Enzymatic Properties Of Recombinant WPDI Overexpressed In Pichia Pastoris And Its Application In The Flour Processing

In recent years,the import of low gluten flour has been further expanded with the increasing consumption of biscuit year by year.Addition of weak gluten agents could reduce the strength of medium gluten flour to prepare the dough for biscuit-baking,but either food safety risks or decreased sensory quality of biscuit might be produced when adding weak glutn agent.Therefore,the development of weak gluten agents with safe,healthy and no side effects properties is an important alternative to improve the processing quality of biscuit for the bakery industry.Recombinant wheat protein disulfide isomerase(wPDI)expressed in E.coli decreased the strength of gluten by breakage the disulfide bonds among gluten.In this paper,P.pastoris system for overexpressing recombinant wPDI was constructed to develop a novel biological weak-gluten agent.The recombinant wPDI expressed in P.pastoris was prepared to compare the enzymatic properties with wPDI expressed in E.coli.Moreover,the effects of recombinant wPDIs on farinograph properties and biscuit baking qualities were investigated.The main research contents and results are as follows:(1)The construction of P.pastoris system for overexpressing recombinant w PDI.The base sequences including four thioredoxin domains of wPDI were subcloned from pMD19-T-wpdi,and then the recombinant plasmid pPIC9K-wpdi was successfully constructed.Moreover,the recombinant plasmid was smoothly transferred into competent cells,P.pastoris strain GS115,and a recombinant P.pastoris strain expressing wPDI with relatively high efficiency was screened by analysis of positive transformants and identification of Western Blot.(2)Expression and purification optimization for recombinant wPDI expressed in P.pastoris.The optimal conditions for expression of recombinant wPDI were obtained: the expression medium was BMMY,the concentration of methanol was 1%(v/v),the induction time was 72 h,the pH of the induced culture medium was 6.5,the amount of loaded medium was 10%(v/v),and the OD600 was 2.Additionally,the recombinant wPDI with higher purity was acquired after ammonium sulphate precipitation and anion exchange chromatography.(3)Comparison the properties of recombinant wPDIs expressed in P.pastoris with that in E.coli.Relative to the recombinant wPDI expressed in E.coli,recombinant wPDI expressed in P.pastoris had higher disulfides reductase activity but lower disulfides oxidase activity and molecular chaperone activity.Both recombinant wPDIs had the same optimum reaction temperature(30 ?)and reaction pH(pH 8)for disulfides reductase activity.However,the thermal stability of recombinant wPDI expressed in P.pastoris was better than that of recombinant wPDI expressed in E.coli.Additinally,the recombinant wPDI expressed in P.pastoris showed a higher alkaline-pH resistance,while recombinant wPDI expressed in E.coli exhibited a higher acid-pH tolerance.Moreover,the conformation of the recombinant wPDI expressed in P.pastoris was more compact than that of the recombinant wPDI expressed in E.coli.(4)The effects of recombinant w PDIs expressed in P.pastoris and E.coli on the processing quality of flour.Recombinant wPDI expressed in P.pastoris presented a stronger ability to weak the processing quality of flour than that of wPDI expressed in E.coli at the same level.Both recombinant wPDIs could effectively refined the surface structure,improve the specific volume and texture characteristics of biscuits.Moreover,the recombinant wPDI expressed in P.pastoris exhibited a better ability to improve texture qualities of biscuit than that of wPDI expressed in E.coli.