RESUMO
Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the "common" TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose-response, by limited structure-activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.
Assuntos
Descoberta de Drogas/métodos , Estabilidade Enzimática/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas , Triose-Fosfato Isomerase/química , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Células HEK293 , Humanos , Mutação , Triose-Fosfato Isomerase/antagonistas & inibidores , Triose-Fosfato Isomerase/deficiência , Triose-Fosfato Isomerase/genéticaRESUMO
Garlic is a well-known example of natural self-defence system consisting of an inactive substrate (alliin) and enzyme (alliinase) which, when combined, produce highly antimicrobial allicin. Increase of alliinase stability and its activity are of paramount importance in various applications relying on its use for in-situ synthesis of allicin or its analogues, e.g., pulmonary drug delivery, treatment of superficial injuries, or urease inhibitors in fertilizers. Here, we discuss the effect of temperature, pH, buffers, salts, and additives, i.e. antioxidants, chelating agents, reducing agents and cosolvents, on the stability and the activity of alliinase extracted from garlic. The effects of the storage temperature and relative humidity on the stability of lyophilized alliinase was demonstrated. A combination of the short half-life, high reactivity and non-specificity to particular proteins are reasons most bacteria cannot deal with allicin's mode of action and develop effective defence mechanism, which could be the key to sustainable drug design addressing serious problems with escalating emergence of multidrug-resistant (MDR) bacterial strains.
Assuntos
Liases de Carbono-Enxofre/metabolismo , Fenômenos Químicos , Dissulfetos/metabolismo , Alho/enzimologia , Ácidos Sulfínicos/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/ultraestrutura , Biocatálise/efeitos dos fármacos , Soluções Tampão , Dissulfetos/química , Estabilidade Enzimática/efeitos dos fármacos , Liofilização , Concentração de Íons de Hidrogênio , Cinética , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Estereoisomerismo , Ácidos Sulfínicos/química , Temperatura , Fatores de TempoRESUMO
Herein acid phosphatase isoenzyme was extracted from the C. murale seedlings. The purification was accomplished by chromatographic techniques and passing through DEAE-cellulose and Sephadex G-100 column. The specific activity of acid phosphatase 5.75 U/mg of protein was obtained with 66 purification fold 15.8% yield and molecular mass was 29 kDa with very faint bands corresponding to 18 kDa and 14 kDa. The maximal activity at pH 5.0 and 50 °C best illustrated by first order kinetics. When temperature was raised (55 °C to 75 °C), the deactivation rate constant was increased from 0.001 to 0.014 min-1, while half-life was decreased from 693 to 49 min-1. The results of activity collected at different temperature were then used to estimate, activation energy of hydrolysis reaction (Ea = 47.59 kJmol-1). A high Z-value (18.86 °C min-1) was obtained indicating a less sensitivity towards temperatures. The residual activity examinations were carried out from 55 °C to 75 °C and assessing the Deactivation Energy (Ed 116.39 kJmol-1), Enthalpy change (ΔH° 113.55kJmol-1), Entropy change (ΔS° 110.33kJmol-1) and change in Gibbs free energy (ΔG° 10.02 kJmol-1). Taken together, thermodynamic parameters confirm the high stability of enzyme and show potential commercial applicability.
Assuntos
Fosfatase Ácida/química , Chenopodium/química , Cinética , Extratos Vegetais/química , Fosfatase Ácida/genética , Entropia , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Peso Molecular , Extratos Vegetais/farmacologia , Plântula/química , Temperatura , TermodinâmicaRESUMO
Lipase based formulations has been a rising interest to laundry detergent industry for their eco-friendly property over phosphate-based counterparts and compatibility with chemical detergents ingredients. A thermo-stable Anoxybacillus sp. ARS-1 isolated from Taptapani Hotspring, India was characterized for optimum lipase production employing statistical model central composite design (CCD) under four independent variables (temperature, pH, % moisture and bio-surfactant) by solid substrate fermentation (SSF) using mustard cake. The output was utilized to find the effect of parameters and their interaction employing response surface methodology (RSM). A quadratic regression with R2 = 0.955 established the model to be statically best fitting and a predicted highest lipase production of 29.4 IU/g at an optimum temperature of 57.5 °C, pH 8.31, moisture 50% and 1.2 mg of bio-surfactant. Experimental production of 30.3 IU/g lipase at above conditions validated the fitness of model. Anoxybacillus sp. ARS-1 produced lipase was found to resist almost all chemical detergents as well as common laundry detergent, proving it to be a prospective additive for incorporation.
