Modification of the reaction system of Ara h 2 catalyzed by MTGase: Products and reaction conditions analysis

Abstract

Peanut (Arachis hypogaea) is listed among the eight major food allergens, in which Ara h 2 is the major allergen. The microbial transglutaminase (MTGase)‐catalyzed cross‐linking reaction reduces the allergenicity of Ara h 2. However, deamidation might occur and influence the cross‐linking reaction. In this work, native and reduced Ara h 2 were catalyzed by MTGase. In addition to intermolecular cross‐linking, intramolecular cross‐linking and deamidation were proven to occur simultaneously. Moreover, intramolecular cross‐linking sites were identified using mass spectrometry and the PLINK software. The reactions moved toward different directions because of different reduction conditions and different protein concentrations. Practical applications Cross‐linking is a common processing method used to improve the textural and functional properties of protein. Deamidation reaction might occur during processing and influence the cross‐linking reaction. The two types of reactions have a competitive relation, because both of them occur on glutamine residue. In Ara h 2 and MTGase system, protein concentration and reduction content influence the direction of reaction. The results can be applied to modify the reaction system of Ara h 2 catalyzed by MTGase. In addition, the relationship between structure and properties changes on Ara h 2 was discussed based on analysis of cross‐linking sites.

Crosslinked Recombinant-Ara h 1 Catalyzed by Microbial Transglutaminase: Preparation, Structural Characterization and Allergic Assessment

“…As seen in Figure 1 C, the average particle size decreased when the protein concentration was lower than 0.4 mg/mL, illustrating the possibility of intramolecular crosslink or deamidation happened inside of the protein molecules. This result is consistent with the former studies [ 23 , 24 ]. The significant increase in particle size occurred when the concentration of rAra h 1 was higher than 0.7 mg/mL, illustrating the intermolecular crosslinks happened during MTG-catalyzed reactions.…”

“…Deamidation would happen when the water becomes the acyl acceptor; glutamine residues could be converted to glutamic acid residues at this time [ 41 ]. The structural characteristics of products mainly depend on the specific reaction conditions, like substrate concentration, temperatures, and pH et al According to Wu’s research [ 23 ], in MTG-catalyzed aggregation, intermolecular crosslinking was more likely to happen when the concentration of reduced Ara h 2 was higher than 0.7 mg/mL. Comparably, intramolecular crosslinking was more likely to be induced when the concentration of reduced Ara h 2 ranged from 0.2 to 0.4 mg/mL.…”

“…According to the research which used MTG-catalyzed reactions to lower the antigenicity of peanut proteins [ 23 , 24 ], the reaction time was normally set between 4 and 5 h. Here, we tested the influence of different reaction times (1–5 h) on the structure and antigenicity of cross-linked rAra h 1. As shown in Figure 1 J, the content of monomer decreased significantly when the reaction time reached 1 h. However, the content of the high-molecular-weight polymer did not increase along with the time extension.…”

“…Transglutaminase (TGase, EC 2.3.2.13) has been extensively utilized in modern food industries like meat packing and Tofu production [ 21 ], for it could catalyze acyl transfer reactions between the γ-carboxyamide of glutamine residues (acyl-donors) and various primary amines (acyl acceptors) [ 22 ]. The ε-(γ-glutamyl) lysine isopeptide bonds are formed when the ε-amino groups of lysine residues act as acyl acceptors [ 23 ], and cross-linking between protein molecules would subsequently happen. Recently, there is some research beginning to use microbial transglutaminase (MTG) in desensitization research.…”