Directed-Mutagenesis of Flavobacterium meningosepticum Prolyl-Oligopeptidase and a Glutamine-Specific Endopeptidase From Barley

Abstract

Wheat gluten proteins are the known cause of celiac disease. The repetitive tracts of proline and glutamine residues in these proteins make them exceptionally resilient to digestion in the gastrointestinal tract. These indigested peptides trigger immune reactions in susceptible individuals, which could be either an allergic reaction or celiac disease. Gluten exclusion diet is the only approved remedy for such disorders. Recently, a combination of a glutamine specific endoprotease from barley (EP-B2), and a prolyl endopeptidase from Flavobacterium meningosepticum (Fm-PEP), when expressed in the wheat endosperm, were shown to reasonably detoxify immunogenic gluten peptides under simulated gastrointestinal conditions. However useful, these "glutenases" are limited in application due to their denaturation at high temperatures, which most of the food processes require. Variants of these enzymes from thermophilic organisms exist, but cannot be applied directly due to their optimum activity at temperatures higher than 37 • C. Though, these enzymes can serve as a reference to guide the evolution of peptidases of mesophilic origin toward thermostability. Therefore, a sequence guided site-saturation mutagenesis approach was used here to introduce mutations in the genes encoding Fm-PEP and EP-B2. A thermostable variant of Fm-PEP capable of surviving temperatures up to 90 • C and EP-B2 variant with a thermostability of up 60 • C were identified using this approach. However, the level of thermostability achieved is not sufficient; the present study has provided evidence that the thermostability of glutenases can be improved. And this pilot study has paved the way for more detailed structural studies in the future to obtain variants of Fm-PEP and EP-B2 that can survive temperatures ∼100 • C to allow their packing in grains and use of such grains in the food industry.

Gluten-degrading bacteria: availability and applications

“…This peptide is the largest and most toxic degradation product. It contains six partially overlapping immunodominant CD epitopes that induce gluten-specific immune responses and intestinal inflammation Cavaletti et al 2019) in the core of bread during baking, where temperatures generally do not exceed 100°C (Osorio et al 2020).…”

New Insights into Non-Dietary Treatment in Celiac Disease: Emerging Therapeutic Options

“…Likewise, therapies focused on the regulation of the immunostimulatory effects have been described for other related pathologies, and due to their efficacy, their indications have been extended to CD. Sphingomonas capsulata (SC-PEP) [70,71] Aspergillus niger (AN-PEP) [72] Gluten hydrolytic enzyme cocktail SC-PEP and EPB-2 (ALV003) [73] FM-PEP and EPB-2 [74] Subtilisin derived from Rothia mucilaginosa (Sub-A) [75] Cysteine endopeptidase derived from Hordeum vulgare (EP-B2) [21] Elastase derived from Homo sapiens (CEL-3B) [22] Table 1. Cont.…”

Prediction and characterization of the T cell epitopes for the major soybean protein allergens using bioinformatics approaches

“…In order to development of soybean hypoallergenic products, proteases that can hydrolyze the above five amino acid sites (I, V, S, N, and Q) can be selected. For example, the endopeptidase Glu-C can hydrolyze glutamate (E) and glutamine (Q) [52][53][54] can be used to specifically destroy the digestionresistant T cell epitopes "SKDNVISQIPSQVQE" (aa 558-572) (P11827) and EEQRQQEGVIVE" (aa 201-212) (P25974).…”