Assuntos
Anoxybacillus/enzimologia , Proteínas de Bactérias/biossíntese , Detergentes/química , Lipase/biossíntese , Modelos Estatísticos , Anoxybacillus/genética , DNA Bacteriano/genética , Detergentes/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Fermentação , Concentração de Íons de Hidrogênio , Índia , Mostardeira/química , Filogenia , Óleos de Plantas/química , RNA Ribossômico 16S/genética , TemperaturaRESUMO
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD-dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single-nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has been linked to a higher risk for several types of cancer and poor survival rate after anthracycline-based chemotherapy. In this study, we show that a small molecular chaperone (N-(2-bromophenyl)pyrrolidine-1-sulfonamide) repopulates the native wild-type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro.
Assuntos
Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Neoplasias/genética , Sequência de Aminoácidos , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Ligantes , Simulação de Acoplamento Molecular , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/química , Conformação ProteicaRESUMO
A 61.3 kDa Phenol hydroxylase (PheA) was purified and characterized from Pseudomonas sp. KZNSA (PKZNSA). Cell free extract of the isolate grown in mineral salt medium supplemented with 600 ppm phenol showed 21.58 U/mL of PheA activity with a specific activity of 7.67 U/mg of protein. The enzyme was purified to 1.6-fold with a total yield of 33.6%. The purified PheA was optimally active at pH 8 and temperature 30 °C, with ≈95% stability at pH 7.5 and temperature 30 °C after 2 h. The Lineweaver-Burk plot showed the vmax and Km values of 4.04 µM/min and 4.03 µM, respectively, for the substrate phenol. The ES-MS data generated from the tryptic digested fragments of pure protein and PCR amplification of a ≈600 bp gene from genomic DNA of PKZNSA lead to the determination of complete amino acid and nucleotide sequence of PheA. Bioinformatics tools and homology modelling studies indicated that PheA from PKZNSA is likely a probable protein kinase UbiB (2-octaprenylphenol hydroxylase) involving Lys and Asp at positions 153 and 288 for binding and active site, respectively. Characterization and optimization of PheA activity may be useful for a better understanding of 2,4-dichlorophenol degradation by this organism and for potential industrial application of the enzyme.
Assuntos
Oxigenases de Função Mista/química , Oxigenases de Função Mista/isolamento & purificação , Modelos Moleculares , Pseudomonas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Fenômenos Biofísicos , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Íons , Metais/farmacologia , Filogenia , Pseudomonas/genética , RNA Ribossômico 16S/genética , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
Intracellular copper/zinc superoxide dismutase (icCuZnSOD) is a member of superoxide dismutase family that is capable of catalyzing the superoxide radicals into either hydrogen peroxide (H2O2) or ordinary molecular oxygen (O2). Unlike mammals, the study of icCuZnSOD in aquatic animals is still in the infancy stage. Here, we identified the cDNA of na-iccuznsod from yellow drum (Nibea albiflora, Richardson 1846) and obtained its fusion protein for the first time. The mRNA expressions of na-iccuznsod were investigated in different tissues, and the dominant distribution was found in head-kidney, followed by brain, liver, heart, and gill. The effects of ammonia-N/nitrite-N on the mRNA expressions of na-iccuznsod were investigated. Na-iccuznsod transcription levels showed a general tendency of an initial up-regulation followed by a down-regulation in liver, gill, and head-kidney when yellow drum were exposed to ammonia-N/nitrite-N at the lethal concentration 50 at 96 h post-treatment, suggesting the important role of Na-icCuZnSOD in eliminating reactive oxygen species (ROS) induced by ammonia-N/nitrite-N. In addition, the characteristics of Na-icCuZnSOD protein and its comparative analysis with Na-ecCuZnSOD were investigated. Na-icCuZnSOD protein showed high enzyme stabilities over a wide range of temperature (10 to 60 °C) and pH (4.9 to 11.0), indicating its broad in vitro applications in many industries. Furthermore, the comparative analysis of Na-icCuZnSOD and Na-ecCuZnSOD gives a new perspective for the study of their structure-function relationship. Collectively, the present study will advance our understanding of the toxicity of ammonia-N/nitrite-N on yellow drum through testing the mRNA expression of iccuznsod gene, and broaden our knowledge of the protein characteristics of icCuZnSOD from fish.
