之L-aspartate β-decarboxylase基因定序流程圖....................................................................87圖二十四. A. faecalis CCRC 11585之L-aspartate β-decarboxylase基因的核酸與預測之胺基酸序列.................................90圖二十五. A. faecalis CCRC 11585之L-aspartate β-decarboxylase基因下游預測的terminator結構...........................................91圖二十六. P. dacunhae CCRC 12623與A. faecalis CCRC11585之L-aspartateβ-decarboxylase基因的核 酸序列比對......... 94圖二十七. P. dacunhae CCRC 12623與A. faecalis CCRC 11585之L-aspartate β-decarboxylase基因的胺基酸序列比對........ 95圖二十八. L-aspartate β-decarboxylase與轉胺胺基酸序列之比對..........................................................................................98圖二十九. 質體pQE-asdA1之構築................................................................................................................................................... 99圖三十. 以PCR與限制截切確認重組質體pQE-asdA1........................................................................................................... 100圖三十一. L-aspartate β-decarboxylase於E. coli Nova-Blue/pQE-asdA1活性分布...................................................................... 101 附 錄 圖 次 頁次附錄圖一、P. dacunhae CCRC 12623之L-aspartate β-decarboxylase基因序列.......................................................................... 103

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