Phenylmethylsulfonyl Fluoride Research Articles

Cathepsin B- and L-like Protease Activities Are Induced During Developmental Barley Leaf Senescence.

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. Previously cysteine proteases have been found to be highly expressed during leaf senescence in different plant species. Using biochemical and immunoblotting approaches, we characterized developmental senescence of barley (Hordeum vulgare L. var. 'GemCraft') leaves collected from 0 to 6 weeks after the onset of flowering. A decrease in total protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunits occurred in parallel with an increase in proteolytic activity measured using the fluorogenic substrates Z-RR-AMC, Z-FR-AMC, and casein labeled with fluorescein isothiocyanate (casein-FITC). Aminopeptidase activity detected with R-AMC peaked at week 3 and then decreased, reaching a low level by week 6. Maximal proteolytic activity with Z-FR-AMC and Z-RR-AMC was detected from pH 4.0 to pH 5.5 and pH 6.5 to pH 7.4, respectively, while two pH optima (pH 3.6 to pH 4.5 and pH 6.5 to pH 7.4) were found for casein-FITC. Compound E-64, an irreversible cysteine protease inhibitor, and CAA0225, a selective cathepsin L inhibitor, effectively inhibited proteolytic activity with IC50 values in the nanomolar range. CA-074, a selective cathepsin B inhibitor, was less potent under the same experimental conditions, with IC50 in the micromolar range. Inhibition by leupeptin and phenylmethylsulfonyl fluoride (PMSF) was weak, and pepstatin A, an inhibitor of aspartic acid proteases, had no effect at the concentrations studied (up to 0.2 mM). Maximal proteolytic activity with the aminopeptidase substrate R-AMC was detected from pH 7.0 to pH 8.0. The pH profile of DCG-04 (a biotinylated activity probe derived from E-64) binding corresponded to that found with Z-FR-AMC, suggesting that the major active proteases are related to cathepsins B and L. Moreover, immunoblotting detected increased levels of barley SAG12 orthologs and aleurain, confirming a possible role of these enzymes in senescing leaves.

Identification and biochemical characterization of a novel halolysin from Halorubellus sp. PRR65 with a relatively high temperature activity.

Extracellular proteases from haloarchaea, also referred to as halolysins, are in increasing demand and are studied for their various applications in condiments and leather industries. In this study, an extracellular protease encoding gene from the haloarchaeon Halorubellus sp. PRR65, hly65, was cloned and heterologously expressed in E. coli. The novel halolysin Hly65 from the genus Halorubellus was characterized by complete inhibition of phenylmethanesulfonyl fluoride (PMSF) on its enzyme activity. Experimental determination revealed a triad catalytic active center consisting of Asp154-His193-Ser348. Deletion of the C-terminal extension (CTE) resulted in loss of enzyme activity, while dithiothreitol (DTT) did not inhibit the enzyme activity, suggesting that Hly65 may function as a monomer. The Km, Vmax and Kcat for the Hly65 were determined to be 2.91mM, 1230.47 U·mg-1 and 1538.09 S-1, respectively, under 60°C, pH 8.0 and 4.0M NaCl using azocasecin as a substrate. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and reorganization of halolysins to generate mutants with new physiological activities.

Assessing the Efficacy of Protease Inactivation for the Preservation of Bioactive Amphibian Skin Peptides.

The skin of amphibians is a rich source of peptides with a wide range of biological activities. They are stored in secretory granules in an inactive form. Upon stimulation, they are secreted together with proteases into the skin. Once activated, they rapidly exert their biological effects, including fighting microorganisms and predators, while their excess is immediately destroyed by the released proteases. To keep bioactive peptides in their initial form, it is necessary to inhibit these enzymes. Several inhibitors for this purpose have previously been mentioned; however, there has not been any reliable comparison of their efficiency so far. Here, we studied the efficiency of methanol and hydrochloric and formic acids, as well as phenylmethylsulfonyl fluoride, in the inhibition of nine frog peptides with the known sequence, belonging to five families in the secretion of Pelophylax esculentus. The results demonstrated that methanol had the highest inhibitory efficiency, while phenylmethylsulfonyl fluoride was the least efficient, probably due to its instability in aqueous media. Possible cleavages between certain amino acid residues in the sequence were established for each of the inhibitors. These results may be helpful for future studies on the nature of proteases and on prediction of the possible cleavage sites in novel peptides.

