The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response.

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation.

Abatement of the binding of human hexokinase II enzyme monomers by in-silico method with the design of inhibitory peptides.

The hexokinase II enzyme is bound to the (VDAC1) channel in the form of a dimer and prevents the release of cell death factors from mitochondria to the cytoplasm. Studies have shown that blocking the binding of hexokinase II enzyme to (VDAC1) led to the initiation of apoptosis in cancer cells. No peptide has been designed so far to inhibit hexokinase II. The aim of this study was to inhibit the dimerization of enzyme subunits in order to inhibition the formation of (VDAC1) and the hexokinase II complex. In this study, the molecular dynamics simulation of the enzyme in monomer and dimer states was investigated in terms of RMSF, RMSD and radius of gyration. The following process involves extracting and designing variable-length peptides from the interacting segments of enzyme monomers. Using molecular dynamics simulation, the stability of the peptide was determined in terms of RMSD. Molecular docking was used to investigate the interaction between the designed peptides. Finally, the inhibitory effect of peptides on subunit association was measured using dynamic light scattering (DLS) technique. Our results showed that the designed peptides, which mimic common amino acids in dimerization, interrupt the bona fide form of the enzyme subunits. The result of this study provides a new way to disrupt the assembly process and thereby decreased the function of the hexokinase II. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00201-8.

Supramolecular Peptide Self-Assemblies Facilitate Oral Immunization.

Oral immunization is a promising strategy for preventing and treating gastrointestinal (GI) infections and diseases, as it allows for direct access to the disease site. To elicit immune responses within the GI tract, however, there are many obstacles that oral vaccines must surmount, including proteolytic degradation and thick mucus barriers. Here, we employed a modular self-assembling peptide nanofiber platform to facilitate oral immunization against both peptide and small molecule epitopes. Synthesizing nanofibers with d-amino acids rendered them resistant to proteases in vitro, whereas l-amino acid nanofibers were rapidly degraded. Additionally, the inclusion of peptide sequences rich in proline, alanine, and serine (PAS), increased nanofiber muco-penetration, and accelerated nanofiber transport through the GI tract. Oral immunization with PASylated nanofibers and mucosal adjuvant generated local and systemic immune responses to a peptide epitope but only for l-amino acid nanofibers. Further, we were able to apply this design to also enable oral immunization against a small molecule epitope and illustrated the therapeutic and prophylactic effectiveness of these immunizations in mouse models of colitis. These findings demonstrate that supramolecular peptide self-assemblies have promise as oral vaccines and immunotherapies.

Genetically encoded libraries and spider venoms as emerging sources for crop protective peptides.

Agricultural crops are targeted by various pathogens (fungi, bacteria, and viruses) and pests (herbivorous arthropods). Antimicrobial and insecticidal peptides are increasingly recognized as eco-friendly tools for crop protection due to their low propensity for resistance development and the fact that they are fully biodegradable. However, historical challenges have hindered their development, including poor stability, limited availability, reproducibility issues, high production costs, and unwanted toxicity. Toxicity is a primary concern because crop-protective peptides interact with various organisms of environmental and economic significance. This review focuses on the potential of genetically encoded peptide libraries like the use of two-hybrid-based methods for antimicrobial peptides identification and insecticidal spider venom peptides as two main approaches for targeting plant pathogens and pests. We discuss some key findings and challenges regarding the practical application of each strategy. We conclude that genetically encoded peptide library- and spider venom-derived crop protective peptides offer a sustainable and environmentally responsible approach for addressing modern crop protection needs in the agricultural sector.

Application of experimental design as a statistical approach to recover bioactive peptides from different food sources.

Bioactive peptides (BAPs) derived from samples of animals and plants have been widely recommended and consumed for their beneficial properties to human health and to control several diseases. This work presents the applications of experimental designs (DoE) used to perform factor screening and/or optimization focused on finding the ideal hydrolysis condition to obtain BAPs with specific biological activities. The collection and discussion of articles revealed that Box Behnken Desing and Central Composite Design were the most used. The main parameters evaluated were pH, time, temperature and enzyme/substrate ratio. Among vegetable protein sources, soy was the most used in the generation of BAPs, and among animal proteins, milk and shrimp stood out as the most explored sources. The degree of hydrolysis and antioxidant activity were the most investigated responses in obtaining BAPs. This review brings new information that helps researchers apply these DoE to obtain high-quality BAPs with the desired biological activities.

