Protease Enzyme Research Articles

Phenotypic, Molecular Identification and Virulence Assessment of Cronobacter spp Isolated from Clinical Samples of Children Under Two Years in Mosul City/Iraq

Cronobacter spp. is known to be a foodborne causative agent for a variety of diseases in both humans and animals. This study focused on isolating Cronobacter species from 150 clinical samples of children under two years (60 from blood and stool samples, 30 from CSF samples) collected from Ibn al Atheer and Al Khanssa Hospitals. The phenotypic identification of bacterial isolates were performed through culture, microscopic, and biochemical examinations and vitek-2 system. The results revealed a mixture of bacteria in 6.5% of the clinical specimens. The identity of isolates was 99% using the vitek-2 system for identifying Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, and Cronobacter pulveris. Out of 150 specimens there were 8 (5.33 %) of specimens gave positive for Cronobacter spp which included: C. sakazaki 2/60 blood specimens (3.3%), C. sakazaki 2/60 stool specimens (3.3%), C. malonaticus 2/60 stool specimens (3.3%), 1/60 C. muytjensii and C. pulveris 1/60 (3.3%). The eight isolates phenotypically identified as Cronobacter were confirmed at the molecular level through 16S rRNA sequencing and submitted to NCBI under the accession numbers (OR825874, OR825875, PP126443, PP126444, PP126445, PP126455). The virulence profile of these isolates showed that 7/8 (87.5%) of Cronobacter strains exhibited haemolysin activity, 5/8 strains (62.5%) were able to produce the protease enzyme and 2/8 (25%) of Cronobacter strains were positive for lipase and lecithinase. All strains lacked the ability to produce a slime layer. The results also showed the ability of Cronobacter strains to produce biofilm by using the tube method and microtiter plate method but with different levels.

Growth performance and enzymatic activities in monosex tilapia (Oreochromis niloticus) supplemented with Najas indica along with the compound identification of the extracts.

Recent research has looked at various macroalgae species as dietary components or feed additives for a variety of fish species due to their nutritional value. The objective of this study was to examine the impact of Najas indica, a macroalgae extract, on the growth performance, proximate composition, and metabolic activities of monosex tilapia (Oreochromis niloticus), while also isolating the compounds present. Three distinct solvents (n-hexane, ethyl acetate, and ethanol) were used to extract bioactive compounds from a coarse powder of macroalgae after drying and grinding, and gas chromatography-mass spectrometry (GC-MS) analysis was used to detect bioactive compounds. The extracts were combined with commercial feed (0.4%) and applied to the treatment with three replications and a control containing 50 fingerlings per tank for 5 weeks. The findings indicated a significant increase in the final weight, weight gain, specific growth rate (SGR), and survival rate among the treated fish, whereas the feed conversion ratio (FCR) was observed to decrease in comparison to the control group. Significantly higher levels of protein and lipids were found in treated fish, whereas moisture and ash levels were significantly lower compared to control fish. In treated fish, the digestive enzyme amylase was significantly higher, but the protease enzyme reduced significantly. The antioxidant enzyme superoxide dismutase activity (SOD) was significantly higher in the treatment group, whereas the catalase (CAT) enzyme did not differ significantly. A total of 47 bioactive compounds were identified in N. indica, among which the prominent compounds included n-hexadecanoic acid, neophytadiene, phytyl palmitate, d-mannitol, and heptanoic acid. The results obtained from this study indicate that the utilization of N. indica macroalgae extract has the potential to serve as an additional dietary component, therefore, enhancing the growth performance and metabolic functions of fish.

Antioxidant peptides from silver carp steak by alkaline protease and flavor enzyme hydrolysis: Characterization of their structure and cytoprotective effects against H2O2-induced oxidative stress.

