臺灣博碩士論文加值系統

最初之研究方向在於選殖耐熱性之N-Carbamoyl-D-amino acid amidohydrolase基因，根據N-Carbamoyl-D-amino acid amidohydrolase胺基酸序列的高保留區域，設計退化型引子，以嗜熱菌Bacillus kaustophilus CCRC 11223及嗜熱菌Bacillus sp. TS23的染色體作為模版，利用Nested PCR，各可分別從染色體中找到兩種長度均為250bp之不同DNA序列片段，將其與Agrobacterium radiobacter N-Carbamoyl-D-amino acid amidohydrolase胺基酸序列的高保留區域比對之相似度，源自B. kaustophilus DNA序列之片段為58%及60%，而源自Bacillus sp. TS23 DNA序列之片段為50%及56%。然而以這些片段作為探針(probe)，於B.kaustophilus 及Bacillus sp. TS23的染色體基因庫，進行篩選，均無法選殖到此基因。因此利用所得之B. kaustophilus DNA序列之片段設計primer，利用PCR方法由B.kaustophilus的染色體基因庫進行篩選，卻選殖到leucine aminopeptidase (EC 3.4.11.1)基因之片段，探討其成因，發現所設計之引子的3''端末7個核酸序列與leucine aminopeptidase 基因上的核酸序列完全相同，而導致此結果。由於目前並無商業化耐熱性leucine aminopeptidase產品，而此耐熱性之leucine aminopeptidase卻有工業應用的潛力，因此將研究方向調整為選殖耐熱性之leucine aminopeptidase基因;隨後以此leucine aminopeptidase基因之片段，由B. kaustophilus library篩選耐熱性leucine aminopeptidase基因，共篩選出2個含有leucine aminopeptidase基因的純株(clone)。經DNA定序後，發現是同一個leucine aminopeptidase基因。將此段基因選殖入 Escherichia coli 菌體內，可表現leucine aminopeptidase的活性。此 ORF 全長為1,494bp，可轉譯出含497個胺基酸殘基的蛋白質、其分子量為53.7kDa，為一胞內酵素。經過胺基酸殘基序列的比對，與B. subtilis leucine aminopeptidase 的胺基酸相似度為62%，由文獻中已知之胺基酸序列進行比對得知此B.kaustophilus leucine aminopeptidase具有兩個zinc center。
將B. kaustophilus之leucine aminopeptidase ORF 選殖入pQE30 載體，轉形入E. coli Nova Blue，回收此酵素，經酵素活性分析後，發現此酵素之最適反應溫度為65℃，最適pH值為pH 10，在無金屬離子下則此酵素不具催化能力，而Co2+、Mn2+及Ni2+等離子能提昇酵素活性，顯示其可能是metalloezyme。L-leucine-p-nitroanilide為基質時，此酵素之Km為0.028mM， Kcat為7.90min-1。

Original research direction is cloning the thermostable N- Carbamoyl-D-amino acid amidohydrolase(nca) gene. Therefor, designing the degenerate primer is based on highly consensus amino acid sequence region from N- Carbamoyl-D-amino acid amidohydrolase. To use of nested PCR with genomic DNA from thermophilic bacterium Bacillus kaustophilus CCRC11223 and Bacilius sp. TS23 as template DNA would find two fragments with 250 bp in length but different in DNA sequences. Using those DNA fragments was found in good alignment with conserved amino acid sequence region from the Agrobacterium radiobacter N- Carbamoyl-D-amino acid amidohydrolase gene. The similarities of two DNA sequences from B. kaustophilus were 58% and 60%, and two other DNA sequences from Bacillus sp. TS23 were 50% and 56% with A.rediobacter nca, respectivily. Using those DNA fragments as probe to screen two genomic libraries of B. kaustophilus and Bacillus sp. TS23 would not find nca gene.
So we use the B. kaustophilus DNA fragment obtained previously to design a pair of specific primers and screen B. kaustophilus genomic library with PCR amplification. But all were found only the leucine aminopeptidase(lap) (EC 3.4.11.1) DNA fragments. To discus the reason, we find the last 7 nucleic acid bases on 3'' end of designed primer was identical to the DNA sequence of leucine aminopeptidase gene. Because it is not found in any commercial application, and the thermostable leucine aminopeptidase has its industrial potential. So we switch the reseach direction to clone the thermostable leucine aminopeptidase gene.
Using this DNA fragment of leucine aminopeptidase as probe to screen the thermostable leucine aminopeptidase gene from B. kaustophilus genomic library. We got 2 pure clones that both contain the leucine aminopeptidase gene. After DNA sequencing , we found they are the same sequence of leucine aminopeptidase gene. Then we transformed this gene into E. coli and found to expression the enzymatic activity of leucine aminopeptidase. The ORF of leucine aminopeptidase gene is 1,494 bp and translated into a peptide of 497 residues with molecule weight 53.7 kDa and its enzyme was found to be intracellular. The similarity was found to be 62% in amino acid sequence comparison between B. subtilus and B. kaustophilus. Amino acid alignment with the know lap genes were found this B. kaustophilus leucine aminopeptidase has two zinc cneters.
The B. kaustophilus leucine aminopeptidase gene was cloned into pQE30 vector and then expressed in E. coli Nova Blue. With the recovery of this pure enzyme, we find the optimal temperature is 65℃ and optimal pH is 10. In the situation with no metal, this enzyme was not found any catalytic activity. But the addition of Co2+、Mn2+及Ni2+ will promote the enzymatic acyivity know as a metalloezyme. When useing L-leucine-p-nitroanilide as substrate, the km is 0.028 mM and kcat is 7.90min-1.

