A new microbial gluten-degrading prolyl endopeptidase: Potential application in celiac disease to reduce gluten immunogenic peptides

Abstract

Gluten is a complex of proteins present in barley, wheat, rye and several varieties of oats that triggers celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and therefore, proline-rich digestion-resistant peptides contain multiple immunogenic epitopes. Prolyl endopeptidases (PEP) hydrolyse internal proline residues on the carboxyl side of peptides and have been proposed for food gluten detoxification and as oral enzyme supplementation for celiacs. The aim of this study was to identify new gluten-degrading microbial enzymes with the potential to reduce gluten immunogenicity by neutralizing its antigenic epitopes. Using a gluten-degrading colony screening approach, a bacterial isolate (2RA3) displaying the highest glutenase activity was selected, characterized and its genome completely sequenced. The identification through 16S rDNA gene sequencing showed a 99,1% similarity to Chryseobacterium taeanense . Hydrolysis of gluten immunogenic peptides (GIP) was further monitored, over a 48-hour period, by colony encapsulation in gliadin-containing microspheres, followed by detection with the G12 anti-GIP monoclonal antibody. Glutenase activity was detected in the extracellular medium of 2RA3 cultures, where gel electrophoresis and gliadin zymography revealed the presence of a ~50 kDa gluten-degrading enzyme. Nano-ESI-Q-TOF of the excised active band identified 7 peptides contained in the protein product predicted for an open reading frame (ORF) in the 2RA3 genome. Based on sequence similarity to the PEP family, the new enzyme was named PEP 2RA3. The PEP 2RA3 coding sequence was PCR-amplified from C . taeanense 2RA3, cloned and expressed in Escherichia coli as a C-terminally His-tagged recombinant protein and purified by Ni-NTA affinity chromatography. The recombinant protein, with predicted molecular mass and isoelectric point of 78.95 kDa and 6.8, respectively, shows PEP activity with standard chromogenic substrates, works optimally at pH 8.0 and 30°C and remains stable at pH 6.0 and 50°C, indicating a potential use in gluten-containing food process applications. The ability of the recombinant enzyme to degrade GIP in beer into smaller peptides was confirmed.

Microbiome of root vegetables—a source of gluten-degrading bacteria

“…It has been shown that the human digestive tract has bacteria that can successfully break down and eliminate peptide fragments that are resistant to human peptidases (Caminero et al 2014;Herrán et al 2017). Extracellular protease genes are abundant in free-living bacteria (Nguyen et al 2019), and the most powerful proteases that degrade gluten are produced by microorganisms isolated from the surrounding environment (Mitea et al 2008;Amador et al 2019). Knowing that the diversity of human microbiome is decreasing as more people move away from nature (Grönroos et al 2019), it is tempting to hypothesize that there is a direct connection between gluten intolerance and a decrease in the available microorganisms capable of gluten destruction.…”

“…Both approaches use peptidases from various sources such as fungi, bacteria, and germinated cereal grains (Curiel et al 2014;Guandalini and Assiri 2014;Wolf et al 2015;Scherf et al 2018;Schulz et al 2018). Postproline cutting prolyl endopeptidases from Sphingomonas capsulata, Flavobacterium meningosepticum, Myxococcus xanthus, Aspergillus niger, Flammulina velutipes, and Chryseobacterium taeanense, among others, have been pursued as drug candidates for the enzymatic treatment of gluten to treat celiac disease (Shan et al 2004;Mitea et al 2008;Schulz et al 2018;Amador et al 2019). Although many digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes, prolyl endopeptidase from Aspergillus niger is able to degrade toxic gluten epitopes (Janssen et al 2015).…”

Characterization of Bacillus cereus AFA01 Capable of Degrading Gluten and Celiac-Immunotoxic Peptides

“…2RA3 revealed genes encoding proteases of the S9 family. A prolyl oligopeptidase was identified, and it degraded gluten immunogenic peptides in beer [36]. Then, we analyzed the genes catalyzing the hydrolysis of the C-N bond in the three B. cereus strains, and the number of those genes was similar among the three strains.…”

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

“…Gluten-degrading enzymes seem to hold the most promise as attractive therapies for helping patients with CD to avoid accidental gluten ingestion and to promote better overall health. A prerequisite is that such enzymes should be active under gastro-duodenal conditions, quickly neutralize the T-cell-activating gluten peptides and be safe for human consumption [ 67 , 68 , 70 , 109 ].…”