Phenylmethylsulfonyl Fluoride Research Articles

A Novel Anticancer Effect of Ephedra alata Decne in Breast Cancer Cells

Cancer is a class of diseases characterized by uncontrolled cell growth. One of the main aims of developing new therapies is to use natural resources to induce apoptosis. LC-ms/ms analysis of a methanolic extract of Ephedra alata (E.A.) allowed the identification of 20 secondary metabolites, including flavonoids, phenolic acids, and proanthocyanidins. Antiproliferative effect was assessed by crystal violet assay. Antimigration effect was tested by wound healing assay and apoptosis induction was determined by annexin binding assays, Hoechst staining, ROS production, and activation of apoptotic proteins. The results indicated that exposure of breast cancer cells to E.A. extract significantly reduced cell viability in a dose and time-dependent manner and inhibited the migration of 4T1 cells at a low dose. Moreover, treatment of cells with E.A. extract induced apoptosis, as it was detected by Annexin V/7 AAD, Hoechst staining, ROS production, and the activation of caspases. Abbreviation: BSA bovine serum albumin DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid LC-ms/ms liquid chromatography-mass spectrometry NAC N-acetyl-l-cysteine PARP poly(ADP-ribose) polymerase PMSF phenylmethylsulfonyl fluoride RIPA radioimmunoprecipitation assay buffer ROS reactive oxygen species RPMI Roswell park memorial institute SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

PMSF and SFN Reduce Alpha‐synuclein Aggregation in a Yeast Model of Parkinson’s Disease

Parkinson’s Disease, PD, is the second most common neurodegenerative disease in humans. PD is marked by Lewy body formation in the brain, which disturbs the dopamine transfer system across neurons. Previous studies have shown that the protein, α‐synuclein, is a major contributor in the formation of Lewy bodies, which are a pathological hallmark of PD. In this study, we modeled α‐synuclein aggregation in the budding yeast, Saccharomyces cerevisiae and treated the cells with phenylmethylsulfonyl fluoride (PMSF) in one trial, and sulforaphane (SFN) in another. Our data suggest that a 4mM concentration of PMSF and a 200μg/ml concentration of SFN significantly reduces α‐synuclein aggregation. We are currently working to uncover the mechanism of action of these drugs.

Low Concentration of the Neutrophil Proteases Cathepsin G, Cathepsin B, Proteinase-3 and Metalloproteinase-9 Induce Biofilm Formation in Non-Biofilm-Forming Staphylococcus epidermidis Isolates.

Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and metalloproteinase-9 (MMP-9) from neutrophils were tested on the biofilm induction in commensal (skin isolated) and clinical non-biofilm-forming S. epidermidis isolates. From 81 isolates, 53 (74%) were aap+, icaA−, icaD− genotype, and without the capacity of biofilm formation under conditions of 1% glucose, 4% ethanol or 4% NaCl, but these 53 non-biofilm-forming isolates induced biofilm by the use of different neutrophil proteases. Of these, 62.3% induced biofilm with proteinase-3, 15% with cathepsin G, 10% with cathepsin B and 5% with MMP -9, where most of the protease-induced biofilm isolates were commensal strains (skin). In the biofilm formation kinetics analysis, the addition of phenylmethylsulfonyl fluoride (PMSF; a proteinase-3 inhibitor) showed that proteinase-3 participates in the cell aggregation stage of biofilm formation. A biofilm induced with proteinase-3 and DNAse-treated significantly reduced biofilm formation at an early time (initial adhesion stage of biofilm formation) compared to untreated proteinase-3-induced biofilm (p < 0.05). A catheter inoculated with a commensal (skin) non-biofilm-forming S. epidermidis isolate treated with proteinase-3 and another one without the enzyme were inserted into the back of a mouse. After 7 days of incubation period, the catheters were recovered and the number of grown bacteria was quantified, finding a higher amount of adhered proteinase-3-treated bacteria in the catheter than non-proteinase-3-treated bacteria (p < 0.05). Commensal non-biofilm-forming S. epidermidis in the presence of neutrophil cells significantly induced the biofilm formation when multiplicity of infection (MOI) 1:0.01 (neutrophil:bacteria) was used, but the addition of a cocktail of protease inhibitors impeded biofilm formation. A neutrophil:bacteria assay did not induce neutrophil extracellular traps (NETs). Our results suggest that neutrophils, in the presence of commensal non-biofilm-forming S. epidermidis, do not generate NETs formation. The effect of neutrophils is the production of proteases, and proteinase-3 releases bacterial DNA at the initial adhesion, favoring cell aggregation and subsequently leading to biofilm formation.

