Protease Enzyme Research Articles

Cloning and Production of Protease Enzyme from Aeribacillus pallidus P18 Strain

Proteins are essential for the proper functioning of cells. The techniques of cloning and protein production have facilitated the advancement of various fields and the creation of specific proteins for industrial and therapeutic uses. The bacterium Aeribacillus pallidus, which is able to survive in extreme conditions, is being studied with a view to identifying its robust enzymes. The objective of this study was to clone the protease gene from the A. pallidus P18 strain into the SUMO vector and produce recombinant protein in Escherichia coli BL21 for protein production. The protease enzyme gene from the A. pallidus P18 strain was isolated and amplified by using PCR. The PCR product was transferred into the SUMO expression vector and amplified in One Shot® Mach1TM-T1R bacteria, followed by colony PCR. Plasmid isolation was performed after positive colony selection. Gene integration was confirmed by cross-PCR using the gene forward, and vector reverse primers. For expression, the plasmid was transferred to E. coli BL21 cells. Two cultures were induced with different IPTG concentrations (0.5 mM and 1 mM) to optimize protein production. Bacterial cells were lysed, and SDS-PAGE analysis was conducted. Purification involved cell lysate preparation and purification using a ProbondTM column. SDS-PAGE and Coomassie Brilliant Blue G-250 staining confirmed successful purification. The results of this study indicate that the optimal product for protein production is that derived from a culture induced with 1 mM IPTG. Upon completion of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure, the weight mass of the produced protein was determined to be 37 kDa, as indicated by the result of the gel stained with Coomassie brilliant blue G-250. This research successfully cloned the protease enzyme gene from the A. pallidus P18 strain using the pET-SUMO vector, performed purification and achieved the targeted result of protein production.

Optimization of the biofuel production by idealized fermentation of the animal manure, chicken wastes, and sewage sludge

Abstract This study aims to optimize an economic procedure to produce biogas and bio-ethanol from different organic wastes such as sewage sludge (SS) and/or cattle dung (CD) and/or poultry manure (PM). The experiment was carried out at a wastewater treatment plant in Egypt. Each waste type was mixed with the starter, CaCO3, and water then loaded in a fermenter and kept for 35 days at 35 °C under the anaerobic digestion. The evolved volume of the biogas and the content of methane CH4 were measured daily while the cellulase and protease enzymes were tested every four days. Results have indicated that the digester containing the SS has produced the greatest biogas volume (L) 27.45 Lb/D/d (liters biogas/digester/day), 0.61 Lb/D contents’ volume/d, and cumulative 606.30 Lb/D during the 16th day. Significant CH4 volume percentages produced during the 17th day were 72.07, 71.16, and 71.11% while the produced bio-ethanol alcohol was 2.47, 2.32, and 1.99% from the SS, CD, and PM, respectively. The procedure efficiency is prominent by the production of the biogases and in-situ activating enzymes all in one reactor that was periodically monitored for its reactants and product content. No need for the pre-treatment of wastes as raw materials or chemical additives and the fermented residue can be further tested for soil fertilization. These wastes can be promising for bio-energy production being economic and environment friendly.

Harnessing Nature's Toolbox: Protease Enzymes in Biotechnology and Industrial Applications

Harnessing Nature's Toolbox: Protease Enzymes in Biotechnology and Industrial Applications

Production of Nattokinase from Bacillus amyloliquefaciens MRS18: A Bacterial Strain Isolated from Fermented Beans.

