Enzymatic Activity Of Protease Research Articles

Effect of Matricaria aurea Essential Oils on Biofilm Development, Virulence Factors and Quorum Sensing-Dependent Genes of Pseudomonas aeruginosa.

The emergence of drug-resistant microorganisms presents a substantial global public health threat. The increase in pathogens resistant to commonly prescribed antibiotics underscores the urgent requirement to explore alternative treatment strategies. This study adopts a novel approach by harnessing natural resources, specifically essential oils (EO), to combat bacterial pathogenicity. The primary aim of this research was to analyze the chemical composition of the aerial part of the Matricaria aurea (M. aureas) EO and evaluate its potential for inhibiting quorum sensing (QS) and disrupting biofilm formation in Pseudomonas aeruginosa (P. aeruginosa). The gas chromatography-mass spectrometry (GCMS) analysis unveiled that α-bisabolol oxide A constituted the predominant portion, comprising 64.8% of the total, with β-bisabolene at 6.3% and α-farnesene at 4.8% following closely behind. The antibiofilm efficacy was observed at concentrations of 0.3, 0.15, and 0.08 mg/mL, demonstrating negligible effects on cell viability. Furthermore, the EO from M. aurea effectively inhibited the formation of P. aeruginosa biofilms by diminishing aggregation, hydrophobicity, and swarming motility. Significantly, the EO treatment resulted in a conspicuous decrease in the production of pyocyanin, rhamnolipid, and extracellular polymeric substances (EPS), along with a reduction in the enzymatic activity of protease and chitinase. The EO effectively hindered QS by disrupting QS mechanisms, resulting in a marked decline in the secretion of N-Acyl homoserine lactone (AHL) molecules and the expression of phazA1 and aprA genes. This investigation offers compelling evidence supporting the potential of M. aurea EO as a promising therapeutic candidate for addressing infectious diseases induced by biofilm formation.

Screening and Identification of a Strain with Protease and Phytase Activities and Its Application in Soybean Meal Fermentation.

The aims of the study were to degrade the anti-nutritional factors (ANFs) such as phytic acid, glycinin, and β-conglycinin and improve the values of soybean meal (SBM). Firstly, in this study, a strain PY-4B which exhibited the best enzymatic activities of protease (403.3 ± 17.8 U/mL) and phytase (62.9 ± 2.9 U/mL) was isolated and screened among the isolates. Based on the analysis of physiological and biochemical characteristics and 16S rDNA sequence, the strain PY-4B was identified and named as Pseudomonas PY-4B. Next, Pseudomonas PY-4B was applied to fermentation of SBM. The results showed that the contents of glycinin and β-conglycinin were decreased by 57-63%, and the phytic acid was remarkably degraded by 62.5% due to the fermentation of SBM by Pseudomonas PY-4B. The degradation of glycinin and β-conglycinin resulted in increase of contents of water-soluble proteins and amino acids in fermented SBM. Moreover, Pseudomonas PY-4B exhibited no hemolytic activity and slight inhibitory effect on the growth of pathogen Staphylococcus aureus and the wide range of pH tolerance (3 to 9). In summary, our study indicates that isolated strain Pseudomonas PY-4B is a safe and applicable strain and has the ability to effectively degrade the ANFs (phytic acid, glycinin, and β-conglycinin) in SBM by fermentation.

ACTIVIDAD ENZIMÁTICA Y PROCESO DE INFECCIÓN INICIAL DE LA PLANTA DE GUARANÁ (Paullinia cupana) POR CEPAS PATOGÉNICAS Y ENDOFÍTICAS DE Colletotrichum guaranicola

