# Microbial Peptides: Key Players in Reducing Gluten Immunogenicity Through Peptide Degradation
## 3 Treatment for Gluten Elimination: Pros and Cons
Clinical trials with AN-PEP revealed safety and partial gluten degradation, though efficacy as a monotherapy was limited [60; 61]. New formulations combining AN-PEP with other proteases have shown synergistic effects in reducing gluten immunogenicity in human gastrointestinal simulations [62]. 2. Glutamine-Specific Cysteine Endoprotease (EP-B2):  
EP-B2 is a glutamine-specific cysteine protease that cleaves peptide bonds adjacent to glutamine residues in gliadin peptides [63; 64]. Its catalytic mechanism is mediated by a Cys-His-Asn triad (Cys25, His159, and Asn175), analogous to that found in papain-like cysteine proteases [65]. EP-B2 specifically cleaves peptide bonds following glutamine residues, which are abundant in toxic gliadin peptides. Kinetic analyses using chromogenic and fluorogenic gliadin substrates revealed Michaelis constants (Km) of 30-100 \(\upmu\)M and turnover numbers (kcat) indicative of efficient cleavage under gastric pH conditions. These findings, together with detailed structural and kinetic characterizations, provide crucial insights into the enzyme's functional performance and support its potential application in oral enzyme therapy for CeD and related disorders. EP-B2's mechanism complements PEPs by targeting peptide bonds not cleavable by prolyl-specific enzymes, facilitating more thorough gluten degradation. Oral EP-B2 administration reduced gluten-induced pathology in gluten-sensitive macaques, demonstratingtherapeutic promise [66]. Heterologous expression and fermentation production of EP-B2 have enabled scalable enzyme supply for clinical and industrial applications [67]. The studies have identified novel EP-B2 isoforms with altered substrate specificity and improved stability under gastrointestinal conditions, expanding the toolbox for enzyme therapy in CeO [68]. 3. Gluten-Degrading Enzymes in Human Saliva:  
Human saliva contains microbially derived gluten-degrading enzymes, including subtilisin-like serine proteases from _Rothia_ species, such as _Rothia aeria_ and _Rothia mucaliagnosa_[69; 70; 71]. These enzymes initiate gluten degradation during oral processing by cleaving immunodominant epitopes like the 33-mer and 26-mer peptides [69; 72]. The presence of these enzymes contributes to the partial detoxification of gluten in the oral cavity and opens possibilities for novel probiotic or enzymatic therapies harnessing oral microbiota [70; 73]. Emerging metaproteinic analyses have revealed a broader diversity of oral microbial glutenases, including metalloproteinases and additional serine proteases, with potential for enhanced gluten degradation. Manipulation of oral microbiota composition is being explored to increase endogenous glutenase activity [73]. Beyond their initial hydrolytic action, the complex interplay between salivary glutenases, particularly those of microbial origin, and the oral microbiota warrants attention. These microbial peptidases can influence the oral and gut flora composition and metabolic activity, for instance, by altering peptide availability [74; 75]. Conversely, metabolites produced by the oral microbiota may modulate the expression or efficiency of these glutenases. Understanding these bidirectional interactions within the oral cavity is essential for a comprehensive view of early gluten processing and its potential implications downstream in the gastrointestinal tract. Beyond these groups, insect-derived peptidases, such as proyloligopeptides from _Tenebrio molitor_ and postglutamine peptidases from _Tribolium castaneum_, have demonstrated the ability to degrade gluten peptides effectively, offering alternative enzyme sources for industrial or therapeutic applications [76; 77]. Plant-derived enzymes, particularly cysteine proteases like EP-B2, have also been explored for gluten degradation. The EP-B2 from barley is a prime example of an enzyme adapted to degrade gliadin peptides in the acidic gastric environment (Table 1). The food industry leverages these enzymes to produce gluten-reduced or gluten-free products.