Nattokinase (NK) is a naturally occurring fibrinolytic protease enzyme obtained from the traditional Japanese food called Natto and has several uses in the pharmaceutical and medical industries. Nowadays, the most often used thrombolytic agent in the clinical field is NK, in part because it is less expensive than other thrombolytic medicines. The objective of this study is to investigate the screening, isolation and characterization of the NK enzyme-producing Bacillus strain from fermented Soya beans. The sample of fermented soya beans were tested for the presence of fibrinolytic protease- producing bacteria, followed by the screening, extraction, characterization and clot lysis assays. A total of three isolates were screened for caseinolytic activities by casein hydrolysis assay. Out of those isolates, MRS18 was found to be potent in producing the enzyme proteinase. To determine the taxonomy and phylogeny of these isolates, biochemical and molecular characterization has been carried out. Bacillus amyloliquefaciens MRS18 has been found with the highest caseinolytic activity. The clot lysing ability of the potent strain Bacillus amyloliquefaciens was found to be 61.7% after 120 min, and on further purification, by ammonium sulphate precipitation method, the lysis percentage was found to be 656% after 120 min. From the results of the present study, we concluded that Bacillus amyloliquefaciens isolated from the fermented soya beans produced an NK enzyme that exhibits immense potential to lyse blood clots.

The covalent immobilization of β-galactosidase from Aspergillus oryzae and alkaline protease from Bacillus licheniformis on amino-functionalized multi-walled carbon nanotubes in milk

This study aimed was to covalently immobilize β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis on amino-functionalized multi-walled carbon nanotubes. In this study, a two-level factorial design was employed to investigate the impact of seven continuous variables (activation pH, glutaraldehyde molarity, activation time (0–8 h), buffer solution pH (8-0), buffer solution molarity, MWCNT-NH2-glutaraldehyde quantity, and stabilization time (0–180 h)) on the immobilization efficiency and enzymatic activity of protease and β-galactosidase. Furthermore, the effect of time on the percentage of enzymatic activity was examined during specific intervals (24, 48, 72, 96, and 120 h) of the immobilization process. The analysis of variance results for protease enzymatic activity revealed a notable influence of the seven variables on immobilization efficiency and enzymatic activity. Additionally, the findings indicate that activation time, buffer pH, MWCNT-NH2-glutaraldehyde quantity, and stabilization time significantly affect the activity of the protease enzyme. The interplay between buffer pH and stabilization time is also significant. Indeed, both activation time and the quantity of MWCNT-NH2-glutaraldehyde exert a reducing effect on enzyme activity. Notably, the influence of MWCNT-NH2-glutaraldehyde quantity is more significant (p < 0.05). In terms of beta-galactosidase enzymatic activity, the study results highlight that among the seven variables considered, only the glutaraldehyde molarity, activation time, and the interplay of activation time and the quantity of MWCNT-NH2-glutaraldehyde can exert a statistically significant positive impact on the enzyme's activity (p < 0.05). The combination of activation time and buffer solution molarity, as well as the interactive effect of buffer pH and MWCNT–NH2–glutaraldehyde, can lead to a significant improvement in the stabilization efficiency of the protease of carbon nanotubes. The analysis of variance results demonstrated that the efficiency of covalently immobilizing β-galactosidase from Aspergillus oryzae on amino-functionalized multi-walled carbon nanotubes is influenced by the molarity of glutaraldehyde, buffer pH, stabilization time, and the interplay of activation time + buffer pH, buffer pH + activation time, activation time + buffer molarity, and glutaraldehyde molarity + MWCNT-NH2-glutaraldehyde (p < 0.05). Through the optimization and selection of optimal formulations, the obtained results indicate enzyme activities and stabilization efficiencies of 64.09 % ± 72.63 % and 65.96 % ± 71.77 % for protease and beta-galactosidase, respectively. Moreover, increasing the enzyme stabilization time resulted in a reduction of enzyme activity. Furthermore, an increase in pH, temperature, and the duration of milk storage passing through the enzyme-immobilized carbon nanotubes led to a decrease in enzyme stabilization efficiency, and lactose hydrolysis declined progressively over 8-h. Hence, the covalent immobilization of β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis onto amino-functionalized multi-walled carbon nanotubes is anticipated to be achievable for milk applications.

