Study On Wheat Seed Peroxidase WP1 And Its Effects On The Mixing Properties Of Dough

Wheat flour is the ideal raw material for many food products. Recent years, how to improve its dough and breadmaking quality has become a research hotspot in the field of wheat breeding and food industry. The breeders attempt to improve flour processing quality by changing the components of wheat grain and their quantity; and the researchers of food industry attempt to incorporating exogenous components which alter the functionality of dough. Wheat peroxidase 1 is a major peroxidase of wheat grain, which is present in all wheat varieties. WP1 is a typical class III peroxidase which can catalyze the conversion of a large number of phenolic compounds. But, the exact enzymatic property, biological function of WP1 is still unknown. The question we try to give an answer in this thesis is that how WP1 effect on seed developing and dough properties.In this study, WP1 was purified to homogeneity by chromatography, its catalytic properties was analyzed completely, and its effects on flour processing was analyzed by farinograph tests. The cDNA of wheat peroxidase 1(wp1) was cloned and its expression pattern and tissue location during seed developing was analyzed carefully. The gene of WP1 was expressed in E. coli, P. pastoris and K. lactis system, and the functional recombinant enzyme was obtained by affinity purification. The results were listed as below:1,The wheat grain major peroxidase WP1 was step wise purified from flour by water extraction, AMS fractional precipitation, SP HP ion-exchange column chromatography and gel filtration chromatography. The purification resulted in a final enrichment of WP1 by 154-fold, with an activity yield of 8.7%. The final specific activity of WP1 for the H2O2-dependent oxidation of ABTS was 2174 U·mg-1. SDS-PAGE result shows that the molecular weight of WP1 is about 36 kDa. WP1 can catalyze the oxidation of ABTS, Ferulic acid and N-acetyl tyrosine, and the optimal pH for them is 4.15,5.45 and 6.30, respectively. WP1 is thermostability and its peroxidase activity can be promoted by Ca2+.2,The cDNA of wheat peroxidase 1(wp1) was amplified from immature seeds by RT-PCR. The cDNA of Mp1 codes a protein of 358 amino acids, which has a high sequence similarity(92%) with barley peroxidase 1 (BP1). Predicted structure indicates that WP1 belonged to classⅢperoxidase and it has their typical heme binding site and two Ca2+ binding sites.3,Farinograph tests were conducted with the base flour and the base flour with addition of seven combinations of WP1,H2O2,Ca2+ and EDTA. We found the combination of H2O2 showed higher values than the control for development time, dough stability and farinograph quality number. This improving effect can be promote by Ca2+ and inhibit by EDTA; the combination of WP1/H2O2 showed remarkable higher values than the combination of H2O2 for development time, dough stability and farinograph quality number. This improving effect can be promote by Ca2+ and inhibit by EDTA too. This indicates WP1 improve the processing properties of flour, and this effect depends on H2O2 as electron acceptor.4,We obtain the recombination WP1 by expression it in prokaryotic system. In this study we construct His-tag and MBP fusion expression vectors. MBP-WP1 is soluble, and its percentage is up to 34.6%. MBP-WP1 was purified by mylase affinity chromatography and it can catalyze the oxidation of ABTS. His-tagged WP1 existed as inclusion body, and its percentage is up to 18.2%. The target protein was purified by Ni-NTA resin affinity chromatography under denatured condition. Pure WP1 was refolded by gradient urea dialysis, then was mixed with equal volume of adjuvant and used as antigen to immune rabbit to prepare polyclonal antibody. The result of ELISA shows that the titer of rabbit anti-WP1 antiserum is higher than 1:625 000. The result of western blotting demonstrated that the prepared WP1 polyclonal antibody can be used to detect WP1 with high specificity.5,The expression pattern of WP1 during seed developing was analyzed by real-time quantitative PCR and western blotting. Within the first few days after pollination the expression level of wp1 is relative lower. Soon after, the expression of wpl increased rapidly, and it reach the top at 13th day. Before seeds mature, its expression level keep in higher. Using immunohistochemical localization assay we found that WP1 mainly distributed in the extracellular part of endosperm and aleurone layer cell.6,The recombinant K. lactis strain with multi-copy of wp1 was obtained. High activity recombinant WP1 was secreted into fermentation liquor (1050 U/L). The specific activity of recombinant protein in culture medium with lactose is two fold of that with glucose. The recombinant P. pastoris strain of wpl was obtained too. Optimized initial concentration of the recombinant strain is 1.5OD, and the optimized concentration of methanol is 1%, the yield of enzyme reach to top 96 h after inducing (8700U/L). His-tag fusion WP1 was purified by Ni-NTA affinity chromatography.From protein purification to gene cloning, prokaryotic expression, antibody preparation, yeast expression, expression profile analysis, immunohisto-localization assay and farinograph tests, wheat grain peroxidase WP1 was studied widely. We first prove that endogenetic peroxidase WP1 can improve flour processing properties, and give an explanation how WP1 does effect on dough and breadmaking quality. At the same, we first functional expression wp1 in two type of yeast, which indicate we can introduce wp1 into baker's yeast to improve the quality of breadmaking.