臺灣博碩士論文加值系統

轉麩氨醯胺 (Transglutaminase；TGase) 是種醯基轉移酵素，它可以催化由蛋白質上麩胺酸 (glutamine) 側鏈所提供的 -carboxyamide 基進行醯基轉移反應。目前轉麩氨醯胺在食品加工及醫學的應用上深具潛力，且是種高價位的酵素，因此本研究希望能尋求新的微生物來源之轉麩氨醯胺，並在酵素特性及大量生產上做進一步的探討。首先利用來自食工所菌種中心之九株 Streptomyces菌株，進行胞外TGase酵素活性偵測，另一方面利用已發表之 Streptomyces TGases 基因序列為基礎，以南方墨點雜交法及聚合鏈鎖反應確認其中除 Streptomyces aureoversale 及 Streptomyces luteoverticillatus 以外都具有 TGase 基因。且此七株菌TGase 基因的部份 DNA 序列轉譯成胺基酸後，經比對發現都有 80% 左右之相同性；然而不是具有 TGase 基因之放線菌都具有 TGase 酵素活性。本研究選擇其中 Streptomyces rectiverticillatus 菌株，篩選得到含完整 TGase基因的選殖株，發現其完整 TGase 基因共有 1257 個鹼基對，可以轉譯出含 418 個胺基酸分子量為 46.8 kDa 的蛋白。經胺基酸序列比對，與 S. mobaraense 完整 TGase 序列的相同性為 69.8%，mature TGase 部份則具有 78.2% 相同性。進一步利用基因重組方式構築含 S. rectiverticillatus與 S. septatus TGase 基因的重組質體，結果發現可以表現出之可溶性蛋白，但利用 Trypsin 切割後只有S. septatus mature TGase具有 TGase 活性。利用 PCR 方式分別增幅出含完整 S. rectiverticillatus TGase 基因的 DNA 片段，分別黏接於不同 pET 表現載體上，在 E. coli 宿主，可以表現出可溶性蛋白，但是偵測不出 TGase 活性。在原菌胞外 TGase 純化上，培養後的S. rectiverticillatus胞外粗酵素液初步偵測不到 TGase 活性，經過 50% 硫銨濃度初步分割，利用疏水性管柱色層分析以 25% 飽和度硫銨濃度沖洗後，可以初步分離得到比活性為 5.75 U/mg 的 TGase 蛋白，顯示於原菌中產生之 TGase 具有活性。

Transglutaminases (TGase；glutaminyl-peptide： amine -glutaminyltransferase, EC 2.3.2.13) are enzymes that catalyze an acyl transfer reaction in which -carboxyamide groups of protein-bound glutamine residues are the acyl donors. In recent years, studies on using TGase for food processing and the potential applications of TGase in medical treatment are increased. In addition, TGase is a high unit price enzyme. Thus, it would be worthwhile to find a new microbial source of TGase and further characterize the highly expressed recombinant microbial Transglutaminase. In this study, nine Streptomyces sp. obtained from CCRC (Taiwan) were analyzed. The presences of the TGase genes in the chromosome of these nine streptomyces sp. were identified by PCR reaction and Southern blot analyses. Results showed that seven out of the nine tested strains, except S. aureoversale or S. luteoverticillatus, have TGase genes. The amino acid sequence deduced from the PCR products of these Streptomyces species share about 80% identities. However, TGase activity was not detected in the cultures of every Streptomyces sp.. The DNA fragment containing the intact TGase gene was isolated from the constructed S. rectiverticillatus partial genomic library. The ORF of the cloned TGase gene has 1257 base pairs from which a 418 amino acids protein with molecular weight of 46.8 kDa could be encoded. The amino acid sequences of the intact and mature part of S. rectiverticillatus TGase share 69.8% and 78.2 % identities with that of S. mobaraense, respectively. The pro- and mature TGase genes of S. rectiverticillatus were separately amplified by PCR and subcloned into pET vector. The TGase protein could be overexpressed with soluble form in BL21(DE3), but no activity could be detected. On the other hand, transglutaminase activity can only be detected in the construct which has pro region of S. mobaraense and S. septatus mature TGase and when the S. mobaraense pro-region was removed by trypsin digestion. To test the TGase activity of S. rectiverticillatus, the extracellular proteins of S. rectiverticillatus were fractionation by ammonium sulfate precipitation and applied to hydrophobic chromatography. The fraction with TGase activities were eluted with the buffer containing 25% ammonium sulfate. The specific activity of the partial purified S. rectiverticillatus TGase is 5.75 U/mg.