IN SILICO STUDIES OF TUMOUR TARGETED PEPTIDE DERIVED FROM PROTEASE PRODUCED BY BACILLUS CEREUS HN FOR TARGETING WNT SIGNALLING PATHWAY USING MOLECULAR DOCKING AND MOLECULAR DYNAMICS

Background:In todays world, its becoming increasingly difficult to ignore the role of cancer in social health. Breast cancer remains the second global cause of death in women. Alterations disrupt normal cell function, cause cancerous cells to over-proliferate, and avoid mechanisms that might typically control their growth, division, and migration. A disruption in signalling pathways, a mutation that might lead to overexpression of certain genes that can, in turn, activate the signalling pathway and send it into an override mode that leads to diseases such as cancers. These signalling pathways can be targeted not only for use as biomarkers for the early detection of certain cancers but also for targeted treatment of cancers. The prospect of the development of new substances with antitumoral potential is of great importance for thetreatment of breast cancer. Result:The objective of this work is to design a peptide from a laboratory-produced Protease enzyme isolated from Bacillus cereus HNand use that as a drug or ligand that could potentially bind to the LRP5 protein and inactivate the Wntsignalling pathway in triple-negative breast cancer cells. Conclusion:The work was aimed at computational modelling of a safe peptide which followed the pharmacokinetic parameters and conducting docking analysis to assess the binding free energy between LRP 5 and the designed peptide. The docking results show that the ligand is a good candidate for novel drug development. The models created and analysed in this work will most certainly help in future research on targeting the Wntsignalling pathway and its components.

Systematic review: How to obtain Fibrinolytic Enzymes from Fungi

Fibrinolytic complications such as stroke and peripheral occlusive disease due to vascular occlusion are significant causes of poor prognosis and death. Given the ability of mushroom fibrinolytic enzymes to dissolve blood clots and prevent side effects, their use as a potential thrombolytic therapy has attracted great interest. Serine protease, metalloprotease, and serine metalloprotease are protease enzymes that can affect the fibrinolytic mechanism by dissolving blood clots and increasing blood flow. This systematic review aimed to disclose all information regarding the production, purification, and characterization of fibrinolytic proteases from fungi. Searches were conducted on online media databases, including Google and journal sites (NCBI, PubMed, and others, using the keywords "(Fibrinolytic Enzymes) OR (Fibrinolytic Proteases) AND (Fungi or Mushrooms)" from 2012-2023. This systematic review observed reviews from the purification and characterization steps, indicating the need for attention to the manufacturing process and enzymatic applications. The production of these enzymes by fungi is associated with developments in pharmaceutical applications.

Isolasi Dan Identifikasi Bakteri Proteolitik Dari Tanah Mangrove Di Pantai Pandaratan Kecamatan Sarudik Kabupaten Tapanuli Tengah

Proteolytic bacteria are bacteria that can degrade proteins by producing extracellular protease enzymes. Proteolytic bacteria can be found in mangrove soil. This is because the dry weight of mangrove leaf litter contains 61% protein. The purpose of this study was to determine the presence of proteolytic bacterial isolates from mangrove soil in Pandaratan Beach, Sarudik District, Central Tapanuli Regency and to characterize them. This study used a laboratory experimental method which was analyzed descriptively. Soil sampling was carried out at two location points I (sandy) and location II (muddy) which were isolated using SMA media with the streak plate method. Characterization includes macroscopic and microscopic observations as well as biochemical tests. The results of the study found that there were 15 isolates of proteolytic bacteria isolated from the mangrove soil of Pandaratan Beach, Central Tapanuli. The highest activity test of the proteolytic index (IP) was obtained 0.82 mm by the Micrococcus (L1) and 0.09 mm lowest by Brevibacillus (L2). A total of 11 genera of proteolytic bacteria were found, including Curtobacterium (P1 and L7), Bacillus (P2, P4, and P7), Enterococcus (P3), Brevibacillus (P5 and L2), Carnobacterium (P6), Paenibacillus (P8), Micrococcus (L1), Vagococcus (L3), Marinococcus (L4), Halobacillus (L5), Sporosarcina (L6).