Characterization and application of a crude bacterial protease to produce antioxidant hydrolysates from whey protein

Bacillus sp. CL14 crude protease was partially characterized and applied to obtain antioxidant whey protein isolate (WPI) hydrolysates. Optimal activity occurred at pH 9.0 and 60 °C. Ca2+, Mg2+, and Mn2+ (5 mM) enhanced activity (12–26%), whereas Co2+, Cu2+, Fe2+, and Zn2+ inhibited it (50–94%). At 1% (v/v), Tween 20 and Triton X-100 enhanced activities (21–27%), β-mercaptoethanol decreased it (15%), and dimethyl sulfoxide (DMSO) had no effect. Sodium dodecyl sulfate (SDS; 0.1%, w/v) increased activity by 36%. Complete inhibition by phenylmethylsulfonyl fluoride (PMSF), and 85% inhibition by ethylenediaminotetraacetic acid, indicates its serine protease character and the importance of cations for activity/stability. With 5 mM Ca2+, protease was optimally active at 65 °C and completely stable after 20 min at 40–55 °C. Crude protease preferentially hydrolyzed WPI and soy protein, followed by casein. WPI hydrolysis was then performed (55 °C, pH 9.0, 5 mM Ca2+) for 0–180 min. Contents of trichloroacetic acid (TCA)-soluble proteins in WPI hydrolysates (HWPI) increased from 29% (0 min) to 50–52% (60–180 min), accompanied by enhanced radical scavenging activity (14%, 0 min; ∼34%, 60–180 min) and Fe2+-chelating ability (56%, 0 min; ∼74%, 45–180 min). CL14 protease might represent an alternative biocatalyst to obtain antioxidant hydrolysates from WPI and, potentially, from other food proteins.

Extracellular proteases and laccases produced by Pleurotus ostreatus PoB: the effects of proteases on laccase activity.

Laccases are enzymes produced by plants and white rot fungi, such as Pleurotus ostreatus, with industrial applications. Fungal laccases have been widely studied, and investigations, such as those involving recombinant DNA technology or adding inducers, have been made to increase laccase production. On the other hand, it has been proposed that extracellular proteases could decrease laccase activity when both types of enzymes are produced by P. ostreatus. The aim of this work was to evaluate the effects of proteases on the activity of extracellular laccases produced by P. ostreatus PoB in submerged culture. Results showed that P. ostreatus PoB produced alkaline, acidic, and neutral proteases. Protease activity was quantified, and the highest activity at alkaline pH (9.0) was 5.63 IU/L (192 h), that at acidic pH (2.0) was 3.38 IU/L (192 h), and that at neutral pH (7.0) was 6.20 IU/L (312 h). The protease activity decreased in the presence of different protease inhibitors, as phenylmethylsulfonyl fluoride (PMSF), EDTA, pepstatin A, and a cocktail of protease inhibitors. Laccase activity was determined in cultures with and without protease inhibitors. In the control culture (without inhibitor), the highest laccase specific activity was 99.88 IU/mg protein. In cultures with PMSF, pepstatin A, or a cocktail of protease inhibitors, laccase activity increased by approximately 1.35-fold (138 IU/mg protein) with respect to the control culture. The inhibitor EDTA did not produce a positive effect on extracellular laccase activity. These results suggest that laccase activity is affected by the actions of acidic and neutral extracellular proteases.

Structure-based discovery of small molecules improving stability of human broadly-neutralizing anti-HIV antibody 2F5 in plant suspension cells.

The production of biopharmaceuticals in engineered plant-based systems is a promising technology that has proven its suitability for the production of various recombinant glyco-proteins that are currently undergoing clinical trials. However, compared to mammalian cell lines, the productivity of plant-based systems still requires further improvement. A major obstacle is the proteolytic degradation of recombinant target proteins by endogenous plant proteases mainly from the subtilisin family of serine proteases. In the present study, the authors screened for putative small molecule inhibitors for subtilases that are secreted from tobacco BY-2 suspension cells using an in silico approach. The effectiveness of the substances identified in this screen was subsequently tested in degradation assays using the human broadly-neutralizing anti-HIV monoclonal antibody 2F5 (mAb2F5) and spent BY-2 culture medium as a model system. Among 16 putative inhibitors identified by in silico studies, three naphthalene sulfonic acid derivatives showed inhibitory activity in in vitro degradation assays and are similar to or even more effective than phenylmethylsulfonyl fluoride (PMSF), a classical inhibitor of serine proteases, which served as positive control.

