Cathepsin G Research Articles

Antioxidant Peptides from Hizikia fusiformis: A Study of the Preparation, Identification, Molecular Docking, and Cytoprotective Function of H2O2-Damaged A549 Cells by Regulating the Keap1/Nrf2 Pathway

Hijiki (Hizikia fusiformis) is a seaweed native to warm-temperate and subtropical regions that has a high edible value and economic value, with a production of about 2 × 105 tons/year. Current research has clearly shown that the pharmacological activities of active ingredients from hijiki have covered a broad spectrum of areas, including antioxidant, hypoglycemic, antiviral, anticoagulant, anti-inflammatory, intestinal flora modulation, anti-aging, antineoplastic and antibacterial, and anti-Alzheimer’s disease areas. However, no studies have reported on the production of antioxidant peptides from hijiki proteins. The objectives of this study were to optimize the preparation process and explore the cytoprotective function and mechanisms of antioxidant peptides from hijiki protein. The results indicated that papain is more suitable for hydrolyzing hijiki protein than pepsin, trypsin, alkaline protease, and neutral protease. Under the optimized parameters of an enzyme dosage of 3%, a material–liquid ratio of 1:30, and an enzyme digestion time of 5 h, hijiki hydrolysate with a high radical scavenging activity was generated. Using ultrafiltration and serial chromatographic methods, ten antioxidant oligopeptides were purified from the papain-prepared hydrolysate and identified as DGPD, TIPEE, TYRPG, YTPAP, MPW, YPSKPT, YGALT, YTLLQ, FGYGP, and FGYPA with molecular weights of 402.35, 587.61, 592.64, 547.60, 532.53, 691.77, 523.57, 636.73, 539.58, and 553.60 Da, respectively. Among them, tripeptide MPW could regulate the Keap1/Nrf2 pathway to significantly ameliorate H2O2-induced oxidative damage of A549 cells by increasing cell viability and antioxidant enzyme (SOD, CAT, and GSH-Px) activity, decreasing ROS and MDA levels, and reducing the apoptosis rate. Molecular docking experiments show that HFP5 (MPW) exerts its inhibitory effect mainly through hydrogen bonds and hydrophobic interactions with the Kelch domain of the Keap1 protein, eventually facilitating the translocation of Nrf2 to the nucleus. Therefore, antioxidant peptides from hijiki can be applied to develop algae-derived health foods for treating diseases associated with oxidative stress.

Novel Insights Into Peptide-Calcium Chelates From Lentinula edodes: Preparation and Its Structure, Stability, and Calcium Transport Analysis.

Peptide-Ca chelates are innovative calcium supplements. Lentinula edodes possesses nutritional advantages for preparing calcium-binding peptides (CBPs), although there are limited studies on this subject. Therefore, this paper investigated the optimal condition for preparing Lentinula edodes CBPs and Lentinula edodes peptide-calcium chelates (LP-Ca), along with analyzing their microstructure, calcium-binding mechanisms, stability, and calcium transporting efficacy. The optimal protease and hydrolysis time for preparing CBPs were neutral protease and 3 h, respectively. The optimized parameters for LP-Ca preparation were as follows: pH9, time 50 min, mass ratio of peptide/CaCl2 5:1, and temperature 65°C. The chelates contain 4.23% ± 0.01% Ca. After chelation, Glu, Asp, Lys, Ser, His, and Cys were enriched. LP-Ca possessed a rough and porous structure, exhibiting a pronounced calcium signal. -COO-, C=O, and N-H groups were contributed to the chelation, with calcium primarily existing in an amorphous form. LP-Ca exhibited enhanced thermal stability and retained most of the calcium (62.33% ± 4.51%) after digestion, and calcium transportation was enhanced in the LP-Ca group (9.57 ± 0.60 μg). Collectively, LP-Ca are studied for the first time and the study is of great significance for developing novel calcium supplements.

A novel digestive protease chymotrypsin-like serine contributes to anti-BmNPV activity in silkworm (Bombyxmori).

