Exopeptidase

Synopsis

Exopeptidase: A Comprehensive Encyclopedic Reference

1. Identity and Classification

1.1 Biochemical Definition

An exopeptidase is any peptidase that catalyzes the cleavage of the terminal (or the penultimate) peptide bond; the process releases a single amino acid, dipeptide, or tripeptide from the peptide chain. The term is used both to describe a functional enzyme class found naturally in the human body and to refer to the same class of enzymes when delivered exogenously as a dietary supplement or food enzyme.

Exopeptidases are a class of proteolytic enzymes that remove amino acids from the termini of peptides and proteins, with two major subclasses being aminopeptidases and carboxypeptidases. They function by cleaving amino acids one at a time or, in some cases, two or three residues at once.

1.2 Nomenclature and Enzyme Commission Classification

Depending on whether the amino acid is released from the amino or the carboxy terminal (N-terminus or C-terminus), an exopeptidase is further classified as an aminopeptidase or a carboxypeptidase, respectively.

According to the International Union of Biochemistry and Molecular Biology (IUBMB) Enzyme Nomenclature, the major exopeptidase sub-subclasses are formally organized as follows:

Those acting at a free N-terminus liberate a single amino-acid residue (aminopeptidases, EC 3.4.11), or a dipeptide or a tripeptide (dipeptidyl-peptidases and tripeptidyl-peptidases, EC 3.4.14).
The exopeptidases acting at a free C-terminus liberate a single residue (carboxypeptidases, EC 3.4.16–18) or a dipeptide (peptidyl-dipeptidases, EC 3.4.15).
Other exopeptidases are specific for dipeptides (dipeptidases, EC 3.4.13), or remove terminal residues that are substituted, cyclized, or linked by isopeptide bonds (omega peptidases, EC 3.4.19).

Depending on the character of the residual amino acid at the active site, carboxypeptidases are clustered under three classifications: serine carboxypeptidases, cysteine carboxypeptidases, and metallo carboxypeptidases. The carboxypeptidases are allocated on the basis of catalytic mechanism: the serine-type carboxypeptidases (EC 3.4.16), the metallocarboxypeptidases (EC 3.4.17), and the cysteine-type carboxypeptidases (EC 3.4.18).

A particularly well-characterized member of the exopeptidase family with significant dietary supplement and pharmaceutical relevance is dipeptidyl peptidase IV (DPP-IV). Approximately 766 amino acids compose human Dipeptidyl Peptidase IV (DPP-4), a ubiquitous serine exopeptidase that cleaves X-Pro/X-Ala dipeptides and regulates incretin hormones central to glucose homeostasis. It is known by alternative names including Dipeptidyl peptidase-4, DPP-4, CD26, and ADCP2, and is classified as a serine exopeptidase; type II transmembrane glycoprotein that forms active homodimers.

Another specific and well-studied exopeptidase in the food enzyme and supplement context is leucyl aminopeptidase (EC 3.4.11.1). The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the genetically modified Aspergillus oryzae strain NZYM-BU by Novozymes A/S. Leucyl aminopeptidases catalyze the hydrolysis of the peptide bonds of N-terminal amino acid residues of proteins or peptides, with a preference for leucine, resulting in the release of amino acids.

1.3 Relationship to Endopeptidases

Endopeptidases initiate the digestion of food proteins, generating new N- and C-termini that are substrates for the exopeptidases that complete the process. This sequential interplay — endopeptidase action first, exopeptidase action second — is foundational to complete protein digestion. Endopeptidases cleave proteins/peptides into shorter peptides but do not systematically cleave protein into absorbable fractions.

2. Natural Sources and Distribution

2.1 Endogenous Human Sources

An aminopeptidase, an enzyme in the brush border of the small intestine, will cleave a single amino acid from the amino terminal, whereas carboxypeptidase, which is a digestive enzyme present in pancreatic juice, will cleave a single amino acid from the carboxylic end of the peptide.

Exopeptidases are key for completing protein digestion, including proline-rich peptides, and are mostly produced by enterocytes of the intestinal brush border membrane. The human brush border membrane contains a diverse range of complementary exopeptidases, many of which, unlike gastric and pancreatic peptidases, have proline specificity.

DPP-4 is expressed on epithelial and endothelial surfaces (kidney, intestine, liver, lung), on activated T lymphocytes, and exists as a proteolytically shed soluble form (sDPP-4) in plasma.

