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In order to investigate the application potential of prolyl endopeptidase (PEP) in the enzymatic preparation of bone collagen peptides, the amino acid sequence characteristics of bovine bone collagen (BBC), porcine bone collagen (PBC) and chicken bone collagen (CBC) were comparatively analyzed, and their potential enzymatic cleavage sites and theoretical hydrolysis degrees were predicted. Collagen hydrolyzed by PEP at 55 ℃ and pH 8.0 was characterized by hydrolysis degree, molecular mass distributionand scanning electron microscopy (SEM). Infrared spectroscopy, X-ray diffraction (XRD) and circular dichroism (CD) spectroscopy were used to explore the structural changes of collagen during enzymatic digestion. The results showed that PEP could hydrolyze the three collagens. The hydrolysis degree of PBC was the highest (51.35%), followed by those of BBC (22.81%) and CBC (29.81%). The molecular masses of the three collagen hydrolysates were mostly distributed below 500 Da. Spectroscopic analysis showed that PEP destroyed the triple helix structure of collagen, and then degraded it. Therefore, PEP can efficiently enzymatically hydrolyze collagen into small molecule peptides, which provides a basis for the enzymatic preparation of functional collagen-derived peptides.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Sea fennel and seaside arrowgrass are two abundant but underutilized halophytes along the Atlantic and Mediterranean coasts. This study investigated the antioxidant capacity and the potential antihypertensive (Angiotensin Converting Enzyme I, ACE-I inhibition), hypoglycaemic (Dipeptidyl Peptidase IV, DPP-IV inhibition), and nootropic (Prolyl Endopeptidase, PEP inhibition) activity of their polyphenol extracts. They had a high phenol content (21–24 mEq GA/g), antioxidant capacity evaluated using the ABTS (17–2 mg ascorbic acid/g) and FRAP (170–270 mM Mohr's salt/g) assays, and effective ACE-inhibiting properties (80–90% inhibiting activity at final concentration of 0.5 mg/mL). Additionally, the sea fennel extract displayed high DPP-IV inhibitory capacity (73% at 1 mg/mL), while the seaside arrowgrass extract exhibited potent Prolyl endopeptidase inhibitory capacity (75% at 1 mg/mL). Fractionation by HPLC concentrated the bioactive molecules in two fractions, for which the composition was analyzed by LC-MS/MS. Different chlorogenic acids seemed to play an important role in the bioactivity of sea fennel extract, and different flavonoids, mainly apigenin, luteolin and chrysoeriol, in the bioactivity of the seaside arrowgrass extract. Given their potential health benefits, these extracts could serve as valuable bioactive ingredients and could potentially encourage the cultivation of these species in regions where traditional crops face challenges in growth. [ABSTRACT FROM AUTHOR]

Abstract Background Prolyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost. Results Here, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii. Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (

Background: Prolyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost. Results: Here, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii. Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (< 10 mg/kg) and thereby, reduced turbidity, which would be beneficial for improving the non-biological stability of beer. Conclusion: Our research provides a promising approach for industrial production of AN-PEP and other enzymes (proteins) from renewable lignocellulosic biomass, which provides a new idea with relevant researchers for the utilization of agricultural residues. [ABSTRACT FROM AUTHOR]

This study provides a comprehensive comparison among the most common gluten-free (GF) brewing practices, with a focus on the impact of each treatment on physicochemical parameters and consumer acceptability of the final beer. In addition, the influence of a longer cold maturation on the natural reduction of the gluten content was investigated. Prolyl endopeptidase addition was found to be the most effective treatment in reducing gluten levels (−75.93%), followed by silica gel (−53.09%), longer cold maturation (−4.32%), and tannins (−1.85%). Nonetheless, none of the treated beer samples was gluten-free (gluten content > 20 ppm) due to the high nitrogen content of the original wort. The silica gel application treatment affected the physicochemical and sensory characteristics of the final beer the least. According to the difference from control test results, no significant difference in terms of overall liking, appearance, odor/aroma, or taste was observed between the silica gel-treated sample and control beer (p > 0.05). On the other hand, the application of enzymes and tannins significantly affected the appearance and the beer odor/aroma. Nevertheless, all beer samples received positive sensory acceptance scores. [ABSTRACT FROM AUTHOR]

Celiac disease (CD) is an inflammatory disorder of the small intestine. Gluten peptides are supposed to be responsible for the reaction, the best-researched of which is the so-called '33-mer'. Analogous peptides in secalins (rye) and hordeins (barley) have been described. This study presents the degradation of gliadins, glutenins, hordeins and secalins purified from the respective flours using a prolyl endopeptidase from the Basidiomycete Flammulina velutipes (FvpP). The flour fractions were incubated with the enzyme, and the cleavage sites were determined using high-resolution nLC-qTOF-MS/MS. For the wheat samples, eight cleavage sites in the 33-mer peptide were shown, and all of the six described epitopes were successfully cleaved. For the commercially available prolyl-specific endopeptidase from Aspergillus niger (An-Pep), which was used as a control, only two cleavage sites that cleaved three of the six epitopes were identified. For the secalins, four prolyl-specific cleavage sites in the CD-active peptide QPFPQPQQPIPQ were found for the FvpP but none for the An-Pep. The CD-active peptide QPFPQPEQPFPW in C-hordein was cleaved at three prolyl-specific positions by the FvpP. The study proves the usability of FvpP to degrade CD-inducing peptides in real-grain flour samples and indicates its higher effectiveness compared with An-Pep. A clinical study would be required to assess the therapeutic or preventive potential of FvpP for CD. [ABSTRACT FROM AUTHOR]

Cellulomonas sp. HM71, a human gut microbe possesses metabolic machinery to catabolize antigenic gluten, hence, holds promises as microbial therapy to treat gluten-derived celiac disease. However, its efficacy, safety, and survivability in the gastrointestinal ecosystem await functional elucidation. The current study is designed to characterize Cellulomonas sp. HM71 for its physiological, genomic, and probiotic properties. The morphological and physiological assessment indicates it as a coccus-shaped gram-positive bacterium growing optimally at 30°C in a neutral environment (pH 7.0). Cellulomonas sp. HM71 showed continuous growth even in stressful environments (salinity up to 3% NaCl and 6% KCl), variable temperature (25°C to 35°C) and pH (5-9), antibiotics, and gastric and intestinal conditions. The Cellulomonas sp. HM71 genome harbors diversified genetic machinery to modulate humongous metabolic potential for the host. This was substantiated by the hemolytic and CaCo-2 cell line assay which confirms its cellular adherence and biosafety. Notably, genome analysis did not identify any pathogenic islands. Probiotic characterization indicates its potential to overcome waterborne infections and digestion-related disorders. Cumulatively, Cellulomonas sp. HM71 can be considered a probiotic strain for improving human health because of the highlighted functions.

