Streptomyces mobaraense轉醯胺麩胺酸基酶基因之選殖與其在E. coli宿主之表現

Cloning of Transglutaminase from Streptomyces Mobaraense and Its Expression in Escherichia coli

陳珍妮(Jen-Ni Chen) ； 林建谷(Chien-Ku Lin) ； 林榮流(Long-Liu Lin)

《弘光學報》 47期 (2005/11) Pp. 51-59

https://doi.org/10.6615/HAR.200511.47.06

摘要

轉醯胺麩胺酸基酶(Transglutaminase; TGase)是一種廣泛於食品質地及官能特性改良之酵素。本研究以Streptomyces mobaraense的轉醯胺麩胺酸基酶(TGase)基因序列為藍本設計出引子，再以該菌的基因體DNA作為模板，以PCR技術複製出TGase基因。回收PCR產物和pET-27(+)載體以HindIII 及 NcoI二種限制選殖切割後，黏結成表現質體pET-SmTG。表現質體pET-SmTG先送入選殖宿主E.coi Nova blue選殖成功後，再轉型入表現宿主E. coli BL21(DE3)。轉型成功的菌體經IPTG再37℃進行誘導後，以SDS-PAGE分析MTGase在細胞內德表現位置，再利用輕和性His-Bind-Resin管柱純化轉醯胺麩胺酸基酶(MTGase)並分析其酵素活性。結果顯示，經PCR製造出的成熟tgl基因序列和原始序列完全相同，且有相當MTGase分子量39KD的蛋白被表現出，並成功純化出結構為雙體的變性TGase蛋白，而變性蛋白經透析復性卻偵測不出酵素活性。此表現蛋白經細胞定位分析得知：MTGase只被表現再cytoplasmic區域且以inclusion body形式存在。若表現培養溫度至25℃，再native狀態可稍微增加TGase溶解度及一些活性，但整體酵素活性偏低。在未來研究中，嘗試引入chaperone 及 trigger factor基因，以協助快速表現蛋白之正確folding，將是一重要的改善之道。

關鍵字

MTGase ； 基因選殖

並列摘要

Microbial transglutaminase(MTGase) has been widely used for improvement of food functionality. In this study, mature transglutaminase gene was amplified from Streptomyces mobaraense by PCR. PCR product and cloning vector pET-27b(+) are both double digested by HindIII and NcoI, and then ligated to construct an expression plasmid, pET-SmTG. This expression plasmid was first transformed into cloning host, E coli Novablue and into expression host, E coli BAL21(DE3) subsequently. The validity of insert obtained from transformant was confirmed by DNA sequencing. After induction of IPTG, expressed protein of MTGase was detected via the accumulated 39D-protein on SDS-PAGE. The expressed proteins were then purified using Ni-NTA His-bind affinity column. However, neither intracellular nor extracellular MTGase activity was detected significantly. Localization of expressed MTGase protein showed that these enzymes were secreted in cytoplasmic region only and existed in an inactive form of inclusion body. Therefore, the incorporation of chaperone and trigger factor genes for assisting exact folding of MTGase might be an important approach for further study.

並列關鍵字

Transglutaminase ； Streptomyces mobaraense ； Gene cloning

參考文獻

Motoki, M., and Segyro, K. (1998), Transglutaminase and its use for good processing. Trends in food Science and Technology, 9, pp204-210

Kuraishi.C., Sakamoto, J. and Soead, T. (1996), The usefulness of transglutaminase for good processing in Biotechnology for improved foods and Flavors. American Chemical Society (ACS) Symposium Series 1037, pp29-38.

Matheis, G. and Whitaker, J.R. (1987), A review: enzymatic cross-linking of Proteins applicable to foods. Journal of Food Biochemistry, 11, pp309-327

Takagaki, Y., Narakawa, K,, and Uchio, R. (1991), Coating of vegetable and fruits with transglutaminase and proteins for preservation, Jpn kokai Tokkyo koho JP 03272639

Yamauchi, K., Uenikawa, S., Enomoto, A., Tanimoto, H., Oohatak, M., and Motoki, M. (1991), Transglutaminase for reducing allergenicity of food proteins and peptides and method of reducing their allergenicity, Jpn kokai Tokkyo koho Jp0327253