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dc.contributor.advisorWen-Hwei Hsuen_US
dc.contributor.authorHsu, Shih-Kuangen_US
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Morphology and chemistry of cell walls of Micrococcus radiodurans. J. Bacteriol 95:641-657.en_US
dc.description.abstract由D. radiodurans菌體中選殖出一條長約1.2 kb之NAAAR基因,可轉譯出375個胺基酸,其分子量約為40 kDa。此酵素在60℃、pH8.0反應條件下具有最佳之酵素活性。NAAAR活性可明顯被二價金屬離子Co2+與Mn2+所提升。基質選擇性分析顯示,NAAAR具有廣泛之作用基質範圍。NAAAR屬於enolase superfamily中MLE (muconate lactonizing enzyme) subgroup類蛋白質,此類蛋白質的酵素催化反應包含有cycloisomerization (MLE)、dehydration [o-succinylbenzoate synthase (OSBS)]與1,1-proton transfer [L-Ala-D/L-Glu epimerase (AEE)]。本研究探討NAAAR在D. radiodurans菌體中扮演之生理功能。利用Dixon plot分析結果得知,NAAAR之活性易被OSBS之類似物 (salicyl hydroxamate) 所抑制,測得其Ki值為0.68 mM,此結果顯示,salicyl hydroxamate與NAAAR之反應基質作用在酵素相同的位置上,當少量之salicyl hydroxamate存在下,即可與NAAAR的反應基質競爭酵素活性部位。以LC/MS/MS與結合酵素反應分析顯示,NAAAR無法催化L-Ala-D/L-Glu dipeptide進行消旋化反應,也就是不具有AEE酵素活性。NAAAR也無法催化cis,cis-muconate進行cycloisomerization反應,顯示無MLE酵素活性。進一步分析D. radiodurans基因體序列,也無法由資料庫中獲取可提供研判生理功能的相關資訊。因此NAAAR在D. radiodurans中真正的生理功能值得再深入探討。依據NAAAR蛋白質結構分析顯示在酵素活性區域中包含有Lys170、Asp195、Glu220、Asp245與Lys269,其中Asp195、Glu220與Asp245推測與二價金屬離子結合有關,而Lys170與Lys269剛好位於活性部位兩相對位置上,可能負責催化消旋反應的進行。以定點突變方式分析Lys170與Lys269兩個殘基,並測試所有變異酵素之生化活性,結果顯示經突變後所有變異酵素之活性明顯降低約200至1400倍左右,因此,推測Lys170與Lys269在酵素催化消旋反應中扮演非常重要的功能。zh_TW
dc.description.abstractThe N-acylamino acid racemase (NAAAR) gene from Deinococcus radiodurans BCRC12827 consists of an 1.2 kb open reading frame, encoding a protein of 375-amino acid residues with a calculated molecular mass of about 40 kDa. NAAAR had maximal activity at 60℃and pH 8.0. The high enzyme activity could be observed by the addition of 2mM Co2+ and Mn2+ ion. Substrate specificity analysis revealed that the NAAAR has a broad substrate range. The NAAAR is a member of the MLE (muconate lactonizing enzyme) subgroup of the enolase superfamily, catalyzing the reactions including cycloisomerization (MLE), dehydration [o-succinylbenzoate synthase (OSBS)], and 1,1-proton transfer [L-Ala-D/L-Glu epimerase (AEE)]. Dixon plot analysis showed that NAAAR activity was competitively blocked by the OSBS inhibitor, salicyl hydroxamate, with a Ki of 0.68 mM, indicating that NAc-Met and salicyl hydroxamate bind to the same substrate site of NAAAR. Based on the amino acid sequences identity and protein structure, we proposed that the NAAAR might has AEE function. LC/MS/MS analysis and coupling enzyme assay revealved that NAAAR cannot catalyze the racemization of L-Ala-D/L-Glu. The NAAAR also showed no cycloisomerization activity to cis,cis-muconate. Our data indicated that the physiological function of NAAAR in D. radiodurans is still unclear. Analysis of NAAAR 3-D structure and site-directed mutagenesis implied Lys170 and Lys269 located at opposite side of the active site might be involved in the reversible racemization reaction.en_US
