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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李佳音 | |
dc.contributor.author | Nai-Chen Wang | en |
dc.contributor.author | 王迺琛 | zh_TW |
dc.date.accessioned | 2021-06-13T03:15:48Z | - |
dc.date.available | 2011-07-31 | |
dc.date.copyright | 2006-07-31 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-31 | |
dc.identifier.citation | Abe K, Hayashi H, Maloney PC.1996. Exchange of aspartate and alanine. J Biol Chem 271, 3079-3084.
Abe K, Ohnishi F, Yagi K, Nakajima T, Higuchi T, Sano M, Machida M, Sarker RI, Maloney PC. 2002. Plasmid-encoded asp operon confers a proton motive metabolic cycle catalyzed by an aspartate-alanine exchange reaction. J Bacteriol 184, 2906-2913. Alberts AW and Vagelos PRI. 1966. Acyl carrier protein. VIII. Studies of acyl carrier protein and coenzyme A in Escherichia coli pantothenate or β-alanine auxotrophs. J Biol Chem 241, 5201-5204. Bowers WF, Czubaroff B, Haschemeyer RH. 1970. Subunit structure of L-aspartate β-decarboxylase from Alcaligenes faecalis. Biochemistry 9, 2620-2625. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254. Cellini B, Bertoldi M, Voltattorni CB. 2003. Treponema denticola cystalysin catalyzes β-desulfination of L-cysteine sulfinic acid and β-decarboxylation of L-aspartate and oxalacetate. FEBS Lett 554, 306-310. Chen CC, Chou TL, Lee CY. 2000. Cloning, expression and characterization of L-aspartate β-decarboxylase gene from Alcaligenes faecalis CCRC 11585. J Ind Microbiol 25, 132-140. Chibata I, Kakimoto T, Kato J, Shibatani T, Nishimura N. 1967. Crystalline aspartic β-decarboxylase of Pseudomonas dacunhae. Biochem Biophys Res Commun 26, 662-667. Chibata I, Kakimoto T, Kato J, Shibatani T, Nishimura N. 1968. On the activation mechanism of L-aspartate β-decarboxylase from Pseudomonas dacunhae by α-ketoglutarate. Biochem Biophys Res Commun 32, 375-379. Chibata I, Takimoto T, Kato J. 1965. Enzymatic production of L-alanine by Pseudomonas dacunhae. Appl Microbiol 13, 638-645. Cronan Jr JE. 1980. β-Alanine synthesis in Escherichia coli. J Bacteriol 141, 1291-1297. Crawford LV. 1958. Studies on the aspartic decarboxylase of Nocardia globerula. Biochem J 68, 221-225. El-Rahmany TA. 1994. Comparison of L-aspartate 4-carboxy-lyases of Cunninghamella elegans and Penicillium citrinum. Microbiol Res 149, 253-257. Graber R, Kasper P, Malashkevich VN, Sandmeier E, Berger P, Gehring H, Jansonius JN, Christen P. 1995. Changing the reaction specificity of a pyridoxal-5’-phosphate-dependent enzyme. Eur J Biochem 232, 686-690. Graber R, Kasper P, Malashkevich VN, Strop P, Gehring H, Jansonius JN, Christen P. 1999. Conversion of aspartate aminotranferase into an L-aspartate β-decarboxylase by a triple active-site mutation. J Biol Chem 274, 31203-31208. Kakimoto T, Kato J, Shibatani T, Nishimura N, Chibata I. 1969. Crystalline L-aspartate β-decarboxylase of Pseudomonas dacunhae. I. Crystalization and some physicochemical properties. J Biol Chem 244, 353-358. Krupka HI, Huber R, Holt SC, Clausen T. 2000. Crystal structure of cystalysin from Treponema denticola: a pyridoxal 5’-phosphate-dependent protein acting as a haemolytic enzyme. EMBO