Silver carp steak is a rarely utilized silver carp processing byproduct. This study aimed to optimize a dual enzymatic method to extract antioxidant peptide components from silver carp steak and characterize their structure and in vitro antioxidant activity through ultrafiltration purification, response surface methodology, molecular docking, and radical scavenging activity analysis. The optimal extraction conditions for silver carp steak antioxidant peptides (SCSAP) were determined as 1:6 solid-liquid ratio, 1500 U/g alkaline protease addition, 4 h alkaline protease hydrolysis time, 1946 U/g flavor enzyme addition, and 2.5 h flavor enzyme hydrolysis time. The <3 kDa SCSAP component (SCSAP-3kDa) showed the strongest antioxidant activity, with its 1,1-diphenyl-2-trinitrophenyl hydrazine (DPPH) radical scavenging rate, ABTS radical scavenging rate, hydroxyl radical scavenging rate, metal ion chelating rate, and reducing capacity reaching 88.75%, 91.21%, 67.02%, 69.07%, and 0.985, respectively. Moreover, the three peptides (PF-7, GP-8, and YF-10) of 100 µg/mL could protect HepG2 cells from oxidative stress damage by reducing the oxidative damage level and activating Keap1-Nrf2-ARE pathways, enabling an increase of superoxide dismutases (SOD) activity, and a decrease of malondialdehyde (MDA) content and reactive oxygen species (ROS) level. The integrated results indicate the enormous potential of SCSAP-3kDa as a functional food ingredient in the food industry. PRACTICAL APPLICATION: This study selected the antioxidant capacity of silver carp steak peptides as the index and developed a facile dual enzymatic hydrolysis method to obtain three antioxidant peptides (PF-7, GP-8, and YF-10) with biological activity, providing a theoretical basis for bioavailability of antioxidant peptides from silver carp steak and contributing to their application in new functional foods.

Optimizing extraction of collagen from hazardous leather shaving dusts by crude protease enzyme: Chrome recovery and reuse for leather rechroming

This study explores an eco-friendly method to achieve 99.98 % dechroming (reducing Cr from 7700 to 4.71 ppm) of Chrome-tanned leather shaving dust (CSD), through enzymatic hydrolysis using crude protease. The enzyme exhibited strong activity (>40 U mL−1) across a temperature range of 50 °C to 65 °C and a pH range of 5 to 11. The pH of the mixture containing CSD, water, MgO, and enzyme was maintained at 8.9 during hydrolysis. The temperature, hydrolysis time, and enzyme concentration were optimized using response surface methodology (RSM). Under optimal conditions of 17.57 % enzyme concentration, 62.01 °C, and a hydrolysis time of 7 h, the predicted collagen yield was 31.66 %. Laboratory experiments conducted with these predicted parameters resulted in a collagen recovery of 30.9 %, achieving an accuracy of 97.6 % compared to the predictions. In contrast, hydrolysis with MgO alone achieved only 87.01 % dechroming with about 2 % collagen yield, highlighting the enzyme's superior effectiveness. The enzymatic cleavage of peptide bonds in the leather matrix enhanced collagen solubilization. Characterization techniques, including FT-IR, NMR, XRD, DSC, and TGA, confirmed successful extraction and purity, with the collagen being rich in glycine and proline. Cytotoxicity results demonstrated its non-toxic nature, indicating potential applications in biomedical fields, tissue engineering, food, pharmaceuticals, and cosmetics. The recovered chromium sulfate was reused for rechroming goat wet blue leather, with chrome content in crust leather treated with recovered basic chromium sulfate (rBCS) and fresh basic chromium sulfate (BCS) being 3.23 % and 3.35 %, respectively, both meeting industry standards for goat crust leather. SynopsisAn eco-friendly method for dechroming chrome-tanned leather shaving dust (CSD) and extracting collagen was established. The process achieved 99.98 % dechroming and 30.9 % collagen yield using crude protease enzyme. Characterization confirmed the collagen's purity, and recovered chromium sulfate was reused for rechroming goat leather, meeting industry standards.

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.

Identifying Novel Inhibitors for Dengue NS2B-NS3 Protease by Combining Topological similarity, Molecular Dynamics, MMGBSA and SiteMap Analysis.