Assuntos
Amônia/toxicidade , Cobre/metabolismo , Regulação Enzimológica da Expressão Gênica , Nitritos/toxicidade , Perciformes/genética , Estresse Fisiológico/genética , Superóxido Dismutase/genética , Zinco/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Estabilidade Enzimática/efeitos dos fármacos , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Filogenia , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Temperatura , Fatores de Tempo , Poluentes Químicos da Água/toxicidadeRESUMO
A novel extracellular protease (SAPRH) was hyper-produced (9000â¯U/mL) from Bacillus safensis RH12, a newly isolated enzyme from a Tunisian offshore oil field. The enzyme was purified to homogeneity, using salt-precipitation, heat-treatment and FPLC anion-exchange chromatography. The purified enzyme was a monomer of molecular mass of ~28â¯kDa. The NH2-terminal 23 amino-acid sequence of SAPRH showed high homology with those of Bacillus-proteases. SAPRH displayed optimal activity at pHâ¯9 and 60⯰C. It was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), indicating that it belongs to the serine-proteases family. Moreover, SAPRH was extremely stable at a broad range of temperature and pH retaining 85% of its activity at 50⯰C and 75% at pHâ¯11. The enzyme exhibited excellent stability and compatibility with surfactants and commercial detergents, revealing 90% stability with SDS and 100% stability with Class commercial laundry detergent. One of the most distinctive properties is its catalytic efficiency, which is higher than that of Alcalase 2.5â¯L, typeDX (commercial enzyme) and SAPB from B. pumilus CBS. Interestingly, the results of the wash performance analysis demonstrated considerably good de-staining at 40⯰C for 30â¯min with low supplementation (500â¯U/mL). Accordingly, such a protease could be considered as a good detergent-additive in detergent industry.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Detergentes/farmacologia , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Cálcio/farmacologia , Corantes/metabolismo , Fibra de Algodão , Interações Medicamentosas , Endopeptidases/biossíntese , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Polímeros/farmacologiaRESUMO
Here, CuO nanoparticles were synthesized using Sambucus nigra (elderberry) fruit extract. Further, the binding of proteinase K, as a model enzyme with green synthesized nanoparticles was investigated. The results demonstrated that the structural changes in enzyme were induced by the binding of nanoparticles. These changes were accompanied by the decrease in the Michaelis-Menten constant at 298â¯K. This means that the enzyme affinity for the substrate was increased. Thermodynamic parameters of protein stability and protein-ligand binding were estimated from the spectroscopic measurements at 298-333â¯K. Depending on the temperature, CuO nanoparticles showed a dual effect on the thermodynamic stability and binding affinity of enzyme. Nanoparticles increase the stability of the native state of enzyme at room temperature. On the other hand, nanoparticles stabilize the unfolded state of enzyme at 310-333â¯K. An overall favorable Gibbs energy change was observed for the binding process at 298-333â¯K. The enzyme-nanoparticle binding is enthalpically driven at room temperature. It was concluded that hydrogen bonding plays a key role in the interaction of enzyme with nanoparticles at 298-310â¯K. At higher temperatures, the protein-ligand binding is entropically driven. This means that hydrophobic association plays a major role in the proteinase K-CuO binding at 310-333â¯K.