Anthracnose of the guarana plant (Paullinia cupana) is a disease caused by Colletotrichum guaranicola Albuq. Few studies have analyzed the origin, the form of penetration and the colonization of endophytic fungi in the guarana plant. This study aimed to determine differences in the enzymatic activities of endophytic and pathogenic strains of C. guaranicola during the initial infection process in leaves of clones sensitive to guarana anthracnose. Fourteen pathogenic and nine endophytic strains of C. guaranicola were isolated from guarana leaves with and without anthracnose symptoms, respectively. The enzymatic activities of protease, amylase, polyphenol oxidase, lipase, pectinase, and cellulolytic activities were evaluated for each fungal strain. The experimental design for each enzymatic assay was completely randomized with five replicates. The enzymatic evaluation was based on the measurement of halo produced by the enzyme reaction in each assay. Conidia were evaluated during germination and appressoria formation. Results did not detect enzymatic difference between pathogenic and endophytic strains. Data prior to germination showed differences between endophytic and pathogenic strains. Colonization of plant tissues by pathogenic strain was evidenced after the appearance of disease symptoms 48 hours after inoculation (hai), while for endophytic strains the presence of appressoria was observed on the surface of the epidermis 48 hai, and colonization of cells was not observed.

Effect of Rhamnolipids on Pathogenicity Characteristics of Microorganisms in Organic Compost

Background: Rhamnolipids are hydrophilic glycolipids, often classified as biosurfactants. They are produced by different bacterial species. Rhamnolipids are extensively studied in biological research because of their interesting features like antimicrobial, antifungal, and antiviral activities. Materials and Methods: To study the antimicrobial effect of rhamnolipid, we conducted a crosssectional study on the eight different pathogenic bacterial strains from November 2019 to June 2020. These bacterial strains were isolated from the organic compost. Both disk diffusion and broth microdilution methods were used to evaluate the inhibitory effects of rhamnolipids on these pathogenic bacteria. Also, protease and amylase enzyme activities were evaluated in these eight bacterial isolates. Results: For Bacillus stearothermophilus, within the area of 31.5 mm, no growth was observed, hence proving the inhibitory effect of rhamnolipid. After calculating the minimum inhibitory (MIC) and minimum lethal concentrations (MLC) for each bacterial strain, it has been found that the studied bacteria were more susceptible to rhamnolipids than most of the antibiotics. Strains were also quantified for their enzymatic activity of proteases and amylases. The bacterial strains of Bacillus stearothermophilus, Brevibacillus brevis, Bacillus licheniformis, and Bordetella petrii showed maximum protease activity. Whereas Escherichia coli, Enterococcus faecalis, Enterobacter aerogenes, and Pseudomonas aeruginosa showed high amylase activity. Conclusion: Rhamnolipids can be used as a potential antimicrobial agent for treatment of infections.

Dissenting degradation: Deubiquitinases in cell cycle and cancer.

Since its discovery forty years ago, protein ubiquitination has been an ever-expanding field. Virtually all biological processes are controlled by the post-translational conjugation of ubiquitin onto target proteins. In addition, since ubiquitin controls substrate degradation through the action of hundreds of enzymes, many of which represent attractive therapeutic candidates, harnessing the ubiquitin system to reshape proteomes holds great promise for improving disease outcomes. Among the numerous physiological functions controlled by ubiquitin, the cell cycle is among the most critical. Indeed, the discovery that the key drivers of cell cycle progression are regulated by the ubiquitin-proteasome system (UPS) epitomizes the connection between ubiquitin signaling and proliferation. Since cancer is a disease of uncontrolled cell cycle progression and proliferation, targeting the UPS to stop cancer cells from cycling and proliferating holds enormous therapeutic potential. Ubiquitination is reversible, and ubiquitin is removed from substrates by catalytic proteases termed deubiquitinases or DUBs. While ubiquitination is tightly linked to proliferation and cancer, the role of DUBs represents a layer of complexity in this landscape that remains poorly captured. Due to their ability to remodel the proteome by altering protein degradation dynamics, DUBs play an important and underappreciated role in the cell cycle and proliferation of both normal and cancer cells. Moreover, due to their enzymatic protease activity and an open ubiquitin binding pocket, DUBs are likely to be important in the future of cancer treatment, since they are among the most druggable enzymes in the UPS. In this review we summarize new and important findings linking DUBs to cell cycle and proliferation, as well as to the etiology and treatment of cancer. We also highlight new advances in developing pharmacological approaches to attack DUBs for therapeutic benefit.

