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標題: Cloning and Characterization of the Transglutaminase Gene from Streptomyces kentuckense
Streptomyces kentuckense轉麩氨醯胺基因之篩選與酵素特性之研究
作者: 吳師誠
Wu, Shih-Chen
關鍵字: Transglutaminase;轉麩氨醯胺;Specific activity;比活性
出版社: 分子生物學研究所
轉麩氨醯胺酶 (transglutaminase, amine γ-glutaminy transferase, EC2.3.2.13, TGase) 是種醯基轉移酵素,可催化蛋白分子間及蛋白分子內鍵結的形成,此種由轉麩氨醯胺酶所催化的聚合作用,在醫藥方面如血液凝固、細胞分化及增生及食品加工上有多方面的應用。本研究即從S. kentuckense菌株中選殖含完整TGase基因的DNA片段 ,並將選殖的基因放入表現載體,以E. coli進行具活性之TGase蛋白的生產,並對由原菌純化後之TGase酵素進行特性分析。首先將菌株在28℃進行活化後培養四天,經次培養20至24小時後可得最大酵素活性 (1.3 U/ml),利用 Mono S陽離子交換樹脂管柱進行層析,可分離得到純化的TGase蛋白,酵素比活性為38.5 U/mg。在特性分析方面,TGase 酵素最適反應溫度與pH值分別為45℃及6.0,且以放置在pH為6及 -20℃下在保存上最為穩定。加入還原劑及EDTA會提升TGase酵素活性,而銅、鉛、鋅等金屬離子與IAA、NEM及PMSF抑制劑會明顯抑制TGase酵素活性。在熱力學上的分析,來自豬血漿第十三凝固因子對熱的耐受性優於S. kentuckense TGase蛋白。在基因選殖方面,利用S. kentuckense之mautre TGase基因為探針,對所構築之 S. kentuckense 染色體部分基因庫進行菌落雜交,篩選到一含2.6 kb染色體 DNA片段之質體,內含完整TGase基因,將其命名為pBNTGA。完成其中1536鹼基對的DNA序列分析,其中包含一完整ORF (open reading frame) 起始於第281 核苷酸 (ATG) 至第1522 核苷酸 (TAA) 間,轉譯後為413胺基酸。與發表之S. mobaraense成熟TGase的胺基酸序列具有約80.1% 的同質性。針對S. mobaraense及S. kentuckense菌株之染色體DNA,以PCR增幅出1.13 kb的TGase基因DNA片段,在E. coli中進行蛋白表現。收取可溶型蛋白利用蛋白處理後,可獲具TGase酵素活性之重組蛋白。重組的S. mobaraense TGase酵素比活性與原菌株相似,而S. kentuckense TGase重組蛋白之酵素比活性為20 U/mg 。

Transglutaminase (amine -glutaminy transferase, EC;TGase) is an acyl-transfer enzyme that catalyzes the inter- and intramolecular cross-linking of proteins. TGase is involved in several biological phenomena, such as blotting clotting, cellular differentiation and proliferation, and has potential for food protein modification and medical utilization. In this study, extracellular TGase protein from culture fluid of S. kentuckense was purified and characterized. Moreover, a novel TGase gene from S. kentuckense was cloned and sequenced. Recombinant TGase with enzymatic activity was overexpressed in E. coli and purified to electrophoretical homogeneity. Results showed that maximum TGase activity could be detected in the cultural filtrate after subculture for 20 to 24 hours at 28℃. A simple Mono S column chromatography was developed to purify the S. kentuckense TGase and the specific activity of purified enzyme was 38.5 U/mg. The optimal temperature and pH of S. kentuckense TGase were 45℃ and 6.0, respectively, and the purified enzyme was stable for more than eight months when stored at -20℃. The TGase activity of S. kentuckense was slightly increased by adding reducing agents and EDTA, but inhibited by Cu2+, Zn2+, Pb2+, IAA, NEM and PMSF. The intact TGase gene was cloned from the constructed S. kentuckense genomic library. Results of 1536 bp DNA sequence analysis revealed one intact ORF begins at nt 281 (ATG) and stops at nt 1522 (TAA), from which a 413 amino acids protein could be translated. The deduced amino acid sequence of the mature S. kentuckense TGase showed 80.1% identity with that of S. mobaraense. Active TGase proteins could be obtained from the E. coli expressed S. kentuckense and S. mobaraense TGases. The specific activity of purified recombinant TGase of S. mobaraense is similar to native TGase but that of S. kentuckense was 20 U/mg.
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