Serine proteases (SPs) are important proteases in the digestive system of lepidopteran insects. They play important roles in protein digestion, coagulation, signal transduction, hormone activation, inflammation and development. Blood-borne pyosis caused by Bombyx mori nuclear polyhedrosis virus (BmNPV) has caused serious harm to sericulture. At present, the scientific problems of BmNPV infection and silkworm resistance to BmNPV infection have been the focus of many scientists, but the molecular mechanism needs further research and exploration. Based on the results of label-free quantitative protein proteomics of the midgut digestive juice of different resistant strains in our laboratory, we successfully screened a differentially expressed candidate protein (DEP), B. mori chymotrypsin-like serine protease (BmCLSP), and comprehensively analyzed the biological characteristics and anti-BmNPV function of BmCLSP. The open reading frame (ORF) of BmCLSP is 891 bp, encoding 296 amino acid residues. The analysis of the domain structure showed that there was a signal peptide and a trypsin-like serine protease domain, Tryp_SPC, in the BmCLSP protein. Semi-quantitative and real-time fluorescence quantitative PCR analysis showed that the BmCLSP gene was highly expressed in the fifth instar larvae of silkworm, and specifically expressed in the midgut. The expression level of BmCLSP in the BmNPV resistant strain A35 was higher than that in the sensitive strain P50. Virus amplification analysis showed that the relative expression level of VP39 was significantly lower than that of the control group after infection of silkworm larvae and BmN cells with BmNPV treated with recombinant BmCLSP at an appropriate concentration. Furthermore, our overexpression of BmCLSP in BmN cells significantly inhibited the expansion of BmNPV. In summary, the results of this study indicate that BmCLSP has anti-BmNPV activity in silkworm, and can significantly inhibit the proliferation of BmNPV in silkworm. It offers a promising avenue for silkworm anti-virus breeding.

CTSG is a prognostic marker involved in immune infiltration and inhibits tumor progression though the MAPK signaling pathway in non-small cell lung cancer

PurposeThis study aims to investigate the biological roles and molecular mechanisms of Cathepsin G (CTSG) in the progression of non-small cell lung cancer (NSCLC).MethodsWestern blotting and immunohistochemistry analyses of clinical samples were performed to determine the expression levels of CTSG in patients with NSCLC. Bioinformatic analysis of clinical datasets was conducted to evaluate the correlation between CTSG and lymph node metastasis, tumor stage, and immune cell infiltration. Gain-of-function assays and tumor implantation experiments were employed to determine the effects of CTSG on malignant behaviors of NSCLC cells. Transcriptome sequencing and subsequent bioinformatic analysis were performed to explore the signaling pathways regulated by CTSG. Western blotting and qPCR were utilized to assess the influence of CTSG on the MAPK and EMT signaling pathways.ResultsCTSG is expressed at low levels and serves as a prognostic marker in NSCLC. The downregulation of CTSG expression was associated with lymph node metastasis, tumor stage, and immune cell infiltration. CTSG inhibits NSCLC cell proliferation, migration, and invasion as well as tumor growth in nude mice. There exists a significant correlation between CTSG expression and endoplasmic reticulum function, cell cycling, and the IL-17 signaling pathway. CTSG suppresses the MAPK and EMT signaling pathways in NSCLC cells. Moreover, DNA methylation and histone deacetylation have been identified as crucial mechanisms contributing to the decreased expression of CTSG.ConclusionCTSG inhibits NSCLC development by suppressing the MAPK signaling pathway and is also associated with tumor immunity.

Enhancing Digestibility and Intestinal Peptide Release of Pleurotus eryngii Protein: An Enzymatic Approach.

Pleurotus eryngii is a tasty and low-calorie mushroom containing abundant high-quality protein. This study aims to improve the digestibility of P. eryngii protein (PEP) and hence to facilitate its development as a healthy alternative protein. The extracted PEP was pretreated with 1000-5000 U of papain, neutral protease and alkaline protease. The Chyme collected from in vitro simulated gastrointestinal digestion was analyzed by fluorescence microscopy and protein particle analyzer, and the endpoint profiles of peptides and amino acids were determined by UHPLC-MS/MS and NanoLC-MS/MS. The particle size curve and fluorescence microscopy images jointly supported that protease hydrolysis improved decomposition and dispersion of PEP during digestion, particularly in the gastric phase. The impact on Zeta potential was minimal. Proteases effectively increased the abundance of amino acids after digestion, particularly L-isomer Lys and Arg Maximum release was achieved when pretreated with 5000 U of alkaline protease, reaching 7.54 times that of control. Pretreatments by proteases also notably increased digestive yields of 16,736-19,870 peptides, with the maximum reaching 1.70 times that of the control, which mainly consisted of small peptides composed of 7-15 amino acids with molecular weight below 800 Da. The findings indicated that protease hydrolysis, especially pretreatment with 5000 U of alkaline protease, effectively enhanced the digestibility of PEP, which shed light on providing enzymatic approaches for improving bioavailability and developing healthy fungal proteins.