Dietary supplementation of Astragalus fermentation products improves the growth performance, immunological characteristics, and disease resistance of crucian carp (Carassius auratus)

The fermentation products of Astragalus have been acknowledged for their ability to enhance immune functions. This study assessed the impact of incorporating Astragalus, fermented with Lactobacillus plantarum and Bacillus coagulans, on crucian carp’s growth, disease resistance, and immunological characteristics. The experimental groups were fed with common feed (C), C + Astragalus (A), A + Lactobacillus plantarum (AL), A + Bacillus coagulans (AB), and AL + Bacillus coagulans (ALB). The fermented products were mixed with common feed at a 1:99 ratio, and crucian carp were fed 2% of their body weight for four weeks, with sampling conducted on days 3, 7, 14, 21, and 28. Disease resistance was evaluated using Aeromonas hydrophila (A. hydrophila) at a concentration of 0.2 mL (1.0×107 CFU/mL). The final weights in the AL, AB, and ALB groups significantly increased compared to the C group. The ALB group exhibited elevated serum albumin levels, alkaline phosphatase, intestinal lipase, protease enzyme, C3, and IgM gene expression compared to the C group. At the same time, alanine aminotransferase, aspartate aminotransferase, and glucose contents were significantly reduced. The survival rate significantly increased in all experimental groups after treatment with A. hydrophila. In conclusion, Astragalus products fermented with Lactobacillus plantarum and Bacillus coagulans could effectively improve crucian carp’s growth, disease resistance, and immune response.

Alpha and gamma mangostins inhibit wild-type B SARS-CoV-2 more effectively than the SARS-CoV-2 variants and the major target is unlikely the 3C-like protease

BackgroundAnti-SARS-CoV-2 and immunomodulatory drugs are important for treating clinically severe patients with respiratory distress symptoms. Alpha- and gamma-mangostins (AM and GM) were previously reported as potential 3C-like protease (3CLpro) and Angiotensin-converting enzyme receptor 2 (ACE2)-binding inhibitors in silico. ObjectiveWe aimed to evaluate two active compounds, AM and GM, from Garcinia mangostana for their antivirals against SARS-CoV-2 in live virus culture systems and their cytotoxicities using standard methods. Also, we aimed to prove whether 3CLpro and ACE2 neutralization were major targets and explored whether any additional targets existed. MethodsWe tested the translation and replication efficiencies of SARS-CoV-2 in the presence of AM and GM. Initial and subgenomic translations were evaluated by immunofluorescence of SARS-CoV-2 3CLpro and N expressions at 16 h after infection. The viral genome was quantified and compared with the untreated group. We also evaluated the efficacies and cytotoxicities of AM and GM against four strains of SARS-CoV-2 (wild-type B, B.1.167.2, B.1.36.16, and B.1.1.529) in Vero E6 cells. The potential targets were evaluated using cell-based anti-attachment, time-of-drug addition, in vitro 3CLpro activities, and ACE2-binding using a surrogated viral neutralization test (sVNT). Moreover, additional targets were explored using combinatorial network-based interactions and Chemical Similarity Ensemble Approach (SEA). ResultsAM and GM reduced SARS-CoV-2 3CLpro and N expressions, suggesting that initial and subgenomic translations were globally inhibited. AM and GM inhibited all strains of SARS-CoV-2 at EC50 of 0.70–3.05 μM, in which wild-type B was the most susceptible strain (EC50 0.70–0.79 μM). AM was slightly more efficient in the variants (EC50 0.88–2.41 μM), resulting in higher selectivity indices (SI 3.65–10.05), compared to the GM (EC50 0.94–3.05 μM, SI 1.66–5.40). GM appeared to be more toxic than AM in both Vero E6 and Calu-3 cells. Cell-based anti-attachment and time-of-addition suggested that the potential molecular target could be at the post-infection. 3CLpro activity and ACE2 binding were interfered with in a dose-dependent manner but were insufficient to be a major target. Combinatorial network-based interaction and chemical similarity ensemble approach (SEA) suggested that fatty acid synthase (FASN), which was critical for SARS-CoV-2 replication, could be a target of AM and GM. ConclusionAM and GM inhibited SARS-CoV-2 with the highest potency at the wild-type B and the lowest at the B.1.1.529. Multiple targets were expected to integratively inhibit viral replication in cell-based system.

