IngredientesProlyl endopeptidase (PE), also known as prolyl oligopeptidase or post-proline cleaving enzyme, is an enzyme that in humans is encoded by the PREP gene. It is a large cytosolic enzyme that belongs to a distinct class of serine peptidases. The enzyme carries multiple designations in the scientific literature, reflecting its long and complex nomenclature history.
Common synonyms and designations include:
The name "post-proline endopeptidase" was recommended by IUBMB in 1978, and then changed to prolyl endopeptidase in the supplement to Enzyme Nomenclature in 1981. Although prolyl oligopeptidase was recognized as a serine peptidase as early as 1977, it was commonly found to be activated by thiol compounds, and for a time the thiol-dependent activity was recognized by a separate EC number. On the basis of the oligopeptidase nature of the reaction catalyzed and the amino acid sequence homology with other oligopeptidases, the name prolyl oligopeptidase (POP) was proposed.
Two distinct EC numbers appear in the literature, reflecting different forms and classifications: Prolyl endopeptidases (PEP, EC 3.4.21.24) hydrolyse internal proline residues in peptides. A related but technically distinct classification, EC 3.4.21.26, is also used in current databases for the broader prolyl oligopeptidase family.
Natural sources: Prolyl endopeptidase has been purified from a number of plant (carrots, mushrooms), microbial (Flavobacterium meningosepticum) and animal tissues. In animals, the enzyme is found ubiquitously throughout the body; however, prolyl endopeptidase is generally found in highest concentrations within the CNS.
Prolyl oligopeptidase (PREP) is a serine protease (EC 3.4.21.26) that is ubiquitously expressed throughout the body, with the highest protein levels in brain, kidney, testis, and thymus; however, the expression levels do not necessarily correlate with PREP enzymatic activity.
Microbial and biotechnological sources investigated for supplement and food-processing applications include:
A number of studies have examined degradation of wheat gluten and to some extent barley gluten and components by prolyl endopeptidase initially extracted from Flavobacterium meningosepticum and other microbes, such as Xanthomonas sp., Aeromonas hydrophila, Sphingomonas capsulata, Halobacterium halobium S9, Lactobacillus helveticus, Myxococcus xanthus, Aspergillus niger, and Aspergillus oryzae.
Molecular characteristics: Prolyl endopeptidase (PEP), also known as prolyl oligopeptidase, is a highly conserved 76 kDa post-proline serine protease. The endogenous human enzyme is encoded by the PREP gene on chromosome 6.
Common commercial preparations: Among proposed enzyme-based treatments for gluten-related conditions, only AN-PEP with a tradename "Tolerase® G" is commercially available. In the supplement marketplace, AN-PEP is also found under the branded product name GliadinX. Capsule preparations have also been developed in which the dry enzyme powder is blended with excipients, encapsulated in a hard gelatin capsule, and enteric coated using Eudragit L30-D55 polymer coat, which provides sufficient resistance to gastric conditions and rapid release under duodenal conditions.
Prolyl endopeptidase is not a traditional herbal or botanical remedy with centuries of use in any healing tradition. It is an enzyme identified entirely through twentieth-century biochemical research. There is no known record of deliberate, preparation-based traditional medicinal use in any culture.
Prolyl oligopeptidase or prolyl endopeptidase (POP or PREP) was first described 50 years ago as an oxytocin-cleaving enzyme, and it was further characterized as a peptidase able to cleave short peptides at the C-side of an internal proline.
More precisely, in 1971, Walter Roderich and colleagues discovered the first prolyl oligopeptidase extracted from human uterine tissue, showing it inactivates oxytocin by cleaving the bond between proline and leucine. In 1976, Walter then discovered and extracted a prolyl endopeptidase from lamb kidney. Then in 1978, Yoshimoto and Tsuru found prolyl endopeptidase activity in a pathogenic bacterium, Flavobacterium meningosepticum, with Pro-X activity similar to the lamb kidney PEP.
The enzyme was first described in the cytosol of rabbit brain as an oligopeptidase, which degrades the nonapeptide bradykinin at the Pro-Phe bond.
Because of its specificity, and the occurrence of internal proline residues in several neuropeptides, PREP was soon regarded as a peptidase relevant in neuropeptide metabolism, with great drug target potential for neurological disorders' therapy. Accordingly, synthesis and testing of PREP inhibitors became the attention of academia and industry, especially since evidence emerged that PREP inhibition improved cognitive decline and dementia in several animal models.