DENV NS2B-NS3 protease inhibitors were designed based upon the reference molecule, 4-(1,3-dioxoisoindolin-2-yl)-N-(4-ethylphenyl) benzenesulfonamide, reported by our team with the aim to optimize lead compound via rational approach. Top five best scoring molecules with zinc ids ZINC23504872, ZINC48412318, ZINC00413269, ZINC13998032 and ZINC75249613 bearing 'pyrimidin-4(3H)-one' basic scaffold have been identified as a promising candidate against DENV protease enzyme. The shape and electrostatic complementary between identified HITs and reference molecules were found to be Tanimotoshape 0.453, 0.690, 0.680, 0.685 & 0.672 respectively and Tanimotoelectrostatic 0.211, 0.211, 0.441, 0.442, 0.442 and 0.442 respectively. The molecular docking studies suggested that the identified HITs displayed the good interactions with active site residues and lower binding energies. The stability of docked complexes was assessed by MD simulations studies. The RMSD values of protein backbone (1.6779, 3.1563, 3.3634, 3.3893 & 3.0960 Å) and protein backbone RMSF values (1.0126, 1.0834, 1.0890, 0.9974 & 1.0080 Å respectively) for all top five HITs were stable and molecules did not fluctuate from the active pocket during entire 100ns MD run. The druggability Dscore below 1 indicate the tightly binding of ligand at the active site. Dscore for ZINC23504872 was found to be 1.084 while for the second class of compounds ZINC48412318, ZINC00413269, ZINC13998032 and ZINC75249613, 0.503, 0.484, 0.487 and 0.501 Dscores were observed. In-silico ADMET calculations suggested that all five HITs were possessed the drug likeliness properties and did not violate the Lipinski's rule of five. Summing up, these in-silico generated data suggested that the identified molecules bearing pyrimidin-4(3H)-one would be promising scaffold for DENV protease inhibitors. However, experimental results are needed to prove the obtained results.

Characterization of alkaline protease enzyme produced from marine yeast Candida orthopsilosis AKB-1 and its applications.

The present study has undertaken the isolation of marine yeasts from mangrove sediment samples and their ability to produce alkaline protease enzymes. A total of 14 yeast isolates were recovered on yeast-malt agar (YMA) and yeast extract peptone dextrose (YEPD) agar medium. After screening for proteolytic activity on skim milk agar, marine yeast isolate, AKB-1 exhibited a hydrolysis zone of 18mm. Optimal conditions for the enzyme production from yeast isolate AKB-1 were at 30°C, pH 8, fructose as carbon source, potassium nitrate as nitrogen source, and 25% saline concentration. Under the optimal conditions, the protease enzyme activity of the isolate AKB-1 was observed to be 978IU/mL. The structural and functional analysis was carried out through FTIR and HPLC analysis for the extracted protease enzyme. Furthermore, the enzyme produced was partially purified by solvent extraction using ethyl acetate and ammonium sulfate precipitation (3.4-fold) followed by dialysis (56.8-fold). The molecular weight of the purified enzyme was observed to be around 60kDa using SDS-PAGE. The extracted protein showed good antibacterial activity against six different clinical bacterial pathogens and the highest against Bacillus cereus (16 ± 0.5mm). The extracted protease enzyme was revealed to remove blood stains from cloth within 20min of application similar to the commercial detergent. The marine yeast isolate was further identified as Candida orthopsilosis AKB-1 (Accession number KY348766) through 18S rRNA sequencing, and a phylogenetic tree was generated.

Synergistic Effect of Purified Protease Enzyme with Antibiotics on Pathogenic S. Aureus In-Vitro and In-Vivo

