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研究生:許惠琪
研究生(外文):Hui-Chi Hsu
論文名稱:Streptomyces netropsis 胞外金屬性蛋白酶基因之篩選與酵素特性研究
論文名稱(外文):Cloning and Characterization of an Extracellular Metallopeptidase Gene from Streptomyces netropsis
指導教授:楊明德楊明德引用關係
指導教授(外文):Ming-Te Yang
口試委員:林彩雲曾夢蛟
口試日期:2011-07-22
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:77
中文關鍵詞:轉麩氨醯胺酶轉麩氨醯胺酶轉麩氨醯胺酶
外文關鍵詞:Streptomycesmetallopeptidasetransglutaminase
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Streptomyces 可分泌多種蛋白酶,這些胞外蛋白酶可以將環境中的蛋白質分解,作為營養的來源,近年來研究指出它們也可能參與了菌體的分化。Streptomyces 所分泌的轉麩氨醯胺酶 (Transglutaminase, amine γ-glutaminy transferase, EC 2.3.2.13; TGase) 是種醯基轉移酵素,可催化蛋白分子間及分子內鍵結的形成。分泌至胞外的 TGases 先為酶原 (zymogen) 狀態,需要有蛋白酶參與才能活化 TGases。本研究希望能從 Streptomyces 菌株選殖出具有可活化 TGase 能力的胞外蛋白酶基因,並於 E. coli 及 Corynebacterium 菌體內進行重組蛋白酶之表現,作進一步的探討及應用於活化態的 TGase 的生產。藉由構築 Streptomyces netropsis 染色體部分基因庫,選殖出一完整蛋白酶基因,此 ORF (open reading frame) 起始於第 119 核苷酸 (GTG) 至第 2386 核苷酸 (TGA),共有 2268 bp,推測可轉譯出 755 個胺基酸,分子量為 78.7 kDa 的蛋白質,推衍的胺基酸序列中包含 HEXXH 的序列,為金屬蛋白酶家族的典型序列。更進一步分析顯示,整個蛋白酶包含了一段訊號胜肽、N 端 pro-region、mature region 及 C 端 pro-region。將此選殖之金屬蛋白酶基因 (Sn mp) 於 E. coli 中進行蛋白表現測試,結果顯示僅在帶有 PelB 訊號胜肽的構築,可在胞外偵測到重組 Sn MP 之活性。進一步將 Sn mp 基因轉接到可以在 coryneform bacteria 複製存在之 pMT51 載體,以 Corynebacterium 作為蛋白表現系統之宿主,結果顯示在菌株培養之胞外液,可偵測到重組 Sn MP 的活性,但重組 Sn MP 的表現量不佳。將帶有 Sn mp 基因的轉形菌株 C. glutamicum ATCC 13869(pMT51-PMP) 與生產 proTGase 的 C. glutamicum ATCC 13869-CSmPTGA 菌株共同培養,由胞外液可以明顯觀察到有 mature 形態之 TGase 產生,顯示由 C. glutamicum ATCC 13869(pMT51-PMP) 分泌至胞外的重組 Sn MP 具有活化重組之 S. mobaraensis proTGase 之活性。另外,亦嘗試由培養 S. netropsis 之胞外培養液中純化 Sn MP,首先測試表現 Sn MP 活性最佳的時間點,發現於次培養後 24 小時,已有活性出現,28 小時的時候活化 proTGase 的效果最好,到了32 小時活性有些減弱。而經由 DEAE 陰離子交換樹脂的初步純化,Sn MP 的比活性可由原來胞外液的 0.04 U/mg 提升為 2.16 U/mg。

