Enzymes Trypsin Research Articles

Assessing the chronic toxicity of climbazole to Daphnia magna: Physiological, biochemical, molecular, and reproductive perspectives

The widespread use of climbazole (CBZ) has led to its increased presence in aquatic environments, potentially threatening freshwater ecosystems. However, evidence regarding the harmful effects of CBZ on aquatic organisms remains limited. In this study, Daphnia magna was exposed to CBZ at concentrations of 0, 0.2, 20, and 200 μg/L for 21 days to evaluate its chronic toxicity through assessment of life-history traits, physiological parameters, biochemical analyses, and gene expression. The results indicated that CBZ exposure delayed the days to the first brood, reduced the frequency of molting per adult, decreased the offspring number at first brood, diminished the body length, and decreased both the total number of broods per female and the total number of offspring per female. Additionally, CBZ inhibited the swimming speed, filtration rate, and ingestion rate. Moreover, CBZ altered the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), while increasing malondialdehyde (MDA) levels. Gene expression analysis revealed varied responses in mRNA levels related to metabolic detoxification (cyp360a8, gst, and p-gp), digestive enzymes (α-amylase, α-esterase, and trypsin), energy (ak), oxygen transport (dhb), and reproduction (nvd, cyp314, ecr, vtg, and jhe) following CBZ exposure. These results indicate that the presence of CBZ in aquatic environments can induce toxicity by altering energy acquisition, supply, and metabolism; impairing metabolic detoxification pathways; eliciting oxidative stress; and causing reproductive toxicity in D. magna.

Scanning Transmission Soft X-Ray Microscopy Probes Topical Drug Delivery of Rapamycin Facilitated by Microneedles.

Scanning Transmission X-ray microscopy (STXM) is a sensitive and selective probe for the penetration of rapamycin which is topically applied to human skin ex vivo and is facilitated by skin treatment with microneedles puncturing the skin. Inner-shell excitation serves as a selective probe for detecting rapamycin by changes in optical density as well as linear combination modeling using reference spectra of the most abundant species. The results indicate that mechanical damage induced by microneedles allows this drug to accumulate in the stratum corneum without reaching the viable skin layers. This is unlike intact skin which shows no drug penetration at all and underscores the mechanical impact of microneedle skin treatment. These results are compared to drug penetration profiles of other drugs highlighting the importance of skin barriers. High spatial resolution studies also indicate that the lipophilic drug rapamycin is observed in corneocytes. Attempts in data evaluation are reported to probe rapamycin also in the lipid layers between the corneocytes, which was not accomplished before. These results are compared to recent results on rapamycin uptake in skin where barrier impairment was induced by pre-treatment with the enzyme trypsin and drug formulations leading to occlusion.

Growth and physiological responses of Nile tilapia, Oreochromis niloticus fed dietary fermented sugar beet bagasse and reared in biofloc system

Sugar beet bagasse (SB) is a common agro-industrial waste used in animal feeds. However, its high crude fiber content makes it difficult for fish to digest. Fermentation processes can improve the nutritional value of SB by-products. The study uses Lactobacillus rhamnosus and yeast (Saccharomyces cerevisiae) to ferment SB waste for use as a soybean meal substitute in Nile tilapia fingerlings. This led to an increase in protein, lipid, ash, and nitrogen-free extracts, while the fiber content decreased. Four experimental isonitrogenous (crude protein: 249.2 g kg−1) and isocaloric (gross energy:18.25 MJ kg−1) diets were formulated as follows: a control diet with 0 % LYFSB (L. rhamnosus and yeast fermented SB), and three other diets with LYFSB replacing soybean meal (SBM) at levels of 10 % (LYFSB10 %), 20 % (LYFSB20 %), and 30 % (LYFSB30 %) based on protein content, over a 70-day period. 180 fingerlings of Nile tilapia (average initial weight of 5.19 ± 0.02 g) were placed at random in twelve circular plastic tanks (60 L capacity) with a stocking density of fifteen fish each. Nitrite and ammonia levels decreased as the replacement levels of LYFSB in tilapia diets increased, while nitrate levels showed the opposite trend. The phytoplankton community includes nine species in the biofloc system and four in tilapia gut content, with Chlorophyceae being the most common class, having the highest number in fish-fed diets (7805.67 organism/ml). The study revealed that LYFSB significantly influenced the zooplankton community, with protozoa dominating in biofloc water and rotifer in tilapia gut. LYFSB10 % and LYFSB20 % had the highest bacteria counts. Fish fed with LYFSB10 % and LYFSB20 % diets showed the highest values for specific growth rate (2.51 % day−1) and weight gain (24.92 g fish−1). These diets also exhibited increased activity levels of trypsin, lipase, and amylase enzymes. Increasing the inclusion of LYFSB in fish diets as a replacement for FM improved the intestinal and liver health of fish. The diets with LYFSB10 % and LYFSB20 % showed the highest levels of globulin, total protein, albumin, and IgM, with no significant differences between them. Fish fed LYFSB10 % had lower cholesterol and triglyceride levels. Superoxide dismutase (SOD) and catalase (CAT) activities were highest in fish fed LYFSB10 % and LYFSB20 %, while malondialdehyde (MDA) activity was lowest in the LYFSB30 % diet. The study showed that the biofloc system enhanced water quality and increased the efficiency of tilapia fed LYFSB10 % and LYFSB20 %.

