Expression And Characterization Of Wheat Protein Disulfide Isomerase And Effect On Flour Processing Quality

Protein disulfide isomerase (PDI) is an oxidation-reduction enzyme that belongs to thethioredoxin family and exists in endoplasmic reticulum of all kinds of eukaryote. It has oxidereducate activity of catalyzing the formation of disulfide bonds, isomerase activity ofcatalyzing the refolding of misfolding proteins and chaperon activity of catalyzing proteinfolding even with on disulfide bond. Wheat protein disulfide isomerase (wPDI) participates inthe formation of wheat gluten, but research about the effect of PDI on wheat flour is rare andit is still controversial to identify the effect.In our study, the total RNA was obtained from the albino seedlings of Triticum aestivumcultivars of “zhongyou9507” and reverse transcription was made to clone the cDNA. Weconstructed the recombinant expression vector and high level of recombinant proteins wereexpressed in BL21(DE3). The recombinant proteins were purified using affinitychromatography, followed by the identification and analysis of the characteristics of thepurified proteins. The effect of wPDI on processing quality of wheat flour was alsoinvestigated. The main results are as below:(1) We successfully cloned the gene of wpdi. The total base sequence of wpdi was1548bp. It shows a high similarity of above99%comparing with genes from other breeds of wheat.There is also a high similarity between other plants, such as similarity of96.7%with barley,60.9%with lemon and58.6%with sweet potato.(2) We constructed the recombinant expression vector which was named asPET-30b-wpdi and transferred into BL21(DE3) to induce the production of recombinantproteins. The optimumexpression conditions were as follows: induced under the temperatureof22°C, concentration of IPTG was0.5mmol/L and induced for6hours.(3) After extracting protein by ultrasounic method to obtain the soluble protein, metalchelate affinity chromatography was used to purify wPDI. wPDI showed good isomeraseenzymatic activity，reducation activity and chaperone activity. It can renature sixty percentactivity of denatured RNase and aggregation inhibitory rate of denatured GAPDH was35%.The reduction activity of the enzyme activity was about4.3U/mg. The optimum pH andtemperature of insulin reduction reaction is7.0and30°C, respectivily. The recombinantwPDI maintained a high enzyme activity when pH varied from7to8and temperature varied from25-40°C.(4) wPDI can impact the development time, stability time and mechanical toleranceindex significantly. With the increase addition amount of wPDI, the strength and themechanical stirring performance of wheat gluten improved. And excessive addition of wPDIcould weaken the effect. Bread baking test showed that recombinant wPDI had no significanteffect on bread volume but can improve the softness and chew resistance significantly.