It is well-known that Streptomyces is able to produce many extracellular proteases. These extracellular proteases are mainly involved in assimilating extracellular proteinaceous nitrogen sources. Recently, they have been suggested to be involved in Streptomyces development. In Streptomyces, transglutaminase (amine γ-glutaminy transferase, EC 2.3.2.13; TGase) is an acyl-transfer enzyme which is able to catalyze the inter- and intramolecular cross-linking of proteins. TGases of Streptomyces have been shown to be secreted as a zymogen, and an activation process directed by extracellular proteases in culture medium has been demonstrated. In this study, a novel extracellular metallopeptidase gene (mp) from Streptomyces netropsis was cloned and sequenced. Furthermore, recombinant metallopeptidases, which were able to process and activate the precursor of S. netropsis transglutaminase, were expressed and identified in E. coli and Corynebacterium. The intact metallopeptidase gene was cloned from the constructed S. netropsis genomic partial library. Results of 3919 bp DNA sequence analysis revealed one intact ORF, begins at nt 119 (GTG) and stops at nt 2386 (TGA), from which a 755 amino acids protein (78.7 kDa) could be translated. The deduced amino acid sequence contains a HEXXH sequence, a typical motif of metallopeptidase family. The metallopeptidase is predicted to be translated as a preproprotein consisting of four domains: a signal peptide, N-terminal pro-region, mature region and a C-terminal extension region. Expression of the S. netropsis metallopeptidase gene (Sn mp) in E. coli revealed that a PelB signal peptide fused construct was allowed to obtain an active form recombinant Sn MP in the culture. The Sn mp gene was also cloned into the expression system of Corynebacterium. Unfortunately, expression of recombinant Sn MP was too low to be detected by SDS-PAGE. However, by coexpressing of Sn mp with S. mobaraensis proTGase in C. glutamicum, processed mature TGase could be identified in the culture medium. This result demonstrated that recombinant Sn MP is able to be secreted and processed as an active form protease in C. glutamicum. To purify Sn MP from culture fluid of S. netropsis, time course of proTGase processing activity of extracellular proteases was conducted. Results showed that proTGase processing activity was observed after 24 h of subculture. The highest enzyme activity was found after 28 h of subculture and the activity declined after 32 h of subculture. Moreover, the Sn MP was partially purified by DEAE ion-exchange column, and the specific activity of Sn MP was increased from 0.04 U/mg to 2.16 U/mg.

中文摘要 I
英文摘要 II
縮寫字對照表 III
前言 1

材料與方法
I.材料
一、菌種與質體 7
二、藥品 7
三、酵素 7
四、純化管柱 8
五、引子 8
六、培養基及緩衝液 8
II.實驗方法
一、質體 DNA 之小量萃取 9
二、Streptomyces 染色體 DNA 之萃取 9
三、DNA 片段回收及純化 10
四、DNA 黏合作用 (Ligation) 10
五、製備大腸桿菌之勝任細胞 (Competent cell) 10
六、熱休克轉形作用 (Heat shock transformation) 11
七、製備 Corynebacterium 之勝任細胞 11
八、Corynebacterium 之電穿孔法 (Electroporation) 11
九、菌種甘油保存 11
十、蛋白質凝膠電泳分析 (SDS-PAGE) 12
十一、西方墨點分析 (Western blot) 12
十二、硝酸銀染色 (Silver stain) 12
十三、DNA 探針之製備 13
十四、南方墨點雜交法 (Southern hybridization) 13
十五、Streptomyces netropsis 染色體部分基因庫之構築 14
十六、菌落雜交法 (Colony hybridization) 14
十七、蛋白酶活性測定 (Hydrolysis of casein) 14
十八、聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 15
十九、膠體活性染色分析 (Zymography) 15
二十、Streptomyces netropsis 胞外金屬蛋白酶之純化 16

結果
一、Streptomyces netropsis 胞外金屬蛋白酶基因之選殖 17
1. 聚合酶鏈鎖反應與定序比對分析 17
2. 南方墨點雜交法分析 S. netropsis 染色體 DNA 17
3. 部分基因庫之建構及篩選 18
4. DNA 定序及比對分析 18
二、E. coli 菌體內表現 S. netropsis 胞外金屬蛋白酶 19
1. 重組質體pET21-SPMP 之構築與蛋白表現 19
2. 重組質體pET21-PMP 之構築與蛋白表現 20
3. 重組質體pET26-PMP 之構築與蛋白表現 20
三、C. glutamicum 菌體內表現 S. netropsis 胞外金屬蛋白酶 21
1. 重組質體pMT51-PMP 之構築 21
2. C. glutamicum ATCC 13869(pMT51-PMP) 蛋白表現測試 22
2.1 BHIS 培養液 22
2.2 No. 66 培養液 22
2.3 CM 培養液 23
2.4 TYG 培養液 23
四、於 C. glutamicum 使用不同的訊號胜肽表現重組蛋白酶 23
1. 重組質體 pMT-s949-PMP 之構築 24
2. 重組質體 pMT-s1834-PMP 之構築 24
3. 蛋白表現測試 24
五、共同培養 C. glutamicum ATCC 13869(pMT51-PMP) 與生產胞外重組 Streptomyces TGase 之 C. glutamicum 菌株 25
六、S. netropsis 胞外金屬蛋白酶之純化 26

討論
一、S. netropsis 胞外金屬蛋白酶基因之選殖 28
二、重組 S. netropsis 胞外金屬蛋白酶之表現 28
三、不同培養基對 C. glutamicum 分泌胞外蛋白之影響 30
四、不同 signal peptide 對 C. glutamicum 分泌胞外蛋白之影響 30
五、S. netropsis 金屬蛋白酶之純化 31

參考文獻 32
圖表 37
附錄 65

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