臺灣博碩士論文加值系統

D-amino acid oxidase (DAO)為工業上應用於是製造頭孢菌素類抗生素之先驅物7-aminocephalosporanic acid的重要酵素。DAO在轉化受質後，會產生副產物H2O2，將其某些胺基酸殘基氧化而使之失活。由於 methionine的氧化常於蛋白質傷害中扮演重要角色，因此本研究藉SOEing (splicing by overlapped extension)方法進行點突變，將Rhodosporidium toruloides和Candida boidinii DAO上的methionine residues轉變成leucine residues，以提高其對H2O2之耐受性而延長作用能力。
以單點突變產生R.. toruloides DAO的突變蛋白M59L 、M88L和M213L以及C. boidinii DAO的突變蛋白 M151L和M221L。經檢測R.. toruloides DAO的三個突變蛋白的比活性依次為101.9、84.1和106.7 U/mg，其中M59L和M213L的比活性較wild type DAO的98.6 U/mg為高。此外，酵素動力學分析顯示R.. toruloides M59L以 D-alanine為受質時的kcat/Km值達39.9 s-1 mM-1，高於wild type DAO的kcat/Km值35.7 s-1 mM-1。另方面，C. boidinii突變蛋白卻皆未檢測出任何活性。
H2O2耐受性的分析顯示，R.. toruloides DAO的三個突變蛋白中，M59L和M213L對H2O2耐受性較wild type DAO略佳，M213L又略優於M59L。以100 mM H2O2處理20分鐘後，M59L、M213L和wild type DAO分別殘餘7.88%、12.36%和0.66%的活性。

D-amino acid oxidase (DAO, EC1.4.3.3) is a key enzyme for the production of 7-aminocephalosporanic acid, the precursor of semi-synthetic cepham antibiotics. It was found to be inactivated by H2O2 generated from its own catalysis. Methionine oxidation usually plays an important role in protein damage. Therefore, using the SOEing (splicing by overlapped extension) method, the methionine residues in Rhodosporidium toruloides and Candida boidinii DAO were replaced by leucine residues to improve their resistance to H2O2.
Three R. toruloides mutant DAOs M59L, M88L, and M213L as well as two C. boidinii mutant DAOs were produced. The specific activities of R. toruloides mutant DAOs M59L, M88L, and M213L were 101.9, 84.1, and 106.7 U/mg, respectively. The former two were higher than that of wild type DAO (98.6 U/mg). The kinetic analysis also showed that M59L had a kcat/Km of 39.9 s-1 mM-1 which was better than that of wild type DAO (35.7 s-1 mM-1). On the other hand, C. boidinii mutant DAOs didn’t show any detectable activity.
The resistance to H2O2 of M59L and M213L were slightly better than that of wild type DAO. The treatment with 100 mM H2O2 for 20 minutes resulted in a decrease in the specific activities of M59L, M213L, and wild type DAO by 7.88, 12.36 and 0.66%, respectively.

誌謝 ……………………………………...…………………..………... I
中文摘要 ………………………………...…….……………..……..... II
英文摘要 …………………………………………………...……..….. IV
目錄 ………………………………………………………..…………. VI
圖、表索引 …………………………………………………..…..……. IX
第一章 前言 ………………………………….……………………… 1
1.1. D型胺基酸氧化酵素 ….……………...……………….………. 1
1.1.1. D型胺基酸氧化酵素之催化反應 …………………………. 2
1.1.2. D型胺基酸氧化酵素的生理功能 …………………………. 3
1.1.3. D型胺基酸氧化酵素的生化特性 …………………………. 5
1.1.4. D型胺基酸氧化酵素之應用 ……..…………………………. 8
1.2. D型胺基酸氧化酵素的異源表現 ………….……………....... 12
1.3. 利用基因工程改善酵素特性 ………………………………... 15
1.3.1. 酵素工程 ……….................................................................. 15
1.3.2. 與DAO相關的蛋白質工程的研究 ……………………. 20
1.4. 論文研究目的 ……………………………….……………….. 21
第二章 實驗材料與方法 …………………….…………………….. 23
2.1. 實驗材料 ……………….………...…….................................. 23
2.1.1. 基因和菌種 ………….……………….…...…….………... 23
2.1.2. 實驗儀器 ……………………….……..…………….......... 23
2.1.3. 培養基及藥品 ………….………………………….……... 24
2.1.4. 酵素 …………………………………....………………….. 24
2.1.5. 質體 …….………………………………………….……... 25
2.1.6. 引子 ……………………..…………………..……..……... 26
2.2. 實驗方法 ……..…………......................................................... 26
2.2.1. DAO cDNA基因在E. coli中的誘導表現 ………….…... 26
2.2.1.1. DAO 的誘導表現 ….……………………...………….. 26
2.2.1.2. DAO的純化 …………………………………………... 27
2.2.1.3. DAO活性的測定 ……………………………………... 29
2.2.1.3.1. H2O2 產量的測定 ………………………………… 29
2.2.1.3.2. Pyruvic acid 產量的測定 ……………………….... 30
2.2.1.4. DAO之定量 ……………………………….…………. 30
2.2.1.4.1. 以ε (extinction coefficient)定量 ..……………...... 30
2.2.1.4.2. 以Bradford Assay定量 ………………………..….. 31
2.2.1.5. SDS-PAGE …………………………...……..………… 31
2.2.2. DAO cDNA基因的定點突變 ………………….………... 33
2.2.3. Escherichia coli的轉化作用(transformation) …………… 35
2.2.4. 少量質體DNA的抽取 ………………………………….. 35
2.2.5. Competent cell 之製備 ………………………………….. 36
2.2.6. DAO 與 DAO突變株對H2O2耐受性的表現 ………… 37
2.2.7. DAO 與 DAO突變株的酵素動力學分析 …………….. 37
2.2.8. DAO的DAN shuffling ………………………………….. 37
2.2.8.1. DNA random fragments之製備 …………………….... 37
2.2.8.2. DAO之基因重組 .......................................................... 39
2.2.8.3. Shuffled DAO基因的表現與篩選 …………………… 40
第三章 實驗結果與討論 ……………………………….………….. 41
3.1. R. toruloides和C. boidinii DAO cDNA基因的定點突變 ….. 41
3.2. T. variabilis、R. toruloides和C. boidinii DAO wild type和突變蛋白基因於E. coli之表現 ………………………….………….… 42
3.2.1. R. toruloides wild type DAO和突變蛋白之表現 ............... 42
3.2.2. C. boidinii wild type DAO和突變蛋白之表現 .................. 42
3.2.3. T. variabilis wild type DAO和突變蛋白之表現 ................ 43
3.2.4. Wild type DAO和突變蛋白比活性的比較 ....................... 43
3.3. Wild type DAO 和突變蛋白的酵素動力學 ………………. 45
3.4. Wile type DAO 和突變蛋白的H2O2耐受性比較 …………. 46
3.4.1. Wile type DAO的H2O2耐受性 ………………..……….. 47
3.4.2. 突變DAO對H2O2的耐受性 ……….………………… .. 47
3.5. DAO基因的DNA shuffline …………………………………. 49
3.5.1. DAO基因之重組 …………………………………….…….. 49
3.5.2. Shuffled DAO基因的表現與篩選 ………………………… 49
第四章 結論 ……………………………………………………. … 51
第五章 圖、表 ……………………...……….………………..…… 53
第六章 參考文獻 ………………………………………………….. 82
第七章 附錄 ……………………………………………………….. 97

