Gliadin [KLH]

Protein glutaminase (PG) is an enzyme that specifically catalyzes the deamidation of glutamine residues on proteins or peptides, remarkably improving the solubility, emulsification and foaming properties of food proteins and, thereby, conferring great potential in food industry applications. PG is primarily produced from wild strains ofChryseobacterium proteolyticumand the low enzyme production yield restricts large-scale industrial applications. In this context, by evaluating different cleavage site insertions between the pro-region and mature domain of PG as well as different linkers flanking the cleavage site, anE. coliexpression and purification protocol has been developed to produce active recombinant PG. To simplify the production workflow, we developed a sequential dual expression system. More than 15 mg of pure and active PG was obtained from 1 L of shaking-flask bacteria culture by one-step nickel affinity chromatography purification. The enzymatic characteristics of the recombinant PG protein were similar to those of native PG. For the deamidation effect of recombinant PG, the deamidation degree (DD) of gliadin reached up to 67% and the solubility increased 84-fold. Thus, this study provides a practical approach to mass producing active PG proteins and investigates its potential applications on food proteins.

Gliadins are major allergens responsible for wheat allergies. Food processing is an effective strategy to reduce the allergenicity of gluten. In the present study, we determined the secondary and tertiary structures of gluten and gliadins treated by chemical, physical, and enzymatic means through FTIR, surface hydrophobicity, intrinsic fluorescence spectra, and UV absorption spectra. The results showed that the three treatments of phosphorylation and alcalase and papain hydrolyses significantly changed the conformational structures of gliadins, especially the secondary structure. Then, the potential allergenicity of the phosphorylated and alcalase and papain hydrolyzed gliadins were further characterized, and we observed a significant decrease in the allergenicity through the results of the index of spleen, serum total IgE, gliadin-specific IgE, histamine, and serum cytokine concentrations. An elevation of Th17 cells, the absence of Treg cells, and an imbalance in Treg/Th17 are associated with allergy. On the basis of the expression levels of related cytokines and key transcription factors, we also confirmed that phosphorylation and alcalase and papain hydrolysis could effectively reduce the allergenicity of gliadins by improving the imbalance of both Th1/Th2 and Treg/Th17 in the spleens of sensitized mice. This study suggested that the changes in conformational structure contribute to gliadin hyposensitization and that phosphorylation and alcalase and papain hydrolysis may be promising strategies for the production of wheat products with low allergenicity.