2.2 Animal-Derived Exogenous Sources

The most common source of exogenous proteases used for inflammation is the pancreas of pigs and cows slaughtered for meat, commercially known as pancreatin. Pancreatin contains such proteases as trypsin, chymotrypsin, carboxypeptidase, and elastase. Carboxypeptidases A and B, which are animal-sourced pancreatic exopeptidases, are among the most historically studied. The first generation carboxypeptidases such as pancreatic carboxypeptidase A1 and A2 exhibit their potentiality at the process of digestion of food molecules.

2.3 Plant-Derived Sources

Plant sources of protease, primarily bromelain derived from pineapple, and papain derived from papaya, are the second most studied exogenous sources of proteases for inflammation. While bromelain (EC 3.4.22.32) is technically a cysteine endoprotease, commercial enzyme preparations from pineapple and papaya may contain mixed protease populations including exopeptidase activity.

The presence of aminopeptidases is reported in a wide variation of microbial species that includes bacteria as well as fungi, plants, and animals.

2.4 Microbial and Fungal Sources

Microorganisms represent an excellent source of enzymes owing to their wide biochemical diversity and their susceptibility to genetic manipulation. Fungi produce a wider range of enzymes than do bacteria. For instance, Aspergillus oryzae produces all types of proteases such as acid, neutral, and alkaline proteases.

In the food industry, Aspergilli have been widely used for a long time. Among Aspergilli, Aspergillus niger is the most widely used species in the food industry. Proteolytic enzymes that are physiologically acceptable can be found in certain bacteria, fungi, yeast, and molds. For example, proteases and peptidases may be found in fungi which have been used in the fermentation of many traditional foods and beverages. Suitable species include Fusarium, Rhizopus, Actinomucor, Aspergillus, and Neurospora species.

Enzymes are obtained from microbes such as alcalase (endoprotease), flavourzyme (mixture of proteases having both endo- and exopeptidase activities), protamex, proteinase K, metalloproteases, serine-protease; from plants such as papain, bromelain, ficin (endopeptidases); and from animals like α-chymotrypsin, neutrase, and trypsin.

Proteases and peptidases also can be found in lactic acid bacteria, which are used to produce yogurt and other fermented dairy products.

2.5 Fermented Food Sources

Cultures across Asia have long used koji mold (Aspergillus oryzae) to ferment soybeans, grains, and other plant materials in the production of miso, tempeh, soy sauce, and sake. These traditional preparations often contained natural proteolytic activity, which pre-digested proteins and enhanced digestibility.

While proprietary microbial enzymes are a modern innovation, the concept of using fermented microorganisms to aid digestion is ancient. Traditional cultures around the world have consumed fermented foods (like miso, kimchi, yogurt, kefir, and tempeh), which naturally contain microbial enzymes that help predigest food, improve gut function, and promote microbial balance.

3. Historical and Traditional Use

3.1 Fermentation-Based Traditions in Asia

Although exopeptidases were not isolated or named as such until the modern era, populations in East Asia have used fermented foods containing naturally occurring exopeptidase activity for many centuries. Cultures across Asia have long used koji mold (Aspergillus oryzae) to ferment soybeans, grains, and other plant materials in the production of miso, tempeh, soy sauce, and sake. These traditional preparations often contained natural proteolytic activity, which pre-digested proteins and enhanced digestibility.

In Japan, natto — a fermented soybean preparation — has a documented history of over a millennium. Nattokinase is extracted and highly purified from a traditional soy-based Japanese food called Natto. Natto is a fermented cheese-like food that has been used in Japan for over 1,000 years for its popular taste and as a folk remedy for heart and vascular diseases. Though nattokinase itself is classified differently (as a serine protease/subtilisin-like enzyme), the cultural practice of consuming enzyme-rich fermented foods for digestive and systemic health is the broader tradition within which exopeptidase-containing preparations fit.

3.2 Ayurveda and Traditional Chinese Medicine

In Ayurveda and Traditional Chinese Medicine, although protease enzymes were not identified by name, foods and preparations that supported digestion and protein metabolism — such as fermented tonics, sour herbal elixirs, and bitter-tasting formulas — likely contained naturally occurring enzymes or compounds that enhanced protease activity in the body. In Ayurvedic and Traditional Chinese Medicine, fermented herbs and foods were often used as tonics for digestion and vitality, long before enzymes were chemically identified. Remedies like fermented barley or rice were thought to "kindle digestive fire" and enhance assimilation.

