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.
Highlights
Gluten is the common name for proteins present in all cereals of the Triticeae grass tribe in which the major industrially relevant crops are barley, wheat and rye [1]
We set 0.1% (w/v) gliadin in the target to search for enzymes with improved gluten-detoxification potential
In order to test the optimal gliadin concentration for the bacterial activity the selected strain was inoculated onto plates containing increasing concentrations of gliadin ranging from 0.04 to 0.5% (w/v) since it has been reported that the concentration of certain antigenic peptides builds up in the upper intestinal lumen following ingestion of a typical meal harbouring *10 g of gluten [33]
Summary
Gluten is the common name for proteins present in all cereals of the Triticeae grass tribe in which the major industrially relevant crops are barley, wheat and rye [1]. Celiac disease develops in genetically susceptible individuals and is triggered by the exposure to partially digested gluten proteins [6]. The only available therapy for CD is the complete avoidance of dietary gluten. Sustaining a strictly gluten-free diet (GFD) is very challenging. There is a need for the use of enzymes as additives or as processing aids in the food biotechnology industry, either to detoxify gluten or as non-dietary oral therapies for celiac patients [10]
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