Neprosin was first discovered in the insectivorous tropical pitcher plants of Nepenthes species as a novel protease with prolyl endopeptidase (PEP) activity. Neprosin has two uncharacterized domains of neprosin activation peptide and neprosin. A previous study has shown neprosin activity in hydrolyzing proline-rich gliadin, a gluten component that triggers celiac disease. In this study, we performed in silico structure-function analysis to investigate the catalytic mechanism of neprosin. Neprosin sequences lack the catalytic triad and motifs of PEP family S9. Protein structures of neprosins from Nepenthes × ventrata (NvNpr) and N. rafflesiana (NrNpr1) were generated by ab initio methods and comparatively assessed to obtain high-quality models. Structural alignment of models to experimental structures in the Protein Data Bank (PDB) found a high structural similarity to glutamic peptidases. Further investigations reveal other resemblances to the glutamic peptidases with low optimum pH that activates the enzyme via autoproteolysis for maturation. Two highly conserved glutamic acid residues, which are stable according to the molecular dynamics simulation, can be found at the active site of the substrate cleft. Protein docking demonstrated that mature neprosins bind well with potent antigen αI-gliadin at the putative active site. Taken together, neprosins represent a new glutamic peptidase family, with a putative catalytic dyad of two glutamic acids. This study illustrates a hypothetical enzymatic mechanism of the neprosin family and demonstrates the useful application of an accurate ab initio protein structure prediction in the structure-function study of a novel protein family. • An extensive in silico structure-function analysis of neprosins from Nepenthes species. • Experimental-quality protein structures revealed the regulatory and catalytic mechanisms of a mature neprosin. • Neprosin is a glutamic peptidase with 2 glutamic acids as the putative catalytic dyad. • The first model on the catalytic mechanism of post-proline cleaving activity of neprosin. [ABSTRACT FROM AUTHOR]

Wheat is a major source of nutrition, though in susceptible people it can elicit inappropriate immune responses. Wheat allergy and non-celiac wheat sensitivity are caused by various wheat proteins, including alpha-amylase trypsin inhibitors (ATIs). These proteins, like the gluten proteins which can cause celiac disease, are incompletely digested in the stomach such that immunogenic epitopes reach the lower digestive system where they elicit the undesirable immune response. The only completely effective treatment for these immune reactions is to eliminate the food trigger from the diet, though inadvertent or accidental consumption can still cause debilitating symptoms in susceptible people. One approach used is to prevent the causal proteins from provoking an immune reaction by enhancing their digestion using digestive protease supplements that act in the stomach or intestine, cleaving them to prevent or quench the harmful immune response. In this study, a digestive supplement enriched in caricain, an enzyme naturally present in papaya latex originally designed to act against gluten proteins was assessed for its ability to digest wheat ATIs. The digestion efficiency was quantitatively measured using liquid chromatography-mass spectrometry, including examination of the cleavage sites and the peptide products. The peptide products were measured across a digestion time course under conditions that mimic gastric digestion in vivo, involving the use of pepsin uniquely or in combination with the supplement to test for additive effects. The detection of diverse cleavage sites in the caricain supplement-treated samples suggests the presence of several proteolytic enzymes that act synergistically. Caricain showed rapid action in vitro against known immunogenic ATIs, indicating its utility for digestion of wheat ATIs in the upper digestive tract.

Although prolyl endopeptidase (PREP) has become one of the important proteins to investigate the pathogenesis of Alzheimer's Disease (AD), the activity of PREP in the brain of AD patients is still controversial. This study aims to construct a novel enzyme-activated near-infrared (NIR) fluorescence probe for in-situ detection and imaging of PREP in brain, as well as to reveal the relationship between the activity variation of PREP and the development of AD. For these purposes, based on PREP's unique endopeptidase activity catalyzing prolyl-bond hydrolysis and cleavage, four probes deriving from a good NIR fluorophore (ACM) were designed and synthesized. Observations indicated that the Z-GP-ACM exhibited superior selectivity and sensitivity in detecting PREP. In addition, molecular thermodynamic/kinetic calculations and enzymatic kinetics studies also showed that Z-GP-ACM was an excellent substrate for PREP. Subsequent experiments demonstrated that Z-GP-ACM can be used to measure and image endogenous PREP in living cells and mouse brain. We also revealed for the first time that PREP activity was abnormally increased in hippocampus of AD mice during the disease progression. All these results highlighted that Z-GP-ACM could be a promising tool for exploring the biological functions of endogenous PREP in living systems. [Display omitted] • A specific NIR-PREP fluorescent probe Z-GP-ACM was developed for the first time. • Theoretical calculations and enzyme kinetic studies proposed PREP-activated mechanism. • Z-GP-ACM can be used for in-situ imaging of PREP in Living cells and mouse brain. • Revealed significant differences of PREP activity in different brain regions. • Revealed abnormal elevation of PREP in hippocampus during AD progression. [ABSTRACT FROM AUTHOR]

This study provides a comprehensive comparison among the most common gluten-free (GF) brewing practices, with a focus on the impact of each treatment on physicochemical parameters and consumer acceptability of the final beer. In addition, the influence of a longer cold maturation on the natural reduction of the gluten content was investigated. Prolyl endopeptidase addition was found to be the most effective treatment in reducing gluten levels (−75.93%), followed by silica gel (−53.09%), longer cold maturation (−4.32%), and tannins (−1.85%). Nonetheless, none of the treated beer samples was gluten-free (gluten content > 20 ppm) due to the high nitrogen content of the original wort. The silica gel application treatment affected the physicochemical and sensory characteristics of the final beer the least. According to the difference from control test results, no significant difference in terms of overall liking, appearance, odor/aroma, or taste was observed between the silica gel-treated sample and control beer (p > 0.05). On the other hand, the application of enzymes and tannins significantly affected the appearance and the beer odor/aroma. Nevertheless, all beer samples received positive sensory acceptance scores.