3.3 Traditional Meat Tenderization

The use of proteolytic enzyme-rich plants to tenderize meat represents another layer of traditional use. Papayas contain papain, a proteolytic enzyme that effectively breaks protein down into its amino acids. That is one reason it has traditionally been used to tenderize meat. This practice, observed in Mesoamerican and tropical cultures long before modern food science, exploited the protease activity of papain and related exopeptidases within the fruit.

3.4 The Modern Supplement Era

The isolation and industrial production of microbial enzymes began in earnest in the 20th century, initially for the food and textile industries. Their use in human health supplements expanded in the 1980s and 1990s, particularly as plant-based, vegetarian alternatives to enzymes like pancreatin (from pork) and bromelain (from pineapple).

DPP-IV enzyme activity was biochemically observed in the 1960s; the CD26 antigen was linked to DPP-IV activity in the 1980s, and the gene was cloned in the 1990s. Contemporary dietary supplements containing exopeptidase activity largely derive from fungal fermentation using Aspergillus species, and are typically formulated as multi-enzyme blends.

4. Key Constituents and Active Compounds

4.1 Principal Exopeptidase Types in Supplemental Preparations

When the term "exopeptidase" appears on a dietary supplement label, it generally refers to a preparation containing one or more of the following:

Aminopeptidases (EC 3.4.11): Aminopeptidases put their activity at a free N-terminal point of the protein and as a result of digestion they release a mono amino acid residue, a dipeptide, or a tripeptide.
Leucyl aminopeptidase (EC 3.4.11.1): Leucyl aminopeptidases catalyze the hydrolysis of the peptide bonds of N-terminal amino acid residues of proteins or peptides, with a preference for leucine residues, resulting in the release of free amino acids.
Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5): DPP-4 (also known as CD26) is a membrane-anchored and soluble serine protease that inactivates regulatory peptides such as GLP-1 and GIP, modulates immune signaling, and is the pharmacologic target of the DPP-4 inhibitor class of antidiabetic drugs (gliptins).
Carboxypeptidases (EC 3.4.16–18): In preferred embodiments, an exopeptidase may be an aminopeptidase such as a leucyl aminopeptidase (EC 3.4.11.1); or a carboxypeptidase such as a proline carboxypeptidase (EC 3.4.16.2); carboxypeptidase A (EC 3.4.17.1); carboxypeptidase B (EC 3.4.17.2); and others.

Supplement blends frequently combine exopeptidases with endopeptidases. For example, the Elevase® supplement was composed of alpha-galactosidase, amylase, beta-glucanase, cellulase, diastase, endopeptidase complex, exopeptidase complex, glucoamylase, invertase, lactase, lipase, protease, xylanase, rice dextrin, and rice bran.

4.2 Mechanisms of Action

Exopeptidases execute their enzyme activity by hydrolysis merely on the nitrogen or carbon terminal points of the substrate chain entirely made up of polypeptide. Exopeptidases are further sorted according to the specificity such as the size of the fragment, identity of the liberated fragment, terminus of origin, and the size restriction on the length of the specific peptide chain.

This enzymatic process results in the liberation of free amino acids, which can then be utilized for various biochemical processes within the organism.

The sequential tandem action of different exopeptidases is a key aspect of their function in supplement design. The AMYNOPEP formulation consists of two tandem-acting aminopeptidases (a monoaminopeptidase and dipeptidyl peptidase) that digest peptides from the amino- to carboxy-terminal to generate absorbable single amino acids and dipeptides.

Single exopeptidases are ineffective alone but may be highly effective when combined. This principle underlies most formulation strategies for exopeptidase-based supplements and enzyme therapies.

Regarding carboxypeptidase-type exopeptidases, carboxypeptidases actively participate in the healing of internal and external wounds and blood clotting — physiological roles extending beyond simple protein digestion.

Exopeptidase activity is also exploited in food science. Exopeptidases can be used to cleave the bitter hydrophobic amino acid residue exposed during protein hydrolysis by endopeptidases, which has applications in reducing bitterness in protein hydrolysate products.

5. Scientific Evidence by Area of Use

5.1 Protein Digestion and Amino Acid Bioavailability

The role of exopeptidases in protein digestion is mechanistically well established. Enzymatic protein hydrolysis can enhance the digestive and absorptive processes of proteins by fragmenting larger molecular weight proteins into smaller peptides. However, direct clinical evidence specifically isolating the contribution of supplemental exopeptidases to amino acid bioavailability in healthy individuals remains limited.