The role of the aryl hydrocarbon receptor (AhR) in modulating intestinal ILC3s to optimise gut pathogen resistance in lupus and benefits of nutritional AhR ligands.

BACKGROUND AND AIMS: Dysbiosis is emerging as a potential trigger of systemic lupus erythematosus (SLE). Group 3 innate lymphoid cells (ILC3s) are recognised as key regulators of intestinal homeostasis. The aryl hydrocarbon receptor (AhR) is critical to intestinal ILC3 development and function. This mechanistic review aimed to investigate whether AhR activation of gut ILC3s facilitates IL-22-mediated antimicrobial peptide (AMP) production to enhance colonisation resistance and ameliorate SLE pathology associated with intestinal dysbiosis. Furthermore, nutritional AhR ligand potential to enhance pathogen resistance was explored. METHODOLOGY: This mechanistic review involved a three-tranche systematic literature search (review, mechanism, intervention) using PubMed with critical appraisal. Data was synthesised into themes and summarised in a narrative analysis. RESULTS: Preclinical mechanistic data indicate that AhR modulation of intestinal ILC3s optimises pathogen resistance via IL-22-derived AMPs. Pre-clinical research is required to validate this mechanism in SLE. Data on systemic immune consequences of AhR modulation in lupus suggest UVB-activated ligands induce aberrant AhR signalling while many dietary ligands exert beneficial effects. Data on xenobiotic-origin ligands is varied, although considerable evidence has demonstrated negative effects on Th17 to Treg balance. Limited human evidence supports the role of nutritional AhR ligands in modulating SLE pathology. Preclinical and clinical data support anti-inflammatory effects of dietary AhR ligands. CONCLUSION: Current evidence is insufficient to fully validate the hypothesis that AhR modulation of intestinal ILC3s can enhance pathogen resistance to ameliorate lupus pathology driven by dysbiosis. However, anti-inflammatory effects of dietary AhR ligands suggest a promising role as a therapeutic intervention for SLE.

Identification and molecular mechanisms of novel antioxidant peptides from fermented broad bean paste: A combined in silico and in vitro study.

This study aimed to investigate the antioxidative and cytoprotective activity of antioxidant peptides from fermented broad bean paste (FBBP) and explore their potential molecular mechanisms using a combined in silico and in vitro approach. Seven novel antioxidant peptides (VSRRFIYYL, SPAIPLP, PVPPPGG, KKDGYWWAKFK, LAWY, LGFMQF, and LPGCP) identified by integrated approaches of peptidomics and in silico bioinformatic analysis were synthesized, exhibiting strong antioxidant potential against in vitro radicals. Molecular docking results suggested that these peptides could form stable hydrogen bonds and solvent-accessible surface with key amino acid residues of Keap1, thus potentially regulating the Keap1-Nrf2 pathway by occupying the Nrf2-binding site on Keap1. Additionally, they exhibited strong cellular antioxidant activity and could protect HepG2 cells from AAPH-induced oxidative injury by reducing reactive oxygen species and MDA accumulation. This study firstly unraveled the molecular mechanisms of antioxidant peptides from FBBP, and provided a new theoretical basis for the high-value utilization of FBBP.

Skin barrier-inflammatory pathway is a driver of the psoriasis-atopic dermatitis transition.