Celiac disease (CD) is an inflammatory disorder of the small intestine. Gluten peptides are supposed to be responsible for the reaction, the best-researched of which is the so-called ‘33-mer’. Analogous peptides in secalins (rye) and hordeins (barley) have been described. This study presents the degradation of gliadins, glutenins, hordeins and secalins purified from the respective flours using a prolyl endopeptidase from the Basidiomycete Flammulina velutipes (FvpP). The flour fractions were incubated with the enzyme, and the cleavage sites were determined using high-resolution nLC-qTOF-MS/MS. For the wheat samples, eight cleavage sites in the 33-mer peptide were shown, and all of the six described epitopes were successfully cleaved. For the commercially available prolyl-specific endopeptidase from Aspergillus niger (An-Pep), which was used as a control, only two cleavage sites that cleaved three of the six epitopes were identified. For the secalins, four prolyl-specific cleavage sites in the CD-active peptide QPFPQPQQPIPQ were found for the FvpP but none for the An-Pep. The CD-active peptide QPFPQPEQPFPW in C-hordein was cleaved at three prolyl-specific positions by the FvpP. The study proves the usability of FvpP to degrade CD-inducing peptides in real-grain flour samples and indicates its higher effectiveness compared with An-Pep. A clinical study would be required to assess the therapeutic or preventive potential of FvpP for CD.

Celiac disease (CD), a chronic inflammatory disorder, is triggered by the ingestion of gluten peptide. Wheat gluten contains gliadins and glutenins fractions, where gliadin peptides are the main cause of CD and nonceliac gluten sensitivity development. Keeping a strict gluten-free diet is the only effective treatment for CD. In recent years, lactic acid bacterial and fungal prolyl endopeptidases (PEP) have been proposed as the sources of proteolytic enzymes for the complete elimination of gluten peptides, and have also proved as a potential common therapeutic agent for CD treatment. Considering these indications, a special focus was devoted to AN-PEP-secreted PEP. Herein, we review the proteolytic enzymes produced by microorganisms, especially by the fungal strain, Aspergillus niger (AN), and discuss its beneficial properties against toxic effects of a-gliadin digestion in affected patients. The present review reveals the importance of proteolytic proteases in industrial applications; from an economic perspective, AN-PEP protease is an appropriate choice for making high-quality gluten-free products. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The gut-liver axis is increasingly recognized as being involved in the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD). Prolyl endopeptidase (PREP) plays a role in gut metabolic homeostasis and neurodegenerative diseases. We investigated the role of PREP disruption in the crosstalk between gut flora and hepatic steatosis or inflammation in mice with NAFLD. Wild-type mice (WT) and PREP gene knocked mice (PREPgt) were fed a low-fat diet (LFD) or high-fat diet (HFD) for 16 or 24 weeks. Murine gut microbiota profiles were generated at 16 or 24 weeks. Liver lipogenesis-associated molecules and their upstream mediators, AMP-activated protein kinase (AMPK) and sirtuin1 (SIRT1), were detected using RT-PCR or western blot in all mice. Inflammatory triggers and mediators from the gut or infiltrated inflammatory cells and signal mediators, such as p-ERK and p-p65, were determined. We found that PREP disruption modulated microbiota composition and altered the abundance of several beneficial bacteria such as the butyrate-producing bacteria in mice fed a HFD for 16 or 24 weeks. The level of butyrate in HFD-PREPgt mice significantly increased compared with that of the HFD-WT mice at 16 weeks. Interestingly, PREP disruption inhibited p-ERK and p-p65 and reduced the levels of proinflammatory cytokines in response to endotoxin and proline-glycine-proline, which guided macrophage/neutrophil infiltration in mice fed a HFD for 24 weeks. However, at 16 weeks, PREP disruption, other than regulating hepatic inflammation, displayed improved liver lipogenesis and AMPK/SIRT1 signaling. PREP disruption may target multiple hepatic mechanisms related to the liver, gut, and microbiota, displaying a dynamic role in hepatic steatosis and inflammation during NAFLD. PREP might serve as a therapeutic target for NAFLD.

Early-life stress is a risk factor for the development of behavioral and cognitive disorders in humans and animals. Such stressful situations include social isolation in early postnatal ontogenesis. Behavioral and cognitive impairments associated with neuroplastic changes in brain structures. We have found that after ten weeks of social isolation, male Wistar rats show behavioral abnormalities and cognitive deficit, accompanied by an increase in the relative expression of gene encoding serine protease prolyl endopeptidase (PREP, EC 3.4.21.26) in the brain frontal cortex. The present study aimed to assess synaptophysin (SYP), brain-derived neurotrophic factor precursor (proBDNF), and PREP expression using Western blot in the brain structures – the hippocampus, frontal cortex, and striatum of the rats subjected to prolonged social isolation compared with group-housed animals. Twenty Wistar rats were used for this study (10 males and 10 females). Experimental animals (5 males and 5 females) were kept one per cage for nine months, starting from the age of one month. Ten-month-old socially isolated rats showed memory deficit in passive avoidance paradigm and Morris Water Maze and reactivity to novelty reduction. We used monoclonal antibodies for the Western blot analysis of the expression of SYP, proBDNF, and PREP in the rat brain structures. Social isolation caused a proBDNF expression reduction in the frontal cortex in females and a reduction in PREP expression in the striatum in males. These data suppose that neurotrophic factors and PREP are involved in the mechanisms of behavioral and cognitive impairments observed in the rats subjected to prolonged social isolation with an early life onset. [ABSTRACT FROM AUTHOR]