Novel extracellular lipase gene Lip1728 influences nutrient-dependent performance bacterial quorum sensing of Burkholderia pyrrocinia WZ10-3

Quorum sensing (QS) is a cellular communication mechanism in which bacteria secrete and recognize signaling molecules to regulate group behavior. Lipases provide energy for bacterial cell growth but it is unknown whether they influence nutrient-dependent QS by hydrolyzing substrate. A high-yield lipase-producing strain, Burkholderia pyrrocinia WZ10–3, was previously identified in our laboratory, but the composition of its crude enzymes was not elucidated. Here, we identified a key extracellular lipase, Lip1728, in WZ10–3, which accounts for 99 % of the extracellular lipase activity. Lip1728 prefers to hydrolyze triglycerides at sn-1,3 positions, with pNP-C16 being its optimal substrate. Lip1728 exhibited activity at pH 5.0–10.0 and regardless of the presence of metal ions. It had strong resistance to sodium dodecyl sulfate and short-chain alcohols and was activated by phenylmethanesulfonylfluoride (PMSF). Lip1728 knockout significantly affected lipid metabolism and biofilm formation in the presence of olive oil. Finally, oleic acid, a hydrolysate of Lip1728, influenced the production of the signal molecule N-acyl homoserine lactone (AHL) and biofilm formation by downregulating the AHL synthetase gene pyrI. In conclusion, Lip1728, as a key extracellular lipase in B. pyrrocinia WZ10–3, exhibits superior properties that make it suitable for biodiesel production and plays a crucial role in QS.

Limosilactobacillus reuteri DSM 17938 Inhibition of Biofilm Formation by Prevotella intermedia and Fusobacterium nucleatum Across Salivary pH: An In Vitro Study

Abstract Objectives This study aims to investigate Limosilactobacillus reuteri DSM 17938's antibiofilm effects on Prevotella intermedia and Fusobacterium nucleatum, common causes of alveolar osteitis. It seeks topical alternatives to prevent this condition posttooth extraction. The secondary objective is to assess these effects under different pH conditions (pH 4.5 and pH 7), mimicking oral cavity saliva pH dynamics. Materials and Methods Ethical approval was secured for the saliva collection process involving five healthy adult participants who had undergone wisdom tooth extraction. Saliva samples were diligently collected on the 7th day post-surgery. The unstimulated saliva underwent a series of treatments, including the addition of phenylmethylsulfonyl fluoride (PMSF), pH adjustments, centrifugation, and filtration. The pH levels were re-measured, and subsequent adjustments were made to achieve pH values of 4.5 or 7. Limosilactobacillus reuteri DSM 17938, with a concentration of 1×108 colony-forming units (CFU) per 5 drops, was utilized in the study. Biofilm testing involved incubating saliva samples with varying pH (4.5 or 7) alongside bacterial suspensions (Prevotella intermedia, Fusobacterium nucleatum, or a mixed species). The Interlac suspension was introduced, and plates were anaerobically incubated for 24 hours. Biofilm results were obtained using a spectrometer. The test is conducted in triplicate. Statistical Analysis To scrutinize the impact of pH on biofilm development, the acquired data underwent a two-way ANOVA test in SPSS as part of the statistical analysis. A significance level of p<0.05 was used to determine statistical significance. Results Limosilactobacillus reuteri DSM 17938 significantly reduced biofilm formation across bacterial strains (p = 0.000). Statistical analysis indicated a significant impact of pH on biofilm development (p = 0.000) compared to no saliva samples, with higher formation observed under acidic conditions (pH 4.5). However, the pH levels of 4.5 and 7 did not result in significantly different bacterial biofilm formation (p = 0.529). Conclusion This research highlights Limosilactobacillus reuteri DSM 17938's potency in inhibiting biofilm formation of Prevotella intermedia and Fusobacterium nucleatum. Salivary pH variations significantly influence biofilm development, emphasizing the need to consider pH when assessing probiotic effectiveness. Despite limitations in saliva sample sterilization, this study provides valuable insights into alternative approaches for preventing alveolar osteitis. Further research should explore clinical applications and refine sterilization methods for more accurate results.

