小麦内质网氧化还原酶的分子改造及其改善面粉加工品质的研究

Previous researches in our group found that recombinant wheat endoplasmic reticulum oxidoreductin 1 (wEro1) could significantly improve the flour processing quality, but the catalytic efficiency and the thermostability needed to be improved. In this project, the three-dimensional structure of wild type wEro1 will be determinated by the single-crystal X-ray diffraction, the catalytic mechanisms of wEro1 improving flour processing quality will be investigated by elucidating two catalytic oxidation pathways in the flour reaction system, one is the transfer and exchange of disulfides, the other is the direct oxidation by the hydrogen peroxide which is generated as byproduct. Based on the catalytic mechanisms and three-dimensional structure, wEro1 will be designed rationally to strengthen the ability of improving flour processing quality. For increasing the catalytic efficiency of wEro1, the motifs involved the regulatory disulfides will be mutated to eliminate the feedback regulation of the activity, and the side chain characteristics of the key amino acid residues for substrate-binding will be modified to adjust the substrate specificity. Moreover, the thermostability of wEro1 will be enhanced by the combination of mutagenesis with molecular dynamics simulation. The influence rules of molecular modifications on the catalytic characteristic of wEro1 and the ability of improving the flour processing quality will be investigated, the structure-function relationship of wEro1 strengthening gluten will also be illuminated, and the molecular mechanism for the mutant wEro1 enhancing the gluten networks will be established. The results will not only lay the theoretical and technological foundation for the molecular modification of the enzyme for improving the flour processing quality, play a vital role of the advanced biotechnologies in the food science, but also enrich the academic connotation of the protein structure-based rational design to facilitate the progress in the involved cross-subject combined with food science, structural biology and protein engineering.

项目组前期研究发现，重组小麦内质网氧化还原酶（wEro1）能够显著改善面粉加工品质，但酶催化效率和热稳定性有待改进。本项目应用单晶X射线衍射技术解析wEro1的三维立体结构，从二硫键转移交换途径和过氧化氢直接氧化途径探讨野生型wEro1改善面粉加工品质的催化机理。并以此为基础，对wEro1分子进行理性设计，通过生物突变调控二硫键相关功能域消除酶活性的反馈调控、改变底物结合相关关键氨基酸残基性质调节底物特异性以提高酶催化效率，并结合分子动力学模拟提高wEro1的热稳定性。考察分子改造对wEro1催化性能及改善面粉加工品质功能的影响规律，阐明wEro1结构与功能的内在关系，建立突变型wEro1强化面筋网络的分子机制。研究结果不仅为分子改造面粉改良酶制剂提供理论和技术基础，发挥前沿生物技术在食品科学领域的重要作用，而且可以丰富以结构生物学为基础的蛋白质理性设计的学术内涵，推动相关交叉学科的进步。

内质网氧化还原酶（Ero1）在体内不但能利用氧分子生产二硫键并转移交换给其它内质网蛋白质，而且在催化反应过程中能释放过氧化氢，小麦体内面筋蛋白二硫键来源于小麦Ero1（wEro1）催化反应。本项目在前期研究基础上，分别采用大肠杆菌和毕赤酵母制备了基因重组wEro1，研究了重组酶的酶学性质和催化机制，wEro1能以二硫苏糖或小麦蛋白质二硫键异构酶（wPDI）为底物催化巯基氧化；而且，wEro1催化氧化的wPDI能以变性蛋白质为电子供体，促进变性蛋白质的折叠。单独添加wEro1能显著提高面粉的粉质特性、面团的流变特性以及面包的焙烤品质。添加wEro1能够促进面筋蛋白二硫键交联，强化面筋网络结构，提高面筋蛋白大聚集体（GMP）含量，以及GMP中超大分子量聚集体比例，增加面筋蛋白二级结构β-转角含量，从而改善面粉加工品质。而且，wEro1改善面粉加工品质机理主要与其催化反应产生的过氧化氢氧化面筋交联相关。以序列同源性分析和同源建模结构为指导对wEro1进行理性设计，在9个与酶活相关的关键半胱氨酸残基定点突变体中，获得5个活性提升的正向突变体，其中的2个突变体的活性增加了1倍以上。解析了小麦源谷氧还蛋白（wGrx）的晶体立体结构，发掘了新的立体结构特征和新的催化途径。此外，研究了小麦过氧化氢酶、水溶性抗性糊精等对面粉加工品质的影响规律，探讨了麦醇溶蛋白基凝胶体系的改性和形成机制。研究结果丰富了面粉加工品质、小麦内源酶以及面筋蛋白等相关基础研究的学术内容，推动了面制品加工行业的进步。