J 19, 3168-3178. Meister A, Sober HA, Tice SV. 1951. Enzymatic decarboxylation of aspartic acid to α-alanine. J Biol Chem 189, 577-590. Nishimura JS, Manning JM, Meister A. 1962. Studies on the mechanism of activation of aspartic acid β-decarboxylase by α-keto acids and pyridoxal 5’-phosphate. Biochemistry 1, 442-447. Novogrodsky A and Meister A. 1964. Control of aspartate β-decarboxylase activity by transamination. J Biol Chem 239, 879-888. Novogrodsky A, Nishimura JS, Meister A. 1963. Transamination and β-decarboxylation of aspartate catalyzed by the same pyridoxal phosphate enzyme. J BIol Chem 238, PC1903-PC1905. Palekar AG, Tate SS and Meister A. 1970. Inhibition of aspartate β-decarboxylase by aminomalonate. Stereospecific decarboxylation of aminomalonate to glycine. Biochemistry 9, 2310-2315. Rathod PK and Fellman JH. 1985. Identification of mammalian aspartate-4-decarboxylase. Arch Biochem Biophys 238, 435-446. Rozzell JD. 1991. Method and compositions for the production of L-alanine and derivatives thereof. U.S. Patent No. 5,019,509. Soda K, Novogrodsky A, Meister A. 1964. Enzymatic desulfination of cysteine sulfinic acid. Biochemistry 3, 1450-1454. Tate SS and Meister A. 1969. Regulation of the activity of L-aspartate β-decarboxylase by a novel allosteric mechanism. Biochemistry 8, 1660-1668. Tate SS and Meister A. 1970. Regulation and subunit structure of aspartate β-decarboyxlase. Studies on the enzymes form Alcaligenes faecalis and Pseudomonas dacunhae. Biochemistry 9, 2626-2632. Vacca RA, Giannattasio S, Graber R, Sandmeier E, Marra E, Christen P. 1997. Active-site Arg→Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase. J Biol Chem 272, 21932-21937. Wilson EM and Kornberg HL. 1963. Properties of crystalline L-aspartate 4-carboxy-lyase from Achromobacter sp. Biochem J 88, 578-587. Yamamoto K, Tosa T, Chibata I. 1980. Continuous production of L-alanine using Pseudomonas dacunhae immobilized with carrageenan. Biotechnol Bioeng 22, 2045-2054. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31611 | - |
dc.description.abstract | 本研究自革蘭氏陰性細菌Pseudomonas sp. ATCC 19121 選殖
L-天冬胺酸4-去羧酶(Asd)基因群,成功地於大腸桿菌宿主中表現帶有His-tagged 的可溶重組蛋白,並完成此重組蛋白之特性分析,及其雙功能角色中部份重要胺基酸的研究。Asd 為工業生產丙胺酸的重要酵素,選殖酵素基因長1,593 bp,可轉譯出59,243 Da 之酵素蛋白;Asd 蛋白質序列與其他Asd 之相同性為37-85%,與部份轉胺酶則為39-63%,asd基因在演化上與革蘭氏陽性菌者不同。次選殖asd 至pET 表現系統,建構之Escherichia coli BL21(DE3)pLysS/pES1,經0.4 mM IPTG 誘導5小時,胞內酵素以親和層析管柱純化,產率為33 mg/L,酵素比活性最高可達280 U/mg;動力學分析純化酵素Km 及 Vmax 分別為11.50 mM、0.11 mM/min。測試的二價金屬離子皆抑制酵素活性。此酵素反應活性最佳的條件為45℃、pH 5.0,且酵素在pH 5~7 的穩定性佳;膠體過濾分析實驗中,當移動相之緩衝液由pH 5.0 提高至pH 7.0 時,4.4%的重組酵素由12 聚合體分解為雙體;在pH 8.0 的tris 緩衝液中培養,原子力顯微鏡觀察Asd以雙體形式存在。此重組酵素對D,L-Asp, L-Glu, L-Gln 及 L-Ala 有微量轉胺活性,以L-Asp 為基質時,酵素主要的去羧活性為轉胺活性的2,477 倍。依據與轉胺酶之多序列比對,建構的16 個Asd 突變株,成功利用pET 表現系統大量表現重組蛋白,除AsdT319Y 及AsdD350S 外皆為胞內可溶蛋白,且純化酵素皆可為Asd 原態酵素抗體辨識。其中AsdF203W 突變蛋白,轉胺活性較原態酵素提高37.74%,去羧酶活性減少27.1%;AsdM177L 兩種活性皆提升約16~25%;AsdH336R、AsdD359P 及AsdV474L 去羧活性提高1.7~2.6 倍,轉胺活性下降,對於β-去羧反應的專一性較原態酵素提升。 | zh_TW |
dc.description.abstract | The L-aspartate 4-decarboxylase (Asd) gene was cloned from