Unveiling the chemical profile, synergistic antibacterial and hemolytic effects of Cymbopogon citratus and Tachyspermum ammi leaves

Current study was performed to investigate the antioxidant, synergistic antibacterial and hemolytic potential of aqueous (AC & AT) and ethanolic (EC & ET) extracts of Cymbopogon citratus and Trachyspermum ammi leaves. The investigated extracts were characterized by FT-IR, HPLC, GCMS and ICP analyses. FT-IR data verified the existence of N–H, O–H, C–O, C=O, S=O, C–N, C=N, C–H(aromatic), Si–C and Si–O moieties in extracts. The highest values of TPC (97.09 mg/100g GAE) and TFC (75.76 mg/100gCE) were shown by the ethanolic extract of T. ammi whereas the aqueous extract of C. citratus showed the lowest quantities of TPC (4.07 mg/100g GAE) and TFC (2.43 mg/100g CE). GC-MS analysis showed the existence of 45, 30, 24 and 8 compounds in AC, EC, ET and AT extracts, respectively. The highest concentrations in AC, EC, ET and AT were observed for 3-hydroxy-3-methylbutanoic acid (11.99%), lupeol (23.26%), phytol (15.53%) and phenylmethylsulfonyl fluoride (58.49%). ICP analysis determined the concentration of 26 metals in extracts. AC possessed the highest concentrations of Na, Ca, Mg and K and may find uses as a nutritional/medicinal supplement of these metals. The ethanolic extract of each plant was richer in antioxidant contents; the ethanolic extract of T. ammi being the richest with 69.07% radical scavenging activity relative to BHT (83.54%). Mixtures of plant extracts exhibited higher biofilm inhibitions than their ingredients or ciprofloxacin against S. aureus and E. coli. Hemolytic activities of all the investigated leaf extracts were found in the safe range.

Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Culture Supernatant of Coprinus comatus.

A novel fibrinolytic enzyme was produced by the liquid fermentation of Coprinus comatus. The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn2+, K+, Ca2+, Mn2+, and Mg2+ while being inhibited by Fe2+, Fe3+, Al2+, and Ba2+. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases.

Purification, and characterization of detergent-compatible serine protease from Bacillussafensis strain PRN1: A sustainable alternative to hazardous chemicals in detergent industry

Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of Monopteruscuchia were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz.Bacillus,Priestia,Aeromonas,Staphylococcus, and Serratia demonstrated proteolytic activity. Bacillussafensis strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of B.safensis strain PRN1 includes 72 h (OD600 = 0.56,1303 U/mL), pH 8 (OD600 = 0.83, 403.29 U/mL), 40 °C (OD600 = 1.75, 1849.11 U/mL), fructose (OD600 = 1.22, 1502 U/mL), and gelatin (OD600 = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties.

