小麦蛋白质二硫键异构酶的毕赤酵母表达及重组酶性质研究

为开发天然健康的酶制剂型面粉改良剂，利用毕赤酵母表达了小麦蛋白质二硫键异构酶（wheat protein disulfide isomerase，wPDI）。以克隆质粒pMD19-T-wpdi为基因模板，亚克隆至毕赤酵母表达载体pPIC9K中，并以毕赤酵母GS115为宿主菌进行真核表达，表达产物经硫酸铵沉淀和阴离子交换层析纯化后，与大肠杆菌重组wPDI的酶学性质进行了比对，并利用粉质仪探究了重组wPDI对面粉品质的影响。结果表明，克隆的wpdi基因含有1347个碱基，共编码449个氨基酸，分子量约为50.2 ku。Western blot结果显示，构建的重组酵母表达系统成功表达了重组wPDI；阴离子交换层析获得的wPDI的酶学性质研究表明，酵母重组wPDI表现出了二硫键氧化还原活性和分子伴侣活性，其还原活性高于大肠杆菌重组wPDI，氧化活性和分子伴侣活性低于大肠杆菌重组wPDI。粉质实验结果表明，相对于大肠杆菌重组wPDI，酵母重组wPDI表现出了更强的弱化面粉加工品质能力。研究结果为wPDI的深入研究及其在面制品中的应用奠定了基础。
Wheat protein disulfide isomerase (wPDI) was expressed in Pichia pastoris to develop a novel and healthy enzyme as a flour improver. The recombinant plasmid, pMD19-T-wpdi, was used as a template and subcloned into a P. pastoris expression vector, pPIC9K, and then expressed in P. pastoris GS115, which was used as a eukaryotic host. After the expressed products were purified by ammonium sulfate precipitation and anion exchange chromatography, the enzymatic properties of the recombinant wPDIs from P. pastoris and E. coli were compared, and their effects on the farinograph characteristics were investigated using a farinograph. The results showed that the cloned wpdi gene contained 1347 bp, encoded 449 amino acids, and had a molecular weight of about 50.2 ku. Western blot analysis showed that the recombinant wPDI was expressed in the yeast expression system. The enzymatic characteristics of the wPDI purified with anion exchange chromatography were analyzed. The results revealed that the recombinant wPDI expressed in P. pastoris showed both oxidoreductase activity from the disulfide bonds, as well as chaperone activity. The wPDI expressed in P. pastoris showed higher reductase activity than that expressed in E. coli; however, the oxidase and chaperone activity of the wPDI from P. pastoris were lower than those of the wPDI from E. coli. The results of the farinograph assay showed that the recombinant wPDI expressed in P. pastoris was better able to weaken the processing quality of flour than that expressed in E. coli. These results provided a basis for in-depth studies on wPDI and its application in flour products.