Pseudomonas sp. ATCC 19121, a gram negative bacterium, and expressed as a His-tagged fusion protein in Escherichia coli BL21(DE3)pLysS. Characterization of the recombinant protein was performed in this study. The 1,593-bp asd encodes a protein with a molecular mass of 59,243 Da. Its protein sequence shares 37-85% identity upon other Asds and 39-63% upon some aminotransferases. The asd diverged in evolution from those in gram positive strains. Productivity rate of the C-terminal His-tagged Asd was at 33 mg/L of E. coli transformant culture. The kinetic parameters Km and Vmax of the fusion protein for L-aspartate were 11.50 mM and 0.11 mM/min, respectively. All divalent ions inhibited Asd activity in our study. Optimum temperature for Asd reaction was 45℃. The recombinant Asd exhibited its maximum activity in β-decarboxylation at pH 5.0 and specific activity of 280 U/mg. This enzyme is stable in the pH range of 5 to 7, and 4.4% protein dissociated into dimer from dodecamer when the pH shifted from 5.0 to 7.0 in gel filtration analysis. The recombinant Asd displayed little aminotransferase activity when D,L-Asp, L-Glu, L-Gln and L-Ala were served as substrates. Asd catalyzed the β-decarboxylation of L-aspartate 2,477 times more than transamination reaction. According to the multiple alignment of protein sequences of Asd and aminotransferases, 16 site-directed mutants of Asd were designed and constructed. Proteins were purified from cell lysates except AsdT319Y and AsdD350S, which form inclusion bodies, and analyzed by SDS-PAGE. All the mutant proteins were recognized by polyclonal antibodies of Asd. AsdF203W mutant protein exhibited aminotransferase activity 37.74% higher than the native one, and decreased 27.1% of the decarboxyalse activity. AsdM177L enhanced 16-25% for each activity. AsdH336R, AsdD359P and AsdV474L enhanced decarboxylase activity 1.7 to 2.6 times higher than the native protein; meanwhile, they showed decreased aminotransferase activity. As a result, these 3 mutant proteins are more specific in β-elimination reaction than the native Asd. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T03:15:48Z (GMT). No. of bitstreams: 1 ntu-95-D89623801-1.pdf: 1496621 bytes, checksum: d616a667fb9f988698b4ba92f8c6cf8f (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 目 錄
目錄………………………………………………………………………… i 表次……………………………………………………………………… v 圖次……………………………………………………………………… vi 第一章 前言……………………………………………………………… 1 第二章 文獻回顧………………………………………………………… 4 一、L-天冬胺酸4-去羧酶之簡介 ……………………………………… 4 1. Asd為多功能酵素…………………………………………… 4 2. 輔酶及活化物………………………………………………… 5 3. 抑制劑………………………………………………………… 5 4. 特性研究……………………………………………………… 5 5. 天冬胺酸1-去羧酶…………………………………………… 6 二、L-天冬胺酸4-去羧酶與轉胺酶之生理功能……………………… 6 三、Asd與轉胺酶研究現況…………………………………………… 7 第三章 L-天冬胺酸4-去羧酶之基因選殖及其基因群序列分析………………………………………………………………… 8 一、摘要………………………………………………………………… 8 二、前言………………………………………………………………… 8 三、材料………………………………………………………………… 9 1. 培養基………………………………………………………… 9 2. 菌株與質體…………………………………………………… 9 3. 藥品與試劑…………………………………………………… 9 4. 儀器設備……………………………………………………… 9 5. 寡核苷酸引子……………………………………………… 10 6. 瓊脂膠片電泳使用試劑…………………………………… 10 7. DNA定量使用試劑………………………………………… 11 8. 雜交反應使用之溶液………………………………………… 11 四、方法……………………………………………………………… 11 1. 培養條件…………………………………………………… 11 2. DNA濃度測定……………………………………………… 12 3. 聚合酶連鎖反應…………………………………………… 12 4. 限制酶反應………………………………………………… 12 5. 