Preparation and Characterization of Calcium-Chelated Sea Cucumber Ovum Hydrolysate and the Inhibitory Effect on α-Amylase.

α-amylase can effectively inhibit the activity of digestive enzymes and alter nutrient absorption. The impact of ovum hydrolysates of sea cucumbers on α-amylase activity was investigated in this study. The protein hydrolysates generated using different proteases (pepsin, trypsin, and neutral protease) and molecular weights (less than 3000 and more than 3000) were investigated. The results showed that all three different hydrolysates demonstrated calcium-chelating activity and induced a fluorescence-quenching effect on α-amylase. The sea cucumber ovum hydrolysate with a molecular weight of less than 3000 Da, isolated using trypsin, showed the most effective inhibitory effect on α-amylase, with an inhibition rate of 53.9%, and the inhibition type was identified as mixed forms of inhibition. In conclusion, the generation and utilization of protein hydrolysates from sea cucumber ovum as a functional food ingredient could be a potential approach to add value to low-cost seafood by-products.

Anticoagulant Activity of the Polysaccharide Fromgonad of Abalone Haliotis discus hannai Ino: The Role of Conjugate Protein.

Few studies are concerned with the effect of the conjugat protein on the bioactivities of the abalone gonad polysaccharide (AGP). In this study, a series of treatments, including raw material (female and male) defatting, extraction temperature (25-121 °C), proteolysis, ultrafiltration, and ethanol precipitation, was conducted to investigate the role of the conjugate protein on AGP anticoagulant activity. All AGP extracts significantly prolonged activated partial thromboplastin time (APTT) and thrombin time (TT). The strongest was observed in the female AGPs prepared at 50 and 121 °C. The most active is located at 30-300 kDa by ultrafiltration. After being exposed to neutral protease, quick shortening of APTT and TT was found in all AGPs. Further ethanol precipitating of found the longest APTT in the sediment, which contains most polysaccharides and proteins. Defatting lowered the activity of female AGP but increased that of males. Proteolysis also significantly weakened the clotting factor inhibition effect of the 50 °C female AGP, but heating seemed not affect the effect. Five fractions were obtained after the 50 °C female AGP was subjected to ion exchange column. Fraction V, with the highest protein and medium polysaccharide content, showed the strongest anticoagulant effect and was also much higher than AGSP, which was obtained by multi-step proteolysis. The findings supported positive effect of the conjugate protein in AGP anticoagulant activity.

In silico design and discovery of pan coronavirus small molecule anti-virals targeting 3CLPRO protease

Coronaviruses (CoV), belonging to the family Coronaviridae, were not considered dangerous pathogens until the outbreaks of SARS, MERS, and more recently, COVID-19. The coronaviruses causing these respective diseases/syndromes, SARS, MERS, and SARS-CoV2, share high sequence and structural similarities. COVID-19 continues to have a global impact on human health and the economy. Human-to-human transmission is at the center of COVID-19’s ability to stall the entire world and force lockdowns across the globe. The corona viruses’ positive sense RNA genome is ∼30 kb long and encodes non-structural (ORF1ab) and structural (Spike, Envelope, Membrane, and Nucleo-capsid) proteins. The main viral protease (NSP5) is a Chymotrypsin-like protease (3CLpro) that cleaves on the carboxy side of the glutamine (Q) of the polypeptide sequence motif x-(L/F/M)-Q-(G/A/S)-x. 3CLPRO is highly conserved among coronaviruses and is critical in the replication and viral life cycle. Therefore, 3CLPRO is considered a promising drug target. We have recently reported three natural compounds, flavonoid derivatives, to target the cysteine 145 of the catalytic dyad covalently. Here, we have screened for small molecules with pan coronavirus activity to target 3CLpro. Our rigid body docking studies have identified 30 small molecules with comparable binding affinities to all the beta coronaviruses. Of these, five molecules have showed the possibility of covalently attacking the C145 of the catalytic dyad. MD simulations have revealed compounds 22 and 23 to be the most ideal lead compounds. Interestingly, one of the compounds, 22, identified in the current study has already shown to be an ideal lead compound against SARS-CoV2 3CLPRO.