Assuntos
Biocatálise/efeitos dos fármacos , Cobre/química , Cobre/farmacologia , Endopeptidase K/química , Endopeptidase K/metabolismo , Nanopartículas/química , Basidiomycota/enzimologia , Estabilidade Enzimática/efeitos dos fármacos , Frutas/química , Extratos Vegetais/química , Sambucus nigra/química , TemperaturaRESUMO
Plant peptidase inhibitors play essential roles in the defense systems of plants. A trypsin inhibitor (PHTI) with a molecular mass of 20.5 kDa was isolated from the fresh roots of the medicinal herb, Pseudostellaria heterophylla. The purification process involved ammonium sulfate precipitation, gel filtration chromatography on Sephadex G50, and ion-exchange chromatography on DEAE 650M. The PHTI contained 3.7% α-helix, 42.1% ß-sheets, 21.2% ß-turns, and 33% disordered structures, which showed similarity with several Kunitz-type trypsin inhibitors. Inhibition kinetic studies indicated that PHTI was a competitive inhibitor, with a Ki value of 3.01 × 10-9 M, indicating a high affinity to trypsin. The PHTI exhibited considerable stability over a broad range of pH (2â»10) and temperatures (20â»70 °C); however, metal ions, including Fe3+, Ba2+, Mn2+, and Al3+, could inactivate PHTI to different degrees. Results of fluorescence spectroscopy and circular dichroism showed that Fe3+ could bind to TI with an association constant of 2.75 × 105 M-1 to form a 1:1 complex, inducing conformation changes and inactivation of PHTI. In addition, PHTI could inhibit the growth of the phytopathogens, Colletotrichum gloeosporioides and Fusarium oxysporum, through disruption of the cell membrane integrity. The present study extended research on Pseudostellaria heterophylla proteins and makes PHTI an exploitable candidate as an antifungal protein for further investigation.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Caryophyllaceae/química , Compostos Fitoquímicos , Extratos Vegetais , Raízes de Plantas/química , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologia , Sequência de Aminoácidos , Antifúngicos/isolamento & purificação , Cromatografia em Gel , Dicroísmo Circular , Estabilidade Enzimática/efeitos dos fármacos , Fungos/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Testes de Sensibilidade Microbiana , Peso Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Temperatura , Inibidores da Tripsina/isolamento & purificaçãoRESUMO
In the present study, the immobilization method of physical adsorption combined with covalent crosslinking was developed to avoid the shortcomings of both the noncovalent and covalent coupling methods. For the first time, tyrosinase (TYR) was immobilized on the surface of aminated magnetic nanoparticles (Fe3O4-NH2) by the developed method. TYR was firstly adsorbed on the surface of Fe3O4-NH2 by electrostatic interaction, and then by covalent crosslinking with glutaraldehyde (GA), TYR was firmly immobilized on the supports. The immobilized TYR showed enhanced pH and temperature endurances at the optimum pH of 7.0 and temperature of 35⯰C. TYR reusability was significantly improved after immobilization and it retained 61.4⯱â¯2.3% of its initial activity after 5 repeated cycles. Immobilized TYR also showed improved storage stability (73.2⯱â¯1.1% after 30 days of storage at 4⯰C). In addition, the immobilized TYR showed a higher biological affinity to substrate owing to the stabilization of TYR in its active conformation by electrostatic interaction prior to covalent crosslinking. Finally, the immobilized TYR was used to screen of enzyme inhibitors from 11 traditional Chinese medicines (TCMs) to validate whether this method can be used for enzyme inhibitor screening or not.
Assuntos
Biocatálise , Reagentes de Ligações Cruzadas/química , Enzimas Imobilizadas/metabolismo , Nanopartículas de Magnetita/química , Monofenol Mono-Oxigenase/metabolismo , Adsorção , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Medicina Tradicional Chinesa , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Eletricidade EstáticaRESUMO
Psychrotolerant yeast Rhodotorula sp. Y-23 was isolated from the sediment core sub-samples of Nella Lake, East Antarctica. Isolate was screened for lipase production using plate assay method followed by submerged fermentation. Production optimization revealed the maximum lipase production by using palmolein oil (5% v/v), pH 8.0 and inoculum size of 2.5% v/v at 15 °C. The potential inducers for lipase were 1% w/v of galactose and KNO3 , and MnCl2 (0.1% w/v). Final productions with optimized conditions gave 5.47-fold increase in lipase production. Dialyzed product gave a purification fold of 5.63 with specific activity of 26.83 U mg-1 and 15.67% yields. This lipase was more stable at pH 5.0 and -20 °C whereas more activity was found at pH 8.0 and 35 °C. Stability was more in 50 mM Fe3+ , EDTA-Na (20 mM), sodium deoxycholate (20 mM), H2 O2 (1% v/v), and almost all organic solvents (50% v/v). Tolerance capacity at wider range of pH and temperature with having lower Km value i.e., 0.08 mg ml-1 and higher Vmax 385.68 U mg-1 at 15 °C make the studied lipase useful for industrial applications. Besides this, the lipase was compatible with commercially available detergents, and its addition to them increases lipid degradation performances making it a potential candidate in detergent formulation.
