Formation Of Peptides Research Articles

Investigation of the Plutonium(IV) Interactions with Two Variants of the EF-Hand Ca-Binding Site I of Calmodulin.

Due to its presence in the nuclear industry and its strong radiotoxicity, plutonium is an actinide of major interest in the event of internal contamination. To improve the understanding of its mechanisms of transport and accumulation in the body, the complexation of Pu(IV) to the most common protein calcium-binding motif found in cells, the EF-hand motif of calmodulin, was investigated. Visible and X-ray absorption spectroscopies (XAS) in solution made it possible to investigate the speciation of plutonium at physiological pH (pH 7.4) and pH 6 in two variants of the calmodulin Ca-binding site I and using Pu(IV) in different media: carbonate, chloride, or nitrate solutions. Three different species of Pu were identified in the samples, with formation of 1:1 Pu(IV):calmodulin peptide complexes, Pu(IV) reduction, and formation of peptide-mediated Pu(IV) hexanuclear cluster.

Novel Umami-, Salty-, and Kokumi-Enhancing γ-Glutamyl Tripeptides Synthesized with the Bitter Dipeptides from Defatted Peanut Meal Protein Hydrolysate.

Defatted peanut meal protein hydrolysates (DPMHs) usually have a bitter taste. γ-Glutamylation by Bacillus amyloliquefaciens l-glutaminase was introduced to DPMH to reduce its bitterness and generated a γ-glutamylated product (DPMH-G). Extra l-glutamine (l-Gln) (5% w/w) was added to DPMH, and the mixture was then γ-glutamylated (DPMH-G-Q). Results showed that γ-glutamylation decreased the bitterness of the products and also enhanced their kokumi, umami, and salty taste, especially for DPMH-G-Q. Bitter amino acids and bitter peptides were found to be substrates (acceptors) of the synthesized γ-[Glu](1,2)-AAs and γ-Glu-AA-AAs, respectively. The production yield of γ-[Glu](1,2)-AAs was only 0.69/100 g for DPMH-G and 2.30/100 g for DPMH-G-Q, which was much lower than that of γ-Glu-AA-AAs (5.73/100 g for DPMH-G and 18.72/100 g for DPMH-G-Q). The improvement in taste attributes of DPMH might mainly be due to the consumption of bitter dipeptides and the production of γ-Glu-AA-AAs. In DPMH-G-Q, eight γ-Glu-AA-AAs were identified, including γ-Glu-Ile-Lys, γ-Glu-Ala-Ile, γ-Glu-Leu-Leu, γ-Glu-Phe-Leu, γ-Glu-Thr-Leu, γ-Glu-Ile-Met, γ-Glu-Val-Leu, and γ-Glu-Ser-Tyr, which were first time reported. They all can enhance umami, salty, and kokumi taste with a threshold value between 1.61 ± 0.21-2.16 ± 0.19, 1.65 ± 0.19-2.23 ± 0.20, and 0.67 ± 0.21-1.00 ± 0.22 mM, respectively. Insufficient l-Gln restricted the formation of γ-glutamyl peptides, and this was why DPMH-G had a lower yield and variety than DPMH-G-Q. This also suggested that l-glutaminase is selective to different substrates. Overall, this study provides a new method to reduce the bitterness of protein hydrolysates and also improve the taste by synthesizing γ-glutamyl tripeptides.

Impact of Physicochemical Modifications in Casein Promoted by UV-C on the Peptide Profile of Gastric Digestion and the Transepithelial Transport of Peptides.

