Dataset Information

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.

SUBMITTER: Osorio CE

PROVIDER: S-EPMC7040222 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Directed-Mutagenesis of <i>Flavobacterium meningosepticum</i> Prolyl-Oligopeptidase and a Glutamine-Specific Endopeptidase From Barley.

Osorio Claudia E CE Wen Nuan N Mejías Jaime H JH Mitchell Shannon S von Wettstein Diter D Rustgi Sachin S

Frontiers in nutrition 20200218

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 ...[more]

PMID: 32133368

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Project description:BackgroundLactic acid bacteria are a family of "generally regarded as safe" organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields.ResultsTo expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4-2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding.ConclusionsOverall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

Dataset Information

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

Publications

Directed-Mutagenesis of <i>Flavobacterium meningosepticum</i> Prolyl-Oligopeptidase and a Glutamine-Specific Endopeptidase From Barley.

Similar Datasets

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