Assuntos
Temperatura Baixa , Proteínas Fúngicas/metabolismo , Sedimentos Geológicos/microbiologia , Lipase/metabolismo , Rhodotorula/enzimologia , Solventes/farmacologia , Regiões Antárticas , Detergentes/química , Estabilidade Enzimática/efeitos dos fármacos , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/fisiologia , Concentração de Íons de Hidrogênio , Cinética , Lipase/biossíntese , Lipase/isolamento & purificação , Lipase/fisiologia , Óleos de Plantas/metabolismo , Rhodotorula/classificação , Rhodotorula/genética , Rhodotorula/isolamento & purificação , Especificidade por Substrato , TemperaturaRESUMO
In this study, the coding sequence of the lipase from Proteus sp. SW1 was optimized via codon optimization and subjected to expression in Pichia pastoris GS115. The maximum enzyme yield was 387 mg/L in the supernatants of the shake-flask culture. The purified recombinant lipase exhibited a specific activity of 130 U/mg toward p-nitrophenyl Laurate. Its optimum pH and temperature were 8.0 and 40°C, respectively. It was highly stable and even activated in water-miscible solvents, showing over 102% residual activity after 24 h incubation in ethanol, acetone, isopropanol and acetonitrile. In addition, the enzyme showed promoted activity with the increasing concentrations of methanol/ethanol and exhibited the maximum activity at 80%. In a solvent-free system for biodiesel synthesis with a one-step addition of methanol, the recombinant lipase displayed a 87% conversion rate toward palm oil at the high water content of 80%. The highly improved expression level and activity of the recombinant lipase may contribute to enable its commercial-scale production, and the unique properties would make it a particularly promising biocatalyst for biodiesel production in the future.
Assuntos
Lipase/genética , Lipase/metabolismo , Pichia/genética , Solventes/farmacologia , 2-Propanol/farmacologia , Acetona/farmacologia , Acetonitrilas/farmacologia , Biocombustíveis/provisão & distribuição , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Etanol/farmacologia , Concentração de Íons de Hidrogênio , Lauratos/metabolismo , Lipase/biossíntese , Lipase/isolamento & purificação , Metanol/farmacologia , Óleo de Palmeira/metabolismo , Proteus/enzimologia , TemperaturaRESUMO
Microtubule affinity regulating kinase 4 (MARK4) is a member of AMP-activated protein kinase, found to be involved in apoptosis, inflammation and many other regulatory pathways. Since, its aberrant expression is directly associated with the cell cycle and thus cancer. Therefore, MARK4 is being considered as a potential drug target for cancer therapy. Here, we investigated the mechanism of inhibition of MARK4 activity by citral. Docking studies suggested that citral effectively binds to the active site cavity, and complex is stabilized by several interactions. We further performed molecular dynamics simulation of MARK4-citral complex under explicit water condition for 100ns and observed that binding of citral to MARK4 was quite stable. Fluorescence binding studies suggested that citral strongly binds to MARK4 and thereby inhibits its enzyme activity which was measured by the kinase inhibition assay. We further performed MTT assay and observed that citral inhibits proliferation of breast cancer cell line MCF-7. This work provides a newer insight into the use of citral as novel cancer therapeutics through the MARK4 inhibition. Results may be employed to design novel therapeutic molecule using citral as a scaffold for MARK4 inhibition to fight related diseases.
Assuntos
Progressão da Doença , Simulação de Dinâmica Molecular , Monoterpenos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/química , Monoterpenos Acíclicos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Monoterpenos/química , Monoterpenos/farmacologia , Neoplasias/enzimologia , Análise de Componente Principal , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , TermodinâmicaRESUMO
The objective of the present study was to identify the proteome pattern, isolate and study the functions of selective proteins from Ferula asafoetida root exudate using chromatographic techniques. The root exudate proteins were fractionated using ion-exchange and gel filtration chromatography. A range of bioactive protein fractions were then separated in sufficient quantity which is the focus of this study. Based on studies, here we report three main proteins with molecular weights 14kDa, 27kDa, and 39kDa. The biological and pharmacological activities of both purified and unpurified proteins obtained were extensively studied to understand their significance. The study revelaed that 27kDa protein interestingly stabilized trypsin activity in 24h of time and retained about 64% of the enzyme activity. Analyses confirmed 40°C and pH 8.0 are the optimum temperature and pH respectively. The 39kDa protein remarkably increased the activity of chymotrypsin and the 14kDa protein showed anti-bacterial activity against Pseudomonas aeruginosa. Invariably all of the three purified proteins showed enhanced anti-oxidant activity. In conclusion, results here obtained suggested that the primary metabolites (proteins) in asafoetida are mainly responsible for its versatile biological and pharmacological activities.
Assuntos
Fracionamento Químico/métodos , Ferula/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Quimotripsina/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Peso Molecular , Proteínas de Plantas/química , Raízes de Plantas/química , Tripsina/metabolismoRESUMO
An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+.