Effects of phytase inclusion in diets containing rice protein concentrate (RPC) on the nutrient digestibility, growth and chemical characteristics of rohu (Labeo rohita).

The objective of the current study was to assess the impact of dietary phytase supplementation on Labeo rohita fingerlings and to examine the effects on growth, nutrient digestibility and chemical characteristics of diets containing rice protein concentrate (RPC) as a major protein source. Six experimental diets were made, i.e., a positive control (fishmeal-based diet with no phytase), FM0; a negative control (RPC-based diet with no phytase), RPC0; and four supplemental phytase levels (250, 500, 1000, and 2000 FTU/kg). Fingerlings with an average weight of 9.42 ± 0.02 grams (mean ± SD) were randomly distributed into six experimental groups of three replicates, each containing 25 fish per tank (75 liters of water), provided with experimental diets at a rate equivalent to 5% of their body weight for 90 days, and uneaten feed was collected after 2 hours to determine feed consumption. The feces were collected before feeding to estimate digestibility. Phytase in combination with the RPC-based diet significantly (p < 0.05) enhanced phytate phosphorus in vitro hydrolysis; growth performance; nutrient (crude protein, crude fat, moisture and gross energy) and mineral (P, Ca, Mg, Na, K, Zn, Mn and Cu) digestibility; digestive enzyme (protease, lipase and amylase) activity; and mineral deposition up to 1000 FTU/kg phytase. However, the hepatosomatic and viscerosomatic indices and carcass composition were not influenced (p > 0.05) by phytase supplementation. Increasing phytase supplementation in the RPC-based diets led to a significant (p < 0.05) decrease in the serum biochemical parameters (alkaline phosphatase activity, aspartate aminotransferase, alanine aminotransferase), which resulted in improved liver health. In conclusion, phytase-supplemented RPC-based diets improved the growth, mineral/nutrient digestibility, digestive enzymes, serum biochemistry, and mineral deposition of L. rohita fingerlings up to 1000 FTU/kg. Broken line regression analysis revealed that the optimum phytase concentration in the RPC-based diet for L. rohita was 874.19 FTU/kg.

Effect of anise seed (Pimpinella anisum L.) and protease alone or in combination on growth performance, carcase characteristics, humoral immunity, nutrients digestibility and cecal microbiota in Japanese quails

ABSTRACT The trial investigated the effects of anise seed powder and protease, alone and combined, in Japanese quail diets. 540 seven-day-old quails were divided into four groups: Control (basal diet), Anise Seed Group (1000 mg/kg anise seed powder), Protease Enzyme Group (30,000 IU/kg Bacillus subtilis-derived protease enzyme), and a combination of anise powder and protease. The combination significantly increased weight gain and dressing percentage compared to the control. Quails receiving protease, anise powder, or both showed higher antibody titers against infectious diseases. The population of beneficial Lactobacillus increased in groups receiving protease and the combination. Additionally, the presence of harmful bacteria like Escherichia coli and Salmonella decreased significantly in quails supplemented with both protease enzyme and anise powder. In summary, combining protease and anise powder improved growth performance, immune responses, increased Lactobacillus population, and decreased pathogenic bacteria in quails.