One of the most direct clinical demonstrations in humans involves the Elevase® multi-enzyme blend (which contains an exopeptidase complex). This study was conducted as a randomized, crossover, placebo-controlled design where each participant served as their own control. The post-hoc analysis investigated the impact of a dietary enzyme supplementation blend known as Elevase® on dietary macromolecule digestion in samples from otherwise healthy participants that had previously undergone a small bowel resection, resulting in an ileostomy.

It was demonstrated that after 4 hours, Elevase® significantly increased monosaccharide levels (predominantly glucose and fructose) in the ileostomy samples. In addition, the bile salt taurohyodeoxycholic acid was also increased, suggesting a physiological host response to the macromolecule digestion induced by the enzymatic blend. Overall, these findings suggest Elevase® could accelerate food digestion and potentially increase nutrient availability from the diet.

Evidence strength: Elevase® had no significant effect on cholesterol, triglycerides, glycogen, protein, branched-chain amino acids, and free amino acid levels in ileostomy samples from study participants 4 hours after food ingestion. This finding indicates that statistically significant effects in this exploratory study were selective (carbohydrate-related more than protein-related in this time window), and the study population was a specific ileostomy cohort, limiting generalizability. The evidence for protein/amino acid bioavailability from exopeptidase supplementation in otherwise healthy humans is preliminary.

5.2 Gluten Immunogenic Peptide Degradation and Celiac Disease

This is the area with the most focused, peer-reviewed, human clinical evidence specifically attributable to exopeptidase activity.

Celiac disease is a common autoimmune-like enteropathy caused by an aberrant response to incompletely digested dietary gluten. Gluten immunogenic peptides including the immunodominant 33-mer are thought to be resistant to proteolytic digestion by human gastrointestinal peptidases.

A 2024 published clinical study (Frontiers in Immunology / PubMed PMC11522800) investigated the dual exopeptidase combination AMYNOPEP. The team developed a novel enzyme therapy approach to support gluten peptide digestion using a combination of two tandem-acting exopeptidases, AMYNOPEP, that complement the intrinsic enzymatic activity of intestinal brush border enterocytes. They evaluated the effects of AMYNOPEP supplementation on 33-mer degradation in vitro and in vivo. In a cross-over clinical study, healthy volunteers with no gastrointestinal disorders were given stable isotope (SI) labelled 33-mer peptides in the presence of varying peptide substrates and caloric loads, with and without AMYNOPEP. 33-mer degradation products (SI-labelled single amino acids) were measured in the blood plasma using LC-MS/MS.

Results: AMYNOPEP achieved rapid, complete amino-to-carboxyl terminal degradation of the 33-mer in vitro, generating single amino acids and dipeptides. In healthy volunteers, AMYNOPEP supplementation significantly increased 33-mer degradation and absorption of SI-labelled amino acids even in the presence of competing substrates. Specifically, a 2.8-fold increase in the C_max of stable isotope-labelled amino acids in the presence of wheat gluten was observed.

The absorption kinetics of labelled amino acids derived from 33-mer digestion with AMYNOPEP closely resembled that of SI-labelled X-Proline dipeptides administered without enzyme supplementation, highlighting the rapid hydrolytic activity of AMYNOPEP on polypeptides.

A 2025 systematic review in Alimentary Pharmacology & Therapeutics (Bonner et al., 2025) specifically analyzed the landscape of enzyme therapies for gluten digestion in the context of exopeptidases. Most enzymes on the market or in development were gastric endopeptidases specific for proline or glutamine residues. Gastric enzymes may achieve poor enzyme–substrate exposure due to limited mixing and rapid emptying of water-soluble particles. Moreover, endopeptidases cleave proteins/peptides into shorter peptides but do not systematically cleave protein into absorbable fractions. Natural digestive physiology provides thorough mixing at the intestinal brush border, which produces exopeptidases necessary to fully digest proline-rich peptides. Despite reduced activity in patients with celiac disease, exopeptidases remain underexplored as therapeutic agents.

Another key finding from that review: Among gluten-digesting enzymes, there are a few exceptions to the gastric endoprotease paradigm, namely the single exopeptidase DPP-IV and the endoprotease Caricain acting in the small intestine.

