Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation.

The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.

Combined techniques for revealing the mechanism beneath the inhibition effects of pectin on gluten digestibility using static in vitro gastro-duodenal protocols.

In the current study, how pectin retards the digestibility of wheat gluten was investigated using a static in vitro gastric-duodenal model. The degree of protein hydrolysis was estimated using the o-phthaldialdehyde method, while the in vitro digestograms were mathematically fitted using a single first-order kinetics model. Peptides' profile, free amino acids compositions, gluten-pectin interactions and their effects on enzymatic activities of proteolytic enzymes as well as on the gluten secondary structures under digestive conditions were studied using combined techniques. Results showed that pectin could retard gluten digestibility through 1). preferential absorption to insoluble gluten aggregates by electrostatic interactions; 2). increasing the helix and reducing the ß-sheet content of the solubilized gluten protein fractions in terms of their secondary molecular structures; 3). reducing pepsin activity by forming negatively charged pectin-gluten mixtures which then interacted with the positively charged pepsin molecules. The deeper insight into gluten-pectin interactions and their influences on gluten digestibility under gastrointestinal conditions provides important clues for developing effective forms of dietary fiber to improve the nutritional benefits of plant protein in individuals.

Mechanism for improving the in vitro digestive properties of coconut milk by modifying the structure and properties of coconut proteins with monosodium glutamate.

In this paper, the effect of monosodium glutamate (MSG) on coconut protein (CP) solubility, surface hydrophobicity, emulsification activity, ultraviolet spectroscopy and fluorescence spectroscopy was investigated. Meanwhile, the changes in the in vitro digestive properties of coconut milk were also further analyzed. MSG treatment altered the solubility and surface hydrophobicity of CP, thereby improving protein digestibility. Molecular docking showed that CP bound to pepsin and trypsin mainly through hydrogen bonds and salt bridges. And MSG increased the cleavable sites of pepsin and trypsin on CP, thus further improving the protein digestibility. In addition, MSG increased the Na+ concentration in coconut milk, promoted flocculation and aggregation between coconut milk droplets, which prevented the binding of lipase and oil droplets and inhibited lipid digestion. These findings may provide new ideas and insights to improve the digestive properties of plant-based milk.

Microfluidic-oriented green synthesis of pepsin-doped gold nanoparticles for colorimetric and photothermal dual-readout detection of food hazards.

Gold nanoparticles (AuNPs)-based immunochromatographic assay has gained popularity as a rapid detection method for food hazards. Synthesizing highly stable AuNPs in a rapid, simple and environmentally friendly manner is a key focus in this field. Here, we present a green microfluidic strategy for the rapid, automated, and size-controllable synthesis of pepsin-doped AuNPs (AuNPs@Pep) by employing glucose-pepsin as a versatile reducing agent and stabilizer. Through combining the colorimetric and photothermal (PoT) properties of AuNPs@Pep, both "signal-off" and "signal-on" formats of microfluidic paper analytical devices (PADs) were developed for detection of a small molecule antibiotic, florfenicol, and an egg allergen, ovalbumin. Compared to the colorimetric mode, a 4-fold and 3-fold improvement in limit of detection was observed in the "signal-off" detection of florfenicol and the "signal-on" detection of ovalbumin, respectively. The results demonstrated the practicality of AuNPs@Pep as a colorimetric/PoT dual-readout probe for immunochromatographic detection of food hazards at different molecular scales.

Effects of Ultrasonic Power on the Structure and Rheological Properties of Skin Collagen from Albacore (Thunnus alalunga).

The effects of ultrasonic power (0, 150, 300, 450, and 600 W) on the extraction yield and the structure and rheological properties of pepsin-soluble collagen (PSC) from albacore skin were investigated. Compared with the conventional pepsin extraction method, ultrasonic treatment (UPSC) significantly increased the extraction yield of collagen from albacore skin, with a maximum increase of 8.56%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that peptides of low molecular weight were produced when the ultrasonic power exceeded 300 W. Meanwhile, secondary structure, tertiary structure, and X-ray diffraction analyses showed that the original triple helix structure of collagen was intact after the ultrasonic treatment. The collagen solutions extracted under different ultrasonic powers had significant effects on the dynamic frequency sweep, but a steady shear test suggested that the collagen extracted at 150 W had the best viscosity. These results indicate that an ultrasonic power between 150 and 300 W can improve not only the extraction yield of natural collagen, but also the rheological properties of the collagen solution without compromising the triple helix structure.