Fragment-based virtual screening identifies novel leads against Plasmepsin IX (PlmIX) of Plasmodium falciparum: Homology modeling, molecular docking, and simulation approaches

Despite continuous efforts to develop safer and efficient medications, malaria remains a major threat posing great challenges for new drug discovery. The emerging drug resistance, increased toxicities, and impoverished pharmacokinetic profiles exhibited by conventional drugs have hindered the search for new entities. Plasmepsins, a group of Plasmodium-specific, aspartic acid protease enzymes, are involved in many key aspects of parasite biology, and this makes them interesting targets for antimalarial chemotherapy. Among different isoforms, PlmIX serves as an unexplored antimalarial drug target that plays a crucial role along with PlmV and X in the parasite’s survival by digesting hemoglobin in the host’s erythrocytes. In this study, fragment-based virtual screening was performed by modeling the three-dimensional structure of PlmIX and predicting its ligand-binding pocket by using the Sitemap tool. Screening identified the fragments with the XP docking score ≤ −3 kcal/mol from the OTAVA General Fragment Library (≈16,397 fragments), and the selected fragments were chosen for ligand breeding. The resulting ligands (≈69,858 ligands) were subsequently subjected to filtering based on the QikProp properties along with carcinogenicity testing performed using CarcinoPred-EL and then docked in the SP (≈14,078 ligands) as well as XP mode (≈3,104 ligands), and compared with that of control ligands 49C and I0L. The top-ranked ligands were taken further for the calculation of the free energy of binding using Prime MM–GBSA. Overall, a total of six complexes were taken further for MD simulation studies performed at 100 ns to attain a better understanding of the binding mechanisms, and compounds 3 and 4 were found to be the most efficient ones in silico. The analysis of compound 3 revealed that the carbonyl group present in position 1 on the isoindoline moiety (Arg554) was responsible for inhibitory activity against PlmIX. However, the analysis of compound 4 revealed that the amide linkage sandwiched between the phenyl ring and isoquinoline moiety (Lys555 and Ser226) as well as carbonyl oxygen of the carbamoyl group present at position 2 of the pyrazole ring (Gln222) were responsible for PlmIX inhibitory activity, owing to their crucial interactions with key amino acid residues.

Antifouling activities of proteinase K and α-amylase enzymes: Laboratory bioassays and in silico analysis

The application of enzymes as antifoulants is one of the environment-friendly strategies in biofouling management. In this study, antifouling activities of commercially available proteinase K and α-amylase enzymes were evaluated using barnacle larva and biofilm-forming bacteria as test organisms. The enzymes were also tested against barnacle cement protein through in silico analysis. The results showed that both enzymes inhibited the attachment of bacteria and settlement of barnacle larvae on the test surface. The lowest minimum inhibitory concentration of 0.312 mg ml−1 was exhibited by proteinase K against biofilm-forming bacteria. The calculated LC50 values for proteinase K and α-amylase against the barnacle nauplii were 91.8 and 230.96 mg ml−1 respectively. While α-amylase showed higher antibiofilm activity, proteinase K exhibited higher anti-larval settlement activity. Similarly, in silico analysis of the enzymes revealed promising anti-settlement activity, as the enzymes showed good binding scores with barnacle cement protein. Overall, the results suggested that the enzymes proteinase K and α-amylase could be used in antifouling coatings to reduce the settlement of biofouling on artificial materials in the marine environment.

Separation and identification of bacteria producing protease from the intestine of Sillago sihama and Rastrelliger kanagurta

The importance of marine enzymes, especially protease enzymes, made us investigate the presence of these enzymes in the intestines of two fish species of the Persian Gulf (Sillago sihama and Rastrelliger kanagurta). Our investigations showed that initially 29 strains of bacteria were isolated of which 13 colonies had a specific growth in culture medium, and finally only seven strains were capable of producing a strong aura. T1/2 HR08 and S6B1 HR09 are two strains with the largest diameter of the halo around the bacteria, along with 5 other bacterial strains that were selected for more research. The results from the absorption of the supernatant by the spectrophotometer showed that the two strains T1/2 HR08 and S6B1 HR09 had the highest level of enzyme activity and were selected for identification accordingly. Also, the studies conducted with the 16S rRNA gene showed that these bacteria belonged to two genera, Kocuria and Aeromonas, from the Micrococcaceae and Aeromonadaceae families, and the highest similarity of HR08 and HR09 strains, with a similarity of 99% was shown to Kocuria rhizophila strain 8d-S5 (MN062066.1) and Aeromonas salmonicida strain SHY16- (CP038102.1), respectively. Generally, based on the results, these bacterial strains could be a good and ideal source for the production of the protease enzyme, which is used in many different industries.