Partial purification and characterization of protease extracted from kinema

Proteases are large group of highly demanded enzymes having huge application in food and pharmaceutical industries. Numerous sources, including plants, microorganisms, and animals, can be used to obtain protease. Due to its affordability and safety consideration, fermented foods have recently attracted more attention as a source of microbial protease. The present study aimed to extract protease from kinema, partially purify the extracted protease following dialysis after precipitation with ammonium sulfate, and determine general characteristics of protease. The kinema having highest proteolysis activity after three days of control fermentation (Temperature 30±2 °C, RH 66 ± 2%) was taken for the study. About 2.45 fold of purification with overall recovery of 63.21% was achieved after precipitation with ammonium sulfate at 30–70% saturation level followed by dialysis of crude extracted protease. The dialysed kinema protease had specific activity of 7.90 U/mg. The enzyme remained actively functional across a wider pH (5–9) and temperature (40-60 °C) range. SDS-PAGE and Zymogram confirmed the presence of three major active bands respectively of 29.04 kDa, 36.09 kDa and 46.35 kDa in the kinema protease extract. The enzyme kinetics data on casein, fitted to Mechaelis Mentens’ plots showed the protease had Vmax of 1.001 U/ml with corresponding Km value of 0.825 mg/ml. Metal ions such as iron, mercury and aluminium showed the inhibition effect whereas presence of sodium, zinc, and calcium shows the activation effect on protease performance. The enzyme was active over various natural substrates; showing maximal activity on casein, and subsequent to bovine serum albumin, gelatin, hemoglobin and whey protein respectively. Furthermore, molecular weight distribution of the protease extract and activity inhibition with ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride, suggesting the protease from kinema could be a metal dependent serine protease or mixture of them.

Physicochemical cell disruption of Bacillus sp. for recovery of polyhydroxyalkanoates: future bioplastic for sustainability

Polyhydroxybutyrate (PHB) is known for wide applications, biocompatibility, and degradability; however, it cannot be commercialized due to conventional recovery using solvents. The present study employed mechanical cell-disruption methods, such as Pestle and mortar, sonication, and glass bead vortexing, for solvent-free extraction of PHA from Bacillus sp. Different time intervals were set for grinding (5, 10, 15 min), sonicating (1, 3 and 5 min), and vortexing (2, 5 and 8 g glass beads with 5, 10 and 15 min each) hence studying their effect on cell lysis to release PHA. Tris buffer containing phenylmethyl sulfonyl fluoride (PMSF) (20 mM Tris–HCl, pH 8.0, 1 mM PMSF) was employed as a lysis buffer to study its action over Bacillus cells. Its presence was checked with the above methods in cell lysis. Sonicating cells for 5 min in the presence of lysis buffer achieved a maximum PHA yield of 45%. Cell lysis using lysis buffer yielded 35% PHA when vortexing with 5 g glass beads for 15 min. Grinding cells for 15 min showed a maximum yield of 34% but lacked a lysis buffer. The overall results indicated that the action of lysis buffer and physical extraction methods improved PHA yield by %. Therefore, the study sought to evaluate the feasibility of applying laboratory methods for cell disruption. These methods can showcase possible opportunities in large-scale applications. The polymer yield results were compared with standard sodium hypochlorite extraction. Confirmation of obtained polymers as polyhydroxy butyrate (PHB) was made through FTIR and 1HNMR characterization.

Production and characterization of an organic solvent activated protease from haloalkaliphilic bacterium Halobiforma sp. strain BNMIITR

An unusual haloalkaliphilic bacterium known as Halobiforma sp. strain BNMIITR, which was noticed to produce an extracellular alkaline protease, was found in a soil sample from Northern India's Sambhar Lake. On the generation of protease, the effects of dietary elements including nitrogen and carbon sources, amino acids, and growth conditions like temperature and pH were investigated. When low-cost agricultural by-products were employed as nitrogen sources, the manufacturing of enzymes was significantly boosted. In the present study, protease production was enhanced by 2.94 fold and 2.17 fold. By solvent precipitation and Hydrophobic interaction chromatography (HIC) on Phenyl Sepharose 6 Fast Flow matrix, the enzyme was purified 31.67 fold. It was determined that the apparent molecular mass was 21 kDa. The pH range where the enzyme was most stable was 6.0–12.0, with a temperature of 50 °C as optimum. When there was alkaline earth metals and heavy metals, protease was discovered to be active. It was evident that the enzyme was a serine type of protease because it was active in the presence of a variety of surfactants, oxidizing and reducing chemicals, and phenylmethylsulfonyl fluoride (PMSF) completely inhibited activity. Enzyme exhibited a wide range of substrate specificity. Amazingly, enzyme remained stable both in polar and nonpolar solvents. The most interesting aspect of this enzyme is enhanced activity in polar solvents like dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). It was discovered that the protease was stable and compatible with a number of widely available detergents.