In 2009, Ehren et al. found that more complete clearance of gluten epitopes could be achieved by combining aspergillopepsin with dipeptidyl peptidase IV (DPP-IV) from Aspergillus oryzae. Dipeptidyl peptidase IV is another candidate used to support resolving gluten digestion. DPP-IV is part of the exopeptidase family and often used in foods and supplements as a debittering agent.

Evidence strength: The AMYNOPEP crossover human study (2024) is the most rigorous clinical evidence to date for a defined exopeptidase combination on gluten peptide degradation in humans. However, this study was conducted in healthy volunteers, not celiac patients, and is thus a proof-of-concept pharmacokinetics study rather than a therapeutic efficacy trial in the target population. To date, there have been limited research and clinical studies aimed at developing exopeptidase-based enzyme therapies, perhaps due to a limited understanding of the human brush border membrane proteome and lack of suitable in vitro models. Overall evidence in actual celiac patients from exopeptidase-specific supplementation remains preliminary, and no exopeptidase preparation has received regulatory authorization for the treatment of celiac disease.

5.3 Exopeptidases as Part of Multi-Enzyme Digestive Supplements

Digestive enzymes can selectively degrade proteins, carbohydrates, and lipids; and their supplementation alongside food may accelerate the breakdown of complex food matrices, facilitate greater nutrient absorption, decrease food sensitivities, and aid in the management of certain disease states.

Exopeptidase-containing blends have been evaluated as part of comprehensive digestive enzyme supplements. The Elevase® study described above used a crossover, placebo-controlled design including an exopeptidase complex among its many constituents. This ileostomy study paradigm offers superior data when compared to that generated in artificial gut digestion models, preclinical animal models, or conventional clinical studies using stool analyses, as it allows real-time access to samples in situ in the small intestine where the majority of nutritional absorption takes place.

Evidence strength: Because the Elevase® product contains many enzyme types, the specific contribution of its exopeptidase complex to any observed outcomes cannot be isolated. The study was exploratory and used a small sample of ileostomy patients, limiting its applicability. Independent clinical trials specifically testing exopeptidase-only supplements on digestive outcomes in broad healthy populations are lacking in the peer-reviewed literature.

5.4 DPP-IV Activity and Glucose / Incretin Metabolism

DPP-IV, as an exopeptidase, is the target of an entire class of approved pharmaceutical drugs (gliptins/DPP-4 inhibitors) for type 2 diabetes, but these are drugs — not dietary supplements — that inhibit the enzyme to prolong the action of glucagon-like peptide-1 (GLP-1). The enzyme itself, when delivered exogenously as a food enzyme or supplement, functions in a different role: primarily as a digestive aid for proline-containing peptides. DPP-4 as an endogenous enzyme is not an approved oral dietary supplement; therapeutic modulation is achieved clinically with prescription DPP-4 inhibitors (e.g., sitagliptin 100 mg, linagliptin 5 mg).

Some exogenous exopeptidases have been studied in human and commercial settings, including DPP-IV and separately, leucyl aminopeptidase, demonstrating favorable safety profiles of these enzymes. DPP-IV is already available as an over-the-counter dietary supplement in several products, and a food enzyme leucyl aminopeptidase recently underwent safety evaluation showing no safety concerns under conditions of use in eight food manufacturing processes.

Evidence strength: The use of exogenous DPP-IV as a dietary supplement for digestive health is mechanistically plausible and is commercially available; however, robust clinical trials in humans demonstrating clinical benefit from supplemental DPP-IV enzyme (as opposed to DPP-4 inhibitor drugs) are not currently available in the peer-reviewed literature.

5.5 Antihypertensive Bioactive Peptide Production

Exopeptidases have been employed in the production of antihypertensive peptides derived from food protein hydrolysates, particularly whey protein. This efficiency has been linked to the presence of high exopeptidase activity. Indeed, the exopeptidases cleave peptides from their C- or N-terminal extremities, allowing a reduction in the bitterness due to terminal hydrophobic amino acid residues. Research into ACE (angiotensin-converting enzyme) inhibitory peptides generated using exopeptidase hydrolysis has produced in vitro findings suggesting potential antihypertensive activity from exopeptidase-processed whey protein hydrolysates; however, this line of evidence pertains to the use of exopeptidases as food processing tools and the resulting bioactive peptides as the active agents — not exopeptidases per se as the ingested supplement. Human clinical data in this area are not yet robust.