The Effect of Hypertonic Osmotic Stress and Its Duration on the Level and Activity of Executioner Caspase Enzymes in Blood Anuclear Cells (Platelets)

Background: Caspases are protease enzymes critical for apoptosis. Caspase-3 and caspase-7, known as executioner caspases, are present in anuclear cells such as platelets. Platelet mitochondria can engage in the intrinsic pathway of apoptosis during stress and cellular damage. Hypertonic osmotic stress influences not only cell shape but also induces chemical changes within the cell. This study examines the function of executioner caspases in platelets under hypertonic osmotic stress due to the absence of a caspase-independent apoptosis pathway in these nucleus-free cells. Methods: Platelets were exposed to osmotic stress in a hypertonic environment (1.7% sodium chloride), with an isotonic environment (0.9% sodium chloride) serving as the control. Both environments were maintained at 37°C for durations of 15 minutes and 24 hours. Following this, platelets were lysed using ultrasound waves, and cellular membranes were removed by centrifugation. The supernatants were then used to measure caspase-3 level (ng/mL) using an ELISA method and caspase-3/7 activity (nmol/min/L) using a colorimetric method. Results: The study confirmed the presence of executioner caspases in platelets under non-stressed isotonic conditions. Following 15 minutes and 24 hours of hypertonic osmotic stress, caspase-3 level increased as determined by ELISA. However, caspase-3/7 activity initially decreased. With extended stress duration from 15 minutes to 24 hours, both the level and activity of these enzymes increased significantly (P-value &lt; 0.05). Conclusions: Exposure to a hypertonic osmotic environment containing 1.7% sodium chloride increases caspase-3 level in anuclear cells such as platelets, while initially reducing caspase-3/7 activity. Prolonging the duration of stress to 24 hours enhances both the level and activity of these enzymes.

Exploring novel peptides in adzuki bean and mung bean hydrolysates with potent antibacterial activity

SummaryThis study delved into the antimicrobial potential of hydrolysates from adzuki bean and mung bean protein concentrates, created using Alcalase and Flavourzyme enzymes. Targeting common foodborne pathogens (Salmonella Typhimurium, Salmonella Enteritidis, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis) and Escherichia coli, the research underscored the crucial role of peptidase in generating antibacterial peptides. Fractions F2 of Flavourzyme hydrolysed adzuki bean (ABF) and F4 of Flavourzyme hydrolysed mung bean (MBF) showed the highest antibacterial activity against S. Typhimurium and S. aureus, respectively, whereas F2 of Alcalase hydrolysed mung bean (MBA) exhibited very poor activity against S. aureus. TripleTOF LC–MS/MS analysis revealed small cationic antibacterial peptides, with lysine being prominent against S. aureus and proline–proline against S. Typhimurium. These findings highlighted the significant contribution of protease enzymes in fostering antibacterial peptide production, offering valuable insights for developing effective antimicrobial agents in the context of food safety.

Characterization of thermophilic bacteria from Ie Seum Hot Springs, Aceh Besar, Indonesia as producers of protease enzyme

