Study On Transglutaminase Treated Ara H 2 Reaction System

Peanut is listed among eight major food allergens by Food and Agriculture Organization(FAO).Peanuts allergn can cause a severe allergic reaction,even endager life,and the allergic reaction generally does not disappear with the growth of the age.With the improvement of people’s living standards,more and more attention has been paid on peanut allergy,therefore,more and more research focused on reducing sensitization ability of peanut allergens.In this study,since Ara h 2 is an important allergen in peanut protein,it has a great practical application to reduce the sensitization of Ara h 2.The present study explored the reaction system of MTGase-catalyzed Ara h 2 cross-linking,the reactions affected by reduction and protein concentration were also investigated.The changes of the structure of the MTGase-catalyzed products were analyzed,the digestibility and potential allergenicity of products were also evaluated.Moreover,the cross-linked products were separated to obtain low molecular weight polymers and high molecular weight polymers,and the differences in the structure and properties of that were analyzed.The methods,results and conclusions were addressed as follows.1.The product of reduced Ara h 2 treated by MTGase was analyzed by SDS-PAGE electrophoresis,dimer and other polymers were clearly observed in the SDS-PAGE profile,indicating that Ara h 2 could inter-crosslink when catalyzed by MTGase after reduction.In addition,monomer Ara h 2 showed a higher electrophoretic migration compared with that of native Ara h 2 after MTGase treatment,the high electrophoretic mobility is mainly due to the Stokes radius value,which should be low when the intramolecular cross-linking occured.Due to the removal of ammonia,deamidation can increase the pH of the protein solution,it was found that the pH of protein solution of native and reduced Ara h 2 treated by MTGase increased,the number of removed ammonia from both reduced and native Ara h 2 treated by MTGase was more than two per molecule protein,these results demonstrated that deamination also occurred in the reaction treated by MTGase.There are three reactions to the MTGase-catalyzed reduced Ara h 2: intermolecular cross-linking,intramolecular cross-linking and deamidation,only intramolecular cross-linking and deamidation occured in the reaction of MTGase-catalyzed native Ara h 2.2.The reactions affected by protein concentration,reduction and temperature were investigated.In the reaction of MTGase-catalyzed reduced Ara h 2,the high-concentration protein benefited the intermolecular reaction,and low-concentration protein favored the appearance of intramolecular cross-links.And the reduction was more conducive to the exposure of glutamyl residues,which is helpful for deamidation.In the reaction of MTGase-catalyzed native Ara h 2,the reaction was not affected by protein concentration,but the deamidation increased with the increasing of temperature.3.The intra-crosslinking sites were determined by mass spectrometry,and the changes of structure and surface hydrophobicity of Ara h 2 were detected by circular dichroism,UV spectroscopy and fluorescence spectroscopy.For enzymatic native Ara h 2,only one cross-linked peptide was observed.18 cross-linked peptides,which involved five different epitopes,were identified for reduced protein,of these,13 fragments contained allergen epitopes,and overlaped 5 linear epitopes.The changes of structure of native Ara h 2 treated by MTGase was not obvious,while after reduction,the α-helix content decreased and β-sheet content increased.The α-helix content of low molecular weight polymers was higher than that of the high molecular weight polymers,the intension of UV absorption of the low molecular weight polymers was lower than that of the high molecular weight polymers,and the surface hydrophobicity was higher than that of the high molecular weight polymers,indicating that the structure of the intra-crosslinked protein become compact.4.The effect of MTGase on the digestibility of Ara h 2 was analyzed by simulates stomach and intestine digestion in vitro.Native Ara h 2 was resistant to gastric digestion,but Ara h 2 could digested by trypsin after gastric digestion,and some 13 kDa fragments still existed after 80 min digestion.The digestibility of native Ara h 2 treated by MTGase did not alter,while the reduction and cross-linking treatment could accelerate the digestibility of Ara h 2,the reduced and cross-linked products could be digested into 3.5 kDa fragments after gastrointestinal digestion.The digestibility of the high molecular weight polymers was higher than low molecular weight polymers,as the intra-crosslinking protein with compact structure could reduce the digestion rate of cross-linked products.5.The potential allergenicity of MTGase-mediated Ara h 2 was evaluated by competitive ELISA.The MTGase-treatment did not substantially alter the IgG binding capacity of Ara h 2,and the reduction treatment could decrease the ability of IgG binding capacity,but not significantly,while the cross-linking could,and the IgG binding capacity of high molecular weight polymers was lower than that of low molecular weight polymers.Similar to the binding capacity of IgG,the high molecular weight polymers had the lowest IgE binding capacity.After gastrointestinal digestion,the IgG binding capacity of high molecular weight polymers still was the lowest.In the third chapter,it was found that when the protein concentration was higher,the reaction benefited the intermolecular cross-linking,therefore,in order to modify the direction of reaction to get the product with low allergenicity,the high protein concentration can be applyed to promote the reaction to intermolecular cross-linking.