Types Of Proteases Research Articles

Hydrolyzed proteins: availability, bioactivity, safety, applications in feed production

Recent years have witnessed a growing interest in the role of peptides in animal nutrition. These peptides are typically formed from ingested proteins in the gastrointestinal tract, but the types of resultant peptides can vary greatly with the physiological conditions of the animals and the composition of the diets. Therefore, feed manufacturers add hydrolysates obtained by enzymatic or bacterial hydrolysis of proteins from an animal or plant source to obtain peptides aimed at increasing the availability of easily digestible protein or peptides with specific biological activity, such as antioxidant or antimicrobial agents. The processing of agricultural crops, animals, poultry and fish in the food industry results in the formation of large amounts of secondary waste in solid or liquid form. These wastes are a good source of cheap proteins, which when used as raw materials in the hydrolysis process, are converted from useless waste into valuable ones. Enzymatic hydrolysis of proteins produces peptides and free amino acids, which are absorbed much faster than the original protein, due to the presence of specialized transporters in the intestinal mucosa. Also, depending on the type of protease, the peptides formed as a result of hydrolysis have beneficial biological effects on animal health. This article discusses the most important hydrolysates used in feed animal and their availability for absorption, as well as the bioactivity and safety of bioactive peptides for animal health.

Identification of potent TMPRSS4 inhibitors through structural modeling and molecular dynamics simulations

TMPRSS4, a transmembrane serine protease type II, is associated with various pathological illnesses. It has been found to activate SARS-CoV-2, enhance viral infection of human small-intestinal enterocytes and is overexpressed in different types of cancers. Therefore, this study aims to disover potential TMPRSS4 inhibitors that have better binding affinity than the approved inhibitors: 2-hydroxydiarylamide and tyroserleutide. Since no 3D-structure is known for TMPRSS4, structural models for the TMPRSS4 serine protease domain were developed. The modeled structures were validated and subjected to molecular dynamics simulations. FDA-approved, clinical/preclinical drugs and natural products were docked to the pocket of TMPRSS4. Moreover, through a systematic analysis, MD simulations and MM-GBSA binding free energy calculations revealed that the best candidates Ergotamine, S55746, NPC478048, Lifirafenib, and NPC77101 are highly stable drug candidates in complex with TMPRSS4, displaying low RMSD and RMSF values with strong binding stability. Among these compounds, Ergotamine showed the most favorable binding energy (-33.73 kcal/mol). Overall, our in silico results revealed that these compounds could act as potent TMPRSS4 inhibitors and need to be validated by future experimental studies.

The biological and psychological impact of the Coronavirus disease-19 pandemic on the characteristics of the menstrual cycle.

The Coronavirus disease-19 (COVID-19) pandemic was declared in March 2020 by the World Health Organization. The severe acute respiratory syndrome-coronavirus-2 virus enters host cells through angiotensin-converting enzyme 2 receptors and transmembrane serine protease type II that are expressed in pulmonary alveoli, as well as in hepatocytes, endothelium, ovaries, uterus, vagina, thyroid, and other tissues. In addition to viral injury, the COVID-19 pandemic, through protective measures such as social isolation and lockdown, has promoted a scenario of psychosocial stress, especially in women. In this context of isolation, anxiety, fear, and mental distress, there is dysregulation of the hypothalamic-pituitary-adrenal axis and subsequent gonadal side effects. Furthermore, studies report an association between COVID-19 and temporary menstrual cycle alterations such, as increased cycle duration, decreased cycle duration, increased menstrual flow, dysmenorrhea, and amenorrhea. Regarding COVID-19 vaccination, menstrual irregularities have been observed in about half of the women, predominantly with a decrease in cycle duration and increased menstrual flow, but without fertility sequelae. The aim of this study was to review the most up-to-date information on the relationship between the COVID-19 pandemic and menstrual irregularities.