The supplemental, dietary, and food-processing use of exogenous PEP enzymes—particularly AN-PEP—emerged in the early 2000s, driven by research into enzymatic gluten detoxification as a potential adjunct or aid for people with celiac disease and gluten sensitivity.
PEPs belong to the serine protease family (clan SC, family S9), which is a group of peptidases that can hydrolyze peptides smaller than 30 residues. PREP cleaves peptide bonds at the C-terminal side of proline residues. Its activity is confined to action on oligopeptides of less than 10 kD, and it has an absolute requirement for the trans-configuration of the peptide bond.
Prolyl endopeptidase is thought mechanistically to act as a serine protease, cleaving peptide bonds by a mechanism similar to other serine proteases such as α-chymotrypsin, trypsin, and subtilisins. The PEP mechanism is thought to be an induced-fit mechanism, where the native enzyme exists in a conformationally flexible open state but is changed to a closed state upon substrate binding.
The acid prolyl endopeptidase catalyses the hydrolysis of peptide bonds at the C-terminal side of proline residues, and to a lesser extent of alanine residues, of peptides and proteins, releasing peptides and amino acids.
Prolyl oligopeptidase is a serine protease found in bacteria and mammals. It cleaves on the carboxyl side of proline residues in oligopeptides, but cannot cleave proteins. Prolyl oligopeptidase will not cleave Pro–Pro bonds, nor N-blocked peptides of the sequence Z-Pro–X.
The enzyme is involved in the maturation and degradation of peptide hormones and neuropeptides such as alpha-melanocyte-stimulating hormone, luteinizing hormone-releasing hormone (LH-RH), thyrotropin-releasing hormone, angiotensin, neurotensin, oxytocin, substance P and vasopressin.
Known PREP substrates include the neuropeptide substance P and thymosin-β4, the precursor to the bioactive peptide Ac-SDKP. In addition, PREP also regulates many additional peptides, including bioactive peptides and proline-rich peptides (PRPs).
Administration of the PEP inhibitor S17092 to rats reverses scopolamine-induced amnesia and increases brain levels of the proline-containing peptides substance P, α-melanocyte-stimulating hormone, thyrotropin-releasing hormone, and arginine-vasopressin. Cleavage of bradykinin and angiotensin I-II by PEP suggests that it is involved in blood pressure regulation.
PEP can also function intracellularly by regulating the inositol 1,4,5-trisphosphate pathway and cell transport mechanisms through association with microtubules.
Prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein (aSyn), a key protein involved in development of Parkinson's disease and other synucleinopathies. PREP inhibitors reduce aSyn aggregation, but the mechanism has remained unknown. Using protein-fragment complementation assays and microscale thermophoresis, PREP has been shown to interact directly with aSyn in both intact cells and in a cell-free system. PREP enhances the formation of soluble aSyn dimers in live neuroblastoma cells.
The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Mammalian enzymes cannot effectively break down proline- and glutamine-rich protein sequences, resulting in incomplete degradation of gluten in the intestinal lumen. This exposes the intestinal mucosa to immunogenic gluten peptides, which in celiac disease (CeD) will reactivate the disease and gluten-specific T cells.
The A. niger prolyl endoprotease works optimally at pH 4–5, remains stable at pH 2, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory epitopes.
AN-PEP, a prolyl endopeptidase isolated from Aspergillus niger, is capable of degrading gluten and related peptides in the stomach itself before they reach the small intestine, as it is resistant to degradation by gastric juices.
Gluten immunogenic peptides are highly resistant to digestion by intestinal brush border (BBM) peptidases, with only dipeptidyl carboxypeptidase I able to act upon them at a very limited rate. Treatment of these peptides with prolyl endopeptidase results in cleavage at internal proline residues, which in turn generates new recognition sites for brush border aminopeptidases.
This is the area with the greatest volume of research on exogenous PEP as a dietary supplement. The evidence ranges from robust in vitro and preclinical work to several human clinical trials, with mixed but cautiously promising results.
Preclinical and in vitro evidence: Prolyl endopeptidase specifically hydrolyses peptide linkages occurring downstream of a proline residue. It was hypothesized that it can be used to degrade the peptide sequences responsible for celiac disease, as all of the toxic epitopes are proline-rich. Multiple in vitro studies across various microbial PEP forms have confirmed broad gluten-degrading capacity.