Abstract. Sabaria E, Yasmin Y, Ismail YS, Bessania MA, Putri I, Fitri L. 2024. Characterization of thermophilic bacteria from Ie Seum Hot Springs, Aceh Besar, Indonesia as producers of protease enzyme. Biodiversitas 25: 1867-1874. Thermophilic bacteria are microorganisms that can survive in high-temperature environments exceeding 75°C. The Ie Seum Hot Springs in Aceh Besar, Indonesia are known to be a place where these thermophilic bacteria inhabit. In such extreme conditions, proteins and enzymes are often denatured, making these bacteria of interest due to their ability to adapt and produce enzymes such as proteases. Proteases are called proteolytic enzymes that can be applied both in the economic and medical fields. This study aimed to isolate and identify thermophilic bacteria species that have the highest potential for protease production in Ie Seum Hot Spring, Aceh Besar by analyzing the 16S rRNA gene and characterizing them using biochemical tests. Based on the result, seven thermophilic bacterial isolates, namely BT1, BT2, BT3, BT4, BT5, BT6, and BT7 were obtained, each showing different colony characteristics. The biochemical tests also revealed differences metabolic activity of each isolate. Of these isolates, BT4 exhibited the highest proteolytic index (4.65). Additionally, the 16S rRNA gene sequence analysis showed that the BT4 strain belongs to the genus Bacillus and has high similarities to Bacillus sp., B. licheniformis and B. sonorensis. These findings suggest that BT4 has significant potential as a source of thermophilic protease enzymes.

Predicting drug-Protein interaction with deep learning framework for molecular graphs and sequences: Potential candidates against SAR-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the COVID-19 disease, which represents a new life-threatening disaster. Regarding viral infection, many therapeutics have been investigated to alleviate the epidemiology such as vaccines and receptor decoys. However, the continuous mutating coronavirus, especially the variants of Delta and Omicron, are tended to invalidate the therapeutic biological product. Thus, it is necessary to develop molecular entities as broad-spectrum antiviral drugs. Coronavirus replication is controlled by the viral 3-chymotrypsin-like cysteine protease (3CLpro) enzyme, which is required for the virus's life cycle. In the cases of severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV), 3CLpro has been shown to be a promising therapeutic development target. Here we proposed an attention-based deep learning framework for molecular graphs and sequences, training from the BindingDB 3CLpro dataset (114,555 compounds). After construction of such model, we conducted large-scale screening the in vivo/vitro dataset (276,003 compounds) from Zinc Database and visualize the candidate compounds with attention score. geometric-based affinity prediction was employed for validation. Finally, we established a 3CLpro-specific deep learning framework, namely GraphDPI-3CL (AUROC: 0.958) achieved superior performance beyond the existing state of the art model and discovered 10 molecules with a high binding affinity of 3CLpro and superior binding mode.

In vitro and in vivo antifungal efficacy of individual and consortium Bacillus strains in controlling potato black scurf and possible development of spore-based fungicide

Potato is a major crop whose yield and quality are impacted by the pathogenic fungus Rhizoctonia solani. Here, a control approach based on the use of endophytic bacillus spores and bacterial consortia was assessed as an innovative solution to improve tuber productivity and health. Endophytic potato bacteria extracted from several organs (leaves, stems and roots) evaluated for their antagonistic activity against R. solani by the double culture technique and their ability to rot potato slices. Additionally, the ability of Bacillus strains to generate hydrolytic enzymes and produce lipopeptides (using ESI-MS/MS mass spectrometry) was assessed. Then, the effectiveness of selected bacterial strains, either individually or in consortium (after carrying out compatibility tests) in the suppression of R. solani was evaluated in the greenhouse. Six endophytes not affecting potato tubers and having an antagonistic effect against R. solani were identified: B. halotolerans SpS5, B. velezensis KnL15, B. aryabhattai FaR1, B. amyloliquefaciens LiR9, B. haynesii ReR10 which produce several types and combinations of lipopeptides belonging to different families (iturins, fengycins and surfactins) as well as hydrolytic enzymes (chitinase, protease and cellulase). The vegetative and sporulating strain SpS5, as well as the 5 other Bacillus and their Sps5-based consortia, were found to be very effective in increasing the yield of healthy, high-quality tubers and reducing disease severity. Based on the results mentioned above, a microbial fungicide from the spores of (i) B. halotolerans SpS5 for which we sequenced the whole genome or (ii) bacterial consortia could be developed to combat black scurf potato and other fungal infections.