臺灣博碩士論文加值系統

Alcaligenes faecalis DA1是本實驗室自土壤中篩選所得，為一能生產高活性N-acyl-D-amidohydrolase之菌株，簡稱D-aminoacylase。分子量約為52 kDa，每一分子內含有2個鋅原子，目前已完成基因選殖及定序，並將基因構築於大量表現載體上。
本論文乃依據先前實驗之化學修飾結果，利用定位突變將Cys97, Cys145, Cys208, Cys309皆改為Ala，並純化各突變酵素，發現C97A酵素完全失去活性，亦失去所結合之2 g.atom之Zn2＋，且結構改變，推測C97A參與Zn2＋結合，為活性表現及結構穩定所必須。並經由序列比對及反應機制之推測，選擇His221, Asp366及Asp66等殘基，以定位突變法改為Ala，結果酵素活性亦完全失去，證實該三殘基亦和催化活性密切相關。
同時構築A. faecalis之partial genomic library，以選殖D-aminoacylase 之上下游基因，並利用chromosome walking讀出其序列，經由比對發現其上游約30 bp處為ABC peptide transporter基因，下游約85 bp處為一個長636 bp之transcription regulator基因，再向下游約20 bp處為一1.1 kb之一種racemase，這些基因之功能角色及是否形成operon，則尚待證實。

The gene (daa) encoding the D-aminoacylase of Alcaligenes faecalis DA1 has been cloned, sequenced, and expressed. To ascertain the role of cysteine residues in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine. Mutation of Cys97 to Ala completely abolished enzyme activity. The C97A mutant did not contain the two g..atom of zinc per mole of protein found in the wild-type. The circular dichroism spectropolarimeter indicated a global conformation change of the C97A enzyme. From these studies, Cys97 appears to be important for enzyme activity and seems to contribute to zinc binding and might thus play a substantial role in maintenance of enzyme structural stability. Mutation of His221, Asp366, and Asp66 to Ala also completely abolished enzyme activity, indicated that these three residues might also contribute to zinc binding and enzyme catalysis. Sequence analysis revealed a incomplete ABC peptide transporter gene located at upstream of the daa gene , a helix-turn-helix motif-containing transcription regulator gene (636 bp) and a 1.1 kb racemase gene located downstream of the daa gene. The relationship of these genes is under investigation.

第一章、緒言………………………………………………………1-1
第一節、D型胺基酸的重要性………………………………1-1
第二節、生產胺基酸的方法…………………………………1-3
第三節、D-Aminoacylase之研究現況………………………1-6
第四節、本論文研究之目的與策略…………………………1-8
第二章、材料與方法………………………………………………2-1
第一節、材料…………………………………………………2-1
1. 化學藥品與酵素………………………………………2-1
2. 儀器……………………………………………………2-1
3. 菌種……………………………………………………2-2
4. 培養基及培養液………………………………………2-2
5. 緩衝液與實驗試液……………………………………2-3
6. 質體……………………………………………………2-3
第二節、方法…………………………………………………2-4
1. 自大腸桿菌抽取質體…………………………………2-4
2. 以PCR法進行DNA定位點突變……………………2-5
3. 大腸桿菌之轉形法……………………………………2-6
4. 酵素之生產……………………………………………2-7
5. 酵素粗萃取液之製備…………………………………2-7
6. 酵素活性測定法………………………………………2-7
7. 自大腸桿菌中純化出大量表現的酵素………………2-9
8. 西方墨點法…………………………………………… 2-10
9. 蛋白質含量的測定…………………………………… 2-11
10.圓形二色光譜儀……………………………………… 2-11
11. 以DTNB法測定蛋白質含SH殘基之數目………… 2-11
12. 金屬元素分析…………………………………………2-12
13. A. faecalis DA1 genomic DNA 之抽取………………2-12
14. 核酸探針之製備………………………………………2-13
15. 南方雜交反應…………………………………………2-13
16. 構築Partial genomic DNA library ……………………2-14
17. 選殖菌株之篩選………………………………………2-15
第三章、結果………………………………………………………3-1
第一節、Cysteine對於D-aminoacylase 酵素之角色探討…3-1
1. DA1 D-aminoacylase基因定位點突變………………3-1
2. 酵素動力學之探討……………………………………3-3
3. 酵素之熱安定性………………………………………3-3
4. 利用圓形二色光譜儀預測酵素之二級結構…………3-4
5. 測定蛋白質所含之SH殘基數目………………………3-5
6. 鋅含量分析……………………………………………3-6
第二節、DA1 D-aminoacylase其metal ligands 之推測……3-6
第三節、Catalytic triad之推測………………………………3-8
第四節、D-Aminoacylase基因上下游序列之探討…………3-9
1. Partial genomic library之構築…………………………3-9
2. 上下游基因之定序…………………………………… 3-10
第四章、討論………………………………………………………4-1
第一節、Zinc ligands與zinc角色之探討……………………4-1
1. Zinc對於D-aminoacylase之角色………………………4-1
2. D-Aminoacylase之metal ligands………………………4-5
第二節、Catalytic triad之探討…………………………………4-6
第三節、D-Aminoacylase之上下游基因之探討……………4-7
1. ABC transporter之探討………………………………4-8
2. DNA-binding motif-containing regulator………………4-10
3. Racemase…………………………………………………4-10
4. D-Aminoacylase與其上下游基因關係之推測…………4-11
第五章、參考文獻…………………………………………………5-1

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