Alkaline Proteases from Rose Snapper (Lutjanus guttatus): Evaluation of Their Stability to Chemical Denaturants and Potential Application to Hydrolyze Seafood Waste Proteins.

Large quantities of by-products are generated after processing of rose snapper (Lutjanus guttatus), such as viscera, head, tail, skin, and bones, which are considered a potential source of valuable molecules. Therefore, the aim of the present study was the biochemical characterization of alkaline proteases isolated from the intestines of L. guttatus and the evaluation of their stability against different chemical denaturants (salts, surfactants/reducing agents, organic solvents, and commercial detergent formulations). In addition, the efficiency to hydrolyze proteins from rose snapper wastes (head, tail, skin, and muscle trimmings) by Alcalase® and alkaline protease extract (APE) isolated from Lutjanus guttatus intestine was evaluated. The APE exhibited a maximum activity at pH 12 and 45°C and high stability at pH and temperature ranges from 9 to 12 and 10 to 40°C, respectively. Assays with specific protease inhibitors indicated that trypsin and chymotrypsin are the main types of proteases in APE. An 80% of the proteolytic activity was retained in the presence of 25% NaCl and was stable in the presence of the reducing agent DTT; however, it lost around 70% of proteolytic activity in the presence of 2-mercaptoethanol. The enzymatic activity of APE was maintained above 60% in methanol, ethanol, and propanol as well as in liquid commercial detergents. Alkaline proteases from rose snapper exhibited higher hydrolytic efficiency, compared to the microbial enzyme Alcalase when protein from L. guttatus wastes were hydrolyzed. According to these results, the integral exploitation of rose snapper could be reached by proper usage of its by-products, creating a baseline to promote circular economy.

Effect of Fuzheng Tongluo Granules on macrophage pyroptosis in rat model with pulmonary fibrosis based on NLRP3/caspase-1/GSDMD pathway

To investigate the therapeutic effect of Fuzheng Tongluo Granules on idiopathic pulmonary fibrosis(IPF) and its mechanism. Seventy-two SD rats were randomly divided into the control group, model group, pirfenidone group(162 mg·kg~(-1)), and low-, medium-and high-dose of Fuzheng Tongluo Granules groups(2.63, 5.25, 10.5 g·kg~(-1)). Rat model of IPF was induced by a single non-invasive tracheal intubation drip of bleomycin(BLM). The corresponding drugs were given daily by gavage after the 2nd day of modeling, and body mass was recorded. On the 28th day, the samples were collected and weighed, and the lung coefficients were calculated. The pathological changes in the lung tissue were observed by HE and Masson staining, and the hydroxyproline(HYP) content of the lung tissue was detected by alkaline hydrolysis. The contents of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-18(IL-18) of the lung tissue were determined by ELISA. The expression of collagen type Ⅰ(collagen Ⅰ) and α-smooth muscle actin(α-SMA) was observed by immunohistochemistry. The expression levels of NOD-, LRR-and pyrin domain-containing 3(NLRP3), cysteine-requiring aspartate protease type 1(caspase-1), gasdermin D-N(GSDMD-N), and apoptosis-associated speck-like protein containing a CARD(ASC) in the lung tissue were detected by Western blot. Immunofluorescence co-localization was used to observe the expression of GSDMD and CD68. The results show that compared with the control group, the model group showed increased lung coefficient, Ashcroft score, Szapiel score, HYP, TNF-α, IL-1β, and IL-18 content in the lung tissue and elevated protein expression levels of NLRP3, caspase-1, GSDMD-N, and ASC. The expression levels of GSDMD and CD68 were increased, and there was a high degree of co-localization between GSDMD and CD68. Compared with those in the model group, the lung coefficient, Ashcroft score, and Szapiel score decreased in all drug administration groups, and the content of HYP, TNF-α, IL-1β, and IL-18 decreased. The protein expression levels of NLRP3, caspase-1, GSDMD-N, and ASC decreased, and the expression levels of GSDMD and CD68 were reduced. There was a high degree of co-localization between GSDMD and CD68. In summary, Fuzheng Tongluo Granules can effectively reduce pulmonary fibrosis and inflammation levels in rats with IPF, and the mechanism may be related to the down-regulation of the NLRP3/caspase-1/GSDMD pathway to inhibit macrophage pyroptosis.

