| Lipoxygenase (LOX, EC1.13.11.12), which can be easily found in nature, is an oxygenase catalyzing polyunsaturated fatty acid (PUFA) with Cis-1,4 and pentadiene structure. It has been reported that the enzyme is most commonly thought of as a bleaching agent of wheat flour. This action is believed to involve the oxidation of pigments and unsaturated fatty acids by oxygen. Fatty acid radicals produced during the intermediate steps of substrate peroxidation are responsible for oxidative degradation of pigments including β-carotene, xanthophylls and chlorophylls. Also, LOX can increase mixing tolerance and generally enhances dough rheological properties. However, it is very difficult for large-scale production due to the low yield and purity from the plants such as soy bean, recombinant lipoxygenase has therefore become the focus of flour processing.Previously, we successful cloned, expressed and secreted an ana-rLOX from Anabaena sp. PCC 7120 in B. Subtilis. In the work, the mechanism behind the effects mediated by LOX on the protein structure and composition of wheat protein were studied. The lipid regiospecificity of ana-rLOX and formation of lipid peroxidation radicals were characterized, also the modification effects of the enzyme on the wheat proteins were investigated. The protein structure and rheological properties of wheat flour were analyzed, in order to make clear of the mechanism of improving dough rheology in this study. The main results of this study are as follows:1. The relationship between kinetic and structure of ana-rLOXThe ana-rLOX expressed in B. Subtilis was used in this study. The change of secondary structure of ana-rLOX treated by different pH and temperature was analynized. It was found that the optimal pH was 9, but the most stable pH was 7 for ana-rLOX. The optimal temperature for ana-rLOX was 45℃ when pH varied between 6～9. The enzyme activity was positively correlated with the content of helical structure and negative correlated with content of sheet structure. When PH varied between 6～9 and the value of pH increase by every one. The content of helical, sheet and random structure increased by 2.3%,-8.9% and 4.4%; 0.4%,-2.6% and 4.4%;-1.6%,3.7% and -1.6% at 25℃,35℃ and 45℃ respectively.2. Identification peroxidation species catalyzed by ana-rLOXKinetic study showed that LA (linoleic acid), ALA (α-linolenic acid), AA (arachidonic acid), EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) by ana-rLOX catalysis had the substrate specificity, and different Km values indicated thier affinity to substrates:LA>AA>DHA>ALA>EPA. By analysis of Fehling reaction and Xanthine oxidation reaction before and after adding ana-rLOX, it was found that the amounts of free radicals increased significantly by 71.4% in presence of ana-rLOX, indicated that ana-rLOX promoted the formation of LA peroxidation free radicals. Peroxidation radical formation and the region-specificity of ana-rLOX were characterized by using ESI/MS. It was found that ana-rLOX oxygenated at C-13 positions of the substrate LA; at C13, C-16 of ALA; at C-9, C-12, C-15 of AA; at C-12, C-15, C-18 of EPAand at C-14, C-16 of DHA, respectively.2. Effect of ana-rLOX/LA on covalent interactionBy ESR monitoring the generation of free radical from the reaction system of ana-rLOX/LA-cystine and ana-rLOX/LA-tyrosin, it was found the cystine and tyrosine captured the free adical. Three polypeptides, P10338, P10339 and P10340 were artificially synthesized in order to investigate the crosslinking mechanism between the proteins. When the addition of ana-rLOX and LA was 50U,75U,100U,125U,150U to P10339 solution, the content of-SH in P10339 decreased by 32.64%,38.09%,43.12%,46.93%,49.35% respectively, along with the increased of LA. When adding 100 u g of LA in enzymatic reaction system, the content of-SH decreased maxmum.The reactants by radical reaction mediated with ana-rLOX were analyzed by ESI/MS, the ion fragments of m/z 341.67 and m/z 444.95 were found, and were identified as products formed intra and inter molecular with S-S bonds between P10339 by radical reaction mediated by ana-rLOX. In addition, the m/z 609.99 was found, identified as tri-polypeptide product formed covalent bonds among three P10338. And the m/z 816.19 was found, identified as bi-polypeptide product formed covalent bonds between P10340.4. Effect of ana-rLOX/LA on properties of wheat proteinThe FC, EAI of wheat protein was significantly increased when adding ana-rLOX/LA in the wheat proteins. The FC of albumin, globulin and gluten solution increased from 320 ml,292 ml,360 ml to 360 ml,310 ml,385 ml, and the EAI of albumin and globulin solution increased from 4948,5838 to 5838,6019 m2/g before and after the enzymatic reaction, respectively. The average particle size of albumin, globulin and gluten solution respectively decreased from 5.792 μm,2.095 μm,31.736 μm before the enzymic reaction to 3.975μm,1.747μm,15.615μm after the enzymic reaction. The results indicated that the structure of wheat proteins became tighter and more compact due to the oxidation of proteins by the addition of ana-rLOX/LA, evidenced that the particle size of wheat proteins got smaller but larger elasticity.5. Effect of ana-rLOX on property of wheat flourThe improvement effect of ana-rLOX on the rheological property of dough was studied by using farinograph and extensograph measurement. When 30U/g of ana-rLOX was added to wheat flour, the time of dough stability of weak-, medium-and strong-gluten wheat flours, increased 25%,35.7% and 21.4% respectively. When 30U/g ana-rLOX added to the medium-, strong-gluten flour, the AUC increased by 59.7%,56.4% and 85.0%; 21.8%,29.5% and 60.2% at fermentation time 45mins,90mins and 135mins, respectively compared to medium-, strong-gluten flour without ana-rLOX. Various kinds of proteins in dough were extracted to investigate the change of quality characteristics of the flour by adding ana-rLOX. It was found that the glutenin in wheat flours was increased 30.86%. While the level of gliadin, albumin and globulin was decreased 15.25% and 21.57%, with the increase of ana-rLOX activity from 5U per 100 mg flour. The protein composition of was also investigated using HPLC/MS/MS, ana-rLOX could make globulin-3A, globulin 1a and S48186 grain softness protein cross-linking with gliadin and LMW (low-molecular-weight) glutenin, leading to form the protein polymer. |
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