Effects of pH adjustment on the hydrolysis of Al-enhanced primary sedimentation sludge for volatile fatty acid production

Aluminum-based chemically enhanced primary sedimentation has seen increasing use in wastewater treatment plants. However, the derived sludge (Al-sludge) has low biodegradability due to the inhibition of Al-coagulants on organic hydrolysis, which increases the difficulty of sludge treatment and limits the potential for resource recovery. In this study, the effects of pH adjustment on the pretreatment and fermentation of Al-sludge was investigated, with a focus on sludge hydrolysis and production of volatile fatty acids (VFAs). The effects of abiotic and biotic hydrolysis on sludge disintegration, organics solubilization, and hydrolytic enzyme activity were also evaluated. The results show that adjustment of the pH to between 8.0 and 10.0 was more effective than adjustment to between 2.0 and 6.0 for sludge hydrolysis and subsequent VFA production. The enzymatic activities of protease and α-glucosidase showed a positive correlation with the pH values, leading to the highest VFA yield (275 mg-COD/g-VS) for the initial pH 10.0 condition, with 46% improvement over the control reactor (without pH adjustment). Semi-continuous fermentation was found to be more effective than batch fermentation for the biotic hydrolysis rate, kH. Little phosphorus (<0.1 mg/L) was released from the Al-sludge into the fermented sludge liquor, so P removal was not necessary before the VFAs were used. The wastewater treatment process with Al-based chemically enhanced primary sedimentation and pH adjustment for enhanced sludge fermentation can recover 24% of organic carbon from domestic wastewater.

Enzymatic activity of proteases and its isoenzymes in fermentation process in cultivars of cocoa (Theobroma cacao L.) produced in southern Bahia, Brazil

Abstract The fermentation of cocoa seeds envolves microbial processes and the action of enzymes. To identify the possible differences in the cocoa fermentation process, with regards to proteolysis, this study has the objective of determining protease activity (under predetermined conditions) and its isoenzymes in two cocoa cultivars (PH-16 and HRT-1188) in different cocoa fermentation times, in addition to establishing the microbial load (molds and yeasts and aerobic mesophilic). Protease and its isoenzymes were extracted and partially purified and the enzymatic activities determined by spectrophotometry. The results showed that the proteases activity was higher at 66h of fermentation for both cultivars. When the isoenzymes activity was evaluated, the results demonstrated similar activity behavior for both cultivars, with regards to the isoenzymes aminopeptidase and carboxypeptidase, although the behavior of the endoprotease isoenzyme activity proved to be a little different for TSH-1188 cultivar. Concerning microbiological analyses, the results indicate that the period after molds and yeast counting reduction is consistent with the period of protease activity increase.

Protease, Amylase and Lactase Enzyme Stability in Gastroval® Capsules after Incubation at Acidic pH and Elevated Temperature

The stability of plant-derived enzymes present in digestion-alleviating food supplement capsules Gastroval® was studied by determining the survival of enzymatic activity of proteases, α-amylases, and lactases (β-galactosidase) after an exposure to acidic pH and elevated temperature. Most of the enzymatic activity after the prolonged (2 h) exposure to acidic conditions (pH 2.0) and elevated temperature (50°C) remained unchanged or slightly reduced, thus indicating that the enzymes would pass the stomach without losing their entire enzymatic activity and efficiently alleviate food digestion in the intestine.

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors.

Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1.

Changes in protein content and activity of proteases and acid phosphatases in needles of Norway spruce (Picea abies L. Karst) seedlings treated with 2,4,5-T

Effect of 2, 4, 5-T in different concentrations and time of herbicide applications on protein biosynthesis and enzymatic activity of proteases and acid phosphatases in needles of spruce seedlings was investigated. 2, 4, 5-T in all concentrations tested and applied on the seedlings during phase of intensive growth caused significant increase in soluble protein content. After spraying the seedlings with 100 and 300 mg ·l&lt;sup&gt;-1&lt;/sup&gt; of 2, 4, 5-T before bud development a small decrease in soluble protein level was noted. The herbicide caused marked disturbances in the activity of enzymatic systems of proteases and acid phosphatases. No effect of 2, 4, 5-T on dry matter accumulation was observed.

Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus

The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence.

The Cockroach and Allergic Diseases

The cockroach represents one of the most common sources of indoor allergens worldwide, and 40%-60% of patients with asthma in urban and inner-city areas possess IgE antibodies to cockroach allergens. In Korean homes, four cockroach species have been found, of which the most commonly encountered is the German cockroach. The pathogenic mechanism underlying the association between cockroach allergens and allergic diseases has not been fully elucidated. Allergenicity is associated with the cockroach allergens themselves, enzymatic protease activity, and ligands for pattern recognition receptors. Although allergen-specific adaptive immune responses orchestrate the cockroach allergic response, recent data suggest that the innate immune system is also a critical contributor to pathogenesis. We review the current evidence for the demographics of cockroach exposure and sensitization, characteristics of cockroach allergens, and inflammatory responses to cockroach allergens initiated through protease-dependent pathways.

Survivin and heat shock protein 25/27 colocalize with cleaved caspase-3 in surviving reactive astrocytes following excitotoxicity to the immature brain

Following immature excitotoxic brain damage, distinct patterns of caspase activation have been described in neurons and glial cells. Neuronal cells show activation of the mitochondrial apoptosis pathway, caspase-3 cleavage and apoptotic cell death, while reactive astrocytes show caspase-3 cleavage that is not always correlated with enzymatic protease activity and does not generally terminate in cell death. Accordingly, the aim of the present study was to evaluate the astrocytic colocalization of cleaved caspase-3 and several anti-apoptotic proteins of the inhibitor of apoptosis proteins family (IAPs), such as survivin and cellular inhibitor of apoptosis-2 (cIAP-2), and the heat shock proteins (HSPs) family, Hsp25/27 and Hsc70/Hsp70, which can all prevent caspases from cleaving their substrates. At several survival times ranging from 4 h to 14 days after cortical excitotoxic damage induced by N-methyl- d-aspartate (NMDA) injection at postnatal day 9 in rat pups, single and double immunohistochemical techniques were performed in free floating cryostat sections and sections were analyzed by confocal microscopy. Our results show that survivin and Hsp25/27 are primarily expressed in reactive astrocytes of the damaged cortex and the adjacent white matter. In addition, both molecules strongly colocalize with cleaved caspase-3. Survivin is primarily located in the nucleus, like cleaved caspase-3; while Hsp25/27 is cytoplasmic but very frequently found in cells showing nuclear caspase-3. cIAP-2 was mostly found in damaged neurons but also in some glial scar reactive astrocytes and showed fewer correlation with caspase-3. Hsc70/Hsp70 was only expressed in injured neurons and did not correlate with caspase-3. Thus, we conclude that primarily survivin and Hsp25/27 may participate in the inhibition of cleaved caspase-3 in reactive astrocytes and may be involved in protecting astrocytes after injury.

Novel bioadhesive chitosan-EDTA conjugate protects leucine enkephalin from degradation by aminopeptidase N.

To develop a novel bioadhesive polymer that protects peptide drugs from luminal degradation by aminopeptidase N and to evaluate the system in vitro on porcine mucosa. EDTA was covalently bound to chitosan in order to combine the bioadhesive properties of the polymer with the well known capacity of EDTA to complexed metal ions which are essential for the enzymatic activity of proteases. The inhibitory effect of this polymer conjugate was evaluated by using leucine enkephalin (Leu enkephalin) as a model drug. The degree of Leu enkephalin degradation caused by aminopeptidase N (EC 3.4.11.2), as well as porcine mucosa, in the presence of the polymer conjugate, was quantified by HPLC analysis. The chitosan-EDTA conjugate is capable of binding 2.01 +/- 0.12 mmole of zinc per gram of polymer at pH 6.5 (n = 3; +/-S.D.). As zinc is an essential co-factor for aminopeptidase N, enzyme activity (48 mU/ml) could be completely inhibited under the use of 1.0% chitosan-EDTA conjugate. The inhibitory effect of 1.0% chitosan-EDTA conjugate on the degradation of Leu enkephalin on porcine mucosa within 3 h at 37 degrees C was even 2.9-fold higher than that of a recently developed zinc complexing bacitracin-poly(acrylic acid) conjugate of the same concentration. The novel polymer conjugate is more bioadhesive than unmodified chitosan and is easily hydratable in water and basic aqueous solutions exhibiting quick swelling properties. The bioadhesive polymer conjugate described here seems to be a useful tool in overcoming enzymatic degradation by aminopeptidase N.

