Expression of Wheat Protein Disulfide Isomerase in Pichia pastoris and the Properties of the Recombinant Enzyme
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Abstract:
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.