Preparation of a novel enzyme-modified cheddar cheese: Molecular mechanism of cheese flavor compensation by synergistic action of cell-free extracts and enzyme systems

A novel enzyme-modified cheddar cheese was prepared and the molecular mechanism of cheese flavor compensation by synergistic action of cell-free extracts and enzyme systems was investigated. By comparing five different protease-peptidase combinations, the group of neutral protease and flavor protease was found to increase the total leucine, valine, and isoleucine content (17.056 ± 0.136 g/kg) and the soluble nitrogen content was up to the level of a 12-month-matured cheese. Molecular docking and molecular dynamics simulations demonstrated their mode of action on four monomeric caseins. Adding a cell-free extract resulted in volatile flavor substances in the enzyme-modified cheese that were closest to those in the 12-month-matured cheese. This might be due to the flavor compensation effect of the conversion of leucine to 3-methylbutyraldehyde by transaminases and decarboxylases, and the conversion of 3-methylbutyric acid to 3-methylbutyraldehyde by ketoacid dehydrogenase and aldehyde dehydrogenase. This is essential for the enzyme modified cheddar cheese preparation.

Recombinant Chymotrypsin-Like Peptidase from Tenebrio molitor with a Non-Canonical Substrate-Binding Site

We characterized an alkaline chymotrypsin-like serine peptidase from the yellow mealworm Tenebrio molitor with a non-canonical substrate-binding subsite for its possible application as a component (an additive) in various biological products. The enzyme was obtained as a recombinant preparation. Purification was carried out using affinity chromatography on Ni2+-NTA agarose. The specificity constants (kcat/KM) for the chymotrypsin substrates, Glp-AAF-pNA, Suc-AAPF-pNA, and Ac-Y-pNA were 7, 4.2 and 0.9 (µM∙min)–1, respectively. Optimum of the proteolytic activity was observed at pH 9.0. The enzyme was stable at the alkaline pH range, and in the presence of BSA also in the acidic region. Peptidase was inhibited by synthetic inhibitors such as PMSF, TPCK, chymostatin, while EDTA, E-64, and pepstatin had no effect on the enzyme activity. The purified enzyme showed high stability over time in the presence of BSA. The short life cycle of the insect and the production of a large number of peptidases in the midgut with high catalytic activity and stability can make T. molitor an excellent alternative source of industrially important enzymes for application as components (additives) in various biological products (e. g., stain removers, detergents, etc.).

Enhancement of Digestive Enzyme Activity in Enterococcus faecalis Using ARTP Mutagenesis.

Enterococcus faecalis is used as a probiotic in animal and human food supplements. Atmospheric and room temperature plasma (ARTP) systems have frequently been used to screen for effective mutant probiotics. In this study, E. faecalis was treated with ARTP, and high-yielding digestive enzyme mutant strains were obtained by measuring the activities of α-amylase, lipase, and neutral protease. A total of 833 mutant strains were obtained after 40-60 s of ARTP treatment, and after screening for digestive enzyme activity, EF-448, EF-798, and EF-804 were obtained. The three strains demonstrated an 180% increase in α-amylase activity, a 30% increase in lipase activity, and a more than 40% increase in neutral protease activity. Furthermore, the enzyme activities remained stable after nine generations. In addition, the strains exhibited high auto-aggregation capacity (over 91%) and high cell hydrophobicity (over 93%). After exposure to simulated intestinal fluid for 6 h, the survival rates of EF-448 and EF-798 were 85.71% and 82.32%, respectively. Moreover, the three mutant strains retained antioxidant capacity and DPPH free radical scavenging ability, and there was no hemolysis. A safety experiment has shown that there is no mortality of Macrobrachium rosenbergii within 14 days after receiving injections of mutant strains at different concentrations. In conclusion, this study obtained three mutant strains with high production of digestive enzymes and stable inheritance through ARTP mutagenesis of E. faecalis, providing an efficient microbial resource.