In silico characterization, phylogeny and molecular docking of the Bowman-Birk type protease inhibitor in species from Phaseoleae tribe

In silico characterization, phylogeny and molecular docking of the Bowman-Birk type protease inhibitor in species from Phaseoleae tribe

A novel alkali and thermotolerant protease from Aeromonas spp. retrieved from wastewater

Enzymes are integral to numerous industrial processes, with a growing global demand for various enzyme types. Protease enzymes, in particular, have proven to be cost-effective, stable, and compatible alternatives to traditional chemical processes in both industrial and environmental applications. In this study, an alkaline protease-producing strain of Aeromonas spp. was isolated from a wastewater treatment plant in Iran. The protease production was confirmed by culturing the strain on casein agar medium. The bacterium was identified through morphological, biochemical, and 16 S rRNA sequencing analyses. The optimal culture medium for bacterial growth and enzyme production was obtained using peptone, salt, yeast extract, galactose, and CaCl₂ at an initial pH of 8. Maximum protease production was achieved after 20 h of incubation at 40 °C. To partially purify the enzyme, the supernatant of the bacterial culture medium was first centrifuged, and the enzyme was precipitated using ammonium sulfate, followed by dialysis. Zymography revealed the production of one type of protease during bacterial growth. The partially purified protease exhibited optimal activity at pH 8.5 and maximum stability at pH 9. The optimum temperature for maximum enzyme activity was observed at 50 °C, with 100% residual activity retained for 1 h at 0 °C. The effect of metal ions on enzyme activity was assessed, revealing that KCl induced the most significant effects (p < 0.0001) on enzyme activity. Chemical amino acid modifiers and inhibitors, such as EDTA, DEPSI, and IAA, did not exhibit significant inhibition. In contrast, PMSF and HNBB significantly (p < 0.0001) reduced enzyme activity, suggesting that the enzyme could be classified as a serine protease. The protease also demonstrated high stability in the presence of 2% SDS, showing no signs inactivation. The alkaline pH optimum, thermal stability, and resistance to SDS exhibited by the protease produced by the Aeromonas strain are particularly promising characteristics that warrant further investigation. Based on preliminary tests and the enzyme’s characteristics, this protease can be recommended for various applications, pending further studies.

Naphthalen-1-ylethanamine–containing small molecule inhibitors of the papain-like protease of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has not yet been eradicated. SARS-CoV-2 has two types of proteases, a main protease (Mpro) and a papain-like protease (PLpro), which together process two translated non-structural polyproteins, pp1a and pp1ab, to produce functional viral proteins. In this study, effective inhibitors against PLpro of SARS-CoV-2 were designed and synthesized using GRL-0048 as a lead. A docking simulation of GRL-0048 and SARS-CoV-2 PLpro showed that GRL-0048 noncovalently interacts with PLpro, and there is a newly identified binding pocket in PLpro. Structure-activity relationship studies were next performed on GRL-0048, resulting in the development of several inhibitors, specifically compounds 1, 2b, and 3h, that have more potent inhibitory activity than GRL-0048.

Isolation of Bacillus altitudinis 5-DSW with Protease Activity from Deep-Sea Mineral Water and Preparation of Functional Active Peptide Fractions from Chia Seeds.