Exoproduction and Biochemical Characterization of a Novel Serine Protease from Ornithinibacillus caprae L9T with Hide-Dehairing Activity.

This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9T) production from Ornithinibacillus caprae L9T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80°C) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70°C and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag+, Ca2+ and Sr2+, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9T protease in industrial applications, especially in leather processing.

Production, purification and characterization of a novel antithrombotic and anticoagulant serine protease from food grade microorganism Neurospora crassa

A novel thrombolytic enzyme was produced by food grade microorganism Neurospora crassa using agro-industrial by-products as substrates. Process parameters were optimized using Plackett–Berman and Box–Benhken design. Under the optimized fermentation conditions, high fibrinolytic activity of 403.59 U/mL was obtained. It was purified with a specific activity of 3572.4 U/mg by ammonium sulfate precipitation and SP Sepharose chromatography. The molecular weight of the enzyme was approximately 32 kDa. It exhibited maximum activity at 40 °C and pH 7.4. Its activity was enhanced by Cu2+, Na+, Zn2+, and completely inhibited by phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, which indicates it could be a serine protease. The enzyme could degrade fibrin clot directly without the need of plasminogen activator, and effectively cleaved Aα, Bβ, γ chains of fibrinogen. It could inhibit the formation of blood clots in vitro and acts as an anticoagulant. Compared to heparin the purified enzyme showed extended anticoagulant activity. Blood clots were dissolved effectively and dissolution rate was increased with time. Based on these results, this novel enzyme has the potential to be developed as a thrombolytic agent.

Determination of the fibrinogenolytic activity of Montivipera raddei (Raddeʼs mountain viper) venom

Snake venom fibrinogenolytic enzymes have diagnostic and therapeutic value and are important for snakebite pathology. In the present study, the fibrinogenolytic activity of Montivipera raddei venom was investigated. Crude venom was incubated with human fibrinogen for different time periods at 37?C. An inhibition study was carried out using different protease inhibitors. The fibrinogenolytic activity was assessed by SDS-PAGE and fibrinogen zymography. An HPLCbased method was used to obtain confirmatory data. Montivipera raddei venom predominantly cleaved the A? chain of fibrinogen in a time-dependent manner. A very slight decrease in band intensity of the B? chain was observable after a longer incubation time. Cleavage of fibrinogen was confirmed by HPLC. Zymography revealed that the venom contained 50 and 75 kDa fibrinogenolytic enzymes. Ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline inhibited the overall fibrinogenolytic activity, while phenylmethylsulfonyl fluoride (PMSF) only inhibited the degradation of the B? chain. These results indicated that metalloproteinases were major fibrinogenolytic enzymes in the venom. The inhibitor study suggested the presence of serine proteinases that broke down the B? chain. With this study, the fibrinogenolytic activity of M. raddei venom was shown for the first time. The results will be useful for further isolation and characterization studies.

PROTEOLYTIC ACTIVITY IN THE HEART OF RATS WITH HYPERHOMOCYSTEINEMIA.

The aim: To investigate the distribution of proteolytic activity and cytokine profile in the heart of rats with hyperhomocysteinemia. Materials and methods: A total of 60 albino non-linear male rats was used in the study. Hyperhomocysteinemia was induced by intragastric administration of DL-homocysteine thiolactone. Total proteolytic activity was measured using casein as a substrate. To determine the activity of metal-dependent and serine proteases, ethylenediaminetetraacetic acid, and phenylmethylsulfonyl fluoride were used. The level of matrix metalloproteinases, tissue inhibitor of metalloproteinases-1, and cytokines was studied by enzyme-linked immunosorbent assay. Results: It was found an increase in the total proteolytic activity in the heart of young, adult, and old animals. In addition, the redistribution of proteolytic activity was revealed - the portion of metal-dependent enzymes increased in all groups while the percentage of serine proteases decreased in the old animals with hyperhomocysteinemia. The state of mild inflammation, evidenced by the increased level of some pro-inflammatory cytokines, was found in the heart of young and old animals with hyperhomocysteinemia. Conclusions: The pathogenesis of hyperhomocysteinemia is accompanied by a change in the proteolytic activity in the heart as well as a change in the cytokine profile.

