Study On The Mechanisms For Recombinant Wheat Endogenous WPDI And WErol Affecting The Flour Processing Quality

Wheat is one of three major food crops in the world,and the yield of wheat in China accounts for one-sixth of world production.The processing quality of flour in China can hardly meet the need of consumers for high quality flour products due to genes,growth environment and storge conditions of wheat.The improvement of flour processing quality is a critical problem which is being studied in the field of cereal chemistry and processing.Using enzymes as flour improvers instead of the chemical improvers is an interesting alternative to improve processing quality of flour.Nowadays,many enzymes such as glucose oxidase,lipoxygenase and transglutaminase have been employed to improve processing quality of flour.In the present study,two recombinant thiol oxidoreductases from wheat,protein disulfide isomerase(wPDI)and endoplasmic reticulum oxidoreductinl(wErol),were focused as the potential flour improvers.The effect mechanism of wPDI on processing quality of flour was firstly investigated,and then the modified wPDIs with different activities were prepared.Moreover,the effects of the modified wPDIs on flour processing quality were explored.On the basis of these studies,wErol was expressed in E.coli and its functional roles with or without wPDI on dough were investigated.The main research contents and results are as follows:(1)The effect of recombinant wild-type wPDI on the processing quality of flour.The effects of wPDI on processing quality of different flours were investigated by farinographic and rheological analysis,scanning electron microscopy,Fourier transform infrared spectroscopy and texture profile analysis(TPA).The addition of wPDI,which exhibited both enzymatic activities(including oxidoreductase activity and isomerase activity)and molecular chaperone activity,weakened the dough strength,indicated by decreased dough development time,dough stability time,and viscoelastic moduli(G’,G").The weakened dough could be attributed to the reduction of disulfide bonds among gluten by reducase activity of wPDI,which was supported by the damaged gluten network structures,reduced β-sheet structure level in gluten and improved free sulfhydryl contents of gluten.Moreover,the results of TPA indicated that the addition of wPDI deteriorated the quality of bread baked from the normal high gluten flour while improved that of bread baked from the superhard wheat flour.(2)The preparation of the modified wPDIs and their influences on the flour processing quality.Eight truncated wPDIs and two modified wPDIs were prepared by molecular biological technology and chemical modification method.A truncated protein of wPDI,fragment AB with mainly oxidase activity of disulfide bonds,and two modified wPDIs,mPDI and aPDI with molecular chaperone activity only were selected to investigate their effects on the processing quality of flour.The results showed that AB weakened gluten strength similar to wPDI.Oppositely,both modified wPDIs improved the processing quality of flour,indicated by the prolonged dough stability times and decreased hardness and chewiness of bread.Moreover,the formation of gluten network was facilitated by the modified wPDIs for the increase amount of gluten macropolymer(GMP)and the decrease content of SDS-soluble gluten.(3)The research on the heterologous expression and the characterization of wErol.The pET-28-werolwas constructed,and then successfully expressed in E.coli BL21(DE3).After the metal chelate affinity chromatography and the size exclusion chromatography,the monomer of wErol was obtained with recovery of 44%enzyme activity.The specific activity of wErol catalyzing the reduction of free FAD was 7.8 U/mg.wErol was captable of catalyzing the oxidation of DTT and wPDI,accompanied by the consumption of oxygen and formation of H2O2.Moreover,wErol catalyzing the oxidation of DTT was pH-dependent,and wErol catalyzed the oxidation of wPDI using denatured protein as an electron donor and facilitated its folding.(4)The improvement of wErol on the flour processing quality.The addition of wErol could strengthen the processing quality of flour,indicated by the improved mixing characteristics,viscoelastic properties and bread qualities,the addition of wErol and wPDI did not show synergistic actions on improvement of dough properties yet.The improvement effects of wErol could be attributed to the formed disulfides cross-linking among gluten,the enhanced gluten network,the improved GMP content and the increased macromolecular polymers level in GMP,and the increased content of β-turn structure of gluten.Moreover,the peroxide content improved in dough after adding the wErol,the improvement mechanism should be involved in disulfide cross-linking in gluten by the peroxide which was produced by the catalytic reaction of wErol.