Study on the effect and mechanism of ultrasonic-assisted enzymolysis on antioxidant peptide activity in walnuts

Walnut meal is a large quantity and high-quality resource with great exploitation value. Ultrasonic-assisted enzymolysis (UAE) was utilized in the preparation of peptides from walnut meal protein. Results indicated that by optimizing the UAE process with neutral protease, an ultrasound power of 180 W, a 4.3 h duration and an enzyme dosage of 10 KU/g, the walnut peptides exhibited the most potent antioxidant activity. In comparison to the control group, the WPI treated with ultrasound and neutral enzymes in combination (UNWPI) demonstrated a significant enhancement in their DPPH, ABTS, and ·OH scavenging capabilities, with increases of 234.23 %, 240.22 %, and 69.52 %, respectively. By analyzing the structure of walnut antioxidant peptides with or without ultrasound, it was observed that the underlying mechanism for the increased antioxidant activity was that UAE not only formed more small peptides, but also produced more peptides with hydrophobic amino acids at their terminal ends. Subsequently, six peptides were identified and screened from UNWPI, namely IFW, IIPF, IVAF, IIFY, ILAFF, and IFIP, which exhibited high antioxidant activity and could bind to Keap1 protein through hydrogen bonding, π-alkyl interactions, and π-π stacking interactions. The research results provided theoretical basis and technical support for the preparation of walnut antioxidant peptides and the high-value utilization of walnut meal.

Design and Evaluation of Peptide Inhibitors Targeting the Dimerization of SARS-CoV-2 Main Protease.

The severe acute respiratory syndrome virus 2 (SARS-CoV-2) seriously impacted public health. The evolutionarily conserved viral chymotrypsin-like main protease (Mpro) is an important target for anti-SARS-CoV-2 drug development. Previous studies have shown that the eight N-terminal amino acids (N8) of SARS-CoV Mpro are essential for its dimerization, and are used to design inhibitors against SARS-CoV Mpro dimerization. Here, we established a simple readout assay using SDS-PAGE and Coomassie blue staining to measure inhibitory activity of N8 peptide derived from SARS-CoV-2 Mpro. To optimize its inhibitory effect, we then modified the side-chain length, charge, and hydrophilicity of the N8 peptide, and introduced a mutated Mpro recognition sequence. As a result, we obtained a series of potent peptide inhibitors against SARS-CoV-2 Mpro, with N8-A24 being the most efficient with an IC50 value of 1.44 mM. We observed that N8-A24 reduced Mpro dimerization with an IC50 value of 0.86 mM. Molecular docking revealed that N8-A24 formed hydrogen bond interactions with critical dimeric interface residues, thus inhibiting its dimerization and activity. In conclusion, our study not only discovers a series of peptide inhibitors targeting the SARS-CoV-2 Mpro dimerization, but also provides a promising strategy for the rational design of new inhibitors against COVID-19.

SMILES-based QSAR virtual screening to identify potential therapeutics for COVID-19 by targeting 3CLpro and RdRp viral proteins

The COVID-19 pandemic has prompted the medical systems of many countries to develop effective treatments to combat the high rate of infection and death caused by the disease. Within the array of proteins found in SARS-CoV-2, the 3 chymotrypsin-like protease (3CLpro) holds significance as it plays a crucial role in cleaving polyprotein peptides into distinct functional nonstructural proteins. Meanwhile, RNA-dependent RNA polymerase (RdRp) takes center stage as the key enzyme tasked with replicating the viral genomic RNA within host cells. These proteins, 3CLpro and RdRp, are deemed optimal subjects for QSAR modeling due to their pivotal functions in the viral lifecycle. In this study, SMILES-based QSAR classification models were developed for a dataset of 2377 compounds that were defined as either active or inactive against 3CLpro and RdRp. Pharmacophore (PH4) and QSAR modeling were used for the virtual screening on 60.2 million compounds including ZINC, ChEMBL, Molport, and MCULE databases to identify new potent inhibitors against 3CLpro and RdRp. Then, a filter was established based on typical molecular characteristics to identify drug-like molecules. The molecular docking was also performed to evaluate the binding affinity of 156 AND 51 potential inhibitors to 3CLpro and RdRp, respectively. Among the 15 hits identified based on molecular docking scores, M3, N2, and N4 were identified as promising inhibitors due to their good synthetic accessibility scores (3.07, 3.11, and 3.29 out of 10 for M3, N2, and N4 respectively). These compounds contain amine functional groups, which are known for their crucial role in the binding interactions between drugs and their targets. Consequently, these hits have been chosen for further biological assay studies to validate their activity. They may represent novel 3CLpro and RdRp inhibitors possessing drug-like properties suitable for COVID-19 therapy.