Objective: The goal of this study is the detection of the P. aeruginosa isolate for production the protease, the optimal conditions for produce, extraction, purification and characterization of the enzyme and study the synergistic effect of protease with antibiotics on S. aureus. Method: Among (110) isolates from clinical sources, only (74) isolates identified as P. aeruginosa isolated from Baghdad hospitals. Detection of the optimal isolate for production the protease enzyme and the optimal conditions its produce, purification of enzyme by using ammonium sulfate, ion exchange chromatography and gel filtration, enzyme characterization of PH and temperature activity and stability, the study of synergistic effect of protease with antibiotics on S. aureus. Results: The optimal conditions to produce protease is pH (8), (37) ºC, (BHI) broth, and during (48 hrs.). The precipitation saturation ratio (80%). Ion- exchanges (DEAE and CM) Cellulose and Gel filtration have specific activity ((121.25), (161.6), (660.53)) units/ mg)) respectively. The characterization done by pH and temperature activity purified protease was active in (138.51units/ ml) at pH (8), and stable in pH (8), the temperature for protease activity is (158.21 units/ ml) at (37) ºC. The synergistic effect of purified protease with antibiotics on S.aureus in- vitro,was detected using the agar diffusion method, the effectiveness of the prepared hydrogel types, the results showed S.aureus was more sensitive isolate to prepare (Gel &amp; Protease &amp; Ceftriaxone) the diameter of the inhibition zone reached (44 mm) ,the synergistic effect we noticed when using a (Gel &amp; Ceftriaxone &amp; Protease) healing was observed in time (five days) with wound healing without effect. Conclusion: In this study, it was discovered that mixing (protease + hydrogel + Ceftriaxone) accelerates the wound healing without leaving traces.

Serine protease RAYM_01812 (SspA) inhibits complement-mediated killing and monocyte chemotaxis and contributes to virulence of Riemerella anatipestifer in ducks

ABSTRACT Riemerella anatipestifer (RA) is a significant poultry pathogen causing acute septicemia and inflammation. The function of protease RAYM_01812, responsible for gelatin degradation, is unexplored in RA pathogenesis. To elucidate its role, we generated a deletion mutant ΔRAYM_01812 (ΔRAYM) and complementary CΔRAYM_01812 (CΔRAYM) strain and revealed the protease’s role in extracellular gelatinase activity. By expressing full-length 76 kDa RAYM_01812 protein without signal peptide as well as seven partial structural domains fragments, we evidence that the N-terminal propeptide acts as an enzymatic activity inhibitor and it gets cleaved at A112. Also, we show that the β-fold sheet domain is necessary for enhancing the enzymatic protease activity. Sequential auto-proteolysis forms a stable 40 kDa enzyme. Then, testing the strains in duck sera indicated that the absence or presence of RAYM_01812 results in reduced or enhanced bacterial survival, respectively. Furthermore, we found that the protease is able to cleave IgY antibodies as well as the complement factors C3a and C5a, that the protease reduces C3a- or C5a-mediated monocyte chemotaxis, and results in enhanced membrane attack complex (MAC) formation on the surface of ΔRAYM compared to CΔRAYM. This suggests that RAYM_01812 plays a crucial role in protecting against the serum complement-mediated bactericidal effect through inhibiting MAC formation and monocyte chemotaxis. Animal infection assays showed a 1090-fold reduced virulence of ΔRAYM compared to RA-YM, evidenced by decreased tissue loading and weaker histopathological changes. In conclusion, RAYM_01812 acts as a vital virulence factor, enabling host innate immune defence escape through complement killing evasion and monocyte chemotaxis inhibition.

Biochemical analysis of packing and assembling heptad repeat motifs in the coronavirus spike protein trimer.

During a coronavirus infection, the spike protein undergoes sequential structural transitions triggered by its receptor and the host protease at the interface between the virus and cell membranes, thereby mediating membrane fusion. After receptor binding, the heptad repeat motif (HR1/HR2) within the viral spike protein bridges the viral and cellular membranes; however, the intermediate conformation adopted by the spike protein when drawing the viral and cellular membranes into close proximity remains unclear due to its transient and unstable nature. Here, we experimentally induced conformational changes in the spike protein of a murine coronavirus by incubating the virus with its receptor, followed by exposure to trypsin. We then treated the virus/receptor complex with proteinase K to probe the tightly packed core structure of the spike protein. The conformations of the spike protein were predicted from the sizes of the protease digestion products detected by western blot analysis. Upon receptor binding, two bands (each showing different reactivity with a fusion-inhibiting HR2-peptide) were detected; we propose that these bands correspond to the packed and unpacked HR1/HR2 motifs. After trypsin-mediated triggering, measurement of temperature and time dependency revealed that packing of the remaining unpacked HR1/HR2 motifs and assembly of three HR1 motifs in a trimer occur almost simultaneously. Thus, the trimeric spike protein adopts an asymmetric-unassembled conformation after receptor binding, followed by direct assembly into the post-fusion form triggered by the host protease. This biochemical study provides mechanistic insight into the previously unknown intermediate structure of the viral fusion protein.IMPORTANCEDuring infection by an enveloped virus, receptor binding triggers fusion between the cellular membrane and the virus envelope, enabling delivery of the viral genome to the cytoplasm. The viral spike protein mediates membrane fusion; however the molecular mechanism underlying this process is unclear. This is because using structural biology methods to track the transient conformational changes induced in the unstable spike trimer is challenging. Here, we harnessed the ability of protease enzymes to recognize subtle differences on protein surfaces, allowing us to detect structural differences in the spike protein before and after conformational changes. Differences in the size of the degradation products were analyzed by western blot analysis. The proposed model explaining the conformational changes presented herein is a plausible candidate that provides valuable insight into unanswered questions in the field of virology.