Key clinical trial — PEP pretreatment of food gluten (2005, Gastroenterology): A study sought to determine whether prolyl endopeptidase (PEP) treatment of food gluten would obviate the intestinal dysfunction produced by small amounts of dietary gluten supplement in patients with celiac sprue. Twenty asymptomatic patients with histologically proven celiac sprue completed a randomized, double-blind, crossover study involving two 14-day stages. Each patient consumed a low dose of a gluten supplement daily (5 g; equivalent to 1 slice of bread) in one stage, and gluten pretreated with PEP in the other stage. Despite clinical remission at baseline, 40% of patients had at least one abnormal celiac antibody, 20% had an abnormal urine xylose, and 63% had an abnormal fecal fat test result.
Key clinical trial — AN-PEP in healthy subjects (2015, Alimentary Pharmacology & Therapeutics): AN-PEP efficiently degrades gluten molecules into non-immunogenic peptides in vitro. A pilot study in celiac patients showed that a combination of AN-PEP and gluten was safe and well tolerated.
Key clinical trial — AN-PEP in a meal setting (2017, Scientific Reports): This study investigated the efficacy of AN-PEP in a physiological meal setting. In this randomized placebo-controlled crossover study, 18 gluten-sensitive subjects consumed a porridge containing 0.5 g gluten together with two tablets either containing a high or low dose of AN-PEP, or placebo. Gastric and duodenal content was sampled over 180 minutes, and areas under the curve of gluten concentrations were calculated. The success rate of high dose AN-PEP, defined as at least 50% gluten degradation compared to placebo in the duodenum, was achieved in 10 of 13 comparisons. In the stomach, gluten levels were reduced from 176.9 (median) to 22.0 µg × min/ml in the high dose group and to 25.4 µg × min/ml in the low dose group (both p = 0.001). This study represents strong mechanistic evidence that AN-PEP can substantially degrade gluten in the stomach during a real meal, though the study population was gluten-sensitive subjects, not confirmed celiac patients.
Key clinical trial — AN-PEP in celiac disease patients on long-term gluten-free diet (2024, World Journal of Gastroenterology): This was an exploratory, double-blind, randomized, placebo-controlled trial that enrolled celiac disease patients on a long-term gluten-free diet (GFD). After a 4-week run-in period, patients were randomized to 4 weeks of two AN-PEP capsules (GliadinX; AVI Research, LLC, United States) at each of three meals per day, or placebo. Median GIP concentration in the AN-PEP arm was 44.7% lower than in the run-in period. One-third of patients exhibiting GIP > 0.08 µg/g during run-in had lower or undetectable GIP after AN-PEP treatment. However, the AN-PEP treatment did not significantly reduce the overall GIP stool concentration. However, given the observation of a significantly lower prevalence of patients with severe symptoms in the AN-PEP arm, further clinical research is warranted.
Evidence strength for celiac/gluten: Evidence for gluten degradation in the stomach is strong at the mechanistic and short-term clinical level (multiple controlled trials). Evidence for clinically meaningful benefit in established celiac disease—reduction of intestinal damage, antibodies, or major symptom burden—remains preliminary and mixed. It is well established that AN-PEP is not intended to treat celiac disease, and the enzyme has not been approved for that indication.
One promising approach is the development of endopeptidases that cleave the toxic peptides and would thus act as bona fide glutenases for oral enzyme therapy, reminiscent of lactase tablets for lactose intolerance. Such an approach would also benefit patients suffering from non-celiac gluten sensitivity, which has a worldwide prevalence of up to 13%. However, clinical trial data specifically in non-celiac gluten sensitivity populations remains sparse as of current publications.
Research on endogenous PREP's role in the brain has generated significant interest, particularly in the context of memory and neurodegenerative disorders.
PREP genetrap mice have decreased synaptic spine density in the CA1 region of the hippocampus, reduced hippocampal long-term potentiation, impaired hippocampal-mediated learning and memory, and reduced growth-associated protein-43 levels when compared with wild-type controls. These observations reveal a role for PREP in mediating hippocampal plasticity and spatial memory formation, with implications for its pharmacological manipulation in diseases related to cognitive impairment.
Because of PREP's specificity and the occurrence of internal proline residues in several neuropeptides, PREP was soon regarded as a peptidase relevant in neuropeptide metabolism, with great drug target potential for neurological disorders' therapy. Accordingly, synthesis and testing of PREP inhibitors became the attention of academia and industry, especially since evidence emerged that PREP inhibition improved cognitive decline and dementia in several animal models.
Clinical trial evidence (inhibitors): Although several PREP inhibitors showed beneficial effects in preclinical memory models, and S17092, Z-321, and JTP-4819 were also tested in clinical trials as memory enhancers, their impact on neuropeptide levels remained unclear and clinical efficacy was not sufficient to support further studies.
