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E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance.

ABSTRACT: Gluten proteins are the causative agent of Celiac Disease (CD), a life-long food intolerance characterized by an autoimmune enteropathy. Inadvertent gluten exposure is frequent even in celiac patients complying with a gluten-free diet, and the supplementation of exogenous gluten-digestive enzymes (glutenases) is indeed a promising approach to reduce the risk of dietary gluten boost. Here we describe Endopeptidase 40, a novel glutenase discovered as secreted protein from the soil actinomycete Actinoallomurus A8, and its recombinant active form produced by Streptomyces lividans TK24. E40 is resistant to pepsin and trypsin, and active in the acidic pH range 3 to 6. E40 efficiently degrades the most immunogenic 33-mer as well as the whole gliadin proteins, as demonstrated by SDS-PAGE, HPLC, LC-MS/MS, and ELISA. T lymphocytes from duodenal biopsies of celiac patients showed a strongly reduced or absent release of IFN-? when exposed to gluten digested with E40. Data in gastrointestinal simulated conditions suggest that no toxic peptides are freed during gluten digestion by E40 into the stomach to enter the small intestine, thus counteracting the intestinal inflammatory cascade to occur in CD patients. E40 is proposed as a novel candidate in Oral Enzymatic Therapy for the dietary management of gluten toxicity.

SUBMITTER: Cavaletti L

PROVIDER: S-EPMC6739405 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance.

Cavaletti Linda L Taravella Anna A Carrano Lucia L Carenzi Giacomo G Sigurtà Alessandro A Solinas Nicola N Caro Salvatore De S Stasio Luigia Di LD Picascia Stefania S Laezza Mariavittoria M Troncone Riccardo R Gianfrani Carmen C Mamone Gianfranco G

Scientific reports 20190911 1

Gluten proteins are the causative agent of Celiac Disease (CD), a life-long food intolerance characterized by an autoimmune enteropathy. Inadvertent gluten exposure is frequent even in celiac patients complying with a gluten-free diet, and the supplementation of exogenous gluten-digestive enzymes (glutenases) is indeed a promising approach to reduce the risk of dietary gluten boost. Here we describe Endopeptidase 40, a novel glutenase discovered as secreted protein from the soil actinomycete Act ...[more]

PMID: 31511534

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Project description: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.

Dataset Information

E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance.

Publications

E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance.

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