Withania coagulans root powder effect on growth, hematology, digestive enzyme activity, antioxidant status, serum immune response, and tolerance against Aeromonas hydrophila in Common Carp

AbstractObjectiveThe use of plant‐derived products in aquaculture has garnered considerable attention due to their potential benefits. This study investigated the impact of supplementing Withania coagulans root powder (WCRP) in the diet of Common Carp Cyprinus carpio on various parameters, including growth performance, digestive enzymes, hematology, antioxidant activity, and immunological aspects.MethodsCommon Carp (mean weight ± standard deviation = 11.69 ± 0.48 g) were divided into four groups in triplicate, receiving different WCRP concentrations (0.0, 1.0, 1.5, and 2.0%; labeled as WCRP0, WCRP1.0, WCRP1.5, and WCRP2.0, respectively) over a 60‐day period, followed by an experimental challenge with Aeromonas hydrophila to assess the relative percentage survival (RPS) over 14 days.ResultResults showed that Common Carp receiving WCRP1.5 and WCRP2.0 demonstrated significantly improved growth performance, with reduced feed conversion ratios (FCRs) being particularly evident in WCRP1.5. Polynomial contrasts indicated significant linear and quadratic effects on weight gain and FCR. Additionally, WCRP1.5 and WCRP2.0 supplementation led to significantly higher activity of digestive enzymes (lipase and protease). Hematological parameters, including white blood cells, red blood cells, hemoglobin, and hematocrit, were significantly elevated in fish that were fed WCRP1.5 and WCRP2.0 compared to the control group. Moreover, serum parameters, such as total protein, albumin, globulin, lysozyme, catalase, superoxide dismutase, and total immunoglobulins, were significantly enhanced in WCRP1.5‐ and WCRP2.0‐treated fish. Notably, the WCRP1.5 group showed the lowest serum cortisol levels. The RPS was highest in WCRP1.5 (73.77%), followed by WCRP2.0 (70.43%), compared to the other groups.ConclusionIn conclusion, supplementation with WCRP1.5 and WCRP2.0 effectively improved both growth and health parameters in Common Carp.

Comparing the frequency, antifungal susceptibility, and enzymatic profiles of the oral fungal composition in patients with and without Alzheimer's disease admitted to a neurology clinic.