Prolyl endopeptidases (PEP) from Sphingomonas capsulata (sc) and Myxococcus xanthus (mx) selectively degrade gluten peptides in vitro, offering a potential therapeutic strategy for celiac disease. However, the mechanisms governing the interaction of these enzymes with their substrates remain unclear. In this study, conventional molecular dynamics simulations with a microsecond timescale and targeted molecular dynamics simulations were performed to investigate the native states of mx PEP and sc PEP enzymes, as well as their allosteric binding with a representative substrate, namely, Z -Ala-Pro- p -nitroanilide (pNA). The simulations reveal that the native sc PEP is in an open state, while the native mx PEP is in a closed state. When pNA approaches a closed mx PEP, it binds to an allosteric pocket located at the first and second β-sheet of the β-propeller domain, inducing the opening of this enzyme. Neither enzyme is active in the open or partly-open states. Enzymatic activity is enabled only when the catalytic pocket in the closed state fully accommodates the substrates. The internal capacity of the catalytic pocket of PEP in the closed state determines the maximum size of the gluten peptides that the enzymes can catalyze. The present work provides essential molecular dynamics information for the redesign or engineering of PEP enzymes. [ABSTRACT FROM AUTHOR]

Aspergillus niger prolyl endopeptidase (An-PEP) has become a research focus because of its advantages in specifically cleaving the C-terminal peptide bond of proline residues, especially it was an industrial food-grade acidic PEP. Aqueous two-phase system (ATPS) was first applied for separating An-PEP from fermentation broth. Via response surface method (RSM) experiment, an effectively separation of An-PEP was achieved by ATPS containing27% (w/w) ethanol and 14.5% (w/w) (NH 4) 2 SO 4 at pH 6.0 with the recovery of 90.29 ± 0.23% and purification coefficient of 15.35 ± 0.30. The purified An-PEP was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), fourier transform infrared (FTIR) and fluorescence spectrometry. The optimum temperature and pH of An-PEP were 40 °C and 4.5–5.0, respectively. An-PEP was activated and stabilized by Ca2+ but inhibited by Fe3+. The enzymatic application of purified An-PEP was evaluated by hydrolyzing egg white protein (EWP) to prepare bioactive peptides. The obtained hydrolysates had good scavenging ability of OH and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, angiotensin converting enzyme (ACE) inhibitory activity and anti-gout activity. This research realized a low-cost, high-efficiency and simple separation technology of An-PEP and provided a broader idea for the preparation of bioactive peptides and the application of An-PEP. Unlabelled Image • A neomethod to separate a specific enzymewas developed. • Satisfactory separation efficiency of Aspergillus niger prolyl endopeptidase was achieved by ethanol/ (NH 4) 2 SO 4 ATPS. • Enzyme activity was well maintained during the separation. • Peptides obtained by An-PEP hydrolyzed egg white protein showed biological activities. [ABSTRACT FROM AUTHOR]

Prolyl endopeptidase from an aerobic and Gram-negative thermophile Meiothermus ruber H328 (MrPEP) was purified in native and recombinant forms, but both preparations had comparable characteristics. Production of the native MrPEP was increased 10-fold by adding intact chicken feathers. The gene for MrPEP (mrH_2860) was cloned from the genome of strain H328 and found to have no signal sequence at the N-terminus. MrPEP is composed of two major domains: the β-propeller domain and the peptidase domain with a typical active site motif and catalytic triad. Based on extensive investigations with different types of peptide substrates and FRETS-25Xaa libraries, MrPEP showed strict preferences for Pro residue at the P1 position but broader preferences at the P2 and P3 positions in substrate specificity with stronger affinity for residues at the P3 position of substrate peptides that are longer than four residues in length. In conclusion, the molecular characterization of MrPEP resembles its animal counterparts more closely than bacterial counterparts in function and structure. [ABSTRACT FROM AUTHOR]

Prolyl endopeptidase (PREP), also known as prolyl oligopeptidase (POP), is an enzyme that cleaves short peptides (<30 amino acids in length) on the C-terminal side of proline. PREP is highly expressed in multiple carcinomas and is a potential target for cancer therapy. A potent inhibitor of PREP, Y-29794, causes long-lasting inhibition of PREP in mouse tissues. However, there are no reports on Y-29794 effects on cancer cell and tumor proliferation. Using cell line models of aggressive triple-negative breast cancer (TNBC), we show here that Y-29794 inhibited proliferation and induced death in multiple TNBC cell lines. Cell death induced by Y-29794 coincided with inhibition of the IRS1-AKT-mTORC1 survival signaling pathway, although stable depletion of PREP alone was not sufficient to reduce IRS1-AKT-mTORC1 signaling or induce death. These results suggest that Y-29794 elicits its cancer cell killing effect by targeting other mechanisms in addition to PREP. Importantly, Y-29794 inhibited tumor growth when tested in xenograft models of TNBC in mice. Induction of cell death in culture and inhibition of xenograft tumor growth support the potential utility of Y-29794 or its derivatives as a treatment option for TNBC tumors. [ABSTRACT FROM AUTHOR]

Gluten is a cereal protein that is incompletely digested by human proteolytic enzymes that create immunogenic peptides that accumulate in the gastrointestinal tract (GIT). Although both environmental and human bacteria have been shown to expedite gluten hydrolysis, gluten intolerance is a growing concern. Here we hypothesize that together with food, we acquire environmental bacteria that could impact our GIT with gluten-degrading bacteria. Using in vitro gastrointestinal simulation conditions, we evaluated the capacity of endophytic bacteria that inhabit root vegetables, potato (Solanum tuberosum), carrot (Daucus sativus), beet (Beta vulgaris), and topinambur (Jerusalem artichoke) (Helianthus tuberosus), to resist these conditions and degrade gluten. By 16S rDNA sequencing, we discovered that bacteria from the families Enterobacteriaceae, Bacillaceae, and Clostridiaceae most effectively multiply in conditions similar to the human GIT (microoxic conditions, 37 °C) while utilizing vegetable material and gluten as nutrients. Additionally, we used stomach simulation (1 h, pH 3) and intestinal simulation (1 h, bile salts 0.4%) treatments. The bacteria that survived this treatment retained the ability to degrade gluten epitopes but at lower levels. Four bacterial strains belonging to species Bacillus pumilus, Clostridium subterminale, and Clostridium sporogenes isolated from vegetable roots produced proteases with postproline cleaving activity that successfully neutralized the toxic immunogenic epitopes. Key points: • Bacteria from root vegetables can degrade gluten. • Some of these bacteria can resist conditions mimicking gastrointestinal tract. [ABSTRACT FROM AUTHOR]