Caseins are the main proteins in milk, and their structure and spatial conformation are responsible for their slow digestion rate. The release of bioactive and β-casomorphin peptides from casein digestion may induce allergic responses during consumption. Spectroscopic techniques were used to observe the structural changes in casein conformation induced by Ultraviolet light irradiation (UV-C). Raman spectroscopy results showed more pronounced peaks at 618 and 640 cm-1 for phenylalanine and tyrosine moieties of the photolyzed micellar casein, respectively, suggesting changes in the micelle structure. The decrease in the intensity of Raman signals for tryptophan and tyrosine corroborates to the UV-C-induced modifications of the micelle structure. Particle size distribution showed a decrease in the average micelle size after 15 min of UV-C exposure, while low-temperature, long-time (LTLT) pasteurization led to the formation of large aggregates, as observed by atomic force microscopy. UV-C did not impact the formation or transport of peptides, as observed by using the Caco-2 cell as a model for peptide absorption. However, the absence of the opioid peptide SRYPSY from κ-casein and only 20% of the concentration of opioid peptide RYLGY were noted. This work demonstrated that UV-C can be utilized to induce the physicochemical modification of dairy products, promoting a higher digestion rate and reducing allergenicity.

Constraining and Modifying Peptides Using Pd-Mediated Cysteine Allylation.

Over the past decades, several strategies for inducing and stabilizing secondary structure formation in peptides have been developed to increase their proteolytic stability and their binding affinity to specific interaction partners. Here, we report how our recently introduced chemoselective Pd-catalyzed cysteine allylation reaction can be extended to stapling and how the resulting alkene-containing staples themselves can be further modified to introduce additional probes into such stabilized peptides. The latter is demonstrated by introducing a fluorophore as well as a PEG moiety into different stapled peptides using bioorthogonal thiol-ene and Diels-Alder reactions. Furthermore, we investigated structural implications of our allyl staples when used to replace conformationally relevant disulfide bridges. To this end, we chose a selective binder of integrin α3 β1 (LXY3), which is only active in its cyclic disulfide form. We replaced the disulfide bridge by different stapling reagents in order to increase stability and binding affinity towards integrin α3 β1 .

Fast Preparation of Peptide Tools to address new emerging Viruses: Case example SARS-CoV-2

Abstract The emergence of new virus species or variants poses a major challenge for healthcare systems. Countermeasures, including vaccine development, need to be developed without delay to ensure success. Vaccine and antibody development depend on highly efficient tools for antigen discovery, epitope mapping, diagnostics and (clinical) immune monitoring. Here we present a workflow for peptide tool development that can be rapidly adapted to emerging viruses. It combines bioinformatics algorithms and an ultra-high throughput peptide synthesis method alongside high-content assay formats. Data from different SARS-CoV-2 studies are presented addressing elucidation of both cellular and humoral immune responses. Robust spike-specific cellular immune responses were measured using SARS-CoV-2 Spike PepMix ™peptide pools during infection and/or vaccine response monitoring [ 1]. Using epitope mapping (matrix) pools an immunodominant T-cell epitope was identified that may contribute to the optimization of existing vaccines [ 2]. In addition, the immune response of Covid-19 convalescents against all major SARS-CoV-2 proteins was analyzed [ 2]. Humoral immune response analysis by high-density peptide microarrays identified immunodominant B cell epitopes as starting points for the development of alternative vaccines and diagnostic tests [ 3]. In conclusion, we show that readily available and tailored peptide formats, as exemplified for SARS-CoV-2, are important tools that contribute to the fast development and optimization of vaccines and diagnostic tests for emerging viruses of concern.

Ghrelin receptor in GtoPdb v.2023.1

The ghrelin receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee for the Ghrelin receptor [19]) is activated by a 28 amino-acid peptide originally isolated from rat stomach, where it is cleaved from a 117 amino-acid precursor (GHRL, Q9UBU3). The human gene encoding the precursor peptide has 83% sequence homology to rat prepro-ghrelin, although the mature peptides from rat and human differ by only two amino acids [75]. Alternative splicing results in the formation of a second peptide, [des-Gln14]ghrelin with equipotent biological activity [50]. A unique post-translational modification (octanoylation of Ser3, catalysed by ghrelin Ο-acyltransferase (MBOAT4, Q96T53) [134] occurs in both peptides, essential for full activity in binding to ghrelin receptors in the hypothalamus and pituitary, and for the release of growth hormone from the pituitary [59]. Structure activity studies showed the first five N-terminal amino acids to be the minimum required for binding [4], and receptor mutagenesis has indicated overlap of the ghrelin binding site with those for small molecule agonists and allosteric modulators of ghrelin function [45]. An endogenous antagonist and inverse agonist called Liver enriched antimicrobial peptide 2 (Leap2), expressed primarily in hepatocytes and in enterocytes of the proximal intestine [36, 69] inhibits ghrelin receptor-induced GH secretion and food intake [36]. The secretion of Leap2 and ghrelin is inversely regulated under various metabolic conditions [72]. In cell systems, the ghrelin receptor is constitutively active [46], but this is abolished by a naturally occurring mutation (A204E) that results in decreased cell surface receptor expression and is associated with familial short stature [94].