Studies have shown that changes in the frequency of oral microorganisms may play a key role in the development of Alzheimer's disease (AD). However, no research has been conducted on the oral fungal composition in AD-patients. The present study aimed to investigate the changes in the frequency of oral fungal composition, the antifungal susceptibility, and the enzymatic profiles of oral fungal composition in patients suffering from AD compared to non-AD individuals. In the present analytical cross-sectional study during 12 months, 76 hospitalized patients with AD were matched with 76 individuals without AD. A sterile serum physiology-moistened cotton-tipped swab was used to sample the mouth area. All swabs were cultured on Sabouraud Chloramphenicol Agar. Fungal identified were confirmed through the PCR-sequencing techniques. Enzyme activity index (EAI) for important pathogenic factors including proteinase, esterase and hemolysin was measured using relevant protocols. The susceptibility to 8 antifungal agents (nystatin, voriconazole, itraconazole, fluconazole, posaconazole, amphotericin B, 5-fluorocytosine, and caspofungin) against fungal strains obtained from AD-patients was evaluated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, document M38-A2 for filamentous fungi, and document M27-A4 for yeasts. The results showed that compared to the non-AD individuals, the prevalence of oral fungal composition in AD group was 1.6 times higher. Candida albicans was the most common fungal species isolated from oral swab samples of AD group (n=53, 80%) and non-AD group (n=28, 40%), and the diversity of the oral fungal composition in AD-patients were lower than non-AD individuals. Among the 3 investigated virulence factors, a statistically significant difference was shown in terms of hemolysin activity level between the two studied groups (p<0.05) and the activity level of esterase and proteinase enzymes did not show a significant difference in the two studied groups (p>0.05). The results showed that almost all of the tested isolates were susceptible to nystatin, the most widely prescribed antifungal to treat superficial infections, and only 1.69 % (2/118) of the Candida isolates were resistant to this antifungal drug. Understanding the changes in the frequency of oral fungal composition the antifungal susceptibility, and the enzymatic profiles of oral fungal composition in patients suffering from AD compared to non-AD individuals makes it possible to better understand the etiology of this disease.

Antioxidant activities of Saudi honey samples related to their content of short peptides

This study explored the effect of geographical and floral origins on the antioxidant activities of Saudi honey samples related to their content of short peptides originated from honeybee proteins. The studied antioxidants were the total protein concentration, catalase activity, phenolic acids and flavonoids. The antioxidant activity assays included were the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, the ferric reducing antioxidant power (FRAP) assay and Ascorbic acid Equivalent Antioxidant Capacity (AEAC). The studied honey samples were obtained from the southwestern region of Saudi Arabia, namely Asir (65) and Jazan (25). The floral origins of the honey samples were Acacia (51), Ziziphus (4) and polyfloral (35). The LC/MS technique was used to detect the short peptides and the mascot database was used to identify the short peptides, their precursor proteins and the protease enzymes that produce them. Jazan honey was characterized by high number of short peptides. The short peptides were originated from honeybee proteins by the action of proteases from the honeybees and bacteria. The antioxidant activity of the honey samples increase with the increase of their content of short peptides and proteins. The amino acids type and sequence of the short peptides qualify them to act as antioxidant, antimicrobial, anti-diabetic, anti-hypertension, immunomodulatory and cholesterol lowering peptides.

Comparative Evaluation of Enzymatic Crude Protein Degradation in Selected Legume Forages

For protein evaluation of feedstuffs for ruminants, the Streptomyces griseus protease test offers a purely enzymatic approach to estimate ruminal protein degradation. This study was conducted to determine the enzymatic crude protein (CP) degradability of alfalfa, sainfoin, and common vetch hays, which are commonly used in ruminant nutrition. To estimate CP degradation, fifteen samples from each type of hay were incubated in vitro with a commercial protease extracted from Streptomyces griseus. The incubation was carried out for 1, 4, 24, and 48 hours in a borate-phosphate buffer at pH 8. Significant differences in CP degradability values were found among all three types of hay across all incubation periods. For all incubation periods, sainfoin had the lowest CP degradability values (P &lt; 0.05), due to its high content of cell wall components and condensed tannins (CTs). For incubation periods longer than 1 hour, common vetch had the highest CP degradability values, followed by alfalfa and sainfoin, respectively (P &lt; 0.05). As a result, the use of the protease enzyme extracted from Streptomyces griseus was confirmed as an effective method for estimating the CP degradability of selected legume forages in the laboratory, eliminating the need for animal testing. However, since plant proteins are often embedded within carbohydrate complexes, it is recommended that future tests consider the combined use of protease and carbohydrase, particularly for sainfoin, which is rich in cell wall components and condensed tannins.