接合作用…………………………………………………… 13 6. 勝任細胞製備……………………………………………… 13 7. 轉形作用…………………………………………………… 13 8. 南氏轉印…………………………………………………… 13 9. 菌落轉印…………………………………………………… 14 10. 雜交反應…………………………………………………… 14 11. 呈色反應…………………………………………………… 14 12. 序列分析……………………………………………-……… 14 五、結果……………………………………………………………… 15 1. 核酸探針設計與基因庫篩選………………………………… 15 2. 選殖株序列確認與初步分析………………………………… 15 3. Asd序列分析………………………………………………… 16 4. 演化分析…………………………………………………… 17 六、討論……………………………………………………………… 17 第四章 Asd重組蛋白之表現、純化及特性分析……………………… 19 一、摘要……………………………………………………………… 19 二、前言……………………………………………………………… 19 三、材料……………………………………………………………… 20 1. 菌株與與培養基…………………………………………… 20 2. 藥品與試劑………………………………………………… 20 3. 儀器設備…………………………………………………… 20 4. 蛋白質膠體電泳試劑……………………………………… 21 5. His-tagged蛋白質純化使用溶液………………………… 21 6. 西氏轉印所需配製溶液及試劑…………………………… 22 7. 膠體過濾使用溶液………………………………………… 22 四、方法……………………………………………………………… 23 1. 培養與破菌條件……………………………………………… 23 2. His-tagged Asd重組酵素純化……………………………… 23 3. 蛋白質定量………………………………………………… 23 4. 酵素活性分析………………………………………………… 24 5. 蛋白質膠體電泳……………………………………………… 24 6. 酵素最適溫度與溫度穩定性………………………………… 24 7. 酵素最適酸鹼度與酸鹼穩定性……………………………… 25 8. 還原劑與金屬離子對Asd重組酵素活性之影響…………… 25 9. 膠體過濾分析………………………………………………… 26 10. 酵素濃度與緩衝液置換…………………………………… 26 11. 電子顯微鏡進行酵素形態觀察…………………………… 27 12. 原子力顯微鏡進行酵素形態觀察………………………… 27 13. 基質專一性試驗…………………………………………… 27 14. 轉胺酶活性分析…………………………………………… 28 15. 抗體製備…………………………………………………… 28 16. 西式轉印…………………………………………………… 28 五、結果……………………………………………………………… 29 1. His-tagged Asd表現與純化………………………………… 29 2. 酵素動力學分析……………………………………………… 30 3. 溫度對酵素的影響…………………………………………… 30 4. pH對酵素的影響…………………………………………… 30 5. 金屬離子與其他試劑對酵素活性的影響…………………… 31 6. 基質專一性…………………………………………………… 32 7. 酵素反應專一性……………………………………………… 32 六、討論……………………………………………………………… 32 第五章 Asd酵素之活性改造………………………………………… 35 一、摘要……………………………………………………………… 35 二、前言……………………………………………………………… 35 三、材料……………………………………………………………… 37 1. 菌株與培養基……………………………………………… 37 2. 藥品與試劑………………………………………………… 37 3. 儀器設備…………………………………………………… 37 四、方法……………………………………………………………… 37 1. Asd與轉胺酶序列比對…………………………………… 37 2. 定點突變引子設計………………………………………… 38 3. 定點突變…………………………………………………… 38 4. 定序確認…………………………………………………… 38 5. 突變株酵素蛋白之表現…………………………………… 38 6. 酵素純化……………………………………………………… 39 7. 不溶體(inclusion body)回溶處理…………………………… 40 8. Asd酵素活性分析…………………………………………… 40 9. 轉胺酶活性分析……………………………………………… 40 10. Circular Dichroism光譜分析………………………… 40 五、結果……………………………………………………………… 41 1. 定點突變……………………………………………………… 41 2. 突變酵素純化………………………………………………… 41 3. 酵素活性分析………………………………………………… 42 4. CD光譜分析…………………………………………………… 42 六、討論……………………………………………………………… 43 第六章 總結與結論……………………………………………………… 47 參考文獻…………………………………………………………………… 48 | |
dc.language.iso | zh-TW | |
dc.title | L-天冬胺酸4-去羧酶特性分析與調控雙功能酵素活性中重要胺基酸之研究 | zh_TW |
dc.title | Characterization of L-aspartate 4-decarboxylase and investigation on its critical residues of bifunctional enzyme activities | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 王惠鈞,朱文深,胡小婷,黃健雄 | |
dc.subject.keyword | 天冬胺酸去羧酶,轉胺酶,定點突變,雙功能酵素,酸鹼度,單體聚合, | zh_TW |
dc.subject.keyword | L-aspartate 4-decarboxylase,aminotransferase,site-directed mutagenesis,bifunctional enzyme,pH,subunit assembly, | en |
dc.relation.page | 83 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-31 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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