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous immunomodulatory peptide that is generated from thymosin β4 (Tβ4) through stepwise hydrolysis, involving meprin-α and prolyl endopeptidase (PREP). It is well acknowledged that AcSDKP exerts beneficial effects on multiple cardiovascular and renal diseases. However, the functional role of AcSDKP in inflammatory bowel disease (IBD) remains poorly understood. Here, we aimed to assess the content of AcSDKP in patients with IBD and investigate the impact of AcSDKP on intestinal inflammation in IBD. We found that in the inflamed mucosal specimens of patients with ulcerative colitis, the expression levels of Tβ4 and meprin-α were decreased, while PREP was expressed at similar levels to non-inflamed mucosa. In vitro, AcSDKP inhibited the expression of proinflammatory factors in intestinal epithelial cells partially by reducing the activation of MEK-ERK signaling. In vivo studies showed that transgenic mice, with lower levels of AcSDKP, were more vulnerable to dextran sulfate sodium (DSS)-induced colitis and exhibited more severe intestinal inflammatory responses. On the other hand, exogenous AcSDKP infusion significantly attenuated the clinical symptoms and intestinal mucosal inflammation in DSS-induced mice. In conclusion, results from this study demonstrated the anti-inflammatory function of AcSDKP within the intestine and suggest that AcSDKP has a promising therapeutic potential for IBD treatment.

Abstract Background Neurogranin (Ng) is a small 7.6 kDa postsynaptic protein that has been detected at elevated concentrations in cerebrospinal fluid (CSF) of patients with Alzheimer’s disease (AD), both as a full-length molecule and as fragments from its C-terminal half. Ng is involved in postsynaptic calcium (Ca) signal transduction and memory formation via binding to calmodulin in a Ca-dependent manner. The mechanism of Ng secretion from neurons to CSF is currently unknown, but enzymatic cleavage of Ng may be of relevance. Therefore, the aim of the study was to identify the enzymes responsible for the cleavage of Ng, yielding the Ng fragment pattern of C-terminal fragments detectable and increased in CSF of AD patients. Methods Fluorigenic quenched FRET probes containing sequences of Ng were utilized to identify Ng cleaving activities among enzymes known to have increased activity in AD and in chromatographically fractionated mouse brain extracts. Results Human Calpain-1 and prolyl endopeptidase were identified as the candidate enzymes involved in the formation of endogenous Ng peptides present in CSF, cleaving mainly in the central region of Ng, and between amino acids 75_76 in the Ng sequence, respectively. The cleavage by Calpain-1 affects the IQ domain of Ng, which may deactivate or change the function of Ng in Ca2+/calmodulin -dependent signaling for synaptic plasticity. While shorter Ng fragments were readily cleaved in vitro by prolyl endopeptidase, the efficiency of cleavage on larger Ng fragments was much lower. Conclusions Calpain-1 and prolyl endopeptidase cleave Ng in the IQ domain and near the C-terminus, respectively, yielding specific fragments of Ng in CSF. These fragments may give clues to the roles of increased activities of these enzymes in the pathophysiology of AD, and provide possible targets for pharmacologic intervention.

The present work shows a procedure to valorize non-commercial boiled shrimp to produce functional ingredients, using a combined treatment based on enzymatic hydrolysis and subsequent glycation under mild conditions. Antioxidant and prolyl endopeptidase-inhibiting activities were determined as a function of hydrolysis and glycation times (0–120 min and 0–180 min, respectively). The reaction products were characterized by determining the degree of hydrolysis, browning, fluorescent compounds, free amino acids, phenol content, Fourier transform infrared spectroscopy (FTIR), and molecular weight of the different fractions obtained. Enzymatic hydrolysis generated hydrolysates with significant antioxidant and prolyl endopeptidase-inhibiting activities. Glycation under mild conditions was used as a strategy to improve the antioxidant and potential nootropic properties of the hydrolysates. During glycation, the free amino acid content decreased, total phenols and fluorescent compounds increased significantly, and low molecular weight melanoidins were formed. The presence of peptide-glucose conjugates was also confirmed by FTIR. Glycation increased the antioxidant activities of the hydrolysates; however, their prolyl-endopeptidase-inhibiting activity was lost. Results showed that compounds with promising antioxidant (hydrolysis and glycation) and potential nootropic (hydrolysis) activities and applications in food systems were obtained from the biotechnological strategy used.

Inhibition of acetylcholinesterase (AChE) is a promising strategy for the management of Alzheimer's disease (AD). Prolyl endopeptidase (PEP) is a post-proline cleaving enzyme that plays an important role in learning and memory. This study explore the AChE and PEP inhibitory activities of compounds from T. cordifolia. Their affinity for different binding sites in silico was also evaluated. Ellman's and β-naphtyl acetate-fast blue salt (NA-FB) assay methods were used to determine the cholinesterase inhibitory activity. PEP inhibition was determined by colorimetric assay according to Yoshimoto. Isolation of bioactive compounds was done by different chromatographic methods and structures established using spectroscopy. Molecular docking was carried out using MOE software suite modeled structures based on the coordinates of PDB ID: 3IVM and 10CE. The ethyl acetate fraction had the best AChE inhibition with an IC50 value of 1.4574 ± 0.47 mg/mL at 5 mg/mL. Nine compounds identified as β-sitosterol, tinosporide, 8-hydroxycolumbin, oxoglaucine, corydine, liriodenine, N-formylanonaine, palmatine, 1-octacosanol were isolated from different fractions with oxoglaucine being reported for the first time in the genus Tinospora. Oxoglaucine, liriodenine and N-formylanonaine demonstrated good AChE inhibitory activity with IC50 values of 0.8033 ± 0.09 mg/mL, 0.8076 ± 0.07 mg/mL and 0.8190 ± 0.06 mg/mL at 1 mg/mL respectively. Oxoglaucine and corydine at 1 mM inhibited prolyl endopeptidase with IC50 values of 0.7802 ± 3.1 mM and 0.7884 ± 3.17 mM respectively. Molecular docking revealed hydrophobic, hydrogen bonding, and Pi-stacking interactions between the ligands and the active site of enzymes. Compounds from Tinospora cordifolia are a possible lead for the development of drugs that may be useful in the management of AD. [ABSTRACT FROM AUTHOR]