As chronic inflammatory conditions driven by immune dysregulation are influenced by genetics and environment factors, psoriasis and atopic dermatitis (AD) have traditionally been considered to be distinct diseases characterized by different T cell responses. Psoriasis, associated with type 17 helper T (Th17)-mediated inflammation, presents as well-defined scaly plaques with minimal pruritus. AD, primarily linked to Th2-mediated inflammation, presents with poorly defined erythema, dry skin, and intense itching. However, psoriasis and AD may overlap or transition into one another spontaneously, independent of biological agent usage. Emerging evidence suggests that defects in skin barrier-related molecules interact with the polarization of T cells, which forms a skin barrier-inflammatory loop with them. This loop contributes to the chronicity of the primary disease or the transition between psoriasis and AD. This review aimed to elucidate the mechanisms underlying skin barrier defects in driving the overlap between psoriasis and AD. In this review, the importance of repairing the skin barrier was underscored, and the significance of tailoring biologic treatments based on individual immune status instead of solely adhering to the treatment guidelines for AD or psoriasis was emphasized.

Total Chemical Synthesis of Aggregation-Prone Disulfide-Rich Starfish Peptides.

: A relaxin-like gonad-stimulating peptide (RGP), Aso-RGP, featuring six cysteine residues, was identified in the Crown-of-Thorns Starfish (COTS, Acanthaster cf. solaris) and initially produced through recombinant yeast expression. This method yielded a single-chain peptide with an uncleaved C-peptide (His Tag) and suboptimal purity. Our objective was to chemically synthesize Aso-RGP in its mature form, comprising two chains (A and B) and three disulfide bridges, omitting the C-peptide. Furthermore, we aimed to synthesize a newly identified relaxin-like peptide, Aso-RLP2, from COTS, which had not been previously synthesized. This paper report the first total chemical synthesis of Aso-RGP and Aso-RLP2. Aso-RGP synthesis proceeded without major issues, whereas the A-chain of Aso-RLP2, in its reduced and unfolded state with two free thiols, presented considerable challenges. These were initially marked by "messy" RP-HPLC profiles, typically indicative of synthesis failure. Surprisingly, oxidizing the A-chain significantly improved the RP-HPLC profile, revealing the main issue was not synthesis failure but the peptide's aggregation tendency, which initially obscured analysis. This discovery highlights the critical need to account for aggregation in peptide synthesis and analysis. Ultimately, our efforts led to the successful synthesis of both peptides with purities exceeding 95%.

Extracellular Microvesicles Modified with Arginine-Rich Peptides for Active Macropinocytosis Induction and Delivery of Therapeutic Molecules.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 µm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.

The predominant Quillaja Saponaria fraction, QS-18, is safe and effective when formulated in a liposomal murine cancer peptide vaccine.

Extracts of the Chilean soapbark tree, Quillaja Saponaria (QS) are the source of potent immune-stimulatory saponin compounds. This study compared the adjuvanticity and toxicity of QS-18 and QS-21, assessing the potential to substitute QS-18 in place of QS-21 for vaccine development. QS-18, the most abundant QS saponin fraction, has been largely overlooked due to safety concerns. We found that QS-18 spontaneously inserted into liposomes, thereby neutralizing hemolytic activity, and following administration did not induce local reactogenicity in a footpad swelling test in mice. With high-dose intramuscular administration, transient weight loss was minor, and QS-18 did not induce significantly more weight loss compared to a liposome vaccine adjuvant system lacking it. Two days after administration, no elevation of inflammatory cytokines was detected in murine serum. In a formulation including cobalt-porphyrin-phospholipid (CoPoP) for short peptide sequestration, QS-18 did not impact the formation of peptide nanoparticles. With immunization, QS-18 peptide particles induced higher levels of cancer neoepitope-specific and tumor-associated antigen-specific CD8+ T cells compared to QS-21 particles, without indication of greater toxicity based on mouse body weight. T cell receptor sequencing of antigen-specific CD8+ T cells showed that QS-18 induced significantly more T cell transcripts. In two murine cancer models, vaccination with QS-18 peptide particles induced a similar therapeutic effect as QS-21 particles, without indication of increased toxicity. Antigen-specific CD8+ T cells in the tumor microenvironment were found to express the exhaustion marker PD-1, pointing to the rationale for exploring combination therapy. Taken together, these data demonstrate that QS-18, when formulated in liposomes, can be a safe and effective adjuvant to induce tumor-inhibiting cellular responses in murine models with potential to facilitate or diminish costs of production for vaccine adjuvant systems. Further studies are warranted to assess liposomal QS-18 immunogic, reactogenic and toxicological profiles in mice and other animal species.

