小麦蛋白质二硫键异构酶的结构及其改善面粉加工品质的机制

Wheat protein disulfide isomerase (wPDI), with gluten as one of the physiological substrates, is a endogenous enzyme which can catalyze the formation of inter- or intra- molecular disulfides, and facilitate the native folding of protein. The processing qualities of wheat flour can be enhanced by the addition of wPDI, the mechanism of improvement is yet unclear. In this project, the enzymatic activity of wPDI catalyzing the reaction for glutenin subunit will be probed, the interactions between wPDI and glutenin subunit will be investigated by isothermal titration calorimetry (ITC) and fluorecence spectra, the molecular mechanism of action of wPDI on the physiological substrate will be uncovered. Furthmore，the three-dimensional structure of oxidized wPDI, reduced wPDI and the complexes of wPDI binding with glutenin subunit will be determined by single-crystal X-ray diffraction. On the basis of the informations of crystal structures, mutated wPDIs with different structures and special activity will be constructed through site-directed mutagenesis and other molecular biology techniques, the effects of mutated wPDIs on the cross-linking of glutenin subunits will be investigated as well. The role of redox activity, isomerase activity and molecular chaperone activity of wPDI in catalyzing the formation of gluten network structure from glutenin subunit will be dissected to understand the molecular mechanism that wPDI strengthen the gluten network. The results will not only lay the molecular and structural foundation for the improvement of wPDI on the processing quality of flour, but also enrich the fundamental research invovled in the processing quality of flour and protein disulfide isomerases (PDIs). Similarily, the project will facilitate the appliction of PDIs in many industries such as food industry.

小麦蛋白质二硫键异构酶（wPDI）是催化二硫键形成、促进蛋白质正确折叠的内源酶，面筋蛋白是其生理底物之一。添加wPDI能改善小麦面粉的加工品质，但其强化机理尚不明确。本项目研究wPDI催化麦谷蛋白亚基的反应活性，应用等温滴定量热、荧光光谱探讨wPDI与麦谷蛋白亚基的相互作用，揭示wPDI作用于生理底物的分子催化机理；应用单晶X射线衍射解析不同氧化还原状态wPDI及其与麦谷蛋白亚基复合体的立体结构，应用基因定点突变等技术构建不同催化活性和结构特点的突变型wPDI，考察其在催化麦谷蛋白亚基聚集方面的影响，明确wPDI的氧化还原活性、异构酶活性和分子伴侣活性在催化面筋网络形成中的作用，阐明wPDI改善面粉加工品质的分子机制。研究结果不但为应用wPDI改善面粉加工品质奠定分子与结构基础，而且可以丰富面粉加工品质及蛋白质二硫键异构酶（PDI）相关基础研究的学术内容，推动PDI在食品等工业领域的应用

小麦蛋白质二硫键异构酶（wPDI）是催化二硫键形成、促进蛋白质正确折叠的内源酶，面筋蛋白是其生理底物之一。本项目采用基因工程手段分别制备了大肠杆菌和毕赤酵母重组wPDI，两种重组蛋白均具有氧化还原活性、异构活性和分子伴侣活性。应用粉质仪、流变仪、扫描电镜、傅里叶红外光谱以及质构仪等探究了重组wPDI对不同面粉加工品质的影响，wPDI会减弱面筋强度，降低面筋蛋白二级结构β-折叠比例，这一特性可提高超硬质小麦粉面包和饼干的烘焙品质。为了明确wPDI不同活性对面筋网络结构的作用规律，应用生物突变和化学改性的方法获得了8种wPDI截短蛋白和2种改性wPDI。在面制品加工过程中，wPDI的氧化还原活性主要表现为还原面筋网络的二硫键，降低面筋强度；相反，分子伴侣活性则降低了SDS-可溶面筋蛋白含量，提高了面筋蛋白大聚集体（GMP）含量，从而起到强化面筋网络结构的作用。同时，应用单晶X射线衍射技术构建了与wPDI同属硫氧还蛋白家族的小麦谷氧还蛋白（wGrx）的立体结构模型，为研究wGrx的催化机制及其联合小麦硫氧还蛋白（wTrx）改善面粉加工品质提供了结构基础。研究结果丰富了面粉加工品质及蛋白质二硫键异构酶（PDI）等相关基础研究的学术内容，推动了硫氧还蛋白家族蛋白在食品等工业领域的应用。