Effects of a potential host gut-derived probiotic, Bacillus amyloliquefaciens AV5, on the growth, biochemical and metabolic responses, and disease resistance of Nile tilapia (Oreochromis niloticus)

The influence of the dietary probiotic Bacillus amyloliquefaciens AV5 on the growth, biochemical and metabolic responses, and disease resistance of Nile tilapia (Oreochromis niloticus) was evaluated. The commercial diet (GC), a diet containing 106 and 108 CFU/g of B. amyloliquefaciens AV5, denoted as G1 and G2, respectively, was fed for 30 days. Fish were allocated to 9 plastic tanks, and each diet was randomly assigned to triplicate groups of 40 fish (22.4 ± 1.3 g).After 30 days of the feeding trial, the specific growth rate, weight gain rate, and final weight increased significantly in G2 compared to those in GC and G1. Feed conversion ratio was significantly higher in the fish-fed GC group than in the G2 group. However, the survival rate of the fish ranged from 99.13 % to 97.17 %, with no significant difference. Glucose levels did not differ significantly (P < 0.05). Total antioxidant capacity (T-AOC), (superoxide dismutase (SOD), and catalase (CAT), as well as digestive enzymes (trypsin, lipase, and protease), were significantly increased in the G2 group relative to the GC and G1 groups.Significantly lower ALT, AST, CHO, TG, and MDA activities were observed in the G2 group than in the GC group. To evaluate the metabolomic response, PCA, OPLS-DA, and volcano plots revealed a distinct difference between the G2 and GC diets, suggesting that the G2 diet impacted the hepatopancreas of fish. Furthermore, challenge experiments revealed that fish fed B. amyloliquefaciens AV5 exhibited substantially increased resistance (P < 0.05) to S. agalactiae at a concentration of 1x108CFU/g. Therefore, dietary supplementation with B. amyloliquefaciens AV5 enhanced the growth performance, metabolic activity, and disease resistance of O. niloticus.

NIR-Sensitive Squaraine Dye-Peptide Conjugate for Trypsin Fluorogenic Detection.

Trypsin enzyme has gained recognition as a potential biomarker in several tumors, such as colorectal, gastric, and pancreatic cancer, highlighting its importance in disease diagnosis. In response to the demand for rapid, cost-effective, and real-time detection methods, we present an innovative strategy utilizing the design and synthesis of NIR-sensitive dye-peptide conjugate (SQ-3 PC) for the sensitive and selective monitoring of trypsin activity by fluorescence ON/OFF sensing. The current research deals with the design and synthesis of three unsymmetrical squaraine dyes SQ-1, SQ-2, and SQ-3 along with a dye-peptide conjugate SQ-3-PC as a trypsin-specific probe followed by their photophysical characterizations. The absorption spectral investigation conducted on both the dye alone and its corresponding dye-peptide conjugates in water, utilizing SQ-3 and SQ-3 PC respectively, reveals enhanced dye aggregation and pronounced fluorescence quenching compared to observations in DMSO solution. The absorption spectral investigation conducted on dye only and corresponding dye-peptide conjugates in water utilizing SQ-3 and SQ-3 PC, respectively, reveals not only the enhanced dye aggregation but also pronounced fluorescence quenching compared to that observed in the DMSO solution. The trypsin-specific probe SQ-3 PC demonstrated a fluorescence quenching efficiency of 61.8% in water attributed to the combined effect of aggregation-induced quenching (AIQ) and fluorescence resonance energy transfer (FRET). FRET was found to be dominant over AIQ. The trypsin-mediated hydrolysis of SQ-3 PC led to a rapid and efficient recovery of quenched fluorescence (5-fold increase in 30 min). Concentration-dependent changes in the fluorescence at the emission maximum of the dyes reveal that SQ-3 PC works as a trypsin enzyme-specific fluorescence biosensor with linearity up to 30 nM along with the limit of detection and limit of quantification of 1.07 nM and 3.25 nM, respectively.