6. Body Systems and Health Areas Associated with Exopeptidases

6.1 Gastrointestinal System

The gastrointestinal tract is the primary site of exopeptidase action both endogenously and when supplemented. Exopeptidases play a vital role in protein digestion, cellular turnover, and the regulation of peptide hormones. The intestinal brush border is the principal anatomical location of endogenous exopeptidase activity during digestion, particularly for the terminal hydrolysis of short peptides into absorbable amino acids and dipeptides. Natural digestive physiology provides thorough mixing at the intestinal brush border, which produces exopeptidases necessary to fully digest proline-rich peptides.

6.2 Immune System and Mucosal Health

The sensitivity of the intestinal brush border to inflammatory damage and consequent loss of brush border exopeptidase activity in celiac disease patients may further exacerbate gluten immunogenic peptide buildup and ensuing enteropathy in patients. This observation links the status of brush border exopeptidase activity to mucosal immune responses, particularly in the context of gluten-related disorders.

6.3 Cardiovascular System

Carboxypeptidase-type exopeptidases actively participate in healing of internal and external wounds and blood clotting, suggesting a role in hemostatic processes. However, definitive human clinical evidence for the cardiovascular effects of supplemental exopeptidases specifically is absent in the current literature.

6.4 Endocrine and Metabolic Function

DPP-4 (DPP-IV) is a membrane-anchored and soluble serine protease that inactivates regulatory peptides such as GLP-1 and GIP, modulates immune signaling, and is the pharmacologic target of the DPP-4 inhibitor class of antidiabetic drugs (gliptins). The endogenous exopeptidase DPP-IV thus plays a central role in incretin hormone regulation and glucose homeostasis, which is why DPP-4 inhibitors (not supplemental DPP-4 enzyme) are a major pharmaceutical drug class for type 2 diabetes.

7. Dosage Forms and Reported Dosages

7.1 Commercial Supplement Forms

Study products in clinical research have been provided in the form of capsules packaged in identical containers in single servings. The Elevase® supplement, for example, was composed of alpha-galactosidase, amylase, beta-glucanase, cellulase, diastase, endopeptidase complex, exopeptidase complex, glucoamylase, invertase, lactase, lipase, protease, xylanase, rice dextrin, and rice bran. The exopeptidase complex is thus one component of a multi-enzyme capsule formulation.

Enzyme supplement potency is typically expressed in activity units rather than mass. The potency of each enzyme in activity units, not just the weight, should be listed clearly on the label. Standardized activity units vary by enzyme type (e.g., HUT for proteases, LAP for leucyl aminopeptidase, DPPU for dipeptidyl peptidase), meaning gram-weight comparisons between products are not straightforwardly meaningful.

7.2 Dosages Reported in Clinical Studies

For the AMYNOPEP formulation (combination of monoaminopeptidase and DPP-IV exopeptidases), in vitro and in vivo studies were conducted where healthy volunteers with no gastrointestinal disorders were given stable isotope-labelled 33-mer peptides in the presence of varying peptide substrates and caloric loads, with and without AMYNOPEP. The publication (PMC11522800) reports the use of a 1:10 enzyme-to-substrate ratio in the in vitro component. Specific enzyme dosages administered in the human crossover component are not stated in the available abstract and summary data; the full trial protocol should be consulted for precise dosing.

There are countless proteolytic enzyme supplements available, each with different enzyme combinations. Because they may vary widely in composition, the supplement bottle should be consulted for instructions on dosage and safe usage.

No universally agreed-upon or regulatory-body-established recommended dietary intake (RDI) or tolerable upper intake level (UL) for supplemental exopeptidases has been established by the NIH Office of Dietary Supplements, EFSA, or other major health authorities, as these are food enzymes rather than nutrients in the classical sense.

8. Safety Considerations and Interactions

8.1 General Safety of Exopeptidase Food Enzymes

Exopeptidases produced from recognized food-safe organisms such as Aspergillus oryzae have been subject to formal regulatory safety evaluations by the European Food Safety Authority (EFSA). For leucyl aminopeptidase (EC 3.4.11.1) from Aspergillus oryzae strain NZYM-EX (Novozymes), the Panel identified a no observed adverse effect level of 440 mg TOS/kg body weight per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 763.

A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

For leucyl aminopeptidase (EC 3.4.11.1) from the genetically modified Aspergillus oryzae strain NZYM-BU (Novozymes), EFSA's 2024 evaluation found that the genetic modifications do not give rise to safety concerns. Genotoxicity tests did not indicate a safety concern.