Enhanced production, artificial intelligence optimized three-phase partitioning extraction, and in silico characterization of extracellular neutral Bacillus cereus proteinase

The present study applied atmospheric and room temperature plasma (ARTP) mutagenesis to improve Bacillus cereus strain for neutral proteinase production. The selected improved stable strain, Phe−Tyr-ARTP-60-E demonstrated a 2.88-fold enhanced proteinase yield and 2.81-fold enhanced proteinase activity with shortened fermentation time. An artificial neural network-embedded genetic algorithm (ANN-GA) was employed to optimize the enzyme's three-phase partitioning (TPP) recovery using Python software. The optimized solution of the hybrid model recommended 38.05% wv−1 (NH4)2SO4, 1.0:2.0 crude extract/tert-butanol ratio, pH 5.84 at 23.4 °C, for the exclusive partitioning of proteinase in the intermediate phase, with 202% recovery, 13-fold purity with specific activity of 922 Umg−1. Sensitivity analysis of the model revealed that enzyme recovery was most sensitive to crude extract/tert-butanol ratio with total sensitivity (ST) of 0.253 while purification factor was most sensitive to pH with an ST of 0.444. In silico analysis using Expasy ProtParam tool revealed that mutant proteinase had 3 amino acid substitutions; Val-to-Cys; Val-to-His and Ile-to-Lys, at positions 152, 288, and 292, respectively, and 39.2 kDa molecular weight, 6.42 isoelectric point, and 33.35 instability index. The 3-Dimensional structure of the enzyme predicted by ModWeb v r273 Server revealed 39% sequence identity with Pseudoalteromonas lipolytica thermolysin. The macromolecule increased transmittance in lysozyme-coated contact lenses by 99.6% in 45 min. We conclude that ARTP mutagenesis and ANN-GA optimized TPP are effective and efficient recalcitrant strain improvement and product recovery methods, respectively. Further genetic tools and analysis of the neutral proteinase-producing gene may be required for increased improvement toward sustainable application.

Study on the interventional effects of Chlamydomonas reinhardtii peptides on chronic unpredictable mild stress-induced depressive-like model mice through metabolomics and microbiota

Depression is a progressive neurodegenerative disease characterized by high prevalence, high suicide rate and high recurrence rate. In this study, the protein from Chlamydomonas reinhardtii was extracted and neutral protease hydrolysate (NPH) was obtained. Its in vitro MAO-A (Monoamine oxidase A) inhibitory activity and in vivo anti-depressive effects in chronic unpredictable mild stress (CUMS) model mice were investigated. The results demonstrated that NPH can improve depressive behaviour in CUMS model mice by elevating neurotransmitter levels and alleviating hippocampal tissue structure damage. The metabolomic analysis of brain and serum samples showed that their common metabolic pathways associated with anti-depressive effects are mainly alanine, aspartate, and glutamate metabolism, glycerophospholipid metabolism, tryptophan metabolism, and caffeine metabolism. Then, the gut microbiota analysis of feces indicated that 8 species with significant changes were associated with anti-depressive effects. Finally, 5 pairs of highly correlated metabolite-bacterium pairs were identified to modulate depressive behaviours. Taken together, the present data suggests that Chlamydomonas reinhardtii-derived hydrolysate could be used for development of functional foods with potential to improve depression.

In Vitro In Silico Screening Strategy and Mechanism of Novel Tyrosinase Inhibitory Peptides from Nacre of Hyriopsis cumingii.