In silico investigation of therapeutic potential of phytosteroids against SARS-CoV2 infection via inhibition of viral multiplication and attenuation of host cytokine storm

SARS-CoV2 infection overcomes host cell membrane barrier, followed by utilization of host cellular processes for viral multiplication. Simultaneously, it triggers a cytokine storm within and around infected cells and tissues. Anti-inflammatory agents that can potentially inhibit viral penetration and multiplication within the host cells may be ideal drug candidates against COVID-19. Dietary phytosteroids have significant anti-inflammatory potential. Hence, the present study intends to investigate anti-viral potential of three dietary phytosteroids, namely, brassinolide, 28-homocastasterone and 24-epibrassinolide against SARS-CoV2 proteins, S1 spike protein, nucleocapsid protein and main protease enzyme, in addition to host pro-inflammatory proteins, interleukin-1, tumour necrosis factor-α, cyclooxygenase-2 and prostaglandin synthase, as drug targets. Molecular docking studies by AutoDock version 4.0 was performed. Brassinolide, 28-homocastasterone and 24-epibrassinolide exhibits high docking score against all the seven proteins, as compared to hydroxychloroquine. Brassinolide and 28- homocastasterone has maximum binding affinity for pro-inflammatory proteins and SARS-CoV2 proteins.Dietary phytosteroids may potentially attenuate cytokine storm with simultaneous inhibition of host entry and multiplication of SARS-CoV2. In-vitro and in-vivo anti-viral studies of plant steroids may provide a clear path for the identification and development of novel drug candidates against COVID-19, that also provides evidence for the concept of reverse pharmacognosy.

Protease activated receptor 2 as a novel druggable target for the treatment of metabolic dysfunction-associated fatty liver disease and cancer.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is spreading worldwide, largely due to unhealthy lifestyles that contribute to the rise in diabetes, metabolic syndrome, and obesity. In this situation, the progression of injury to metabolic steatohepatitis can evolve to cirrhosis and, eventually, to hepatocellular carcinoma (HCC). It is well known that serine protease enzymes with different functions in cellular homeostasis act as signaling molecules that regulate liver inflammation by activating the protease-activated receptors (PARs) family members, expressed on the cellular plasma membrane. Among them, PAR2 plays a central role in the activation of signaling pathways in response to changes in the extracellular microenvironment. Experimental data have provided evidence that PAR2 is involved not only in inflammatory response but also in insulin resistance, lipid metabolism, and cancer. The major aims of this narrative review are addressed to assess PAR2 involvement in inflammation, metabolism, and liver disease progression and to explore possible therapeutic strategies, based on PAR2 inhibition, in order to prevent its biological effects in the context of MAFLD and cancer.

INVESTIGATION OF THE EFFECT OF ENZYME APPLICATION ON THE STRUCTURE OF WAFERS IN THE FOOD INDUSTRY

The study aimed to assess the impact of 13 different enzymes, including protease, amylase, hemicellulase, and xylanase, on wafer dough and sheets, aiming to identify the most effective enzyme combination. Commercial protease, amylase, hemicellulase, and xylanase enzymes were applied to wafer dough following manufacturer instructions, and their flow behaviors were analyzed. Subsequently, the doughs were baked into wafer sheets, and various parameters such as water activity (aw), moisture content (%), weight loss (%), color parameters (L*, a*, b*), hardness (g), and sensory attributes were evaluated. Protease 4, Hemicellulase 2, and Xylanase 2 enzymes were selected for combination testing based on the analysis of flow behavior and characterization parameters of the wafer doughs. Among the tested combinations, the wafer dough containing 80 ppm Xylanase 2 + 300 ppm Protease 4 enzyme group exhibited promising potential for industrial applications due to its favorable flow behavior, while the resulting wafer sheets demonstrated desirable crispness and a brown-caramel color, suggesting suitability for commercial use.

Integrative in silico evaluation of the antiviral potential of terpenoids and its metal complexes derived from Homalomena aromatica based on main protease of SARS-CoV-2