中英文摘要…………………………………………………………2
前言…………………………………………………………………5
材料與方法…………………………………………………………8
一、菌株及質體…………………………………………………8
二、藥品…………………………………………………………8
三、E. coli質體DNA的抽取………………………………………9
四、DNA洋菜膠體電泳分析……………………………………10
五、DNA片段的回收與純化……………………………………11
六、DNA純度鑑定及定量分析…………………………………11
七、退化型引子的設計…………………………………………12
八、PCR反應……………………………………………………13
(1) PCR…………………………………………………………14
(2) Nested PCR…………………………………………………15
九、DNA黏接反應………………………………………………15
十、質體的轉形作用……………………………………………16
(1) 勝任細胞之製備……………………………………………16
(2) 轉形作用……………………………………………………16
十一、基因的選殖………………………………………………17
(1) 染色體DNA的抽取…………………………………………17
(2) 染色體基因庫的建立………………………………………18
(3) 染色體基因庫的放大………………………………………20
(4) 含phagemid之噬菌體的大量釋出…………………………21
(5) DNA探針的製備……………………………………………22
(6) 溶菌斑雜交法………………………………………………22
(7) 單一選殖株質體釋出法……………………………………24
(8) 限制西每圖譜之製作………………………………………25
(9) 南方轉漬雜交法……………………………………………25
十二、DNA序列分析……………………………………………27
(1) 單股DNA模板的製作………………………………………27
(2) DNA定序……………………………………………………28
(3) 定序膠體電泳分離…………………………………………28
(4)primer extention………………………………………………30
十三、蛋白質之定量……………………………………………31
十四、蛋白質的電泳分析………………………………………32
十五、lap基因的表現……………………………………………34
(1) lap基因於B. kaustophilus內的表現……………………………34
(2) lap基因選殖入E. coli表現載體………………………………35
(3) lap基因的大量表現…………………………………………35
(4) LAP的純化…. ………………………………………………36
(5) 呈色法測定酵素活性………………………………………37
十六、LAP性質之分析…………………………………………38
(1) LAP最適反應溫度…………………………………………38
(2) 金屬離子對LAP活性的影響………………………………38
(3) pH值對LAP活性的影響……………………………………38
(4) LAP對基質的選擇性………………………………………39
(5) LAP的酵素動力學分析……………………………………39
結果………………………………………………………………45
一、N-Carbamoyl-D-amino acid aminohydrolase基因的選殖……40
(一) N-Carbamoyl-D-amino acid aminohydrolase基因探針的
設計……………………………………………………………40
(二) B. kaustophilus染色體基因庫之構築………………………41
(三) nca基因的篩選………………….…………………………42
(A)溶菌斑雜交選殖…………………………………………42
二、利用PCR由基因庫中選殖nca基因的片段…………………43
三、lap基因的選殖………………………………………………43
(一) 溶菌斑雜交選殖……………………………………………43
(二) 含lap基因之DNA片段的限制西每圖譜……………………44
(三) DNA定序……………………………………………………45
四、lap基因在E. coli之表現………………………………………45
五、純化回收LAP酵素…………………………………………46
六、蛋白質的電泳分析…………………………………………47
七、LAP的生化特性……………………………………………47
(1) LAP酵素的最適反應溫度…………………………………47
(2) 金屬離子對LAP酵素活性的影響…………………………48
(3) pH值對LAP酵素活性的影響………………………………49
(4) LAP對基質的選擇性與酵素動力學分析… ………………49
討論………………………………………………………………50
一、B. kaustophilus LAP的胺基酸序列…………………………50
二、金屬離子對LAP活性的影響………………………………50
三、LAP的大量表現……………………………………………51
四、LAP的最適反應溫度………………………………………52
五、pH值對LAP酵素活性的影響………………………………53
六、LAP對基質的選擇性與酵素動力學分析………………….53
圖表……………………………………………………………….54
參考文獻…………………………………………………………97

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