Microbial succession and its effect on the formation of umami peptides during sufu fermentation.

Sufu, a traditional Chinese fermented food, is famous for its unique flavor, especially umami. However, the formation mechanism of its umami peptides is still unclear. Here, we investigated the dynamic change of both umami peptides and microbial communities during sufu production. Based on peptidomic analysis, 9081 key differential peptides were identified, which mainly involved in amino acid transport and metabolism, peptidase activity and hydrolase activity. Twenty-six high-quality umami peptides with ascending trend were recognized by machine learning methods and Fuzzy c-means clustering. Then, through correlation analysis, five bacterial species (Enterococcus italicus, Leuconostoc citreum, L. mesenteroides, L. pseudomesenteroides, Tetragenococcus halophilus) and two fungi species (Cladosporium colombiae, Hannaella oryzae) were identified to be the core functional microorganisms for umami peptides formation. Functional annotation of five lactic acid bacteria indicated their important functions to be carbohydrate metabolism, amino acid metabolism and nucleotide metabolism, which proved their umami peptides production ability. Overall, our results enhanced the understanding of microbial communities and the formation mechanism of umami peptides in sufu, providing novel insights for quality control and flavor improvement of tofu products.

Novel insight into the formation and inhibition mechanism of dipeptidyl peptidase-Ⅳ inhibitory peptides from fermented mandarin fish (Chouguiyu)

Fermented foods are a potential source to produce novel dipeptidyl peptidase-IV inhibitory peptides (D4IPs). In this study, the fermented mandarin fish (Chouguiyu) was used to screen D4IPs and their formation mechanism was studied by metagenomics and peptidomics. A total of 400 D4IPs with DPP-IV inhibition structure and high hydrophobicity were identified. The correlation network map showed that Lactococcus, Bacillus, Lysobacter, Pelagivirga, Kocuria, Escherichia, Streptococcus, and Peptostreptococcus were significantly correlated with the most D4IPs. Four stable D4IPs, including KAGARALTDAETAT, GEKVDFDDIQK, VVDADEMYLKGK, and GQKDSYVGDEAQ were respectively from the precursor proteins parvalbumin, troponin, myosin, and actin, and were mainly formed by the hydrolysis of subtilisin (EC 3.4.21.62), aspartic proteinase (EC 3.4.23.1), thermolysin (EC 3.4.24.27), oligopeptidase B (EC 3.4.21.83), and proteinase P1 (EC 3.4.21.96) from Bacillus, Kocuria, Lysobacter, Lactococcus, and Peptostreptococcus. The inhibition mainly resulted from the hydrogen bond and salt bridge between D4IPs and DPP-IV enzyme. This study provides important information on the proteases and related microbial strains to directionally prepare D4IPs in Chouguiyu.