Sea conch (Rapana venosa) peptide hydrolysate regulates NF‐κB pathway and restores intestinal immune homeostasis in DSS‐induced colitis mice

AbstractInflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract. Sea conch peptide hydrolysate (CPH) was produced by enzymatic digestion of fresh conch meat with trypsin enzyme. To analyze the molecular composition, functional groups, and structural morphology of the hydrolysate, we employed liquid chromatography–mass spectrometry (LC–MS), Fourier‐transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Results confirmed that crude protein could be effectively digested by enzymes to generate peptides. In this study, we evaluated the bioactivities of CPH on dextran sulfate solution (DSS)‐induced colitis in mice. The findings demonstrated that CPH supplementation improved body weight, food and water intake, and colon length. The therapeutic efficacy and immunoregulatory effect of CPH were further determined. Our results exhibited that CPH treatment significantly ameliorated pathological symptoms by enhancing intestinal integrity, mucin production, and goblet cell count. Moreover, the immunoregulatory effect of CPH on mRNA expression levels of different pro‐ and anti‐inflammatory cytokines was determined. Results exhibited a decrease in the expression of pro‐inflammatory cytokines and an increase in anti‐inflammatory cytokines in the colon. Additionally, the CPH administration modulates the nuclear factor kappa B (NF‐κB) pathway, preventing DNA damage and cell death. Assays for apoptosis and DNA damage revealed that CPH reduced oxidative DNA damage and apoptosis. These findings highlight the immunomodulatory and treatment amelioration effect of CPH in reducing the severity of colitis.

Integrated transcriptome and 1H NMR-based metabolome to explore the potential mechanism of Spodoptera litura in response to flupyrimin

Flupyrimin (FLP) is a novel class of insecticide acting on insect nicotinic acetylcholine receptor (nAChR) and shows robust insecticidal activity. However, the toxicological effects of FLP on Spodoptera litura have not been revealed. In this study, the results showed that the larval survival rate decreased significantly with increasing concentration of FLP. The hematoxylin-eosin (HE) staining showed that FLP exposure damages the structure of the larval midgut. Additionally, FLP treatments significantly increased the activities of detoxification (GST and CarE) and digestive (α-Amylase and Trypsin) enzymes and reduced lipase activity. Transcriptome sequencing identified 855, 1493 and 735 differentially expressed genes (DEGs) at 12 h, 24 h and 48 h after exposure to 3 mM FLP, respectively. Gene function enrichment analysis revealed that DEGs were mainly related to fatty acid metabolic, protein processing in the endoplasmic reticulum and drug metabolism-cytochrome P450. The DEGs associated with food digestion and detoxification was validated by reverse-transcription quantitative PCR (RT-qPCR). Furthermore, a total of fifteen energy-related metabolites were identified, among which thirteen metabolisms were significantly influenced after FLP treatment based on 1H NMR-based metabolome analysis, including tyrosine, glucose, trehalose, malate, threonine, proline, glycine, lysine, citrate, alanine, lactate, valine, and leucine. Taken together, these results provide useful information for revealing the toxicological effect of FLP against S. litura.