Cyanopyrrolidine derivatives benzyloxycarbonyl-methionyl-cyanopyrrolidine (ZMetPrdN), benzyloxycarbonyl-phenylalanyl-cyanopyrrolidine (ZPhePrdN), tert-butyl-hydroxycarbonyl-glycyl-cyanopyrrolidine (BocGlyPrdN), tert-butyl-hydroxycarbonyl-methionyl-cyanopyrrolidine (BocMetPrdN) are inhibitors of prolyl endopeptidase (PREP; ЕС 3.4.21.26) with the IC50 values ranged from 2 nM to 12 nM. Besides inhibition of PREP, ZMetPrdN, ZPhePrdN, and BocMetPrdN also inhibited activity of dipeptidyl peptidase IV (DPP-4; ЕС 3.4.14.5) with the IC50 values ranged from 1100 nM to 3200 nM. In the acetic acid writhing test in mice all the compounds demonstrated antinociceptive properties. However, only the cyanopyrrolidine derivatives with aromatic substituents (ZMetPrdN and ZPhePrdN) decreased acute exudative inflammation in animals. Three hours after induction of inflammation, the studied cyanopyrrolidine derivatives increased PREP activity and compensatory decreased DPP-4 activity in serum of mice. Thus, one of the components of the mechanism responsible for realization of the antinociceptive and antiexudative properties of the studied derivatives of cyanopyrrolidine is obviously associated with their ability to inhibit PREP activity. [ABSTRACT FROM AUTHOR]

• Prolyl endopeptidase reduced soy sauce secondary precipitate via degrading B 3 subunit. • Ultrasound-assisted prolyl endopeptidase further reduced soy sauce secondary precipitate. • Ultrasound improved prolyl endopeptidase's structure and thus enhanced its activity. • Degradation rate increase of B 3 subunit is attributed to increase of the enzyme's activity. Prolyl endopeptidase can partially degrade soybean protein B 3 subunit and alleviate soy sauce secondary precipitate. In this study, the influences of ultrasound-assisted prolyl endopeptidase on the degradation of soybean protein B 3 subunit of soy sauce and primary mechanism were investigated using SDS-PAGE, MALDI-TOF-MS, circular dichromatic spectrometer, fluorescence spectra, etc. Results showed that ultrasound-assisted prolyl endopeptidase enhanced 72% degradation rate of B 3 subunit and reduced soy sauce secondary precipitate remarkably, meanwhile significantly increased content of organic taste compounds of soy sauce compared with control (p < 0.05). Sonication markedly reduced percentage of α-helix and increased percentage of random coil, made hydrophobic amino acids inside prolyl endopeptidase exposed to its surface and enhanced its flexibility, which facilitated the binding of prolyl endopeptidase active center with B 3 subunit and finally enhanced the latter's degradation rate and appearance quality of soy sauce. This work laid a foundation for solving soy sauce secondary precipitate. [ABSTRACT FROM AUTHOR]

Background and Aims: Prolyl endopeptidase (PREP) is a serine endopeptidase that participates in many pathological processes including inflammation, oxidative stress, and autophagy. Our previous studies found that PREP knockout exhibited multiple benefits in high-fat diet (HFD) or methionine choline-deficient diet-induced metabolic dysfunction-associated fatty liver disease (MAFLD). However, cumulative studies have suggested that PREP performs complex functions during disease development. Therefore, further understanding the role of PREP in MAFLD development is the foundation of PREP intervention., Methods: In this study, an HFD-induced MAFLD model at different time points (4, 8, 12, and 16 weeks) was used to explore dynamic changes in the PREP proline-glycine-proline (PGP)/N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) system. To explore its potential value in MAFLD treatment, saline, or the PREP inhibitor, KYP-2047, was administered to HFD-induced MAFLD mice from the 10th to 16th weeks., Results: PREP activity and expression were increased in HFD-mice compared with control mice from the 12th week onwards, and increased PREP mainly resulted in the activation of the matrix metalloproteinase 8/9 (MMP8/9)-PREP-PGP axis rather than the thymosin β4-meprin α/PREP-AcSDKP axis. In addition, KYP-2047 reduced HFD-induced liver injury and oxidative stress, improved lipid metabolism through the suppression of lipogenic genes and the induction of β-oxidation-related genes, and attenuated hepatic inflammation by decreasing MMP8/9 and PGP. Moreover, KYP2047 restored HFD-induced impaired autophagy and this was verified in HepG2 cells., Conclusions: These findings suggest that increased PREP activity/expression during MAFLD development might be a key factor in the transition from simple steatosis to steatohepatitis, and KYP-2047 might possess therapeutic potential for MAFLD treatment., Competing Interests: JGF has been an associate editor of Journal of Clinical and Translational Hepatology since 2013. The other authors have no conflict of interests relevant to this publication., (© 2023 Authors.)