Abstract 5331: DNA-encoded macrocyclic compound libraries for challenging targets

Abstract Increasingly drug discovery programs require the development of orally available and cell permeable classes of drugs derived from the chemical space beyond Lipinski’s rule of 5 (bRO5). The chameleon-like physicochemical properties and differentiated binding modes of bRO5 compounds make them suitable starting points, especially for difficult-to-drug targets beyond traditional small molecule drug discovery. The synthesis of macrocyclic (MC) peptide DNA-Encoded Libraries (DEL) aligns with these efforts and has been an important addition to the rapidly growing field of DNA-encoded small molecule library screening. DEL screening of de novo designed bRO5 compounds will aid the exploration of bRO5 chemical space. X-Chem’s macrocyclic compound libraries apply chemistries beyond traditional peptide formation and extend to the inclusion of diverse building block classes. A case study of a successful screening campaign utilizing these MC DEL for Bcl2 will be presented. Citation Format: Ying Zhang, Anthony Keefe, Paolo Centrella, John Cuozzo, Holly Soutter, Daniel Resnicow, Sevan Habeshian, Matt Clark. DNA-encoded macrocyclic compound libraries for challenging targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5331.

Cloning and functional verification of PhAEP gene, a key enzyme for biosynthesis of heterophyllin A in Pseudostellaria heterophylla

This paper aimed to study the role of asparagine endopeptidase(AEP) gene in the biosynthesis mechanism of cyclic peptide compounds in Pseudostellaria heterophylla. The transcriptome database of P. heterophylla was systematically mined and screened, and an AEP gene, tentatively named PhAEP, was successfully cloned. The heterologous function verification by Nicotiana benthamiana showed that the expression of the gene played a role in the biosynthesis of heterophyllin A in P. heterophylla. Bioinformatics analysis showed that the cDNA of PhAEP was 1 488 bp in length, encoding 495 amino acids with a molecular weight of 54.72 kDa. The phylogenetic tree showed that the amino acid sequence encoded by PhAEP was highly similar to that of Butelase-1 in Clitoria ternatea, reaching 80%. The sequence homology and cyclase active site analysis revealed that the PhAEP enzyme may specifically hydrolyse the C-terminal Asn/Asp(Asx) site of the core peptide in the HA linear precursor peptide of P. heterophylla, thereby participating in the ring formation of the linear precursor peptide. The results of real-time quantitative polymerase chain reaction(RT-qPCR) showed that the expression level of PhAEP was the highest in fruits, followed by in roots, and the lowest in leaves. The heterophyllin A of P. heterophylla was detected in N. benthamiana that co-expressed PrePhHA and PhAEP genes instantaneously. In this study, the PhAEP gene, a key enzyme in the biosynthesis of heterophyllin A in P. heterophylla, has been successfully cloned, which lays a foundation for further analysis of the molecular mechanism of PhAEP enzyme in the biosynthesis of heterophyllin A in P. heterophylla and has important significance for the study of synthetic biology of cyclic peptide compounds in P. heterophylla.

Cyclic or Linear? Parameters Determining the Outcome of Glycine Polymerization in Silica Surface Prebiotic Scenarios.

The parameters that determine the formation of linear peptides and cyclic dimers (diketopiperazine, DKP) on silica surfaces of different surface area, silanol and siloxane ring populations, controlled by thermal treatments, are investigated upon glycine deposition from gas and liquid phases. The formed products were characterized by infrared and Raman spectroscopies, X-ray diffraction and thermogravimetric analysis. The results reveal the importance of "nearly-free" silanols to form ester centers as primers for the formation of linear peptides over DKP, on surfaces with medium silanol density (1.4 to 2.7 nm-2 ). Quenched reactivity is seen on isolated silanols (density≤0.7 nm-2 ), while silanols involved in hydrogen bonding (density of 4.5 nm-2 ) weakly interact with Gly resulting in its cyclization to DKP. Deposition of glycine from liquid phase may also form both DKP and linear polymers, depending on its loading and silica surface. These conclusions demonstrate the complexity of glycine surface chemistry in the polymerization reaction and highlight the interest of a surface science approach to evaluate geochemical prebiotic scenarios.

