Purification And Characte Research Of Wheat Seed Peroxidase Associated With Tyrosine Cross-Links

Wheat processing quality has close relationship to wheat protein content and composition. Wheat gluten high molecular weight glutenin, low molecular weight glutenin and gliadins cross-link comprise gluten three dimensional protein netwok structure, which affects wheat flour processing quality directly. It is significant for wheat flour processing quality to elucidate how glutens was formed. In 2001,Tilley postulated that tyrosine cross-link were a very important factor to form gluten, which challenged traditional disulfide interchange theory. All results show that tyrosine cross-link in life-form were catalyzed by peroxidase or an enzyme associated with peroxidase. This study purified a peroxidase from wheat seed(WsPOD). The characters of WsPOD's physi-chemical,absorption spectrum and trypsin enzyme hydrolyzed peptide mass fingerprinting were analyzed. The result show that WsPOD can catalyze tyrosine cross-linking to dityrosine and effect whet flour processing quality. It will be helpful to understand tyrosine cross-link mechanism of gluten network and find another way to improve wheat flour processing quality. It also will be useful to discover a new effectively and safely additive instead of chemical oxidants to foodstuff industry.1. The crude enzyme extraction was acid deposited with acetic acid to Ph4.0 and fractioned with 35-75% saturation solid ammonium sulfate, separated by Source 30S column, Superdex G-75 column, respectively. The enzyme was determined to be homogenous by SDS-PAGE which apparent molecular weight is about 36KD.2. Using guaiacol as substrate, the optimum enzyme catalyzing reaction conditions of the WsPOD was carried out. The result shows that the optimum reaction pH is 5.0, the optimum reaction temperature is 40℃, the optimum reaction concentration of hydrogen peroxide is 14mmol/L. The absorption spectrum scan of the enzyme in UV-1100 ultraviolet-visible Spectrophotometer show that WsPOD is an acid enzyme containing with a haemochrome prosthetic group which has a obvious soret aborption at 399 nm. The WsPOD shows a good stability dealing with high temperature and is able to retain its catalytic activity at elevated temperature. . 3. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF MS) was applied to the analysis of peptide mass fingerprinting (PMF) in WsPOD . The PMF data of WsPOD was analyzed using Mascot search, and there was only one matched which is wheat endosperm enzyme named peroxidase1 and expressing after post anthesis 8—12 days. The conservertive search using scanprosite software show that WsPOD has two conservertive blocks; one is a biocatalytic activity site, the other is a haemochrome prosthetic group link site.4. The compound of tyrosine reacting were analyzed through absorption spectrum scan and high-performance liquid chromatography electrospray ionization-tandem mass spectrometry (HPLC-ESI/MS). The result show there has the production of dityrosine. It suggests that the WsPOD can catalyze tyroine cross-link.5. Taken wheat Luo Yang 8716 as the basic flour, following the method of GB/T 14614-93, the WsPOD was added into the basic flour and mixed, the result show that the WsPOD increased wheat flour mixing time and stable time,and heightened Farinograph Quality Number(FQN) score. It had a positive effect on dough viscidity , elasticity and mixing-resistance.6. The dityrosine were synthesized following the method of Michael Tilley. The dityrosine were purified through HPLC-C18 Revered phase column. It can be used as a standard substance in the next experiments.