In this study, we successfully isolated Bacillus strains with high protease activity from deep-sea mineral water in Korea and used them to obtain functional peptide fractions from chia seeds. The obtained Bacillus strains showed a high similarity of 99% with B. altitude with a long rod type (named B. altitudinis 5-DSW) and high protease activity at 40 °C, and 70% of the activity remained even at 70 °C. The defatted chia seed protein (15-50 kDa) was treated with crude protease from B. altitudinis 5-DSW and digested into small peptides below 20 kDa. The obtained chia seed peptides showed 3 times and 1.5 times higher antioxidant activity in DPPH and ABT radical scavenging assays, respectively. Moreover, chia seed peptides showed enhanced AChE inhibitory activity with an IC50 value of 14.48 ± 0.88 μg/mL and BChE inhibition activity with an IC50 value of 10.90 ± 0.80 μg/mL. Our results indicate that the newly isolated B. altitudinis 5-DSW and chia seed protein hydrolysates have potential applications in biotechnology and functional food development, enhancing the nutritional quality and value-added utilization of chia byproducts.

Engineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis

Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.

A new Bowman-Birk type protease inhibitor regulated by MeJA pathway in maize exhibits anti-feedant activity against the Ostrinia furnacalis.

Jasmonic acid (JA), an important plant hormone, plays a crucial role in defending against herbivorous insects. In this study, we have identified a new Bowman-Birk type protease inhibitor (BBTI) protein in maize that is regulated by the JA pathway and exhibits significant antifeedant activity, which is notably induced by exogenous Methyl Jasmonate and Ostrinia furnacalis feeding treatments. Bioinformatics analysis revealed significant differences in the BBTI protein among different maize inbred lines, except for the conserved domain. Prokaryotic and eukaryotic expression systems were constructed and expressed, and combined with bioassays, it was demonstrated that the antifeedant activity of BBTI is determined by protein modifications and conserved domains. Through RT-qPCR detection of BBTI and JA regulatory pathway-related genes' temporal expression in different maize inbred lines, we identified the regulatory mechanism of BBTI synthesis under the JA pathway. This study successfully cloned and identified the MeJA-induced anti-feedant activity gene BBTI and conducted functional validation in different maize inbred lines, providing valuable insights into the response mechanism of insect resistance induced by the plant JA pathway. The increased expression of the anti-feedant activity gene BBTI through exogenous MeJA induction may offer a potential new strategy for mediating plant defense against Lepidoptan insects.

Association between TMPRSS2 rs2070788 polymorphism and COVID-19 severity: a case-control study in multiple cities of Iran.

Host genetic variations have been identified as potential influencers of COVID-19 infection. This study aimed to examine the association between transmembrane serine protease type 2 (TMPRSS2) rs2070788 single nucleotide polymorphism (SNP) and the prognosis of COVID-19 in Iranian populations. This case-control study was performed on 756 COVID-19 patients and 59 healthy individuals across Iran. Clinical data, blood samples, and the presence of the TMPRSS2 rs2070788: G>A SNP were determined using T-ARMS-PCR. Additionally, serum levels of tumor necrosis factor α (TNF-α), C-reactive protein (CRP), interleukin-6 (IL-6), and IL-1β were evaluated in the collected blood samples. No significant association was found between the genotypes and allele frequencies of TMPRSS2 rs2070788 SNP and susceptibility to or mortality from COVID-19 infection. However, we observed a substantial increase in IL-6 and CRP levels associated with the severity of COVID-19, while no such trend was observed for IL-1β and TNF-α. This study showed a considerable rise in TNF-α and IL-1β serum levels exclusively in COVID-19 patients with TT rs2070788 TMPRSS2 SNP genotype compared to healthy controls. In this study conducted across multiple cities in Iran, no significant association was found between the TMPRSS2 rs2070788 SNP genotypes and COVID-19 severity or mortality.

