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研究生:林成晉
研究生(外文):Cheng-Chin Lin
論文名稱:探討鏈黴菌屬轉麩氨醯胺酶之前胜肽突變對其成熟酵素之影響
論文名稱(外文):Study the effect Streptomyces transglutaminase propeptidemutations on their mature form enzymative activities
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:李天雄鄭貽生
口試委員(外文):Tien-Hsiung LiYi-Sheng Cheng
口試日期:2019-01-29
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:2019
畢業學年度:107
語文別:中文
論文頁數:82
中文關鍵詞:鏈黴菌屬轉麩氨醯胺酶前胜肽突變
外文關鍵詞:Streptomycestransglutaminasepropeptide mutations
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微生物來源之轉麩氨醯胺酶(MTG),因可催化蛋白質形成分子內或分子間共價鍵結,因而被廣泛應用於食品加工、生物醫學、紡織加工等。基因表達蛋白現時會先形成酶原,然後藉由內生性蛋白酶將其前胜肽(propeptide) 切除後,進而形成具有完整催化活性的成熟酵素。轉麩氨醯胺酶之前胜肽扮有雙重角色,既可以幫助成熟酵素進行正確蛋白折疊,同時又具有抑制成熟酵素活性的特點。本研究之目的在篩選可降低前胜肽與成熟酵素作用的突變株,期望能得到具降低抑制能力但保留伴護蛋白功能之前胜肽突變,最終能得到具較高純度之轉麩氨醯胺酶。首先利用定點及隨機突變的方式,在Streptomyces mobaraense (Sm) 及Streptomyces netropsis (Sn) 兩株鏈黴菌之轉麩氨醯胺酶前胜肽上進行胺基酸置換,並將突變之轉麩氨醯胺酶基因構築在表現載體上,於大腸菌中與TVMV蛋白酶基因進行蛋白共表現。將表現之重組轉麩氨醯胺酶經Ni2+-NTA與陽離子交換(SPFF) 管柱進行純化,篩選經純化後比野生型轉麩氨醯胺酶具有更高活性和蛋白產率之突變株。結果顯示,當Sn和Sm轉麩氨醯胺酶前胜肽Ala13被不同的胺基酸取代後,純化的重組Sn和Sm轉麩氨醯胺酶在其酵素比活性的表現上會有差異,相同的是當Ala13以Tyr,Phe和Trp 胺基酸取代後,轉麩氨醯胺酶酵素活性明顯降低。除此之外,Sm 轉麩氨醯胺酶之前胜肽Asp22以Val 取代或Glu31 以Asp 取代後,可產生比野生型轉麩氨醯胺酶具更高的酵素比活性及蛋白產量。所篩選出之Sm轉麩氨醯胺酶突變株A24VN30YA35V,經由酵素動力學分析,其Km 及 Vmax 分別為 9.9±0.3 mM 與35.4±0.6 mol/min/mg,而酵素催化能力Kcat/Km 為 143.4±3.8 min-1M-1,較野生型102.1±10.3 min-1M-1 提高 40 %。
Microbial transglutaminases (MTG) are able to catalyze intra- or intermolecular crosslinking of protein and have been widely used in the food industry, textiles, and biomedicine. They are first synthesized as zymogens, and the pro-peptides are cleaved by endogenous proteases and processed into mature enzymes. The pro-peptide of MTG has been demonstrated to play dual roles as having both molecular chaperone and inhibitor activities. The aim of this study was to identify pro-peptide mutants of Streptomyces mobaraense (Sm) and Streptomyces netropsis (Sn) TGases which could weaken their interactions with mature enzyme as an inhibitor but maintain their function as a chaperone. Site-directed mutagenesis and random mutations generated by error-prone PCR in the pro-peptide regions of the Sm and Sn TGases were constructed. After co-expression of the mutant proTGase and TVMV protease genes in E. coli, recombinant mature TGases with identical amino acid sequences were purified by Ni2+-NTA and SPFF cation exchange columns. The purified recombinant mature TGases with higher enzymatic activities and yields than the corresponding wild type TGases were subjected to be identified. Results revealed that the purified recombinant mature Sn and Sm TGases display variation in their specific activities when pro-peptide Ala13 replaced by different amino acid residues. However, replacement of Ala13 with Tyr, Phe and Trp, showed significant decrease in mature enzyme specific activities. Moreover, mutants of Sm TGase pro-peptide with Asp22 replaced by Val or Glu31 replaced by Asp produced mature enzymes with higher specific activities and facilitated higher yield than that of the wild type. The pro-peptide mutant of Sm TGase A24VN30YA35V was urified and further characterized to reveal a Km and Vmax of 9.9±0.3 mM and 35.4±0.6 μmol/min/mg, respectively. Results showed that the kcat/Km value of this mutant is 143.4±3.8min-1M-1 and higher 40% than that of the Sm-WT (102.1±10.3 min-1M-1).
摘要 i
Abstract ii
目錄 iii
表目錄 v
圖目錄 vi
縮寫字對照表 vii
一、前言 1
(一) 轉麩氨醯胺酶 (TGase) 1
1. 來源與生理功能 1
2. TGase 作用機制 2
3. TGase 之應用 3
(二) Streptomyces TGase 之表達、純化、生產及前人出現的困難處 3
1. S. mobarensis 及 S. netropsis 之 TGase 比較 3
2. Propeptide 扮演之角色 4
3. 前人 propeptide 之探討 5
4. 對 propeptide 之相關研究 6
(三) 研究動機 7
二、材料與方法 9
(一) 實驗材料 9
(二) 實驗方法 9
1. 建構質體 10
2. 重組 TGase 蛋白之表現與純化 11
3. 蛋白質定量 12
4. 酵素活性測定 12
5. TGase 的生化特性分析 13
三、結果 14
(一) Sn TGase propeptide 之定點突變分析 14
1. Sn-PTGA-A13 (I、N、Q、K、G、P) 單點突變株 14
2. Sn TGase propeptide H16位點之突變分析 15
3. Sn TGase propeptide A13、H16及D22多位點之突變分析 16
(二) Sm TGase propeptide突變株分析 16
1. Sm TGase propeptide A13位點之突變分析 16
2. Sm TGase propeptide前段區域突變株分析 17
3. Sm propeptide 之 helix 1 區域突變株分析 17
4. Sm TGase propeptide 之 helix 2 區域突變株分析 18
5. 酵素動力學分析 19
四、討論 21
(一) Sn TGase propeptide 突變株 21
1. Sn TGase propeptide A13突變株探討 21
2. Sn TGase propeptide H16突變株 22
3. 在Sn TGase propeptide多位點突變株 23
(二) Sm TGase propeptide 突變株 23
1. Sm TGaese propeptide A13 突變株探討 23
2. 在Sm TGase propeptide不同位點突變株 24
(三) 酵素動力學分析 26
五、參考文獻 28
六、附錄 69
附錄一、Sn-TGase 3D結構圖。A13 與催化活性中心相對位置。 69
附錄二、3D結構模擬圖。 70
附錄三、Sm-WT 結構模擬及不同來源 TGase propeptide胺基酸序列比對 71
附錄四、純化之Sn-A13X 重組TGases蛋白之熱穩定性分析。 72
附錄五、純化之Sn-H16X 重組TGases蛋白之熱穩定性分析 73
附錄六、純化之Sm-mutants 重組TGases蛋白之熱穩定性分析 74
附錄七、培養基及緩衝溶液 75
附錄八、標準分生實驗方法 80
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