Nanohydroxyapatite/Peptide Composite Coatings on Pure Titanium Surfaces with Nanonetwork Structures Using Oyster Shells.

Titanium and its alloys are extensively applied in artificial tooth roots because of their excellent corrosion resistance, high specific strength, and low elastic modulus. However, because of their biological inertness, their surface needs to be modified to improve the osteointegration of titanium implants. The preparation of biologically active calcium-phosphorus coatings on the surface of an implant is one effective method for enhancing the likelihood of bone integration. In this study, osteoinductive peptides were extracted from oyster shells by using acetic acid. Two peptide-containing hydroxyapatite (HA) composite coatings were then prepared: one coating was prepared by hydrothermally synthesizing an HA coating in the presence of peptides (HA/P/M), and the other coating was prepared by hydrothermally synthesizing HA and then immersing the hydrothermally synthesized HA in a peptide solution (HA/P/S). Characterization results indicated that the composite HA coatings containing oyster shell-based peptides were successfully prepared on the alkali-treated pure titanium surfaces. The HA/P/M and HA/P/S composite coatings were found to exhibit excellent hydrophilicity. Protein adsorption tests confirmed that the HA/P/M and HA/P/S coatings had an approximately 2.3 times higher concentration of adsorbed proteins than the pure HA coating.

SARS-CoV-2 Spike Protein-Derived Cyclic Peptides as Modulators of Spike Interaction with GRP78.

The human glucose-regulated protein GRP78 is a human chaperone that translocactes to the cell surface when cells are under stress. Theoretical studies suggested it could be involved in SARS-CoV-2 virus entry to cells. In this work, we used in vitro surface plasmon resonance-based assays to show that human GRP78 indeed binds to SARS-CoV-2 spike protein. We have designed and synthesised cyclic peptides based on the loop structure of amino acids 480-488 of the SARS-CoV-2 spike protein S1 domain from the Wuhan and Omicron variants and showed that both peptides bind to GRP78. Consistent with the greater infectiousness of the Omicron variant, the Omicron-derived peptide displays slower dissociation from the target protein. Both peptides significantly inhibit the binding of wild-type S1 protein to the human protein GRP78 suggesting that further development of these cyclic peptide motifs may provide a viable route to novel anti-SARS-CoV-2 agents.

Stack-AAgP: Computational prediction and interpretation of anti-angiogenic peptides using a meta-learning framework.

BACKGROUND: Angiogenesis plays a vital role in the pathogenesis of several human diseases, particularly in the case of solid tumors. In the realm of cancer treatment, recent investigations into peptides with anti-angiogenic properties have yielded encouraging outcomes, thereby creating a hopeful therapeutic avenue for the treatment of cancer. Therefore, correctly identifying the anti-angiogenic peptides is extremely important in comprehending their biophysical and biochemical traits, laying the groundwork for uncovering novel drugs to combat cancer. METHODS: In this work, we present a novel ensemble-learning-based model, Stack-AAgP, specifically designed for the accurate identification and interpretation of anti-angiogenic peptides (AAPs). Initially, a feature representation approach is employed, generating 24 baseline models through six machine learning algorithms (random forest [RF], extra tree classifier [ETC], extreme gradient boosting [XGB], light gradient boosting machine [LGBM], CatBoost, and SVM) and four feature encodings (pseudo-amino acid composition [PAAC], amphiphilic pseudo-amino acid composition [APAAC], composition of k-spaced amino acid pairs [CKSAAP], and quasi-sequence-order [QSOrder]). Subsequently, the output (predicted probabilities) from 24 baseline models was inputted into the same six machine-learning classifiers to generate their respective meta-classifiers. Finally, the meta-classifiers were stacked together using the ensemble-learning framework to construct the final predictive model. RESULTS: Findings from the independent test demonstrate that Stack-AAgP outperforms the state-of-the-art methods by a considerable margin. Systematic experiments were conducted to assess the influence of hyperparameters on the proposed model. Our model, Stack-AAgP, was evaluated on the independent NT15 dataset, revealing superiority over existing predictors with an accuracy improvement ranging from 5% to 7.5% and an increase in Matthews Correlation Coefficient (MCC) from 7.2% to 12.2%.

