【摘要】：目的:克隆小麥蛋白質二硫鍵異構酶(wheat protein disulfide isomerase,wPDI)基因,實現(xiàn)其在大腸桿菌中的表達并探究其酶學性質。方法:以小麥種子總RNA為模板,逆轉錄并擴增得到wpdi,并以pET-30b為表達載體、大腸桿菌BL21(DE3)為宿主菌進行原核表達,表達產物經(jīng)金屬螯合層析純化后進行了酶學性質研究。結果:克隆的基因全長1 548 bp,與"Wyuna"品種小麥wpdi基因相似性達99%。構建了pET-30b-wpdi表達載體,獲得w PDI的最佳表達條件為:誘導溫度22℃,誘導時間6 h,誘導劑濃度0.5 mmol/L。該酶含4個硫氧還蛋白結構域,分子質量約為66.2 kD,具有二硫鍵的還原酶和異構酶活性以及分子伴侶活性。結論:對重組wPDI的表達和酶學性質研究,為wPDI在面制品加工及其他方面的應用提供參考依據(jù)。
[Abstract]:Aim: to clone wheat protein disulfide isomerase (wheat protein disulfide isomerasewPDI gene and express it in Escherichia coli and investigate its enzymatic properties. Methods: using total RNA of wheat seed as template, wpdil was obtained by reverse transcription and amplification. PET-30b was used as expression vector and Escherichia coli BL21 (DE3) was used as host strain for prokaryotic expression. The expression product was purified by metal chelate chromatography and its enzymatic properties were studied. Results: the total length of the cloned gene was 1 548 BP, which was similar to the wpdi gene of "Wyuna" wheat. The pET-30b-wpdi expression vector was constructed and the optimal expression conditions of w PDI were obtained as follows: induction temperature 22 鈩，

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