Daily rhythm in feeding behavior and digestive processes in totoaba (Totoaba macdonaldi) under commercial farming conditions

To identify daily changes in the digestive physiology of Totoaba macdonaldi, the feed intake, activity (pepsin, trypsin, chymotrypsin, lipase, amylase, and L-aminopeptidase), and gene expression (aminopeptidase and maltase-glucoamylase) of key digestive enzymes were measured in the intestine and the pyloric caeca. Fish were fed for three weeks every four hours during the light period to apparent satiation, and samples were taken every four hours throughout a 24-h cycle under a 12:12 L:D photoperiod. The feed consumption steadily increased until the third feeding (16:00 h, ZT-8) and decreased significantly towards the end of the day. The activity of pepsin and alkaline enzymes (trypsin, chymotrypsin, lipase, amylase, and L-aminopeptidase) exhibited a pattern dependent on the presence of feed, showing a significant reduction during the hours of darkness (ZT-12 to ZT-24). Expression of the intestinal brush border enzyme (L-aminopeptidase) increased during the darkness period in anticipation of the feed ingestion associated with the subsequent light period. The cosinor analysis used to estimate the feed rhythms for all tested enzymes showed that activity in the intestine and pyloric caeca exhibited significant rhythmicity (p < 0.05). However, no rhythmicity was observed in the intestinal expression of maltase-glucoamylase. Our results demonstrate that some of the behavioral and digestive physiology features of totoaba directly respond to rhythmicity in feeding, a finding that should be considered when establishing optimized feeding protocols.

Effects of dietary supplementation of spray dried hog plum (Spondias pinnata (L.f.) Kurz) fruit powder on growth, digestive enzyme activity, and skin mucus immune parameters of climbing perch (Anabas testudineus (Bloch, 1972)

The effects of dietary supplementation of spray-dried hog plum (Spondias pinnata (L.f.) Kurz) fruit powder (SPP) on growth, digestive enzyme activity, and skin mucus immune parameters of climbing perch (Anabas testudineus (Bloch, 1972)) were examined. The fish (N = 330; 2.00±0.1 g) were assigned to five groups in triplicates. They were fed different SPP levels at 0, 5, 10, 15, and 20 g/kg diet for eight weeks. Flavonoids, terpenoids, phenolic compounds, tannins, steroids, glycosides, and glycosides were detected in SPP. The SPP containing diet significantly improved growth parameters (P &lt; 0.05) without a negative effect on the survival rate (P &gt; 0.05). Intestinosomatic and hepatosomatic indices significantly declined in the fish fed SPP added diets (P &lt; 0.05). There were significant elevations in digestive enzyme activities, amylase, protease, lipase, trypsin, pepsin, and esterase, in the tested fish compared to the control fish (P &lt; 0.05). Additionally, fish fed SPP containing diets showed a significant enhancement of total protein, immunoglobulin M, lysozyme, antiprotease, alkaline phosphatase, and esterase in the skin mucus compared to the control group (P &lt; 0.05). Significantly higher levels of superoxide dismutase, catalase, and total antioxidant capacity were detected in fish fed SPP containing diets compared to the control (P &lt; 0.05). These findings suggest that dietary SPP supplementation can effectively enhance fish growth, digestive enzyme activities, and mucosal immune parameters in the climbing perch. The suitable level of SPP observed in this present study was 13.00 – 15.88 g/kg diet.

Validation of Molecular Markers for Low Kunitz Trypsin Inhibitor Content in European Soybean (Glycine max L. Merr.) Germplasm.

Trypsin inhibitors (TI) in raw soybean grain, mainly represented by the Kunitz trypsin inhibitor protein (KTI), prevent the normal activity of the digestive enzymes trypsin and chymotrypsin in humans and monogastric livestock. The inactivation of TI is achieved through costly and time-consuming heat treatment. Thermal processing also impairs the solubility and availability of the soybean grain protein. Therefore, the genetic elimination of KTI has been proposed as a suitable alternative to heat treatment. The aim of this study was to screen the collection of European soybean cultivars with six genetic markers (one SSR marker and five SNP markers) previously proposed as tightly linked to the KTI3 gene encoding the major Kunitz trypsin inhibitor seed protein of soybean and validate their usability for marker-assisted selection (MAS). The six markers were validated on a subset of 38 cultivars with wide variability in KTI content and in the F2 and F3:5 progenies of two crosses between the known high- and low-KTI cultivars. Three genetic markers (SSR Satt228 and two SNP markers, Gm08_45317135_T/G and Gm08_45541906_A/C) were significantly associated with KTI content in a subset of 38 cultivars. Low-KTI alleles were detected in both low- and high-KTI genotypes and vice versa, high-KTI alleles were found in both high- and low-KTI genotypes, indicating a tight but not perfect association of these markers with the KTI3 gene. The genetic marker SSR Satt228 showed a significant association with KTI content in the F2 progeny, while the SNP markers Gm08_45317135_T/G and Gm08_45541906_A/C allowed significant discrimination between progeny with high- vs. low-KTI progenies in the F3:5 generation. These three markers could be applied in MAS for low-KTI content but not without the additional phenotyping step to extract the desired low-KTI genotypes.