Fibroblast activiation protein alpha (FAP) is considered a diagnostic and prognostic biomarker for various types of cancer. FAP shares substrate specificity with prolyl oligopeptidase (PREP), studied in (neuro)inflammation and neurodegeneration as well as cancer. Current assays inadequately discriminate between FAP and PREP and there is need for an assay that reliably quantitates the FAP/PREP activity ratio in plasma. FAP and PREP activities were measured in human EDTA-plasma in presence of well characterized PREP and FAP inhibitors. A combined kinetic assay was developed in conditions to optimally measure FAP as well as PREP activity with Z -Gly-Pro-AMC as substrate. Limit of detection was 0.009 U/L and limit of quantitation was 0.027 U/L for the combined FAP-PREP assay. Within-run coefficient of variation was 3% and 4% and between-run precision was 7% and 12% for PREP and FAP, respectively. Accuracy was demonstrated by comparison with established end-point assays. Hemolysis interferes with the assay with 1.5 g/L hemoglobin as cut-off value. PREP (but not FAP) activity can increase upon lysis of platelets and red blood cells during sample preparation. With this new assay, on average 67% of the Z -Gly-Pro-AMC converting activity in plasma can be attributed to FAP. Unlabelled Image • There is need for an enzymatic assay for measuring FAP and PREP simultaneaously • Specific PREP and FAP inhibitors are used in combination with a common substrate • 5 mM DTT is required for optimal PREP activity, < 0.01% DMSO for FAP • PREP activity can be influenced by the presence of platelets and hemolysis • On average 67% of the combined activity in EDTA-plasma can be attributed to FAP [ABSTRACT FROM AUTHOR]

Summary: Prolyl endopeptidase (PREP) is a post-proline cleaving enzyme. It is involved in the regulation of multiple inositol polyphosphate phosphatase activity implicated in the pathway of inositol 1,4,5-trisphosphate, resulting in the modulation of cytosolic Ca2+ levels. Besides its peptidase activity, PREP was identified as a binding partner of tubulin, suggesting that it may participate in microtubule-associate processes. In this paper, we evaluated the expression of PREP mRNA and protein by polymerase chain reaction and western blot analyses and its co-localization with tubulin by immunofluorescence in adult mouse seminal vesicles. We showed that both proteins are cytoplasmic: tubulin is localized at the apical half part of the cell, while PREP has a more diffuse localization, showing a prominent distribution at the apical cytoplasm. These findings support our hypothesis of a specific role for PREP in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular its association with tubulin filaments. Moreover, it may regulate Ca2+ levels, and promote the final step of vesicular exocytosis, namely the fusion of the vesicles with the plasma membrane. These results strongly suggest that there is a pivotal role for PREP in vesicle exocytosis, as well as in the physiology of mouse seminal vesicles. [ABSTRACT FROM AUTHOR]

Prolyl endopeptidase (PREP) is an enzyme which cleaves several peptide hormones and neuropeptides on the carboxyl side of proline residues and is involved in many biological processes, including cell proliferation and differentiation, glucose metabolism, learning, memory, and cognitive disorders. PREP has also been identified as a binding partner of tubulin, suggesting the involvement of endopeptidase in microtubule-associate processes, independent of its peptidase activity. Furthermore, several reports have implied PREP participation in both male and female reproduction-associated mechanism. We herein assess a potential association of PREP to the morphogenesis of rat testis, profiling its localization versus tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60 dpp). We show that, in mitotic phases, PREP shares its localization with tubulin in Sertoli cells, gonocytes, and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, and in the cytoplasm of Sertoli cells protrusions, surrounding the germ cells, while, during spermiogenesis, they both localize in the cytoplasm of round and elongating spermatids. We also found that this enzyme has a peculiar nuclear localization, in the proliferating cells in all phases of analysis. Finally, they are expressed in the flagellum of mature gametes, as corroborated by additional immunolocalization analysis on both rat and human sperm. Our data support the hypothesis of the fundamental role of PREP in reproduction and in cytoskeletal organization during mammalian testis morphogenesis and gamete progression. [ABSTRACT FROM AUTHOR]

Prolyl endopeptidase is involved in neurodegeneration, proliferation, neuroinflammation, and neuron differentiation. It has been observed that severity of the neurodegenerative process correlates with an increasing level of prolyl endopeptidase in the brain. According to recent research, prolyl endopeptidase might interact with prion-like proteins, which are believed to play the key role in the development of neurodegenerative diseases. Consequently, prolyl endopeptidase inhibitors might be a promising group of chemicals to study in neurodegenerative diseases, particularly Parkinson's disease. The aim of this research was to determine the effects of benzyloxycarbonyl-methionyl-2(S)-cyanopyrrolidine, a new inhibitor of prolyl endopeptidase, using haloperidol catalepsy test in SHK outbred mice and Parkinson-like syndrome induced by a single systemic injection of neurotoxic 1-methyl-4-phenyl-tetrahydropiridine at a dose of 30 mg/kg in C57Bl/6 inbred mice. As a result, the compound reduces the severity of the main extrapyramidal symptoms: rigidity and motor deficits. However, anticataleptic activity was not found. According to our results, the prolyl endopeptidase inhibitor does not affect the dopaminergic neurons directly but provides possible neuroprotection to the neurons. [ABSTRACT FROM AUTHOR]