縮寫字對照表 3
中文摘要 5
英文摘要 6
前言 7
材料與方法 14
I. 材料 14
一、菌種及質體 14
二、藥品 14
三、酵素 14
四、培養液及緩衝液 14
五、引子 (primers) 15
II. 實驗方法 16
一、染色體DNA之抽取 16
二、E. coli 質體 DNA 的小量抽取 16
三、DNA 的洋菜膠電泳分析 17
四、DNA 片段的回收及純化 17
五、DNA 黏接反應 (ligation) 17
六、引子 (Primer) 的設計 18
七、聚合鏈鎖反應 (polymerase chain reaction, PCR) 18
八、T-vector 之製備 18
九、勝任細胞 (Competent cell) 之製備 19
十、轉形作用 (Transformation) 19
十一、質體快速篩選法( Rapid screen ) 19
十二、DNA 探針的製備 20
十三、南方轉漬雜交法 (Southern hybridization) 20
十四、膠體內雜交法 ( In gel hybridization ) 20
十五、S. rectiverticillatus 染色體 TGase 基因部份基因庫之構築 21
十六、菌落雜交法 (colony hybridization) 21
十七、雙股DNA模板之製備 22
十八、DNA定序分析 22
1. DNA定序反應 (Sanger dideoxy DNA sequencing reaction) 22
2. Automated cycle DNA定序反應 22
十九、菌種甘油保存法 23
二十、Transglutaminase 基因在 E. coli 的表現 23
1. Transglutaminase 蛋白表現載體的構築 23
2. Transglutaminase 蛋白的大量表現 23
3. Transglutaminase 蛋白的純化 23
二十一、SDS-PAGE之蛋白分析 24
1.SDS-PAGE之配製 24
2.蛋白樣本之製備 24
3.泳分析及染色 24
4.西方墨點法 (Western blot) 25
二十二、Transglutaminase 活性的測定 25
二十三、Transglutaminase 酵素活性單位 25
結果 27
一、不同菌株轉麩氨醯胺基因之篩選 27
1. 胞外轉麩氨醯胺蛋白的表現與基因之篩選 27
a.胞外轉麩氨醯胺活性測試 27
b.胞外轉麩氨醯胺之西方墨點法之分析 27
c.南方墨點雜交法之分析 28
d.聚合鏈鎖反應 28
e. DNA序列分析…………………………………………………… 29
二、S. rectiverticillatus TGase基因之選殖……………………………………29
1.部分基因庫之構築 29
2.菌體雜交法 30
3. DNA定序分析 30
三、E. coli菌體內表現S. rectiverticillatus TGase 31
1. mature 部份 31
a. pET-21d 重組質體之構築與表現 31
b. pET-26b 重組質體之構築與表現 32
c. pET-30b重組質體之構築與表現 32
d. pET-32a 重組質體之構築與表現 33
e. pLT7重組體質之構築與表現 34
2. Pro-TGase部份 34
a. pET32a-SrproTGA重組質體之構築與表現 34
b. pET32a-SmproSrTGase、pET32a-SmproSsTGase重組質體之構築與表現 35
四、原菌胞外 TGase 之純化 36
討論 38
參考文獻 43
圖表 51
附錄 87

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