Effect of Chloramphenicol as Antibiotic on the Structure and Function of Pepsin and Its Mechanism of Action.

The interaction between chloramphenicol (CHL) and pepsin (PEP), as well as the impact of CHL on PEP conformation, were investigated using spectroscopic techniques and molecular docking simulations in this study. The experimental results demonstrate that CHL exhibits a static quenching effect on PEP. The thermodynamic parameters indicate that the reaction between CHL and PEP is spontaneous, primarily driven by hydrogen bonding and van der Waals forces. Moreover, the binding distance of r<7 nm suggests the occurrence of Förster's non-radiative energy transfer between these two molecules. In the synchronous fluorescence spectrum, the maximum fluorescence intensity of PEP produced a redshift phenomenon, indicating that CHL was bound to tryptophan residues of PEP. The addition of CHL induces changes in the secondary structure of PEP, as confirmed by the observed alterations in peak values in three-dimensional fluorescence spectra. The UV spectra reveal a redshift of 3 nm in the maximum absorption peak, indicating a conformational change in the secondary structure of PEP upon addition of CHL. Circular dichroism analysis demonstrates significant alterations in the α-helix, ß-sheet, ß-turn, and random coil contents of PEP before and after CHL incorporation, further confirming its ability to modulate the secondary structure of PEP.

Different functional groups of carbon dots influence the formation of protein crowns and pepsin characteristic in vitro digestion.

Application of nanomaterials (NMs) in agriculture poses an ingestion risk to humans and may affect the digestive process. Different fates of NMs with differential charges in the gastrointestinal tract should be considered. In this study, the interaction between three carbon dots (CDs) carried with different functional groups (-NH2, -OH, and -COOH) and pepsin was analyzed through an in vitro digestion model. The results showed that CDs significantly reduced pepsin activity. Among them, CDs-NH2 had the greatest effect, following by CDs-OH, and CDs-COOH. Besides, molecular docking demonstrated the specific binding site of CDs to pepsin, while the most stable binding energy (-8.10 kcal/mol) was formed between CDs-NH2 and pepsin. Further, CDs formed a nanomaterial-protein crown structure with pepsin. The present study enriches the functional group properties of CDs in the digestion and provides new ideas for the potential human health of NMs.

Spectroscopic and molecular docking study of three kinds of cinnamic acid interaction with pepsin.

In this work, under simulated physiological conditions (pH = 2.2, glycine hydrochloric acid buffer solution), the interactions of cinnamic acid (CA), m-hydroxycinnamic acid (m-CA) and p-hydroxycinnamic acid (p-CA) with pepsin were studied by fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FTIR), molecular docking and molecular dynamic simulation (MD). The spectrogram results showed that these three kinds of CA had a strong ability to quench the intrinsic fluorescence of pepsin, and the quenching effects were obvious with the increase of concentration of these three kinds of molecules. The quenching mechanism of CA, m-CA and p-CA on the fluorescence of pepsin was static quenching. In addition, a stable complex was formed between three kinds of CA with pepsin. Thermodynamic data and docking information suggested that three kinds of CA combine with pepsin were mainly driven by electrostatic force and hydrogen bond. The binding constant and the number of binding sites were determined. The interaction of CA, m-CA and p-CA with pepsin was spontaneous, and accompanied by non-radiative energy transfer. The results from CD, FTIR, UV-Vis and synchronous fluorescence spectra measurements manifested that the secondary structure of pepsin was changed by the binding of three kinds of CA. The ß-sheet of pepsin increased after the interaction with three kinds of CA. The assay results of pepsin activity showed that three kinds of CA led to a decrease in pepsin activity within the investigated concentrations. Molecular docking investigation revealed the formation of polar hydrogen bonds as well as hydrophobic interactions between three kinds of CA with pepsin, and the ligand within the binding pocket of pepsin. MD results implied the formation of a stable complex between three kinds of CA and pepsin. The research suggested that cinnamic acid and its derivatives could be a potential effect on the structure and properties of digestive enzyme.

Native and compactly folded in-solution conformers of pepsin are revealed and distinguished by mass spectrometric ITEM-TWO analyses of gas-phase pepstatin A - pepsin complex binding strength differences.

Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle protease. Pepsin has also been study object of protein higher-order structure analyses, but questions about how to best investigate pepsin in-solution conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the in-solution existence of (a) natively folded pepsin (N) which by nanoESI-MS analysis gave rise to a narrow charge state distribution with an 11-fold protonated most intense ion signal, (b) unfolded pepsin (U) with a rather broad ion charge state distribution whose highest ion signal carried 25 protons, and (c) a compactly folded pepsin conformer (C) with a narrow charge structure and a 12-fold protonated ion signal in the center of its charge state envelope. Because pepsin is a protease, unfolded pepsin became its own substrate in solution at pH 6.6 since at this pH some portion of pepsin maintained a compact/native fold which displayed enzymatic activity. Subsequent mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex dissociation reactions in the gas phase confirmed a very strong binding of pepstatin A by natively folded pepsin (N). ITEM-TWO further revealed the existence of two compactly folded in-solution pepsin conformers (Ca and Cb) which also were able to bind pepstatin A. Binding strengths of the respective compactly folded pepsin conformer-containing complexes could be determined and apparent gas phase complex dissociation constants and reaction enthalpies differentiated these from each other and from the pepstatin A - pepsin complex which had been formed from natively folded pepsin. Thus, ITEM-TWO turned out to be well suited to pinpoint in-solution pepsin conformers by interrogating quantitative traits of pepstatin A - pepsin complexes in the gas phase.

Extraction and Characterization of Pepsin- and Acid-Soluble Collagen from the Swim Bladders of Megalonibea fusca.

There is a growing demand for the identification of alternative sources of collagen not derived from land-dwelling animals. The present study explored the use of pepsin- and acid-based extraction protocols to isolate collagen from the swim bladders of Megalonibea fusca. After extraction, these acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) samples respectively were subjected to spectral analyses and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization, revealing both to be comprised of type I collagen with a triple-helical structure. The imino acid content of these ASC and PSC samples was 195 and 199 residues per 1000 residues, respectively. Scanning electron microscopy demonstrated that samples of freeze-dried collagen exhibited a compact lamellar structure, while transmission electron microscopy and atomic force microscopy confirmed the ability of these collagens to undergo self-assembly into fibers. ASC samples exhibited a larger fiber diameter than the PSC samples. The solubility of both ASC and PSC was highest under acidic pH conditions. Neither ASC nor PSC caused any cytotoxicity when tested in vitro, which met one of the requirements for the biological evaluation of medical devices. Thus, collagen isolated from the swim bladders of Megalonibea fusca holds great promise as a potential alternative to mammalian collagen.

Reframing prosegment-dependent folding and limits on natural protein folding landscapes from an evolutionary perspective.

In this perspective, we propose that the folding energy landscapes of model proteases including pepsin and alpha-lytic protease (αLP), which lack thermodynamic stability and fold on the order of months to millennia, respectively, should be viewed as not evolved and fundamentally distinct from their extended zymogen forms. These proteases have evolved to fold with prosegment domains and robustly self-assemble as expected. In this manner, general protein folding principles are strengthened. In support of our view, αLP and pepsin exhibit hallmarks of frustration associated with unevolved folding landscapes, such as non-cooperativity, memory effects, and substantial kinetic trapping. The evolutionary implications of this folding strategy are considered in detail. Direct applications of this folding strategy on enzyme design, finding new drug targets, and constructing tunable folding landscapes are also discussed. Together with certain proteases, growing examples of other folding "exceptions"-including protein fold switching, functional misfolding, and prevalent inability to refold-suggests a paradigm shift in which proteins may evolve to exist in a wide range of energy landscapes and structures traditionally thought to be avoided in nature.

Structural change study of pepsin in the presence of spermidine trihydrochloride: Insights from spectroscopic to molecular dynamics methods.

Spermidine is an aliphatic polyamine that directs a set of biological processes. This work aimed to use UV-Vis spectroscopy, fluorescence spectroscopy, thermal stability, kinetic methods, docking, and molecular dynamic simulations to examine the influence of spermidine trihydrochloride (SP) on the structure and function of pepsin. The results of the fluorescence emission spectra indicated that spermidine could quench pepsin's intrinsic emission in a static quenching process, resulting in the formation of the pepsin-spermidine complex. The results discovered that spermidine had a strong affinity to the pepsin structure because of its high binding constant. The obtained results from spectroscopy and molecular dynamic approaches showed the binding interaction between spermidine and pepsin, induced micro-environmental modifications around tryptophan residues that caused a change in the tertiary and secondary structure of the enzyme. FTIR analysis showed hypochromic effects in the spectra of amide I and II and redistribution of the helical structure. Moreover, the molecular dynamic (MD) and docking studies confirmed the experimental data. Both experimental and molecular dynamics simulation results clarified that electrostatic bond interactions were dominant forces.