Biochemical Properties and Antithrombotic Effect of a Serine Protease Isolated from the Medicinal Mushroom Pycnoporus coccineus (Agaricomycetes).

The purification of a fibrinolytic enzyme from the fruiting bodies of wild-growing medicinal mushroom, Pycnoporus coccineus was achieved through a two-step procedure, resulting in its homogeneity. This purification process yielded a significant 4.13-fold increase in specific activity and an 8.0% recovery rate. The molecular weight of P. coccineus fibrinolytic enzyme (PCFE) was estimated to be 23 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PCFE demonstrated its optimal activity at a temperature of 40 °C and pH 8. Notably, the enzymatic activity was inhibited by the presence of zinc or copper metal ions, as well as serine protease inhibitors, such as phenylmethylsulfonyl fluoride and 4-amidinophenylmethanesulfonyl fluoride. PCFE exhibited remarkable specificity towards a synthetic chromogenic substrate for thrombin. The enzyme demonstrated the Michaelis-Menten constant (Km), maximal velocity (V ), and catalytic rate constant (Kcat) values of 3.01 mM, 0.33 mM min-1 μg-1, and 764.1 s-1, respectively. In vitro assays showed PCFE's ability to effectively degrade fibrin and blood clots. The enzyme induced alterations in the density and structural characteristics of fibrin clots. PCFE exhibited significant effects on various clotting parameters, including recalcification time, activated partial thromboplastin time, prothrombin time, serotonin secretion from thrombin-activated platelets, and thrombin-induced acute thromboembolism. These findings suggest that P. coccineus holds potential as an antithrombotic biomaterials and resources for cardiovascular research.

Thrombolytic protease characterization from leaves and fruit flesh of the jernang rattan plant (Daemonorops draco)

Thrombolytic agents are used for thrombolytic therapy to dissolve blood clots that form in a blood vessel. All currently used thrombolytic agents have unfavorable shortcomings, such as gastrointestinal bleeding, allergic reactions, and thrombolytic agent resistance, treatment for some of which can be quite expensive. As a result, the search for thrombolytic agents derived from plants is currently taking place. Some plants have been discovered to contain protease enzymes with thrombolytic activity; pharmaceuticals derived from plants are believed to be safer. Jernang rattan (Daemonorops draco) is a plant of the Arecaceae family and is known to produce resin. Jernang rattan resin is also known to have antioxidant, antiseptic, antitumor, antimicrobial, and cytotoxic activity, but very limited information on proteolytic activity of the protease from this plant. This research aims to isolate proteases from the leaves and fruit flesh of the rattan jernang plant (D. draco) and to investigate the proteolytic activity of the isolated proteases. The protease was isolated from the leaves and the fruit flesh, and then partially purified by ammonium sulfate precipitation. The radial caseinolytic assay showed that protease in a 60% ammonium sulfate fraction gave a clear zone, with diameters of 1.4 cm and 1.8 cm for the protease isolated from leaves and fruit flesh, respectively. A Folin‐Ciocalteau assay showed that the enzymes isolated were able to hydrolyze casein and release L‐tyrosine, with activity of 0.158 U/mL and 0.174 U/mL for the protease from the leaves and fruit flesh, respectively. A fibrinogenolytic assay showed that the protease from the fruit flesh hydrolyzed the A‐α, B‐β and the γ chain of human fibrinogen, while the protease from the leaves hydrolyzed the A‐α and γ chain. Both proteases were inhibited by 56% by phenylmethylsulfonyl fluoride (PMSF), indicating that the enzymes are serine proteases. Based on the assay results obtained, it can be concluded that proteases isolated from the leaves and fruit flesh have potential as thrombolytic proteases.