High-Efficiency Degradation of PET Plastics by Glutathione S-Transferase under Mild Conditions.

Plastic pollution is a significant environmental concern globally. Plastics are normally considered chemically inert and resistant to biodegradation. Although many papers have reported enzyme-induced biodegradation of plastics, these studies are primarily limited to enzymes of microbial origin or engineered enzymes. This study reveals that poly(ethylene terephthalate) (PET, ∼6000 Da and 100 kDa) particles and plastic bottle debris (PBD, 24.9 kDa) can be efficiently degraded by a mammal-origin natural phase II metabolic isozyme, glutathione S-transferase (GST), under mild conditions. The degradation efficiency of PET plastics reached 98.9%, with a degradation rate of 2.6 g·L-1·h-1 under ambient or physiological conditions at 1 atm. PET plastics can be degraded by GST with varying environmental or biological factors (i.e., temperature, light irradiation, pH, and presence of humic acid or protein). We suggest a novel mechanism for PET degradation other than hydrolysis, i.e., the mechanism of cleavage and release of PET plastic monomers via nitridation and oxidation. This finding also reveals a novel function of GST, previously thought to only degrade small molecules (<1000 Da). This method has been successfully applied in real human serum samples. Additionally, we have tested and confirmed the ability to degrade PET of a mammal-origin natural digestive enzyme (trypsin) and a human-derived natural metabolic enzyme (CYP450). Overall, our findings provide a potential new route to plastic pollution control and contribute to our understanding of the metabolism and fate of plastics in organisms.

Efficient and rapid digestion of proteins with a dual-enzyme microreactor featuring 3-D pores formed by dopamine/polyethyleneimine/acrylamide-coated KIT-6 molecular sieve

The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6’s large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde’s spacing arm, reducing spatial hindrance and enhancing enzyme–substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.

First Feeding of Cuttlefish Hatchlings: Pioneering Attempts in Captive Breeding.

In the last few decades, the cuttlefish market has grown to approximately 14% of the world's fisheries, and operators have begun to express concerns about the decline of this resource. In this context, the production of cuttlefish through aquaculture could offer a diversifying and valuable response to the increasing market demand and help alleviate the environmental pressure on this species. Therefore, the aim of this study is to identify a dry, cost-effective, and easy-to-administer diet that can successfully support the initial phases of cuttlefish growth and provide a similar performance to a krill-based diet, which closely mimics their natural diet. To achieve this objective, cuttlefish hatchlings were distributed among different experimental tanks, each receiving one of the five different diets (namely Diets A to E). Mortality and morphological parameters were monitored until day 10 post hatching, and the two most effective diets (Diets A and B) were chosen for further trials. The results indicated that Diet B had similar survival and growth rates to Diet A, which was based on frozen krill. Histological analysis revealed a comparable degree of gut maturity between the organisms fed the two diets. Likewise, levels of amylase and trypsin enzymes and hsp70, cat, and sod mRNA did not exhibit significant differences between the two groups. In conclusion, our findings provide preliminary evidence supporting the possibility of cultivating cuttlefish in captivity using a pelleted diet, representing a promising starting point for larger-scale breeding efforts.

How D-amino acids embedded in the protein sequence modify its digestibility: Behaviour of digestive enzymes tested on a model peptide used as target

D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH2 (belonging to β-lactoglobulin, position 48–60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved.