Perception of the interaction behavior between pepsin and the antimicrobial drug secnidazole with combined experimental spectroscopy and computer-aided techniques.

Drug-pepsin interaction possibly affects pepsin activity, leads to undesirable shift of its functionality, and likely induces adverse effects in the gastrointestinal tract. The present study aims at exploring the interaction of pepsin with the antiprotozoal/antibacterial drug secnidazole adopting a combination of experimental spectroscopy and computational techniques. For this purpose, different spectroscopic methods including fluorescence, synchronous fluorescence, UV-Visible absorption, and infrared spectroscopy were adopted and coordinated with in silico analysis via molecular docking. The employed synchronized approaches evidenced that; pepsin interacted with secnidazole via static mechanism at stomach pH inferring some consequent conformational changes in the structure of pepsin. Thermodynamic study of drug-pepsin interaction demonstrated that the interaction is spontaneous via van der Waals and hydrogen bonding interaction and the orientation of ligand within pepsin cavity was illustrated by molecular docking. The synchronous fluorescence study proved that tyrosine amino acid residues were involved in the interaction more than tryptophan amino acid residues. Eventually, the combined experimental and molecular docking approaches suggest that secnidazole interacts with pepsin and alter its structure, that finding correlates to gastrointestinal side effects related to secnidazole oral administration.

Exploration of binding mechanism of apigenin to pepsin: Spectroscopic analysis, molecular docking, enzyme activity and antioxidant assays.

Pepsin plays an important role in nutrient metabolism. Apigenin (AP) is a beneficial polyphenol to human health. To enhance the bioavailability of AP and elucidate the inhibitory effect of AP on pepsin, the interaction mechanism of AP with pepsin was investigated using spectroscopic analysis and molecular docking, and the activity of pepsin and antioxidant activity of AP was also evaluated. Specifically, AP performed static quenching of pepsin and had only one binding site on pepsin. More interestingly, the interaction between AP and pepsin was spontaneous, while hydrogen bonds and van der Waals forces were the main binding forces. Generally, synchronous and three-dimensional fluorescence confirmed that AP induced the conformational changes of pepsin, and molecular docking proved the above results and illustrated the specific binding patterns. Specifically, AP inhibited the activity of pepsin, while pepsin decreased the antioxidant activity of AP. These results provided useful information for elucidating the interactions between AP and pepsin.

Investigation of the interaction behavior between quercetin and pepsin by spectroscopy and MD simulation methods.

The ability of a therapeutic compound to bind to proteins is critical for characterizing its therapeutic impacts. We have selected quercetin (Qu), a most common flavonoid found in plants and vegetables among therapeutic molecules that are known to have anti-inflammatory, antioxidant, anti-genotoxic, and anti-cancer effects. The current study aimed to see how quercetin interacts with pepsin in an aqueous environment under physiological conditions. Absorbance and emission spectroscopy, circular dichroism (CD), and kinetic methods, as well as molecular dynamic (MD) simulation and docking, were applied to study the effects of Qu on the structure, dynamics, and kinetics of pepsin. Stern-Volmer (Ksv) constants were computed for the pepsin-quercetin complex at three temperatures, showing that Qu reduces enzyme emission spectra using a static quenching. With Qu binding, the Vmax and the kcat/Km values decreased. UV-vis absorption spectra, fluorescence emission spectroscopy, and CD result indicated that Qu binding to pepsin leads to microenvironmental changes around the enzyme, which can alter the enzyme's secondary structure. Therefore, quercetin caused alterations in the function and structure of pepsin. Thermodynamic parameters, MD binding, and docking simulation analysis showed that non-covalent reactions, including the hydrophobic forces, played a key role in the interaction of Qu with pepsin. The findings conclude of spectroscopic experiments were supported by molecular dynamics simulations and molecular docking results.

A comprehensive insight into the effects of caffeic acid (CA) on pepsin: Multi-spectroscopy and MD simulations methods.

The interaction between caffeic acid (CA) and pepsin was investigated using multi-spectroscopy approaches and molecular dynamic simulations (MDS). The effects of CA on the structure, stability, and activity of pepsin were studied. Fluorescence emission spectra and UV-vis absorption peaks all represented the static quenching mechanism of pepsin by CA. Moreover, the fluorescence spectra displayed that the interaction of CA exposed the tryptophan chromophores of pepsin to a more hydrophilic micro-environment. Consistent with the simulation results, thermodynamic parameters revealed that CA was bound to pepsin with a high binding affinity. The Van der Waals force and Hydrogen bond interaction were the dominant driving forces during the binding process. The circular dichroism (CD) spectroscopy analysis showed that the CA binding to pepsin decreased the contents of α-Helix and Random Coil but increased the content of ß-sheet in the pepsin structure. Accordingly, MD simulations confirmed all the experimental results. As a result, CA is considered an inhibitor with adverse effects on pepsin activity.