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

Atlantic Forest’s and Caatinga’s semiarid soils and their potential as a source for halothermotolerant actinomycetes and proteolytic enzymes

ABSTRACT Actinomycetes are versatile about their metabolism, displaying high capacity to produce bioactive metabolites. Enzymes from actinomycetes represent new opportunities for industrial applications. However, proteases from actinomycetes are poorly described by literature. Thereby, to verify proteolytic potential of actinomycetes, the present study aimed the investigation of bacterial isolates from Caatinga and Atlantic Forest rhizosphere. Fluorescence resonance energy transfer (FRET) peptide libraries were adopted for the evaluations, since they are faster and more qualitative methods, if compared with others described by most reports. A total of 52 microorganisms were inoculated in different culture media (PMB, potato dextrose agar, brain heart infusion agar, Starch Casein Agar and Reasoner’s 2A agar), temperatures (12, 20, 30, 37, 45 and 60°C), and saline conditions (0–4 M NaCl), during 7 days. The actinomycetes named as AC 01, 02 and 52 were selected and showed enzymatic abilities under the peptide probes Abz-KLRSSKQ-EDDnp and Abz-KLYSSKQ-EDDnp, achieving enhanced performance at 30 °C. Biochemical parameters were established, showing a predominance of alkaline proteases with activity under saline conditions. Secreted proteases hydrolysed preferentially polar uncharged residues (Y and N) and positively charged groups (R). Phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid inhibited the proteins, a characteristic of serine (AC 01 e 02) and metalloproteases (AC 52). All selected strains belonged to Streptomyces genera. In summary, actinomycete strains with halophilic proteolytic abilities were selected, which improve possibilities for their use in detergent formulations, food processing, waste management and industrial bioconversion. It is important to highlight that this is the first report using FRET libraries for proteolytic screening from Caatinga and Atlantic Forest actinobacteria.

Assessment of gelatinolytic proteinases in chilled grass carp (Ctenopharyngodon idellus) fillets: characterization and contribution to texture softening.

Texture softening is always a problem during chilling of grass carp fillets. To solve this problem and provide for better quality of flesh, understanding the mechanism of softening is necessary. Gelatinolytic proteinases are suspected to play an essential role in the disintegration of collagen in softening of fish flesh. In the present study, the types and contribution of gelatinolytic proteinases in chilled fillets were investigated. Four active bands (G1, 250 kDa; G2, 68 kDa; G3, 66 kDa; G4, 29 kDa) of gelatinolytic proteinases were identified in grass carp fillets by gelatin zymography. The effect of inhibitors and metal ions revealed that G1 was possibly a serine proteinase, G2 and G3 were calcium-dependent metalloproteinases and G4 was a cysteine proteinase. The effect of the inhibitors phenylmethanesulfonyl fluoride (PMSF), l-3-carboxy-trans-2,3-epoxy-propionyl-l-leucine-4-guanidinobutylamide (E-64) and 1,10-phenanthroline (Phen) on chilled fillets revealed that gelatinolytic proteinase activities were significantly suppressed. Collagen solubility indicated that metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days, and cysteine proteinase revealed its effect after 3 days. Meanwhile, during chilled storage for 11 days, the final values of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively, whereas the increase after Phen treatment was 49.89%. Our study concluded that the disintegration of collagen in post-mortem softening of grass carp fillets was mainly mediated by metalloproteinase and to a lesser extent by serine proteinase and cysteine proteinase. © 2021 Society of Chemical Industry.

A new carboxypeptidase from Aspergillus niger with good thermostability, pH stability and broad substrate specificity

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and the gene was cloned and expressed in Pichia pastoris GS115. In a shake flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 209.3 U mg−1. The optimal temperature and pH for enzyme activity were determined to be 45 °C and 6.0, respectively. After incubation at 40–50 °C or at pH 4.0–8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. The presence of 1–10 mmol L−1 Mg2+ enhanced the activity of CapA, whereas 1–10 mmol L−1 Cu2+, Fe2+, or Co2+, 10 mmol L−1 Mn2+, or 1–10 mmol L−1 phenylmethylsulfonyl fluoride (PMSF) significantly inhibited its activity. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys at the C terminus of proteins, and N-benzyloxycarbonyl-l-phenylalanyl-l-leucine (Cbz-Phe-Leu) was the optimal substrate, for which CapA exhibited Km 0.063 mmol L−1 and kcat/Km 186.35 mmol L−1 s−1. The good thermostability, pH stability and hydrolysis characteristics of CapA provide a solid foundation for application in the food and biotechnology fields.