The impetus started to fade away when further research determined that the role of PREP, as a neuropeptide regulator, was not that clear. This is important context: the cognitive research has focused on PREP inhibitors (drugs that block the enzyme), not on supplementing exogenous PEP enzyme itself. The translational value of this research for oral PEP supplementation remains unestablished.
Previous studies have shown that prolyl oligopeptidase (PREP) negatively regulates autophagy and increases the aggregation of alpha-synuclein (αSyn), linking it to the pathophysiology of Parkinson's disease. PREP accelerates the aggregation of α-synuclein (aSyn), a key protein involved in development of Parkinson's disease and other synucleinopathies. PREP inhibitors reduce aSyn aggregation, but the mechanism has remained unknown.
It is noteworthy that α-Syn is not cleaved by POP. Prolyl endopeptidase-like (PREPL) protein is structurally related to the serine peptidases belonging to the POP family. Given the attention to POP inhibitors as potential drugs to treat synucleinopathies, PREPL represents another potential target to be explored.
Evidence strength for Parkinson's/synucleinopathies: All current evidence is preclinical (cell culture and animal models). No human clinical trials have been conducted on PEP supplementation for Parkinson's disease. The relevant research direction has been inhibiting endogenous PREP activity to reduce αSyn aggregation, not exogenous PEP administration.
Altered levels of serum PEP activity have been observed in patients with post-traumatic stress disorder, depression, mania, schizophrenia, anorexia, and bulimia nervosa.
Prolyl endopeptidase (PEP) is a serine proteinase which may cleave peptides involved in the pathophysiology of major depression, such as arginine vasopressin, beta-endorphin, luteinizing hormone-releasing hormone, thyrotropin-releasing hormone, and possibly corticotropin-releasing hormone. PEP may be involved in activation of cell-mediated immunity, autoimmune and inflammatory responses, which repeatedly occur in severe depression. Serum PEP activity was significantly lower in depressed subjects compared to normal controls, and in melancholic depressed subjects compared to minor and simple major depressed subjects.
Altered PREP activity may be associated with autism spectrum disorders and various psychological diseases such as schizophrenia, mania, and clinical depression. However, there is conflicting information as to the exact role that prolyl endopeptidase plays in the pathophysiology of depression, with earlier studies documenting a decreased activity of the enzyme in depressed patients, but more recent studies demonstrating that inhibition of the same enzyme actually results in alleviation of depressive symptoms.
Evidence strength for psychiatric conditions: Observational and biomarker studies only; no controlled clinical trials of exogenous PEP supplementation for psychiatric conditions have been published. The relationships are complex and contradictory, and no clinical recommendations follow from the current evidence.
AN-PEP has a well-established industrial application in brewing, where it is used to prevent chill-haze (protein-polyphenol turbidity) in beer by degrading proline-rich haze proteins. This application is the basis for the original GRAS and food enzyme safety evaluations by regulatory bodies. The food processing use is distinct from the dietary supplement application, though both rely on the same enzyme class.
Dosage data for PEP-based supplements are entirely from research studies and vary considerably by enzyme source, preparation, and indication. The following dosages are reported as stated in sources; they are not recommendations.
The challenge of dosing is partly enzymatic: the enzymes studied have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine. AN-PEP is exceptional precisely because it is acid-stable and remains active at gastric pH.
The food enzyme acid prolyl endopeptidase is produced with the genetically modified Aspergillus niger strain GEP by DSM Food Specialties B.V. The genetic modifications did not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA.
Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The panel identified a no observed adverse effect level (NOAEL) of 5040 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 5520.
The U.S. FDA also reviewed AN-PEP safety data as part of a GRAS notification process: DSM discussed an unpublished bacterial reverse mutation test and an in vitro chromosomal aberration test in human lymphocytes to support the conclusion that the subject of the notice is neither genotoxic nor clastogenic. DSM also discussed the results from an unpublished 90-day oral (gavage) toxicity study of the acid prolyl endopeptidase enzyme concentrate in rats. DSM stated no treatment-related adverse effects at the highest dose tested.
A separate 2025 EFSA review for a related Aspergillus niger prolyl oligopeptidase (Novozymes strain NZYM-MR, EC 3.4.21.26): Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The panel identified a NOAEL of 1488 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 2671.
A search for the homology of the amino acid sequence of the acid prolyl endopeptidase to known allergens was made and no match was found. The panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low.
Similarly, for the prolyl oligopeptidase from the Novozymes strain, a search for homology of the amino acid sequence to known allergens found no match. The panel considered that a risk of allergic reactions upon dietary exposure cannot be excluded, but that the likelihood is low.