dc.description.tableofcontents表 次 VII 圖 次 VIII 縮寫對照表 X 第一章:Deinococcus radiodurans BCRC12827的N-acylamino acid racemase酵素之生化性質與生理功能 1 中文摘要 2 英文摘要 3 一、緒 論 4 (一) 單一旋光性胺基酸及其應用 4 (二) 光學活性胺基酸的合成 4 (三) Aminoacylase結合N-acylamino acid racemase生產光學活性胺基酸 7 (四) N-acylamino acid racemase的生化特性與功能 7 (五) Enolase superfamily的相關研究 8 (六) N-acylamino acid racemase的生理角色 10 (七) Deinococcus radiodurans的特性 13 (八) 研究目的 14 二、實驗材料與方法 15 (一) 實驗材料 15 (二) 菌株、質體及培養條件 15 (三) D.radioduransi 染色體 DNA 之快速抽取法 15 (四) 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 17 (五) naaar 基因選殖、表現與純化 17 (六) 蛋白質之定量 19 (七) 蛋白質的電泳分析 20 (八) 酵素活性分析 21 (九) o-succinylbenzoate synthase (OSBS) 酵素活性分析 21 (十) L-Ala-D/L-Glu epimerase (AEE) 酵素活性分析 22 (十一) Muconate lactonizing enzyme活性分析 23 (十二) NAAAR之定點突變 23 三、結 果 25 (一) N-acylamino acid racemase基因的選殖與表現 25 (二) 相關N-acylamino acid racemase蛋白質序列分析及比對 25 (三) N-acylamino acid racemase酵素之生化性質 25 (四) N-acylamino acid racemase生理功能之探討 29 (五) 以定向演化法改造NAAAR酵素之活性 37 四、討 論 42 (一) N-acylamino acid racemase基因之表現與生化性質 42 (二) N-acylamino acid racemase之生理功能 42 (三) N-acylamino acid racemase變異酵素之催化機制與基質光學選擇性 47 五、結 論 50 六、參考文獻 51 第二章:利用表現L-aminoacylase和N-acylamino acid racemase的重組E. coli細胞進行光學選擇性合成L-homophenylalanine 56 中文摘要 57 英文摘要 58 一、緒 論 59 (一) 前 言 59 (二) 血管收縮素轉換酶抑制劑的發展 59 (三) L-Homophenylalanine的生合成 64 (四) Aminoacylase 65 (五) 研究目的 67 二、實驗材料與方法 69 (一) 實驗材料 69 (二) laa 基因在 E. coli 菌體中的表現 69 (三) 酵素活性分析 69 (四) 構築naaar和laa雙基因在單一宿主細胞E. coli中的共同表現 73 (五) 利用全細胞生物催化系統轉換生產 L-HPA 74 三、結 果 75 (一) L-aminoacylase與N-acylamino acid racemase的生化性質 75 (二) naaar和laa基因在單一宿主細胞E. coli中的共同表現 75 (三) 利用雙基因在同一宿主細胞中共同表現的全細胞生物催化反應生產L-HPA 79 (四) 利用各別基因表現的宿主細胞生產L-HPA 80 (五) 重複使用全細胞生物催化反應生產L-HPA 80 四、討 論 87 (一) LAA之基質光學選擇性 87 (二) naaar和laa基因在單一宿主細胞E. coli中的共同表現 87 (三) 利用雙基因在同一宿主細胞中共同表現的全細胞生物催化反應生產L-HPA 87 (四) 利用各別基因表現的宿主細胞生產L-HPA 88 五、結 論 90 六、參考文獻 91 第三章:利用carbamoylase酵素法結合N-acylamino acid racemase的消旋化作用進行立體選擇性生合成L-homophenylalanine 96 中文摘要 97 英文摘要 98 一、緒 論 99 (一) 前 言 99 (二) N-Carbamoylamino acid amidohydrolase (N-carbamoylase) 99 (三) 全細胞生物催化反應之應用 100 (四) 增加微生物對物質通透性之方法 101 (五) 研究目的 103 二、實驗材料與方法 104 (一) 實驗材料 104 (二) 菌株、質體與分離純化DNA 104 (三) 基因之構築與表現 104 (四) 酵素活性分析 107 (五) 細胞通透性之處理 107 (六) 全細胞生物催化法生產L-HPA 109 (七) 利用高濃度基質生產L-HPA 109 三、結 果 110 (一) NAAAR酵素性質分析 110 (二) 利用雙基因(NAAAR與LNCA)在同一宿主細胞中共同表現的全細胞生物催化系統生產L-HPA 113 (三) 通透劑的選擇 113 (四) 以具通透性之E. coli全細胞生物催化反應進行生物轉換作用 118 (五) 重複使用具通透性之E. coli全細胞生物催化反應生產L-HPA 121 四、討 論 123 (一) NAAAR與LNCA酵素對基質光學選擇性之分析 123 (二) 利用雙基因在同一宿主細胞中共同表現的全細胞生物催化反應生產L-HPA 123 (三) 通透劑的選擇及最適反應條件 125 (四) 以具通透性之E. coli全細胞生物催化反應生產L-HPA 126 (五) 重複使用具通透性之E. coli全細胞生物催化反應生產L-HPA 127 五、結 論 129 六、參考文獻 130zh_TW
dc.subjectN-acylamino acid racemaseen_US
dc.subjectMuconate lactonizing enzymeen_US
dc.subjecto-succinylbenzoate synthaseen_US
dc.titleBiochemical characterization of N-acylamino acid racemase from Deinococcus radiodurans BCRC12827 and its application in the enantioselective synthesis of L-homophenylalanineen_US
dc.titleDeinococcus radiodurans BCRC12827的N-acylamino acid racemase酵素生化性質之分析及應用此酵素參與光學選擇性合成L-homophenylalaninezh_TW
dc.typeThesis and Dissertationzh_TW
item.fulltextno fulltext-
item.openairetypeThesis and Dissertation-
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