小鼠攝護腺穀胺酸轉氨?在酵母菌中的表現及其特性

Abstract

穀胺酸轉氨?（蛋白質－穀胺醯酸γ-穀胺醯基轉移?，EC2.3.2.13）可催化醯基轉移反應，將兩個蛋白質或勝?經由穀胺醯酸與穀胺醯酸或穀胺醯酸與離胺酸的鍵結而連結起來。我們利用小鼠攝護腺cDNA基因庫中篩選出的小鼠攝護腺穀胺酸轉氨?基因為範本，DPSAC-1及DPSAC-2為引子 (Primer)進行聚合?連鎖反應，將所得之基因片段構築於表現載體pYEX－Sl上，並轉殖進入酵母菌(Saccharomyces cerevisiae) BJ2168中表現。由北方雜合法(Northern Blotting)證實小鼠攝護腺穀胺酸轉氨?基因在酵母菌中能進行轉錄作用，並產生一個約60kDa的小鼠攝護腺穀胺酸轉氨?蛋白。利用hydroxamate的生成反應，計算出穀胺酸轉氨?之k(上標 m)為19.65mM，?之比活性(specific activity)為8.08?mole/min/mg。每公升酵母菌可生產0.9mg的穀胺酸轉氨?。由於穀胺酸轉氨?在食品加工上可改良食品的品質，我們將酵母菌含有穀胺酸轉氨?的培養基上清液加入麵糰中，可使麵糰的抗延展力增加及延展性降低，而改善麵糰的品質。

Transglutaminase (protein-glutamine γ-glutaminyltransferase, EC 2.3.2.13) is an enzyme capable of catalyzing acyl-transfer reactions by introducing covalent cross-links between proteins or peptides via peptide bond formation between glutamine and glutamine or glutamine and lysine. A cDNA encoding rat prostate transglutaminase was amplified by polymerase chain reaction (PCR) with DPSAC-1 and DPSAC-2 primers. The PCR product carrying the rat prostate transglutaminase gene was inserted into pYEX-S1, and expressed in Saccharomyces cerevisiae BJ2 168. The northern blot analysis indicated a RNA of 2-kb was transcribed in yeast, and a protein of about 60 kDa was expressed. The activity of transglutaminase was determined by hydroxamate assay. The k(subscript m) and specific activity of the enzyme was 19.65 mM and 8.08?mole/min/mg, respectively. Zero point nine milligrams of transglutaminase can be produced from one liter of culture medium. The ability of transglutaminase to improve the properties of foods was also examined. After transglutaminase treatment, the dough became less sticky and extensible, and more resistant extension.