Identification and characterization of phage display-selected peptides having affinity to Peste des petits ruminants virus

Phage display is a well-established technique used for selecting novel ligands having affinity to a plethora of targets including proteins, viruses, whole bacterial and mammalian cells as well as lipid targets. In the present study, phage display technology was used to identify peptides having affinity to PPRV. The binding capacity of these peptides was characterized through various formats of ELISA using phage clones, linear and multiple antigenic peptides. The whole PPRV was used as an immobilized target in a surface biopanning process using a 12-mer phage display random peptide library. After five rounds of biopanning, forty colonies were picked and amplified followed by DNA isolation and amplification for sequencing. Sequencing suggested 12 different clones expressing different peptide sequence Phage-ELISA was performed using all 12 phage clones. Results indicated that four phage clones i.e., P4, P8, P9 and P12 had a specific binding activity to PPR virus. Linear peptides displayed by all 12 clones were synthesized using solid phase peptide synthesis and subjected to virus capture ELISA. No significant binding of the linear peptides with PPRV was evident which may be due to loss of conformation of linear peptide after coating. When the four selected phage clones displayed peptide sequences were synthesized in Multiple antigenic peptide (MAP) format and used in virus capture ELISA, the results indicated significant binding of PPRV to the MAPs. It may be due to increased avidity and/or better projection of binding residues in 4-armed MAPs as compared to linear peptides. MAP-peptides were also conjugated on gold nanoparticles (AuNPs). Visual colour change from wine red to purple was observed on addition of PPRV in MAP-conjugated AuNPs solution. This colour change may be attributable to the networking of PPRV with MAP –conjugated AuNPs resulting in aggregation of AuNPs. All these results supported the hypothesis that the phage display selected peptides were capable of binding to the PPRV. The potential of these peptides to develop novel diagnostic or therapeutic agents remains to be investigated.

Aggregation and crystallization of small alkanes.

We present a computer simulation study of the aggregation and ordering of short alkane chains using a united atom model description. Our simulation approach allows us to determine the density of states of our systems and, from those, their thermodynamics for all temperatures. All systems show a first order aggregation transition followed by a low-temperature ordering transition. For a few chain aggregates of intermediate lengths (up to N = 40), we show that these ordering transitions resemble the quaternary structure formation in peptides. In an earlier publication, we have already shown that single alkane chains fold into low-temperature structures, best described as secondary and tertiary structure formation, thus completing this analogy here. The aggregation transition in the thermodynamic limit can be extrapolated in pressure to the ambient pressure for which it agrees well with experimentally known boiling points of short alkanes. Similarly, the chain length dependence of the crystallization transition agrees with known experimental results for alkanes. For small aggregates, for which volume and surface effects are not yet well separated, our method allows us to identify the crystallization in the core of the aggregate and at its surface, individually.

Melatonin in the regulation of human life and its role in development pathology

The aim of our study was to research the literature data available for analysis of the melatonin role in the neurological and mental disorders origin. Materials and methods: to compile a literature review by keywords, articles there were selected and analyzed in the MEDLINE / PubMed and e-library databases from 1993 to 2021. For a detailed analysis, 42 literature sources were selected for the melatonin role in the development of neurological and mental disorders. Results: Melatonin is a hormone with unique adaptive capabilities. Violation of its production, both quantitatively and its rhythm, is the starting point, leading at the initial stages to desynchronizes, followed by the emergence of organic pathology. Consequently, the very fact of a violation of melatonin production can be the cause of various diseases. It was found that the volume of the pineal gland can change with the development of various types of neurological and psychiatric diseases, which is associated with a violation of the synthesis of melatonin and serotonin. Melatonin has been found to have important protective properties in Alzheimer's disease - the ability to inhibit the formation of beta-amyloid peptide in the brain, which is the morphological basis of this disease. The same correlation was found in patients with autism, psychosis and obsessive-compulsive disorder compared with healthy volunteers. Regarding the neuroprotective properties of melatonin, it should be noted its effectiveness in age-related diseases (Alzheimer's, Parkinson's, vascular diseases) Conclusion: Melatonin can be considered as a unique bioregulator, adaptogen and stabilizer of the whole organism and, in particular, the functions of the central nervous system. It has been proven that disruption of melatonin metabolism leads to various neurological and mental disorders.