Biochemical and hemostatic description of a thrombin-like enzyme TLBro from Bothrops roedingeri snake venom

Objective: The current study’s objective is to characterize a new throm-bin-like enzyme called TLBro that was obtained from Bothrops roedingeris snake from a biochemical and hemostatic perspective.Methodology: One chromatographic step was used to purify it, producing the serine protease TLBro. Molecular mass was estimated by SDS-PAGE to be between reduced and unreduced by 35 kDa. Tryptic peptide sequencing using Swiss Prot provided the complete amino acid sequence. Expasy.org by conducting a search that is limited to Crotalinae snake serine proteases and displaying a high degree of amino acid sequence.Results: Ser (182) is inhibited by phenylmethylsulfonyl fluoride (PMSF), and TLBro demonstrated the presence of Asp (88) residues. It also deduced the positions of His (43) and Ser (182) in the set of three coordinated amino acids in serine proteases. It was discovered that this substrate had high specificity for BANA, Michaelis-Menten behavior with KM 0 point85 mM and Vmax 1 point89 nmoles -NA/L/min, and high stability between temperatures (15 to 70°C) and pHs (2 point0 to 10 point0). According to doses and incubation times, TLBro degraded fibrin preferentially on the B-chain; additionally, its activities were significantly diminished after preincubation with divalent ions (Zn2 and Cd2). When incubated with PMSF, a particular serine protease inhibitor, enzymatic activities and platelet aggregation were inhibited.Conclusion: The findings revealed distinct structural and functional differences between the serine proteases, adding to the information and assisting in the improvement of the structure-function relationship.

Molecular Cloning, Characterization, and Application of Organic Solvent-Stable and Detergent-Compatible Thermostable Alkaline Protease from Geobacillus thermoglucosidasius SKF4.

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80°C. In addition, the enzyme showed a half-life of 15 h at 80°C, and there was a 60% increase in its activity at 10 mM Ca2+ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

Study on active capacity and detergent application potential of low-temperature alkaline serine protease produced by new strain Exiguobacterium indicum 1.2.3

Microorganisms have long captivated researchers for their potential to produce enzymes with diverse industrial applications. Efficient production of proteases from new strains is crucial as these enzymes play a vital role in breaking down protein bonds, enabling their use in industrial applications. Therefore, a novel Exiguobacterium indicum 1.2.3 was isolated (Istanbul, Turkiye) and characterized in this study. This strain produced alkaline serine protease, which works in lower temperatures (20–40 °C) with casein as a specific substrate. The protease was utterly stable for 3 h at 30 °C. The enzyme was also highly stable in the pH range of 8–11. The optimum activity was obtained at pH 10. The crude enzyme activity was enhanced by various metal ions and retained 147%, 125%, 124%, and 117% of its activity within 1 mM Ca2+, Mn2+, Cu2+, and Mg2+, respectively. The crude enzyme was inactive with phenylmethylsulfonyl fluoride, indicating a serine residue on the active side. The enzyme exhibited a significant proteolytic effect in the presence of surfactants and oxidizing agents. The addition of Tween 80, Triton X-100, and sodium perborate improved enzymatic activity up to 135%, 109%, and 105%, respectively. According to the washing results, the crude enzyme effectively removed the blood on different types of standard pre-stained textiles at 30 °C. In conclusion, Exiguobacterium indicum 1.2.3 is a promising candidate for protease production, with its diverse applications spanning various industrial sectors, particularly detergents.Graphical

Cloning, expression, and characterization of a surfactant-stable alkaline serine protease (KNBSSP1) from Bacillus safensis PRN1 with remarkable applications in laundry and leather industries

Proteases represent an important category of proteolytic enzymes with vast biotechnological applications in various industries like detergent, leather, agriculture, poultry, pharmaceutical, food, etc. The present study proposes an eco-friendly enzyme-mediated approach as opposed to conventional chemical approaches in detergent and laundry industries that are hazardous to human health and the environment. Accordingly, the knbsSP1gene, which encodes the trypsin-like serine protease KNBSSP1, from Bacillus safensis PRN1 was isolated, cloned into an expression vector (PET-28a), and expressed in the Escherichia coli cells BL21 (DE3). A single Ni-NTA affinity chromatography step was utilized to purify the recombinant protease (rKNBSSP1). The molecular weight of the rKNBSSP1 was approximately ∼33 kDa determined in Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The rKNBSSP1 showed optimal activity at 60 °C and pH 8. It retained above 70% of its enzyme activity at pH 10 and 70 °C. The protease was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate suggesting its inclusion in the serine family of protease. The rKNBSSP1 demonstrated outstanding compatibility and stability with metal ions, commercial detergents, and surfactants, exhibiting 97.8 ± 2.5% stability with Surf Excel, a commercial laundry detergent, and 96.9 ± 2.0% stability with SDS. Interestingly, the protease demonstrated blood-stained removal and goat skin dehairing (8 h) properties. All these remarkable characteristics make this protease a potential eco-friendly candidate in the detergent formulation and leather processing industries.