Neither AN-PEP nor placebo elicited any reported adverse events or concerns from the patients, indicating that the treatment doses were well-tolerated.
Many so-called enzyme preparations currently sold over the counter as celiac disease dietary supplements do not inactivate toxic gluten peptides and thus represent a hazard for coeliacs. This observation, published in Nature Communications, underscores the importance of enzyme form and source when evaluating commercial supplements in this category.
Oral supplementation with prolyl oligopeptidases has been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. AN-PEP is distinguished by its acid stability, but other PEP forms used in supplements may lack this property.
The existing safety data pertain to AN-PEP specifically, derived from genetically modified A. niger strains, and evaluated primarily in the context of food processing. Safety data for long-term use as an oral dietary supplement in clinical populations remain limited, and the current evidence base does not establish a complete safety profile for chronic supplemental use.
Because PEP acts on proline-containing peptides and is involved in the processing of neuropeptides such as vasopressin, angiotensin, substance P, and TRH, theoretical interactions with drugs or physiological processes that depend on these peptides cannot be excluded, though no specific clinical drug-drug interaction studies for oral PEP supplements have been published in the peer-reviewed literature. The enzyme's substrate size limitation (peptides under ~10 kDa) means that intact proteins and large molecules are not its substrates, limiting the scope of potential interactions.
Condiciones de salud que prolil endopeptidasa (PEP) puede ayudar a apoyar.
AN-PEP (Aspergillus niger prolyl endopeptidase) has been shown in randomized clinical trials to substantially degrade gluten in the stomach before it reaches the small intestine, thereby reducing the substrate responsible for gluten-induced GI symptoms including bloating, diarrhea, and abdominal pain. A 2024 exploratory RCT in celiac disease patients found a significantly lower prevalence of severe symptoms in the AN-PEP arm versus placebo.
Celiac disease is a systemic autoimmune disorder triggered by gliadin peptides that activate CD4+ T cells in HLA-DQ2/DQ8-positive individuals, causing autoantibody production against tissue transglutaminase. PEP enzymes target and destroy these T-cell epitopes, and multiple phase I/II human clinical trials have tested PEP-based oral enzyme therapies as adjuncts to the gluten-free diet in celiac disease.
Prolyl endopeptidases (PEPs), especially the Aspergillus niger-derived AN-PEP, cleave proline-rich immunogenic gluten peptides that human gut proteases cannot fully digest. Multiple clinical trials have evaluated AN-PEP in celiac disease patients as an adjunct to a gluten-free diet; a 2024 double-blind RCT observed a significantly lower prevalence of severe symptoms in the AN-PEP arm.
PREP activity changes during aging and is altered in neurodegenerative conditions including Alzheimer's disease and Parkinson's disease. Decreased PREP activity in cerebrospinal fluid has been documented in Parkinson's disease patients, and PREP colocalizes with α-synuclein, β-amyloid, and tau protein in post-mortem human brain tissue. PREP inhibitors have been explored as potential disease-modifying agents for neurodegenerative disease.
Human digestive enzymes cannot cleave the proline-rich bonds in gliadin and other proline-dense dietary proteins, resulting in incomplete digestion and accumulation of potentially bioactive peptides in the gut lumen. Oral PEP supplementation is designed to fill this functional gap, acting as an exogenous digestive enzyme targeting a specific substrate class that mammalian proteases cannot effectively process.
PEP enzymes, particularly AN-PEP, degrade the immunogenic, proline-rich gluten peptides that trigger food sensitivity reactions in susceptible individuals, before they reach the small intestine. Randomized clinical trials in healthy volunteers and gluten-sensitive subjects demonstrate significant gluten degradation in the stomach compartment. This mechanism specifically targets the molecular driver of gluten food sensitivity.
Prolyl endopeptidase (PEP) is a serine protease that cleaves proline-rich gluten peptides resistant to human digestive enzymes, directly addressing the mechanism of gluten sensitivity. A randomized trial (Clin Nutr 2022) showed a proline-specific PEP allowed gluten reintroduction in NCGS patients; additional studies confirm its ability to significantly reduce gluten immunogenic peptides in vivo.
Prolyl endopeptidase (PREP) is expressed in hippocampal regions of the human brain and degrades neuropeptides associated with learning and memory. Genetic knockout mouse models deficient in PREP show reduced hippocampal synaptic spine density, impaired long-term potentiation, and spatial memory deficits. PREP inhibitors improved memory and cognitive function in rodents, monkeys, and early human trials, though clinical translation has been limited.
Sistemas corporales que prolil endopeptidasa (PEP) puede ayudar a apoyar.
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