Urea-mediated warm ponds: Prebiotic formation of carbamoyl amino acids on the primordial Earth

The emergence of prebiotic peptides on the early Earth was a crucial step for the origin of life. Carbamoyl amino acids may have played a vital role in prebiotic peptide formation. Herein, we report carbamoyl amino acids obtained with nearly 100% yield under prebiotic conditions. The reaction mechanism involves nucleophilic substitution with the release of NH3 from urea, which was verified by 15N-labeling approach. Meanwhile, experiments indicated that carbamoyl amino acids can be also well-obtained under UV radiation in the urea pool, which provides a better condition for carbamoyl amino acids to form prebiotic peptides. The study implies that peptide precursors other than classical amino acids may have been present on the primitive Earth.

Brevicillin, a novel lanthipeptide from the genusBrevibacilluswith antimicrobial, antifungal, and antiviral activity

This study was aimed to determine antimicrobial and antiviral activity of a novel lanthipeptide from a Brevibacillus sp. for disinfectant application. The antimicrobial peptide (AMP) was produced by a bacterial strain AF8 identified as a member of the genus Brevibacillus representing a novel species. Whole genome sequence analysis using BAGEL identified a putative complete biosynthetic gene cluster involved in lanthipeptide synthesis. The deduced amino acid sequence of lanthipeptide named as brevicillin, showed>30% similarity with epidermin. Mass determined by MALDI-MS and Q-TOF suggested posttranslational modifications like dehydration of all Ser and Thr amino acids to yield Dha and Dhb, respectively. Amino acid composition determined upon acid hydrolysis is in agreement with core peptide sequence deduced from the putative biosynthetic gene bvrAF8. Biochemical evidence along with stability features ascertained posttranslational modifications during formation of the core peptide. The peptide showed strong activity with 99% killing of pathogens at 12 μg ml-1 within 1 minute. Interestingly, it also showed potent anti-SARS-CoV-2 activity by inhibiting ∼99% virus growth at 10 μg ml-1 in cell culture-based assay. Brevicillin did not show dermal allergic reactions in BALB/c mice. This study provides detailed description of a novel lanthipeptide and demonstrates its effective antibacterial, antifungal and anti-SARS-CoV-2 activity.

The generation and application of antioxidant peptides derived from meat protein: a review

Meat and its products, rich in protein, are one of the important potential sources of antioxidant peptides. However, reviews on the generation and application of meat-derived antioxidant peptides are still limited. To understand the research and application progress of meat-derived antioxidant peptides, the main formation pathways and their commercial applications are exhibited, the research methods for the isolation, purification, and identification are summarized, and the influencing factors, evaluation methods, and intestinal absorption pathways are presented in this work. It is summarized that limited degradation by exogenous and endogenous enzymatic hydrolysis is the main pathway for the production of animal-derived antioxidant peptides. Traditional separation, purification, and identification techniques are also applicable to animal-derived antioxidant peptides. The formation of animal-derived antioxidant peptides is affected by many factors, and the intestinal absorption pathways of antioxidant peptides are different. Finally, insufficient and future development directions are provided.

Site-specific amino acid D-isomerization of Tau R2 and R3 peptides changes the fibril morphology, resulting in attenuation of Tau aggregation inhibitor potency

Tau, a microtubule-binding protein, is a major component of neurofibrillary tangles in the brains of Alzheimer's disease patients. Tau aggregation following fibril formation induces Alzheimer's disease pathogenesis. The accumulation of D-isomerized amino acids in proteins that occurs in several tissues with aging is thought to be implicated in age-related diseases. D-isomerized Asp accumulation has also been found in Tau in neurofibrillary tangles. We previously demonstrated the effects of D-isomerization of Asp within microtubule-binding repeat peptides of Tau, Tau R2, and R3 on the rates of structural transition and fibril formation. Here, we investigated the potency of Tau aggregation inhibitors on fibril formation of wild-type Tau R2 and R3 peptides and D-isomerized Asp-containing Tau R2 and R3 peptides. D-isomerization of Asp within Tau R2 and R3 peptides attenuated the potency of inhibitors. We next investigated the fibril morphology of D-isomerized Asp-containing Tau R2 and R3 peptides by electron microscopy. D-isomerized Asp-containing Tau R2 and R3 fibrils showed significantly different fibril morphology from that of wild-type peptides. Our results indicate that D-isomerization of Asp within Tau R2 and R3 peptides affects fibril morphology, resulting in attenuation of the potency of Tau aggregation inhibitors.