Phenylmethylsulfonyl fluoride pulse and cold storage independently or synergistically alleviate postharvest losses in Dianthus chinensis L.

The present study focuses on examining the effect of phenylmethylsulfonyl fluoride (PMSF) at three distinct temperatures: 5 °C, 10 °C and room temperature (RT, i.e., 25 ± 2 °C) on the postharvest attributes of Dianthus chinensis flower stems. The flower stems with the most mature floral bud at the paint brush stage (i.e., one day before anthesis) were harvested and uniformly re-cut in the laboratory. These stems were then divided into three groups, with each group further categorized into four sets. The three groups of flower stems were stored for 72 h in distilled water (wet stored) at 5 °C, 10 °C and RT, respectively. At each temperature, one set designated as control was kept unpulsed and the other three sets were pulsed for 1 h with 0.05 mM PMSF before, during (i.e., after 35 ½ h) and after storage, respectively. After 72 h wet storage and PMSF pulse, the flower stems were transferred to conical flasks containing 0.05 M sucrose and the effect of different treatments was assessed in the subsequent days at room temperature. Flower stems treated with PMSF pulse or cold storage individually or in combination showed significant improvement of vase life, with the maximum value of 22 days registered for flower stems pulsed with PMSF before wet storage at 5 °C for 72 h, which was 14, 12 and 5 days more than the control at RT, 10 °C and 5 °C, respectively. The improved vase life of flower stems was marked by enhanced floral diameter, higher levels of soluble proteins and sugars as compared to the control. The flower stems with improved longevity maintained lower content of total phenols and higher membrane stability index, besides exhibiting a significant alleviation in the deteriorating effects of lipid peroxidation and lipoxygenase activity on membrane integrity. Further, the flower stems with improved postharvest characteristics were also found to be associated with elevated activity of antioxidant enzymes like superoxide dismutase, catalase, and ascorbate peroxidase.

Selection of a pH- and temperature-stable laccase from Ganoderma australe and its application for bioremediation of textile dyes

By virtue of screening, purification, and properties characterization, this study captures a new pH- and temperature-stable laccase, designated Galacc-F, from Ganoderma australe for dye bioremediating applications. The enzyme was purified to homogeneity by salt precipitation, ionic exchange, and size exclusion chromatography with a final specific activity of 22.214 U mg−1, yielding a purification fold of 23.989 and recovery of 38.44%. Its molecular weight was estimated to be 48.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, Sephadex G-100 column, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, which confirmed its monomeric nature. Galacc-F exhibited high levels of activity and stability over wide ranges of pH (5.0–8.0) and temperature (10–60 °C), which are highly valuable properties in industrial processes. Broad substrate specificity was observed, wherein a better affinity was found for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) with a low value of Km (164.137 μM) and higher kcat/Km ratio (1.663 s−1 μM−1). Activity was stimulated by Cu2+ and β-mercaptoethanol but inhibited by ethylenediaminetetraacetic acid, diethylpyrocarbonate, iodoacetic acid, phenylmethylsulfonyl fluoride, and Hg2+, indicating that Galacc-F is a metalloprotease containing a typical histidine-cysteine-serine catalytic triad. It had high tolerance to surfactants, oxidants, and salts. Additionally, a fabricated protocol for native Galacc-F immobilization onto Fe3O4@Chitosan composite nanoparticles using glutaraldehyde as a crosslinker was developed. Most importantly, the enzyme was determined to be ideal for use in efficient treatment of dye effluents as compared with the laccases requiring redox mediators.

Effective bioconversion of feather-waste Keratin by Thermo-Surfactant Stable Alkaline Keratinase produced from Aspergillus sp. DHE7 with promising biotechnological application in detergent formulations