圖、表索引
表1. 本研究用於增幅DAO cDNA基因的引子 …….…...……… 53
表2. 本研究用於產生點突變的引子 …………………………….. 54
表3. 不同來源DAO的受質專一性比較 .……………….………. 55
表4. R. toruloides、C. boidinii和T. variabilis DAO和突變蛋白的比活性 ….…...…………………….………………………………. 56
表5. R. toruloides、C. boidinii和T. variabilis DAO和突變蛋白的酵素
動力學參數 ………………………………………………….. 57
表6. R. toruloides、C. boidinii和T. variabilis DAO和突變蛋白的H2O2耐受性 ……………………………………………………..… 58
圖1. DAO催化機制 …………. ………………………….………. 59
圖2. D-serine與NO的調控機制 ………………..……………….. 60
圖3. 不同來源DAO胺基酸序列的alignment。conseved amino acid residues以黑底標示 ………………………………………… 61
圖4. DAO於生產動物飼料添加劑之應用 …………………….... 62
圖5. 由cephalosporin C生產7-ACA的酵素反應步驟 …….….. 63
圖6. 以DNA shuffling的方式重組DNA ……………………..… 64
圖7. Family DNA shuffling …………………..…………………… 65
圖8 SOEing 定點突變的操作原理 ………………………..…….. 66
圖9. pET23am的結構圖 ……..……………………….…………… 67
圖10. H2O2標準曲線圖 ……..………………………………..…… 68
圖11. 2.4-dinitrophenylhydrazine的化學結構式(a)以及與酮類的反應式(b) …………………………………………………………… 69
圖12. Pyruvate標準曲線圖 ………………………………………. 70
圖13. BSA標準曲線圖 ……………………………….…………... 71
圖14. R. toruloides wild type DAO和突變蛋白的誘導表現 ...….. 72
圖15. C. boidinii wild type DAO的表現 ………..…….…….....… 73
圖16. T. variabiliswild type DAO和突變蛋白的誘導表現 ……... 74
圖17. R. toruloides DAO 3D結構中之Met殘基位置 ...………… 75
圖18. Wild type R. toruloides (a)、C. boidinii (b)和T. variabilis (c) DAO的H2O2耐受性 …………………………………………….. 76
圖19. R. toruloides M59L的H2O2耐受性 …………....….…….... 77
圖20. R. toruloides M88L的H2O2耐受性 ……………….……… 78
圖21. R. toruloides M213L的H2O2耐受性 …….……………….. 79
圖22. T. variabilis M156L的H2O2耐受性 ……………….……… 80
圖23. 經DNA shuffling重組產生DAO基因片段的電泳分析 …. 81


附錄索引
附錄1. R. toruloides wild type DAO的1/S vs. 1/V的雙倒數圖現 97
附錄2. R. toruloides M59L DAO的1/S vs 1/V的雙倒數圖 …...... 98
附錄3. R. toruloides M88L DAO的1/S vs. 1/V的雙倒數圖 ……. 99
附錄4. R. toruloides M213L DAO的1/S vs. 1/V的雙倒數圖 …. 100
附錄5. C. boidinii wild type DAO的1/S vs. 1/V的雙倒數圖 …. 101
附錄6. T. variabilis wild type DAO的1/S vs. 1/V的雙倒數圖 ... 102
附錄7. T. variabilis M156L的1/S vs. 1/V的雙倒數圖 ………… 103

吳明峰，2003，D型胺基酸氧化酵素基因在Escherichia coli的表現，碩士論文，生物工程研究所，大同大學，臺北。
吳東嶺，2003，D型胺基酸氧化酵素之蛋白質工程，碩士論文，生物工程研究所，大同大學，臺北。
駱彥甫，2003，Candida boidinii D型胺基酸氧化酵素在大腸桿菌中的異源表現研究，碩士論文，生物工程研究所，大同大學，臺北。
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