Combined effects of gelatin extraction methods and hydrolysis protease types on the functional properties of tilapia scale gelatin hydrolysates

SummaryThe combined effects of gelatin extraction methods and protease types on the functional properties of tilapia scale gelatin hydrolysates (TSGH) were investigated. The tilapia scale gelatin (TSG) was extracted after acid/base pretreatments, respectively, and then hydrolysed with different proteases to produce TSGHs. The degree of hydrolysis and the content of trichloroacetic acid soluble peptide were increased with hydrolysis time, while the content of β‐sheet was decreased significantly with the hydrolysis time (P &lt; 0.05). The emulsification stability of acid protease hydrolysate (APH) was higher than that of neutral protease hydrolysate (NPH) and alkaline protease hydrolysate (ALPH). All samples showed good resistance to oxidation: DPPH (20%–64.66%), ABTS (29.08%–63.04%), and iron reducing power (0.017–0.499). The hydrolysates obtained from the base‐pretreated TSG showed higher antioxidant properties than acid‐pretreated TSG. The antioxidants of different protease hydrolysates are ranked by ALPH &gt; NPH &gt; APH. In conclusion, gelatin extraction methods and its hydrolysis conditions had influences on the functional properties of TSGH, and the study of the processing of marine biological wastes is of great significance in realising their high‐value utilisation and reducing the burden on the environment.

Aprotinin (I): Understanding the Role of Host Proteases in COVID-19 and the Importance of Pharmacologically Regulating Their Function.

Proteases are produced and released in the mucosal cells of the respiratory tract and have important physiological functions, for example, maintaining airway humidification to allow proper gas exchange. The infectious mechanism of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), takes advantage of host proteases in two ways: to change the spatial conformation of the spike (S) protein via endoproteolysis (e.g., transmembrane serine protease type 2 (TMPRSS2)) and as a target to anchor to epithelial cells (e.g., angiotensin-converting enzyme 2 (ACE2)). This infectious process leads to an imbalance in the mucosa between the release and action of proteases versus regulation by anti-proteases, which contributes to the exacerbation of the inflammatory and prothrombotic response in COVID-19. In this article, we describe the most important proteases that are affected in COVID-19, and how their overactivation affects the three main physiological systems in which they participate: the complement system and the kinin-kallikrein system (KKS), which both form part of the contact system of innate immunity, and the renin-angiotensin-aldosterone system (RAAS). We aim to elucidate the pathophysiological bases of COVID-19 in the context of the imbalance between the action of proteases and anti-proteases to understand the mechanism of aprotinin action (a panprotease inhibitor). In a second-part review, titled "Aprotinin (II): Inhalational Administration for the Treatment of COVID-19 and Other Viral Conditions", we explain in depth the pharmacodynamics, pharmacokinetics, toxicity, and use of aprotinin as an antiviral drug.

Effect of ‘Procumbenase’ a serine protease from Tridax procumbens aqueous extract on wound healing: A scar free healing of full thickness wounds

Wound healing involves several cellular and molecular pathways. Tridax procumbens activates genetic pathways with antibacterial, antioxidant, anticancer, and anti-inflammatory properties, aiding wound healing. This study purified Procumbenase, a serine protease from T. procumbens extract, using gel filtration (Sephadex G-75) and ion exchange (CM-Sephadex C-50) chromatography. Characterization involved analyses of protease activity, RP-HPLC, SDS-PAGE, gelatin zymogram, PAS staining, mass spectrometry, and circular dichroism. Optimal pH and temperature were determined. Protease type was identified using inhibitors. Wound-healing potential was evaluated through tensile strength, wound models, hydroxyproline estimation, and NIH 3T3 cell scratch analysis. In incision wound rat models, Procumbenase increased tensile strength on day 14 more than saline and Povidone‑iodine. It increased wound contraction by 89 % after 10 days in excision wound models, attaining full contraction by day 15 and closure by day 21. Scarless wound healing was enhanced by 18 days of epithelialization against 22 and 21 days for saline and povidone‑iodine. Procumbenase increased hydroxyproline concentration 2.53-fold (59.93 ± 2.89 mg/g) compared to saline (23.67 ± 1.86 mg/g). In NIH 3 T3 cell scratch assay, Procumbenase increased migration by 60.93 % (50 μg) and 60.57 % (150 μg) after 48 h. Thus, Procumbenase is the primary bioactive molecule in T. procumbens, demonstrates scar-free wound healing properties.