Keratin-rich wastes are troublesome pollutants that their accumulation could lead to environmental problems. Microbial degradation of keratin wastes has emerged as an alternative bio-catalyst to steam-pressure cooking and alkali hydrolysis for the recycling of such residues to value-added products. The current work investigates the purification and biochemical characterisation of an extracellular keratinase from new Aspergillus sp. DHE7 using turkey feathers as substrate. The enzyme was purified 31-fold with a final yield of 37.5%. Purified keratinase is monomeric and its molecular mass was estimated to be 33 kDa as demonstrated on SDS-PAGE. The enzyme was active in wide range of pH (7.0–11) and temperature (37–70 °C) profiles with maximum activity at pH 8.5 and 60 °C. The activity was strongly suppressed by phenylmethylsulfonyl fluoride (93.4% inhibition) and moderately inhibited by EDTA (41.2% inhibition), suggesting that the current keratinase belongs to serine-metalloprotease. Bivalent cations, such as Ca2+, Zn2+, and Mn2+, enhanced enzyme activity by 165.7%, 176.3%, and 194.8%, respectively. The purified keratinase exhibited a wide proteolytic activity towards soluble and insoluble protein substrates displaying its greater activity toward casein, keratin, bovine serum albumin, and gelatin, followed by feathers, goat hair, and wool, and was substantially stable and compliant with surfactants and commercial laundry detergents. The Km, Vmax, Kcat and Kcat/Km values on keratin were 2.2 mg/mL, 2702.7 U/mL, 136.5 s−1 and 62.04 (s−1/mg mL−1), respectively. According to these characteristics, Aspergillus sp. DHE7 keratinase is suggested as a potential candidate for use in detergent formulations (as an additive) and in various biotechnological applications.

Disorders of proteolytic homeostasis in the liver of rats with hyperhomocysteinemia

Introduction: The liver is a key organ of the body that is responsible for maintaining homeostasis. It helps regulate almost all biochemical pathways associated with metabolism, nutrition, energy supply, and the formation of immunity. Consequently, impaired liver function can contribute to the progression of hyperhomocysteinemia and the development of associated complications. The present study aims to investigate the level and activity of metal-dependent and serine proteases in the liver of rats with hyperhomocysteinemia. Methods: A total of 60 albino nonlinear male rats were used in this study. Hyperhomocysteinemia was induced by intragastric administration of DL-homocysteine thiolactone. Total proteolytic activity was measured using casein as a substrate. To determine the activity of metal-dependent and serine proteases, ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride were used. The levels of MMP-1, MMP-2, and MMP-3 were studied by enzyme-linked immunosorbent assay. The fraction of serine proteases was isolated by affinity chromatography on the benzamidine sepharose column. The composition of serine protease fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results: The pathogenesis of hyperhomocysteinemia was found to be accompanied by a proteolytic imbalance manifested in the increase in the total proteolytic activity, the activity of serine proteases and especially the activity of metal-dependent enzymes. The increase in the levels of MMP-1, MMP-2, and MMP-3 indicates the involvement of these enzymes in the amplification of proteolysis mediated by metal-dependent enzymes. Despite a significant increase in the content of the serine proteases, their activity did not increase significantly. This may be because of the proteolytic degradation of serine proteases, which was confirmed by changes in their qualitative composition. The accumulation of low-molecular-weight proteins and the decrease in the part of proteins with a molecular weight of greater than 50 kDa in the fraction of serine proteases were revealed. Conclusion: Collectively, our observations indicate that impaired proteolysis in the liver may be an important determinant in the development of hyperhomocysteinemia-associated disorders.

Feather protein lysate optimization and feather meal formation using YNDH protease with keratinolytic activity afterward enzyme partial purification and characterization

Incubation parameters used for the creation of a protein lysate from enzymatically degraded waste feathers using crude keratinase produced by the Laceyella sacchari strain YNDH were optimized using the Response Surface Methodology (RSM); amino acids quantification was also estimated. The optimization elevated the total protein to 2089.5 µg/ml through the application of the following optimal conditions: a time of 20.2 h, a feather concentration (conc.) of 3 g%, a keratinase activity of 24.5 U/100 ml, a pH of 10, and a cultivation temperature of 50 °C. The produced Feather Protein Lysate (FPL) was found to be enriched with essential and rare amino acids. Additionally, this YNDH enzyme group was partially purified, and some of its characteristics were studied. Crude enzymes were first concentrated with an Amicon Ultra 10-k centrifugal filter, and then concentrated proteins were applied to a "Q FF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranging from 6 to 10 kDa. The maximum enzyme activity was observed at 70 °C and for a pH of 10.4. Most characteristics of this protease/keratinase group were found to be nearly the same when the activity was measured with both casein and keratin-azure as substrates, suggesting that these three protein bands work together in order to degrade the keratin macromolecule. Interestingly, the keratinolytic activity of this group was not inhibited by ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), or iron-caused activation, indicating the presence of a mixed serine–metallo enzyme type.

Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis (Guenée) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins (Cry2A, Cry1C, Cry1Aa, and Cry1Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid (EDTA), PMSF, tosyl phenylalanine chloromethyl ketone (TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). EDTA could significantly inhibit the degradation of Cry2A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1C and Cry1Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect's ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.