Valorization of Fish Waste: Isolation and Characterization of Acid- and Pepsin-Soluble Collagen from the Scales of Mediterranean Fish and Fabrication of Collagen-Based Nanofibrous Scaffolds.

In search of alternative and sustainable sources of collagenous materials for biomedical applications, the scales of five Mediterranean fish species-fished in high tonnage in the Mediterranean region since they represent popular choices for the local diet-as well as those of the Atlantic salmon for comparison purposes, were comparatively studied for their acid- and pepsin-soluble collagen content. Fish scales that currently represent a discarded biomass of no value could be efficiently exploited for the production of a high added-value biomaterial. The isolated collagenous materials, which showed the typical electrophoretic patterns of type I collagen, were morphologically and physicochemically characterized. Using scanning electron microscopy the fibrous morphology of the isolated collagens was confirmed, while the hydroxyproline content, in conjunction with infrared spectroscopy and X-ray diffraction studies verified the characteristic for collagen amino acid profile and its secondary structure. The acid- and pepsin-soluble collagens isolated from the fish scales were blended with the bioactive sulfated marine polysaccharide ulvan and polyethylene oxide and electrospun to afford nanofibrous scaffolds that could find applications in the biomedical sector.

Physicochemical, Structural and Antioxidant Properties of Collagens from the Swim Bladder of Four Fish Species.

This study aimed to isolate and characterize pepsin-solubilized collagen (PSC) from marine and freshwater fish swim bladders. The physicochemical properties, protein pattern, amino acid composition, structure, thermal denaturation temperature, and antioxidant activity of PSC from four different swim bladder sources were investigated and compared. The results demonstrated that the four types of collagen extracted were all type I collagen. The yield of PSC extracted from grass carp (GCSB-PSC), bighead carp (BCSB-PSC), grouper (GSB-PSC), and monkfish swim bladders (MSB-PSC) were 38.98, 27.97, 18.16, and 10.35%, respectively. Compared to the other three PSCs, BCSB-PSC has the highest thermal denaturation temperature (38.60 °C). Based on FTIR spectroscopy and circular dichroism (CD) analysis, the extracted PSCs retained the triple helix and secondary structure well. Antioxidant studies showed that in the swim bladders of four species the swim bladder PSC could scavenge DPPH and ABTS radicals. Overall, swim bladders from marine and freshwater fish can be utilized as raw materials for collagen extraction, and the extracted collagen has potential commercial applications.

In Vitro Antioxidant and Antiaging Activities of Collagen and Its Hydrolysate from Mackerel Scad Skin (Decapterus macarellus).

The skin of mackerel scad fish (Decapterus macarellus) is a new source for pepsin-soluble collagen and its hydrolysate, both of which have never been explored. This study aims to characterize and determine the in vitro antioxidant, antiglycation, and antityrosinase activity of pepsin-soluble collagen (PSC) and hydrolyzed collagen (HC) from mackerel scad skin. PSC was extracted using 0.5 M acetic acid containing 0.1% pepsin for 48 h at 4 °C. The obtained PSC was then hydrolyzed with collagenase type II (6250 U/g) to produce HC. The PSC yield obtained was 6.39 ± 0.97%, with a pH of 6.76 ± 0.18, while the HC yield was 96% from PSC. SDS-PAGE and Fourier Transform Infrared (FTIR) analysis showed the typical features of type I collagen. HC demonstrated high solubility (66.75-100%) throughout the entire pH range (1-10). The PSC and HC from mackerel scad skin showed antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH), with IC50 values of 148.55 ± 3.14 ppm and 34.966 ± 0.518 ppm, respectively. In the antiglycation test, PSC had an IC50 value of 239.29 ± 15.67 ppm, while HC had an IC50 of 68.43 ± 0.44 ppm. PSC also exhibited antityrosinase activity, with IC50 values of 234.66 ± 0.185 ppm (on the L-DOPA substrate), while HC had an IC50 value of 79.35 ± 0.5 ppm. Taken together, these results suggest that the skin of mackerel scad fish has potential antiaging properties and can be further developed for pharmaceutical and cosmetic purposes.