Abstract Celiac disease and non-celiac gluten sensitivity are both related to gluten consumption. In sensitive subjects, gluten triggers an immune-mediated enteropathy for which no therapy is yet available. Beer must be avoided by gluten sensitive subjects, since it is generally made from gluten containing grains such as barley and wheat. In this study we compared two different deglutinization treatments on an American India Pale Ale, brewed on a craft beer pilot plant: an enzymatic process based on Aspergillus niger prolyl endopeptidase and a precipitation process by silica gel. The gluten amount in all stages of brewing, and the effects of treatments on standard quality attributes, chemical composition and sensory profile were evaluated. Both treatments were able to reduce gluten level below 20 ppm, showing a different behavior. Only slight differences were observed in the chemico-physical characteristics of treated and untreated beers, whereas sensory analysis did not highlight any change. Industrial relevance Considering the massive increase of microbreweries and the grown of gluten-free products market, two different deglutinization treatment were compared in pilot plant production of craft beer. Results show how it is possible to produce a gluten-free beer from traditional ingredients. The slight differences observed between treatments had no impact on the sensory profile, allowing to brew high quality gluten-free beers. Highlights • Gluten-free craft beer can be produced from traditional ingredients. • Gluten content decreases in brewing stages. • Beer deglutinization treatments let unaltered the sensory profile of the original recipe. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A prolyl endopeptidase was isolated from the basidiomycete Flammulina velutipes. • Cleavage of gliadin, casein, and gelatin was confirmed by HR-QTOF-MS/MS. • FvpP degraded the antigenic epitope of α -gliadin which triggers celiac disease. • The biochemical properties of FvpP favour application in food processing. Abstract A prolyl endopeptidase, FvpP, was identified in the culture supernatant of the basidiomycete Flammulina velutipes and functionally expressed for gene verification in Aspergillus oryzae. The wild-type FvpP with a molecular mass of 50 kDa under denaturing conditions, exhibited optima at pH 5, and 45 °C and an isoelectric point of 3.8. Five mM Fe2+ and Fe3+ activated the prolyl endopeptidase, while it was not affected by 10 mM CaCl 2 , EDTA, and Pepstatin A. Five mM Cu2+, Mn2+, Zn2+, Ni2+, Co2+ and low Z -Pro-prolinal concentrations decreased the enzyme activity. Concluded from comparative RP-HPLC and high-resolution QTOF-MS/MS analyses of the hydrolytic cleavage products of gliadin, casein and gelatin, FvpP possessed a P1-P1′-substrate specificity similar to the prolyl endopeptidase from Aspergillus niger (An-Pep), although the sequence similarity was only 39%. FvpP and An-Pep (S28.004; MEROPS) are unique in the serine peptidase family S28, because they share more sequence similarity with Pro-X carboxypeptidase (S28.001; MEROPS) and dipeptidyl peptidase II (S28.002; MEROPS) than with prolyl oligopeptidases of the serine peptidase family S09. As a potential degrader of the antigenic epitopes of α -gliadin, FvpP might provide an efficient tool for the processing of wheat and other gluten containing cereals to better digestible foods for celiac patients. [ABSTRACT FROM AUTHOR]

Wheat storage proteins and products of their hydrolysis may cause coeliac sprue in genetically predisposed individuals with high expression of main histocompatibility complex HLA-DQ2 or DQ8, since by consuming wheat, they become exposed to proline- (P) and glutamine (Q)-rich gluten. In bread-making, the hydrolysis of gliadins and coeliac-toxic peptides occurs with varied efficiency depending on the fermentation pH and temperature. Degradation of gliadins catalysed by Lactobacillus acidophilus 5e2 peptidases and a commercial prolyl endopeptidase synthesised by A. niger, carried out at pH 4.0 and 37 °C, reduces the gliadin concentration over 110-fold and decreases the relative immunoreactivity of the hydrolysate to 0.9% of its initial value. Hydrolysis of coeliac-toxic peptides: LGQQQPFPPQQPY (P1) and PQPQLPYPQPQLP (P2) under the same conditions occurs with the highest efficiency, reaching 99.8 ± 0.0% and 97.5 ± 0.1%, respectively. The relative immunoreactivity of peptides P1 and P2 was 0.8 ± 0.0% and 3.2 ± 0.0%, respectively. A mixture of peptidases from L. acidophilus 5e2 and A. niger may be used in wheat sourdough fermentation to reduce the time needed for degradation of proteins and products of their hydrolysis.

Thimet oligopeptidase (Top) is a thiol-sensitive metallopeptidase with increased concentrations in brain and reproductive tissues. Prolyl endopeptidase (Pep) is another endopeptidase with high enzymatic activity in the brain. The two neuropeptidases are involved in the degradation of gonadotropin-releasing hormone. To clarify their roles in reproductive regulation, the spotted scat top (sstop) and pep (sspep) genes were cloned and analyzed. Tissue distribution by RT-PCR showed that sstop and sspep were more highly expressed in reproduction-related tissues than in other tissues. The sstop and sspep mRNA was detected in female hypothalamuses at phase II (oocytes at the perinuclear stage), phase III (oocytes forming the vitelline vesicle) and phase IV (oocytes at lipidic and proteic vitellogenesis stages). Both sstop and sspep were highly expressed in phase III, moderately in phase IV, and the lowest in phase II. The sstop and sspep genes were down-regulated in hypothalamuses after treatment with estradiol both in vitro and in vivo. It is suggested that Sstop and Sspep play significant roles in the regulation of reproduction and these results suggested an ssTop- and ssPep-mediated positive feedback mechanism of ovarian function. [ABSTRACT FROM AUTHOR]

Rats with experimental parkinsonism induced by intraperitoneal daily administration of rotenone (2.75 mg/kg) during seven days are characterized by an increased activity of prolyl endopeptidase (PEP; ЕС 3.4.21.26) in serum and a decreased activity of adenosine deaminase (ADA; EC 3.5.4.4) in serum and in the prefrontal cortex. PEP and ADA activities in other brain structures (in the striatum, hypothalamus, and hippocampus) as well as activity of dipeptidyl peptidase IV (DPP-4, CD26; ЕС 3.4.14.5) in serum and in all the brain structures investigated remained unchanged. Afobazole and levodopa, which exhibit antiparkinsonian activity in this model of parkinsonism, decreased the elevated PEP activity in serum and increased ADA activity reduced in the prefrontal cortex of rats with the experimental pathology. At the same time, treatment with these drugs resulted in a decrease of ADA activity in the other brain structures. [ABSTRACT FROM AUTHOR]

Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase was estimated to be 77 kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro- p -nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation at 63 °C was higher than 75 %. The enzyme was activated and stabilized by Co 2+ and inhibited by Mg 2+ , K + and Ca 2+ followed by Zn 2+ , Na +, Mn 2+ , Al 3+, and Cu 2+ . The K m and k cat values of the purified enzyme for different substrates were evaluated. The ability to degrade immunogenic gluten peptides (PQPQLPYPQPQLPY (a-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein)) was also confirmed by enzymatic assays and mass spectrometric analysis of cleavage fragments. Addition of the enzyme during small scale mashing of barley malt reduced the gluten content. The findings here demonstrate the potential of enzyme use during mashing to produce gluten free beer, and provide new insights into the effects of proline specific proteases on gluten degradation. [ABSTRACT FROM AUTHOR]