Structural dynamics at the active site of the cancer-associated flavoenzyme NQO1 probed by chemical modification with PMSF.

A large conformational heterogeneity of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with various human diseases, has been observed to occur in the catalytic site of the enzyme. Here, we report the X-ray structure of NQO1 with phenylmethylsulfonyl fluoride (PMSF) at 1.6 Å resolution. Activity assays confirmed that, despite being covalently bound to the Tyr128 residue at the catalytic site, PMSF did not abolish NQO1 activity. This may indicate that the PMSF molecule does not reduce the high flexibility of Tyr128, thus allowing NADH and DCPIP substrates to bind to the enzyme. Our results show that targeting Tyr128, a key residue in NQO1 function, with small covalently bound molecules could possibly not be a good drug discovery strategy to inhibit this enzyme.

Isolation and Characterization of Thermus thermophilus Strain ET-1: An Extremely Thermophilic Bacterium with Extracellular Thermostable Proteolytic Activity Isolated from El Tatio Geothermal Field, Antofagasta, Chile.

The present study describes the isolation of an extremely thermophilic bacterium from El Tatio, a geyser field in the high planes of Northern Chile. The thermophile bacterium named Thermus thermophilus strain ET-1 showed 99% identity with T. thermophilus SGO.5JP 17-16 (GenBank accession No. CP002777) by 16S rDNA gene analysis. Morphologically, the cells were non-sporeforming Gram-negative rods that formed colonies with yellow pigmentation. This strain is able to proliferate between 55 and 80 °C with a pH range of 6-10, presenting an optimum growth rate at 80 °C and pH 8. The bacterium produces an extracellular protease activity. Characterization of this activity in a concentrated enzyme preparation revealed that extracellular protease had an optimal enzymatic activity at 80 °C at pH 10, a high thermostability with a half-life at 80 °C of 10 h, indicating that this enzyme can be classified as an alkaline protease. The proteolytic enzyme exhibits great stability towards chelators, divalent ions, organic solvents, and detergents. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), implying that it was a serine protease. The high thermal and pH stability and the resistance to chelators/detergents suggest that the protease activity from this T. thermophilus. strain could be of interest in biotechnological applications.

Antiplatelet mechanism of a subtilisin-like serine protease from Solanum tuberosum (StSBTc-3)

The aims of this study are to characterize the antiplatelet activity of StSBTc-3, a potato serine protease with fibrino (geno) lytic activity, and to provide information on its mechanism of action. The results obtained show that StSBTc-3 inhibits clot retraction and prevents platelet aggregation induced by thrombin, convulxin, and A23187. Platelet aggregation inhibition occurs in a dose-dependent manner and is not affected by inactivation of StSBTc-3 with the inhibitor of serine proteases phenylmethylsulfonyl fluoride (PMSF). In addition, StSBTc-3 reduces fibrinogen binding onto platelets. In-silico calculations show a high binding affinity between StSBTc-3 and human α2bβ3 integrin suggesting that the antiplatelet activity of StSBTc-3 could be associated with the fibronectin type III domain present in its amino acid sequence. Binding experiments show that StSBTc-3 binds to α2bβ3 preventing the interaction between α2bβ3 and fibrinogen and, consequently, inhibiting platelet aggregation. StSBTc-3 represents a promising compound to be considered as an alternative to commercially available drugs used in cardiovascular therapies.