New Method of Obtaining Proteolytic Enzymatic Preparation from Fungal Strain Fusarium gibbosum CNMN FD 12

Proteases, that are able to hydrolyze the peptide bonds in proteins and polypeptides, occupy a key position in diverse fields, such as food, agriculture, animal feed. Considering the increasing demand for microbial proteases, identification of new way for improving the productivity of producers is of great importance. The aim of the present study was to evaluate the effect of a new coordination compound [Fe(H2L)(H2O)2](NO3)3∙2.5H2O (where H2L = 2,6-diacetylpyridine bis(picolinoylhydrazone)) on the activity of proteolytic complex synthesized by the micromycete Fusarium gibbosum CNMN FD 12 under the submerged fermentations in shake flask, as well as in lab-scale fermenter. It was found that [Fe(H2L)(H2O)2](NO3)3∙2.5H2O exerts a significant positive effect on proteolytic activity in F. gibbosum CNMN FD 12 both in flasks and bioreactor and can be used as stimulator and regulator of proteases activity. By varying the concentration of the coordination compound and the duration of cultivation, it is possible to obtain proteolytic complexes enriched in different types of proteases (acid, neutral or alkaline), depending on the practical needs.

Characterization and evaluation of the in vitro and in vivo anti-diabetic activities of camel milk protein hydrolysates derived with different protease digestions

The study aimed to determine the potential anti-diabetic activities of the hydrolysates of camel milk proteins (CMPH). Camel milk proteins were hydrolyzed independently by using 11 proteases, and their anti-diabetic activities were analyzed by in vitro, and in vivo. The results showed that CMPH-Flavourzyme exhibited the best α-glucosidase and dipeptidyl peptidase-IV inhibitory activity, while CMPH-Papain exhibited the highest α-amylase inhibitory activity, and best proliferative effect on streptozotocin-induced NIT-1 cells in vitro. Furthermore, CMPH-Papain exhibited the most effect in decreasing the serum levels of fasting blood glucose, oral glucose tolerance test, pro-inflammatory factors (IL-1β, IL-6) and triglyceride, as well as increasing insulin levels on in vivo streptozotocin-induced diabetic mice. Peptides identification followed with molecular docking and network pharmacology analysis also found the potential anti-diabetic peptides from CMPH-Flavourzyme and CMPH-Papain. Our results indicated the type of protease used for the treatment significantly influences the anti-diabetic activities of the resulting CMPH.

PSII-17 Effect of protease supplementation on amino acids digestibility of soybean meal fed to growing pigs of different ages