Microbial activities in the Lena River delta and Laptev Sea

Total bacteria, bacterial production and bacterial extracellular enzymatic activities were determined from water samples taken in the Lena River delta and adjacent Laptev Sea. These parameters were determined to assess organic matter recycling and ultimate preservation with regard to organic carbon production, transport from continent into the ocean, and sequestration in arctic environments. Total bacterial cells varied in the range 2.0 × 108 − 8.3 × 109 1−1, higher values being encountered in the Lena River and its delta and in the Laptev Sea, at stations influenced by riverine waters. Net bacterial production was highly variable, from high values in the Lena River and its delta (up to 7.8 pmol l−1 h−1) down to the lowest values ( 19). Specific extracellular enzymatic activities of protease, glucosidase, galactosidase and phosphatase were determined with some insight into kinetic parameters, substrate affinity and maximum incorporation rate.

Avian Retroviral Protease and Cellular Aspartic Proteases are Distinguished by Activities on Peptide Substrates

The avian sarcoma/leukemia virus protease (PR), purified from avian myeloblastosis virus has a native molecular mass of 26 kDa, suggesting a dimer structure. The enzymatic activity of PR has been characterized using synthetic peptide substrates. PR is most active at pH 5.5, 35 degrees C and 2-3 M NaCl. Under these conditions PR cleaves decapeptides which are resistant in low ionic strength. This high, nonphysiological, salt concentration also increases the proteolytic activity of a cellular aspartic protease, pepsin. PR and pepsin show additional similarities: they both cleave a synthetic decapeptide at the same Tyr-Pro bond in low and high salt, while the cleavage site preferences of human renin and cathepsin-D in this substrate are altered at high salt concentrations. In addition, iodination of the tyrosine residue in this decapeptide causes an increase in the rates of hydrolysis by both PR and pepsin. However, Km values are too high to be estimated accurately for PR using Tyr-Pro and Tyr(I)-Pro decapeptides as substrates. Comparison of the digestion products of two additional decapeptides, altered in a single amino acid residue, shows that PR cleaves at fewer sites than all three cellular enzymes. Furthermore pepstatin, a strong inhibitor of pepsin, renin, and cathepsin-D has little effect on PR.

Investigations on Changes in Soluble Protein Content and Activity of Proteases and Acid Phosphatases in Needles of Norway Spruce (Picea abies L. Karst) Seedlings Treated with Dalapon

The effect of dalapon (2,2-dichloropropionic acid, sodium salt) in different concentrations on the soluble protein content and enzymatic activity of proteases and acid phosphatases in the needles of spruce seedlings treated with this herbicide before bud development and during bud formation was investigated. The following results have been obtained: 1. Dalapon in all concentration tested increased soluble protein content in needles of spruce seedlings, regardless of the plant growth stage. 2. No effect of dalapon on total nitrogen content could be observed. 3. Dalapon applied on seedlings during bud formation retarded the proteolytic activity of enzymes in the needles. In needles treated with the herbicide before bud development a stimulation of protease activity was observed (measurements were made in newly formed needles). 4. The activity of acid phosphatases can be stimulated or retarded depending on dalapon concentration, plant growth stage, and time of exposure. 4. Dalapon disturbes the metabolism in newly formed plant organs.