微生物轉麩胺醯胺酶之基因選殖及在大腸桿菌中表現

Molecular Cloning of Microbial Transglutaminase and its expression in E.coli

陳珍妮(Jen-Ni Chen) ； 陳錦樹(Chin-Shuh Chen)

《弘光學報》 75期 (2015/06) Pp. 115-124

https://doi.org/10.6615/HAR.201506.75.08

摘要

微生物轉麩胺醯胺基酶(Microbial Transglutaminase; ＭTGase)是一種廣泛應用於食品質地及官能特性改良之酵素。本研究以Streptoverticillium mobaraense 的轉麩胺醯胺基酶(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Hind III及Nco I二種限制酶切割後，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.coi BL21(DE3)。轉型成功的菌體經IPTG在37℃進行誘導後, 以SDS-PAGE分析MTGase在細胞內的表現位置，再利用親和性His-Bind-Resin管柱純化轉麩胺醯胺基酶（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，且成功以變性方式將TGase蛋白純化出來。經表現蛋白之細胞定位分析得知：MTGase只被表現在cytoplasmic區域且以inclusion body形式存在。若降低培養溫度至25℃，在native狀態可稍微增加TGase溶解度及一些活性,但整體酵素活性仍偏低。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 Streptoverticillium 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 BL21(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 ca. 39KD-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

Microbial Transglutaminase ； Gene cloning

參考文獻

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