Escherichia coli YgiC and YjfC Possess Peptide─Spermidine Ligase Activity.

Polyamines and polyamine-containing metabolites are involved in many cellular processes related to bacterial cell growth and survival. In Escherichia coli, the bifunctional enzyme glutathionylspermidine synthetase/amidase (GspSA) controls the production of glutathionylspermidine, which has a protective role against oxidative stress. E. coli also encodes two enzymes with homology to the synthetase domain of GspSA, YgiC, and YjfC; however, these do not catalyze the formation of glutathionylspermidine, and their catalytic function remained unknown. Here, we detail the structural and functional characterization of YgiC and YjfC. Using X-ray crystallography, the high-resolution crystal structures of YgiC and YjfC were obtained. This revealed that YgiC and YjfC possess multiple substitutions in key residues required for binding of glutathione in GspSA. Despite this difference, these enzymes share a similar active site structure to GspSA, suggesting that they catalyze the formation of an alternate peptide─spermidine conjugate. As the physiological substrates of YgiC and YjfC are unknown, this was probed using the peptide triglycine as a model substrate. A combination of enzyme activity assays and mass spectrometry revealed that YgiC and YjfC can function as peptide-spermidine ligases, forming a triglycine-spermidine conjugate. For both enzymes, conjugate formation was only observed in the presence of spermidine, but not other common polyamines, supporting that spermidine or a spermidine derivative is the physiological substrate. Importantly, since YgiC and YjfC are widely distributed in Gram-negative bacterial species, this suggests that these enzymes function in a conserved cellular process, representing a currently unknown aspect of bacterial polyamine metabolism.

Jack Bean (Canavalia ensiformis) Tempeh: ACE-Inhibitory Peptide Formation during Absorption in the Small Intestine.

High blood pressure is the most significant cause of mortality globally. Some fermented foods include ACE-inhibitory peptides that help fight this disease. The ability of fermented jack bean (tempeh) to inhibit ACE during consumption has not been demonstrated yet. This study identified and characterised ACE-inhibitory peptides from jack bean tempeh produced by small intestine absorption using the everted intestinal sac model. Sequentially, the protein extract of jack bean tempeh and unfermented jack bean was hydrolysed using pepsin-pancreatin for 240 min. The hydrolysed samples were then evaluated for the peptide absorption using three-segmented everted intestinal sacs (duodenum, jejunum and ileum). The peptides absorbed from all intestinal segments were mixed in the small intestine. The data showed that both jack bean tempeh and unfermented jack bean had the same peptide absorption pattern, with the highest percentage of peptide absorption in the jejunum, followed by the duodenum and ileum. The absorbed peptides of jack bean tempeh exhibited equally strong activity of ACE inhibition in all intestinal segments, while the unfermented jack bean showed strong activity only in the jejunum. The mixture of the peptides from jack bean tempeh absorbed in the small intestine had higher ACE-inhibitory activity (81.09%) than the unfermented jack bean (72.22%). The peptides produced from jack bean tempeh were identified as pro-drug ACE inhibitors and had the mixed inhibition pattern. The mixture of peptides consisted of seven types of peptides with a molecular mass of 826.86-978.20 Da (DLGKAPIN, GKGRFVYG, PFMRWR, DKDHAEI, LAHLYEPS, KIKHPEVK, and LLRDTCK). This study discovered that consuming jack bean tempeh generated more potent ACE-inhibitory peptides during small intestine absorption than cooked jack beans. Absorbed tempeh peptides have high ACE-inhibitory activity.