Abstract Substantial research is currently conducted focusing on the development of promising antiviral drugs employing in silico screening and drug repurposing strategies against SARS-CoV-2. The current study aims at identifying lead molecules targeting SARS-CoV-2 by the application of in silico and molecular dynamics (MD) approaches from phytoconstituents present in Homalomena aromatica. The main protease (Mpro) enzyme of SARS-CoV-2 is taken as the target protein to perform the docking analysis of 71 molecules reported from H. aromatica by the application of different modules of Discovery Studio 2018. Five molecules were taken as prospective leads namely dihydrocuminaldehyde, p-cymen-8-ol, cuminaldehyde, p-cymene, and cuminol. In the absence of known inhibitors, a comparative study was performed with the compounds reported in the literature and potent terpenoid–metal complexes were taken into account based on known efficacy as anti-viral molecules. After performing the docking studies with Mpro enzyme of SARS-CoV-2, it was observed that the –CDocker Energy of cuminaldehyde thiosemicarbazone was 29.152, indicating a significant affinity toward Mpro. The same was also supported by the MD study. Taken together, our results provided in silico evidence that secondary metabolites derived from H. aromatica could be employed as potent antiviral agents targeting SARS-CoV-2. Our findings warrant further validation of their in vitro and in vivo efficacies prior to their development into bona fide therapeutic agents.

Nattokinase Attenuated Excitatory Amino Acids and Cytokines Release and Restored Cerebral Blood Flow in a Thrombolytic Focal Cerebral Ischemic Rat Model.

Nattokinase (NK), a protease enzyme present in traditional fermented Japanese food, has shown fibrinolytic properties in vitro as well as in cardiac ischemia. In the present study, the Neuroprotective effect of standardized NK was evaluated in the thrombolytic focal cerebral ischemic model. The parameters of behavioural assessment, cerebral blood flow, inflammatory mediators, excitatory amino acids, and immunohistochemistry were measured to support the NK effect. NK was administered at 150 and 300 mg/kg, and its effects were compared with streptokinase (STK) (100μl/rat). Each mg of NK contains 5.5 Units of the enzyme, which can cause lysis of the fibrin. The results indicate that 7 days of treatment of 300 mg NK restored the cerebral blood flow and prevented the release of cytokine and excitatory amino acids. Similarly, neurological scores were reduced, and grip strength increased significantly with NK treatment. The GFAP and synaptophysin staining of the hippocampus (CA1) and cerebrum have shown recovery of neurons from ischemic damage in comparison to vehicle-treated ischemic-reperfused rats. The NK (300 mg/kg) fibrinolytic effect is comparable to STK treatment. To conclude, NK, a serine protease, protects the brain from ischemic degeneration in thrombolytic cerebral ischemia. Consumption of this Japanese food might exhibit prophylactic activity.

Endophytic fungi from &lt;i&gt;Dichrocephala integrifolia&lt;/i&gt;: Diversity, antifungal properties, enzymatic activities, and plant growth promotion

Dichrocephala integrifolia is a wild medicinal plant utilised in traditional healing and Ayurveda to cope up with several health issues by various groups of people around the world. Medicinal plants are associated with diverse fungal endophytes with potential bioactive properties. In this investigation, 26 fungal endophytes were isolated from D. integrifolia and three sterile forms using the Petri plate culture method. The endophytic isolation rate was highest for inflorescence (35.97%) and lowest for stem (15.61%). The highest colonization frequency was shown by F. solani (8.37%) and the lowest by Sterile morphotype 3 (1.36%). Out of the four plant parts, inflorescence was found to be highly infected, displaying an infection rate of 93.43% and the least infection occurred in the stem with 45%. The maximum number of isolated endophytic fungi belongs to the class Sordariomycetes, with a relative occurrence (%) of 71.72%. The Simpson’s diversity index reveals that the leaf endophytes were more diverse (0.94). Qualitative antifungal activity of the sporulating isolates against Curvularia lunata has shown that the maximum number of endophytes possessed Class 3 antagonism. Four isolates were selected based on screening of their antagonistic activity and their antifungal inhibition was calculated against nine fungal phytopathogens. Maximum inhibition (100%) was shown by Trichoderma sp. 2 (S2B2) against Alternaria alternata, A. brassicicola, Colletotrichum capsici, C. lunata, and Ustilaginoidea virens and least inhibition by Gliocladium sp. 1 (19.78%) against C. lunata. The four isolates were found to produce protease, lipase, amylase and cellulase enzymes. The isolates produced ammonia and hydrogen cyanide, but none of the isolates could solubilize phosphate. Potent biocontrol agents are much needed to replace synthetic chemicals and restore soil microflora.