Assuntos
Aspergillus/enzimologia , Poligalacturonase/química , Poligalacturonase/isolamento & purificação , Poligalacturonase/metabolismo , Carbono/metabolismo , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Cromatografia em Camada Fina/métodos , Citrus sinensis/química , Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Estabilidade Enzimática/efeitos dos fármacos , Etanol/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ácidos Hexurônicos/metabolismo , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/métodos , Cinética , Metais/metabolismo , Peso Molecular , Pectinas/metabolismo , Dióxido de Enxofre/metabolismo , Temperatura , Fatores de TempoRESUMO
The raw starch digesting type of amylase (RSDA) presents greater opportunities for process efficiency at cheaper cost and shorter time compared to regular amylases. Chemical modification is a simple and rapid method toward their stabilization for a wider application. RSDA from Aspergillus carbonarius was modified with either phthalic anhydride (PA) or chitosan. Activity retention was 87.3% for PA-modified and 80.9% for chitosan-modified RSDA. Optimum pH shifted from 5 to 7 after PA-modification. Optimum temperature changed from 30°C (native) to 30-40°C and 60°C for PA-modified and chitosan-modified, respectively. Activation energy (kJmol-1) for hydrolysis was 13.5, 12.7, and 10.2 while the activation energy for thermal denaturation was 32.8, 80.3, 81.9 for free, PA-modified and chitosan-modified, respectively. The specificity constants (Vmax/Km) were 73.2 for PA-modified, 63.1 for chitosan-modified and 77.1 for native RSDA. The half-life (h) of the RSDA at 80°C was increased from 6.1 to 25.7 for the PA-modified and 138.6 for the chitosan derivative. Modification also led to increase in D value, activation enthalpy and Gibbs free energy of enzyme deactivation. Fluorescence spectra showed that center of spectral mass decreased for the PA-modified RSDA but increased for chitosan modified RSDA.
Assuntos
Amilases/química , Amilases/metabolismo , Aspergillus/enzimologia , Quitosana/química , Quitosana/farmacologia , Anidridos Ftálicos/química , Amido/metabolismo , Biocatálise , Digestão , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Solanum tuberosum/química , TemperaturaRESUMO
The aim of the present work was to study the ability of a halophilic bacterial laccase to efficient delignification in extreme conditions. Here, a highly stable extracellular laccase showing ligninolytic activity from halophilic Aquisalibacillus elongatus is described. The laccase production was strongly influenced by NaCl and CuSO4 and under optimal conditions reached 4.8UmL-1. The monomeric enzyme of 75kDa was purified by a synthetic affinity column with 68.2% yield and 99.8-fold purification. The enzyme showed some valuable features viz. stability against a wide range of organic solvents, salts, metals, inhibitors, and surfactants and specificity to a wide spectrum of substrates diverse in structure and redox potential. It retained more than 50% of the original activity at 25-75°C and pH 5.0-10.0. Furthermore, the enzyme was found to be effective in the delignification of sugar beet pulp in an ionic liquid that makes it useful for industrial applications.
Assuntos
Bacillus/enzimologia , Beta vulgaris/química , Cromatografia de Afinidade/métodos , Lacase/isolamento & purificação , Lignina/isolamento & purificação , Temperatura , Bacillus/efeitos dos fármacos , Bacillus/crescimento & desenvolvimento , Estabilidade Enzimática/efeitos dos fármacos , Ponto Isoelétrico , Cinética , Lacase/metabolismo , Peso Molecular , Compostos Orgânicos/farmacologia , Filogenia , Sais/farmacologia , Solventes/farmacologiaRESUMO
The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.
Assuntos
Sistemas CRISPR-Cas/genética , Avaliação Pré-Clínica de Medicamentos , Doença de Fabry/tratamento farmacológico , Técnicas de Inativação de Genes , alfa-Galactosidase/metabolismo , Antígenos Glicosídicos Associados a Tumores/metabolismo , Sequência de Bases , Morte Celular/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Fibroblastos/metabolismo , Edição de Genes , Marcação de Genes , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Leupeptinas/administração & dosagem , Leupeptinas/farmacologia , Modelos Biológicos , Proteínas Recombinantes/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Abstract Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 °C, with Km and V max values of chitinase to be 5.6 mg/mL and 0.87 µmol/min, respectively. Ag+, Co2+, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn2+, Cu2+, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.