Pyrazole-thiazole hybrids: Synthesis, computational studies and impact on digestive enzymes

In this study, the syntheses of some pyrazole-thiazole hybrids and their cyanoacrylate derivatives have been reported. 1H NMR, 13C NMR, LC-MS, and IR spectroscopy were used to characterize all the synthesized compounds. DFT studies have been employed to assess the electronic properties of the synthesized compounds. To ensure the necessary metabolization of the synthesized compounds, their safety profiles were assessed using the Vnn-ADMAT tool. Docking studies investigated the interactions between the synthesized compounds (6a-h and 8a-h) and the active sites of gastric enzymes α-Amylase, Trypsin, and Pepsin with binding affinities (kcal mol−1) ranging from 6.0 to 8.1, 6.7 to 8.5, and 7.7 to 8.6, respectively. Both pyrazole-thiazole hybrids and their cyanoacrylate derivatives demonstrated efficient anti-gastric activities. Varying levels of inhibition were observed among the analogs, such as compound 8f exhibiting the highest inhibitory activity at 61.93 % against α-Amylase at 10−7M, compound 6e showed best inhibition at 68.09 % against Trypsin at 10−7M, and compound 6c displayed remarkable inhibition at 69.51 % against Pepsin at 10−7M. The synthesized compounds exhibited selectivity for α-Amylase.

Beneficial effects of bioactive peptides extracted from Spirulina platensis and Gracilaria gracilis algae on bone regeneration/osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells are used in the treatment of many diseases, particularly in the repair of bone injuries. Algae with various medicinal applications are considered important natural resources. There is limited research on the effects of bioactive peptides from algae extraction on mesenchymal stem cells. In this study the impact of bioactive proteins, protein lysates and peptide fractions (<3, <30 and <50 kDa) isolated from two algae species, Spirulina platensis and Gracilaria gracilis on the proliferation and osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs) was investigated. The proteins were extracted ant hydrolyzed with trypsin enzyme to create peptides, which were then separated by ultrafiltration. hAMSCs were exposed to different concentrations of bioactive compounds (100, 300, 500 and 700 µg/ml) for varying time periods. Cell proliferation was assessed using the with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and to evaluate differentiation into bone tissue, the amount of mineral deposition was measured with alizarin red staining, and alkaline phosphatase enzyme activity was determined using a colorimetric method. The expression of Runx2, Osteocalcin, and β-Catenin genes expression was analyzed using RT-qPCR on days 7, 14 and 21 post-treatment. The results indicated that the <3 kDa peptide fraction of S. platensis and G. gracilis had no cytotoxic effects, increased cell proliferation at a concentration of 300 μg/ml, and enhanced the expression of osteogenic marker genes, alkaline phosphatase enzyme a activity, and calcium deposition in the extracellular matrix. In general, fractions that show positive effects on hAMSC differentiation have the potential to treat bone defects and promote osteoregeneration.

Discarded CHO cells as a valuable source of bioactive peptides for sustainable biotechnological applications

Repurposing discarded cells stands as a groundbreaking paradigm shift in sustainable biotechnology, with profound implications across diverse industrial sectors. Our study proposes a transformative concept by harnessing histone proteins from discarded CHO cells to produce bioactive peptides. We systematically isolated and hydrolyzed histones using Trypsin and Neutrase enzymes, optimizing reaction conditions. Ultrafiltration yielded distinct peptide fractions (<3 kDa and 3–10 kDa), which we analyzed for DPP-IV inhibition, antioxidant potential, and other activities. Furthermore, LC-Q-TOF-MS analysis and in silico tools unveiled the structural composition of bioactive peptides within these fractions. Three peptide sequences with high bioactivity potential were identified: KLPFQR, VNRFLR, and LSSCAPVFL. Our findings demonstrated exceptional DPP-IV inhibition, potent antioxidant effects, and effective anti-lipid peroxidation activities, surpassing reference compounds. Hemolytic activity assessment indicated promising biocompatibility, enhancing therapeutic application prospects. Pioneering the strategic repurposing of discarded cells, this research addresses cost-efficiency in cell-based studies and promotes sustainable use of biological resources across sectors. This novel approach offers an efficient, eco-friendly method for bioactive molecule procurement and resource management, revolutionizing cell culture studies and biotechnological applications.