The use of a selective, nontoxic dual-acting peptide for breast cancer patients with brain metastasis.

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the absence of commonly targeted receptors. Unspecific chemotherapy is currently the main therapeutic option, with poor results. Another major challenge is the frequent appearance of brain metastasis (BM) associated with a significant decrease in patient overall survival. The treatment of BM is even more challenging due to the presence of the blood-brain barrier (BBB). Here, we present a dual-acting peptide (PepH3-vCPP2319) designed to tackle TNBC/BM, in which a TNBC-specific anticancer peptide (ACP) motif (vCPP2319) is joined to a BBB peptide shuttle (BBBpS) motif (PepH3). PepH3-vCPP2319 demonstrated selectivity and efficiency in eliminating TNBC both in monolayers (IC50≈5.0 µM) and in spheroids (IC50≈25.0 µM), with no stringent toxicity toward noncancerous cell lines and red blood cells (RBCs). PepH3-vCPP2319 was also able to cross the BBB in vitro and penetrate the brain in vivo, and was stable in serum with a half-life above 120 min. Tumor cell-peptide interaction is fast, with quick peptide internalization via clathrin-mediated endocytosis without membrane disruption. Upon internalization, the peptide is detected in the nucleus and the cytoplasm, indicating a multi-targeted mechanism of action that ultimately induces irreversible cell damage and apoptosis. In conclusion, we have designed a dual-acting peptide capable of brain penetration and TNBC cell elimination, thus expanding the drug arsenal to fight this BC subtype and its BM.

The 12-Membered TNFR1 Peptide, as Well as the 16-Membered and 6-Membered TNF Peptides, Regulate TNFR1-Dependent Cytotoxic Activity of TNF.

Understanding the exact mechanisms of the activation of proinflammatory immune response receptors is very important for the targeted regulation of their functioning. In this work, we were able to identify the sites of the molecules in the proinflammatory cytokine TNF (tumor necrosis factor) and its TNFR1 (tumor necrosis factor receptor 1), which are necessary for the two-stage cytotoxic signal transduction required for tumor cell killing. A 12-membered TNFR1 peptide was identified and synthesized, interacting with the ligands of this receptor protein's TNF and Tag7 and blocking their binding to the receptor. Two TNF cytokine peptides interacting with different sites of TNFR1 receptors were identified and synthesized. It has been demonstrated that the long 16-membered TNF peptide interferes with the binding of TNFR1 ligands to this receptor, and the short 6-membered peptide interacts with the receptor site necessary for the transmission of a cytotoxic signal into the cell after the ligands' interaction with the binding site. This study may help in the development of therapeutic approaches to regulate the activity of the cytokine TNF.

Olive Pomace Extract Contains Low Molecular Weight Peptides and Possesses ACE Inhibitory Activity.

The aim of the present study was to determine the ACE inhibitory activity of aqueous extracts of olive pomace and to understand whether they represent a good source of bioactive LMW peptides for nutritional and pharmacological applications. We produced a water extract from olive pomace (var. Picual) and obtained its low molecular weight (LMW) fraction (<3 kDa). The calculated yield of extraction was 100.2 ± 7.9 mg of LMW peptides per 100 g of olive pomace. The olive pomace LMW fraction possessed strong ACE inhibitory activity (IC50 = 3.57 ± 0.22 µg prot/mL). The LMW fraction (<3 kDa) was analysed by nanoscale liquid chromatography-Orbitrap coupled with tandem mass spectrometry and de novo sequencing. Thirty new peptides, containing between 7-17 amino acids and molecular masses ranging 778-1354 Da, were identified by the Peaks database algorithm using the available Olea europaea (cv. Farga) genome database. Ten new peptides were also identified by Peaks de novo sequencing. The protein sources of twelve peptides detected in the database by Peaks DB were identified by BLAST search. The ACE inhibitory activity of the identified peptides was predicted by BIOPEP software. We conclude that olive pomace possesses ACE inhibitory activity and contains low molecular weight peptides with (predicted) biological activity. Olive pomace may represent a good source of peptides for nutritional and pharmaceutical applications. In our study, it has been shown that olive pomace possesses ACE inhibitory activity and contains low molecular weight peptides with (predicted) biological activity. Olive pomace may represent a good source of peptides for nutritional and pharmaceutical applications. More research is needed in order to identify the in vivo effects of olive pomace bioactive peptides.