For thousands of years, pearl and nacre powders have been important traditional Chinese medicines known for their skin whitening effects. To prepare the enzymatic hydrolysates of Hyriopsis cumingii nacre powder (NP-HCH), complex enzymatic hydrolysis by pineapple protease and of neutral protease was carried out after the powder was pre-treated with a high-temperature and high-pressure method. The peptides were identified using LC-MS/MS and picked out through molecular docking and molecular dynamics simulations. Subsequently, the tyrosinase inhibitory and antioxidant properties of novel tyrosinase inhibitory peptides were investigated in vitro. In addition, the enzymatic activity of tyrosinase in B16F10 cells as well as melanin content and antioxidant enzyme levels were also examined. The results showed that a tyosinase inhibitory peptide (Tyr-Pro-Asn-Pro-Tyr, YPNPY) with an efficient IC50 value of 0.545 ± 0.028 mM was identified. The in vitro interaction results showed that YPNPY is a reversible competitive inhibitor of tyrosinase, suggesting that it binds to the free enzyme. The B16F10 cell whitening test revealed that YPNPY can reduce the melanin content of B16F10 cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that YPNPY could be widely used as a tyrosinase inhibitor in whitening foods and drugs.

Screening and characterization of an antifreeze peptide from sea cucumber intestinal protein hydrolysates

Protein deterioration caused by ice crystals is an important factors affecting the frozen storage of fish. In this study, antifreeze peptides extracted from hydrolysates of sea cucumber intestinal protein with inhibition of protein denaturation were screened and characterized. The peptide Leu-Pro-Glu-Phe-Thr-Glu-Glu-Glu-Lys (LPEFTEEEK), derived from neutral protease hydrolysates of sea cucumber intestinal protein, was investigated for its potential to enhance the quality of salmon fillets during three freeze-thaw cycles. The results showed that the application of LPEFTEEEK effectively maintained the texture of fish fillets, as well as the oxidative and conformation stability of myofibrillar protein during the freezing process. Additionally, molecular dynamics simulations verified that LPEFTEEEK could bind to ice crystals and inhibit their recrystallization, thus preventing organisms from being damaged by freezing. This suggests that LPEFTEEEK holds significant promise as a novel cryoprotective agent for marine-derived antifreeze peptides.

Preparation and stability of angiotensin-I-converting enzyme (ACE) inhibitory peptides from protein hydrolysate of Wuyi rock tea residues

ABSTRACT Wuyi rock tea residues, generated after brewing or aroma extraction, are rich in nutrients. This study aimed to prepare ACE inhibitory peptides from the protein hydrolysate of Wuyi rock tea residues and investigate their stability. The optimal proteolysis conditions for neutral protease were optimized as: a substrate concentration of 2% (w/v), pH 7.0, hydrolysis temperature of 45°C, enzymatic digestion time of 3 h, and enzyme dosage of 6800 U/g. Under these conditions, the ACE inhibition rate of protein hydrolysate of Wuyi rock tea residues reached 70.47 ± 0.29% at a concentration of 0.5 mg/mL. Additionally, the prepared ACE inhibitory peptides demonstrated excellent stability against acid, base, heat, and certain metal ions. These findings suggest that the proteins extracted from Wuyi rock tea residues can serve as a promising source to produce highly active ACE inhibitory peptides. These peptides could be utilized in the development of nutraceuticals, functional foods, and pharmaceuticals.

Physicochemical and stability analysis of mung bean protein hydrolysates with lipid peroxidation inhibition

This study investigated mung bean protein hydrolysates (MBPH) produced using neutral protease, examining their physicochemical properties, stability, and lipid peroxidation inhibition capabilities. The research revealed that MBPH molecular weight ranged from 17 to 26 kDa and perform various functions, including catalytic, nutrient storage, and binding. Stability assessments showed that MBPH are stable at 45 °C and pH of 7.5 but are light-sensitive and unstable in solution or when combined with sugars. Additionally, increased concentrations of digestive enzymes reduce MBPH stability. Antioxidant tests in vitro and in Caenorhabditis elegans confirmed MBPH's ability to neutralizing radicals, enhance antioxidant enzyme activities, and reduce lipid peroxidation, thereby protecting against oxidative damage. Furthermore, in vivo experiments showed that MBPH extend the lifespan of worms and reduced their body lipid content, indicating potential benefits in mitigating cholesterol-related damage. This research demonstrates the potential of MBPH in inhibiting lipid peroxidation.