Changes in Digestive Enzyme Activities during Larval Development of Spotted Seatrout (Cynoscion nebulosus)

The spotted seatrout (Cynoscion nebulosus)—an important commercial species—has a high potential for aquaculture in the Gulf of Mexico. To optimize its feeding during larval rearing, this study aims to evaluate the primary gastric (pepsin), intestinal (leucine aminopeptidase and alkaline phosphatase), and pancreatic (alkaline protease, trypsin, chymotrypsin, amylase, and lipase) enzyme activities from hatching to day 30. A multivariate analysis identified three digestive enzyme development stages during the spotted seatrout larval transformation. The first stage occurred between 1 (mean ± standard error (SE) = 1.73 ± 0.14 millimeter (mm) standard length (SL)) and 3 (2.14 ± 0.07 mm SL) days after hatching (DAH); a period of digestive stability showed the highest activity in amylase and bile salt-dependent lipase. The second stage (from 4 (2.53 ± 0.09 mm SL) to 20 (10.92 ± 0.51 mm SL) DAH) was a period of digestive transition, during which leucine aminopeptidase, chymotrypsin, and alkaline proteases were identified as the predominant enzymes from 4 to 5 DAH. In the third stage—a period of digestive stability—pepsin was the major enzyme that occurred between 25 (16.51 ± 0.81 mm SL) and 30 (25.91 ± 0.82 mm SL) DAH. These results indicate that the spotted seatrout larvae have a digestive system adapted to lipids and carbohydrates at the onset of feeding, with an immediate transition to protein digestion when exogenous feeding begins. Additionally, the digestive system of the spotted seatrout may be considered mature at 25 DAH. Further research is needed to elucidate the mechanisms of digestive tract development in the spotted seatrout larvae.

STUDY OF ANGIOTENSIN-I CONVERTING ENZYME INHIBITORY PEPTIDES DERIVED FROM Ficus pumila var. awkeotsang SEEDS HYDROLYSATE

Hypertension also known as high blood pressure that has associated with multi-metabolic pathways such as the renin-angiotensin-aldosterone system (RAAS). There have been several synthetic ACE inhibitory drugs used clinically as therapeutics for hypertension. However, synthetic drugs showed side effects on human health after long-term use. Therefore, alternative natural ACE inhibitors from food-derived are necessary to develop. The study aimed to examine the activity of angiotensin-I converting enzyme inhibitory peptides derived from Ficus pumila var. awkeotsang seeds hydrolysate and to study the efficiency of enzymatic digestion. This study used four enzymes (trypsin, pepsin, thermolysin, and chymotrypsin) to release bioactive peptides from Ficus pumila var. awkeotsang seeds that could exhibit an antihypertensive effect. The results showed that Ficus pumila var. awkeotsang trypsin hydrolysate has the highest ACEI activity, 90.37%, and the IC50 reached 169±3.5 µg/mL. Then, the hydrolysate produced by trypsin has the highest degree of hydrolysis, 4%, after 8 hours of incubation. Hence, Ficus pumila var. awkeotsang seeds hydrolysate could develop as an alternative natural product that is responsible for ACE-inhibitory activity.

Behavior of enzymatic membranes under pressure: Effect of enzyme location

Enzyme immobilized membranes combine catalysis and separation functions. Their application in large-scale continuous processes requires knowing the behavior under pressure. Also, the effects of enzyme location on the mass transfer limitation, membranes’ stability, and filtration performance should be investigated. In this study, urease (URE) and trypsin (TRY) enzymes were physically immobilized in/on the surface of a polyacrylonitrile (AN69) membrane through layer-by-layer (LbL) self-assembly method using polyethylenemine (PEI) and sodium-alginate (ALG) as cationic and anionic polyelectrolytes respectively. URE, located on the membrane’s surface, degraded urea in a reaction-controlled regime, and its immobilization did not significantly change the hydraulic permeability. On the other hand, the TRY enzyme attached to the membrane’s pores reduced the permeability and degraded the BAPNA in a diffusion-controlled region. In TRY immobilized membranes, the conversion increased linearly with the transmembrane pressure, while in URE immobilized ones, conversion was maximum at 1 bar. Sandwiching the enzymes between two polyelectrolytes resulted in the highest catalytic activities. This configuration maintained most of the URE activity in the long-term filtration, but it did not help prevent TRY’s activity loss.