It is noteworthy that safety evaluations can differ by production strain. In a previous evaluation, the Panel concluded that a food enzyme could not be considered safe when used in five food manufacturing processes, due to insufficient margins of exposure estimated for all age groups — specifically when the production strain identity was uncertain. This underscores that safety is strain-specific, not a class-wide property.

8.2 Human Clinical Safety Data for Exopeptidase Supplementation

Regarding the safety of AMYNOPEP in the clinical crossover study, data on adverse events were collected during the course of study participation, and no volunteers reported adverse events related to the ingestion of study materials (AMYNOPEP or stable isotope-labelled peptides). However, the study was conducted in healthy volunteers over a limited period, and no long-term safety data specific to exopeptidase combinations are available in the published literature.

8.3 Interaction Considerations: DPP-IV and Pharmaceutical DPP-4 Inhibitors

Individuals taking pharmaceutical DPP-4 inhibitor drugs (sitagliptin, saxagliptin, linagliptin, alogliptin, etc.) for type 2 diabetes should be aware that supplemental DPP-IV enzyme and DPP-4 inhibitor drugs target the same enzyme system. The clinical significance of taking exogenous DPP-IV enzyme alongside a DPP-4 inhibitor drug has not been studied; theoretical considerations would apply. DPP-4 as an endogenous enzyme is not an approved oral dietary supplement; therapeutic modulation is achieved clinically with prescription DPP-4 inhibitors.

8.4 Allergenicity

A search for similarity of the amino acid sequence of leucyl aminopeptidase to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Individuals with documented allergies to fungi (particularly Aspergillus species) or to the production substrates used in fermentation should exercise caution, as these enzymes are protein in nature and carry a theoretical risk of sensitization.

8.5 Potential for Altered Peptide Signaling

Because exopeptidases — particularly DPP-IV — are involved in the metabolism of bioactive peptides including incretins (GLP-1, GIP), neuropeptides, and regulatory hormones, supplemental delivery of active exopeptidases in high concentrations could, theoretically, modulate signaling peptide half-lives. This mechanism is well-established for the endogenous enzyme and is why DPP-4 inhibitors are therapeutically effective. The reverse scenario — supplemental active DPP-IV enzyme reducing circulating GLP-1 — is pharmacologically plausible but has not been formally assessed in human supplementation studies.

8.6 Stability Considerations

Earlier enzyme candidates were shown to be inadequate because they could not survive contact with pepsin or the low pH of the stomach. More recently identified Aspergillus niger-derived prolyl endopeptidase (AN-PEP) was shown to not only break down gluten peptides 60 times faster than typical prolyl oligopeptidases, but also remained stable at a gastric pH as low as two, with optimal function at a pH of four to five. Stability at gastric pH is a critical formulation consideration for any orally administered exopeptidase supplement targeting the intestinal brush border.

8.7 Debittering and Food Processing Context

In food manufacturing, exopeptidases are deliberately employed for debittering protein hydrolysates. Exopeptidases can be used to cleave the bitter hydrophobic amino acid residue exposed during the protein hydrolysis by endopeptidases. This function is distinct from therapeutic use but is relevant to the safety dossier for food enzyme approvals.

9. Current Status and Research Landscape

To date, there have been limited research and clinical studies aimed at developing exopeptidase-based enzyme therapies, perhaps due to a limited understanding of the human brush border membrane proteome and lack of suitable in vitro models to study the human brush border membrane, the source of most exopeptidases.

To date, there has yet to be a rational design for exopeptidase supplementation in celiac disease leveraging complementary exopeptidase activity. Future approaches to enzyme therapy design that incorporate exopeptidase combinations stand to enhance or replace a critical enzyme type in the small intestine, while harmonizing with the natural flow of dietary protein and peptide digestion.

Despite reduced activity in patients with celiac disease, exopeptidases remain underexplored as therapeutic agents. The most promising and current area of investigational use is as adjunct enzyme therapy for gluten digestion in celiac disease and non-celiac gluten sensitivity, with the proof-of-concept human study on AMYNOPEP (2024) representing a significant step forward. Broader evidence for other claimed applications — general protein digestion enhancement, antihypertensive peptide delivery, or immune modulation — in human populations remains sparse, preliminary, or limited to in vitro and animal models.