Research Progress of Small Plant Peptides on the Regulation of Plant Growth, Development, and Abiotic Stress.

Small peptides in plants are typically characterized as being shorter than 120 amino acids, with their biologically active variants comprising fewer than 20 amino acids. These peptides are instrumental in regulating plant growth, development, and physiological processes, even at minimal concentrations. They play a critical role in long-distance signal transduction within plants and act as primary responders to a range of stress conditions, including salinity, alkalinity, drought, high temperatures, and cold. This review highlights the crucial roles of various small peptides in plant growth and development, plant resistance to abiotic stress, and their involvement in long-distance transport. Furthermore, it elaborates their roles in the regulation of plant hormone biosynthesis. Special emphasis is given to the functions and mechanisms of small peptides in plants responding to abiotic stress conditions, aiming to provide valuable insights for researchers working on the comprehensive study and practical application of small peptides.

Peptide-Based Recognition Agents of Histamine: A Biopanning Approach with Enhanced Specificity.

Histamine is a biogenic amine that poses a potential threat to public health due to its toxicological effects. In this study, we identified histamine-binding peptides by screening a random 12-mer peptide library, employing a novel biopanning approach that excluded histidine-binding sequences in the final round. This additional step enhanced the selectivity of the peptides and prevented interference from histidine during detection. The binding affinities of synthesized peptides to histamine were assessed using isothermal titration calorimetry (ITC). Among the identified peptides, HBF10 (SGFRDGIEDFLW) and HBF26 (IPLENQHKIYST) showed significant affinity to histamine, with Kavalues of 2.56×104 (M-1) and 8.94×104 (M-1), respectively. Notably, the identified peptides did not demonstrate binding affinity towards histidine, despite its structural similarity to histamine. Subsequently, the surface plasmon resonance (SPR) sensor surface was prepared by immobilizing the peptide HBF26 to investigate the potential of the peptide as a recognition agent for histamine detection. The findings suggest that the identified peptides have an affinity to histamine specifically, showcasing their potential applications as diagnostic agents with specific targeting capabilities.

Characterization and Hydrolysis Mechanism Analysis of a Cold-Adapted Trypsin-Like Protease from Antarctic Krill.

Cold-adapted proteases are capable of efficient protein hydrolysis at reduced temperatures, which offer significant potential applications in the area of low temperature food processing. In this paper, we attempted to characterize cold-adapted proteases from Antarctic krill. Antarctic krill possesses an extremely active autolytic enzyme system in their bodies, and the production of peptides and free amino acids accompanies the rapid breakdown of muscle proteins following the death. The crucial role of trypsin in this process is recognized. A cold-adapted trypsin named OUC-Pp-20 from Antarctic krill genome was cloned and expressed in Pichia pastoris. Recombinant trypsin is a monomeric protein of 26.8 ± 1.0 kDa with optimum reaction temperature at 25 °C. In addition, the catalytic specificity of OUC-Pp-20 was assessed by identifying its hydrolysis sites through LC-MS/MS. OUC-Pp-20 appeared to prefer Gln and Asn at the P1 position, which is an amino acid with an amide group in its side chain. Hydrolysis reactions on milk and shrimp meat revealed that it can effectively degrade allergenic components in milk and arginine kinase in shrimp meat. These findings update the current knowledge of cold-adapted trypsin and demonstrate the potential application of OUC-Pp-20 in low temperature food processing.