Abstract Protease supplementation can increase amino acids (AA) availability; however, the effectiveness of protease in swine diets varies and has been associated with the type and dose of protease, dietary ingredients, and potentially animal age. This study investigated the interactive effect of protease inclusion level and pig age on standardized ileal digestibility (SID) of protein (CP) and AA of soybean meal (SBM) fed to growing pigs. A corn starch–based diet was formulated with SBM as the sole source of AA and containing titanium as an indigestible marker. Protease was supplemented at 0, 15,000; 30,000 and 45,000 NFP/kg of feed (ProAct 360). An N-free diet was used to estimate basal ileal endogenous AA losses. Ten cannulated pigs (body weight = 21 kg ± 2 kg) were assigned to experimental diets in a duplicate 5 × 5 Latin square design. In Phase I (23 to 30 kg-pigs, 90 ± 17 d of age), ileal digesta was collected in 5 periods of 7 d (5 d adaptation and 2 d ileal digesta collection). In Phase II (50 to 65 kg-pigs. 140 ± 17 d of age), using the same diets, ileal digesta was collected in 5 more periods of 7 d. Pigs were fed at 4% of body weight (BW) adjusted at the end of each period. Diets and ileal digesta were analyzed for CP, AA, and titanium and SID was calculated. Orthogonal polynomial contrasts were used to determine the linear and quadratic effects of protease supplementation. The combined data were analyzed as 4 x 2 factorial, including the fixed effects of protease inclusion (excluding N-free diet), age, and their interactions. In phase I, increasing levels of protease resulted in linear improved (P &amp;lt; 0.1) of SID for the majority of indispensable AA (Except Arg, His, Met + Cys, and Trp) and the average of dispensable AA. In phase II, the SID of Ile, Leu, Met, Met + Cys, Val, the average of indispensable AA, and the majority of dispensable AA was quadratically improved. Overall (Phase I and II), the SID of Ile, Leu, Met + Cys, Val, Cys, Glu, Tyr, and the average of dispensable AA was quadratically improved (P &amp;lt; 0.1). In most cases, the supplementation of 30,000 NFP/kg of feed resulted in the greatest AA digestibility improvements. However, the linear response in the first phase for some AA suggests that diets for younger pigs could be supplemented with a greater level (45,000 NFP/kg of feed). There were no interactions between protease levels and age. Younger pigs had improved (P &amp;lt; 0.1) SID of CP and most AA (Except Cys, Gly, and Ser). In conclusion, dietary protease supplementation can improve SID of AA in soybean mean independent of pig age. Pigs age can influence AA digestibility where younger pigs have greater AA digestibility.

Effect of thermal pretreatment and gastrointestinal digestion on the bioactivity of dry-cured ham bone enzymatic hydrolyzates

This study reports the effect of thermal pretreatment and the use of different commercial proteolytic enzymes (Protamex, Flavourzyme, Protana prime, and Alcalase) on the free amino acid content (FAA), peptide profile, and antioxidant, antidiabetic, antihypertensive, and anti-inflammatory potential (DPPH, FRAP, and ABTS assay, DPP-IV, ACE-I, and NEP inhibitory activities) of dry-cured ham bone hydrolyzates. The effect of in vitro digestion was also determined. Thermal pretreatment significantly increased the degree of hydrolysis, the FAA, and the DPP-IV and ACE-I inhibitory activities. The type of peptidase used was the most significant factor influencing antioxidant activity and neprilysin inhibitory activity. Protana prime hydrolyzates failed to inhibit DPP-IV and neprilysin enzymes and had low values of ACE-I inhibitory activity. After in vitro digestion, bioactivities kept constant in most cases or even increased in ACE-I inhibitory activity. Therefore, hydrolyzates from dry-cured ham bones could serve as a potential source of functional food ingredients for health benefits.

Integrated machine learning-based virtual screening and biological evaluation for identification of potential inhibitors against cathepsin K.

Cathepsin K is a type of cysteine proteinase that is primarily expressed in osteoclasts and has a key role in the breakdown of bone matrix protein during bone resorption. Many studies suggest that the deficiency of cathepsin K is concomitant with a suppression of osteoclast functioning, therefore rendering the resorptive properties of cathepsin K the most prominent target for osteoporosis. This innovative work has identified a novel anti-osteoporotic agent against Cathepsin K by using a comparison of machine learning and deep learning-based virtual screening followed by their biological evaluation. Out of ten shortlisted compounds, five of the compounds (JFD02945, JFD02944, RJC01981, KM08968 and SB01934) exhibit more than 50% inhibition of the Cathepsin K activity at 0.1μM concentration and are considered to have a promising inhibitory effect against Cathepsin K. The comprehensive docking, MD simulation, and MM/PBSA investigations affirm the stable and effective interaction of these compounds with Cathepsin K to inhibit its function. Furthermore, the compounds RJC01981, KM08968 and SB01934 are represented to have promising anti-osteoporotic properties for the management of osteoporosis owing to their significantly well predicted ADMET properties.