RESUMEN
L-asparaginase (L-ASNase) is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia and is used to treat acute lymphoblastic leukemia. It is also toxic to the cells of some solid tumors, including melanoma cells. Immobilization of this enzyme can improve its activity against melanoma tumor cells. In this work, the properties of bacterial cellulose (BC) and feasibility of BC films as a new carrier for immobilized L-ASNase were investigated. Different values of growth time were used to obtain BC films with different thicknesses and porosities, which determine the water content and the ability to adsorb and release L-ASNase. Fourier transform infrared spectroscopy confirmed the adsorption of the enzyme on the BC films. The total activity of adsorbed L-ASNase and its release were investigated for films grown for 48, 72 or 96 h. BC films grown for 96 h showed the most pronounced release as described by zero-order and Korsmayer-Peppas models. The release was characterized by controlled diffusion where the drug was released at a constant rate. BC films with immobilized L-ASNase could induce cytotoxicity in A875 human melanoma cells. With further development, immobilization of L-ASNase on BC may become a potent strategy for anticancer drug delivery to superficial tumors.
Asunto(s)
Asparaginasa , Celulosa , Melanoma , Asparaginasa/química , Asparaginasa/farmacología , Asparaginasa/metabolismo , Humanos , Celulosa/química , Melanoma/tratamiento farmacológico , Melanoma/patología , Línea Celular Tumoral , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Enzimas Inmovilizadas/metabolismo , Portadores de Fármacos/química , Antineoplásicos/farmacología , Antineoplásicos/química , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Adsorción , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Pathogenic bacteria have consistently posed a formidable challenge to human health, creating the critical need for effective antibacterial solutions. In response, enzyme-metal-organic framework (MOF) composites have emerged as a promising class of antibacterial agents. This study focuses on the development of an enzyme-MOF composite based on HZIF-8, incorporating the advantages of simple synthesis, ZIF-8 antibacterial properties, lysozyme hydrolysis, and high biological safety. Through a one-pot method, core-shell nanoparticles (HZIF-8) were synthesized. This structure enables efficient immobilization of lysozyme and lactoferrin within the HZIF-8, resulting in the formation of the lysozyme-lactoferrin@HZIF-8 (LYZ-LF@HZIF-8) composite. Upon exposure to light irradiation, HZIF-8 itself possessed antibacterial properties. Lysozyme initiated the degradation of bacterial peptidoglycan and lactoferrin synergistically enhanced the antibacterial effect of lysozyme. All of the above ultimately contributed to comprehensive antibacterial activity. Antibacterial assessments demonstrated the efficacy of the LYZ-LF@HZIF-8 composite, effectively eradicating Staphylococcus aureus at a cell density of 1.5 × 106 CFU/mL with a low dosage of 200 µg/mL and completely inactivating Escherichia coli at 400 µg/mL with the same cell density. The enzyme-MOF composite exhibited significant and durable antibacterial efficacy, with no apparent cytotoxicity in vitro, thereby unveiling expansive prospects for applications in the medical and food industries.
Asunto(s)
Antibacterianos , Escherichia coli , Lactoferrina , Estructuras Metalorgánicas , Pruebas de Sensibilidad Microbiana , Muramidasa , Staphylococcus aureus , Zeolitas , Muramidasa/farmacología , Muramidasa/química , Muramidasa/metabolismo , Lactoferrina/química , Lactoferrina/farmacología , Estructuras Metalorgánicas/química , Estructuras Metalorgánicas/farmacología , Estructuras Metalorgánicas/síntesis química , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Staphylococcus aureus/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Zeolitas/química , Zeolitas/farmacología , Imidazoles/química , Imidazoles/farmacología , Imidazoles/síntesis química , Porosidad , Propiedades de Superficie , Tamaño de la Partícula , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacologíaRESUMEN
The bacterial nanocellulose (BnC) membranes were produced extracellularly by a novel aerobic acetic acid bacterium Komagataeibacter melomenusus. The BnC was modified in situ by adding carboxymethyl cellulose (CMC) into the culture media, obtaining a BnC-CMC product with denser fibril arrangement, improved rehydration ratio and elasticity in comparison to BnC. The proteolytic enzyme bromelain (Br) and antimicrobial peptide nisin (N) were immobilized to BnC matrix by ex situ covalent binding and/or adsorption. The optimal Br immobilization conditions towards the maximized specific proteolytic activity were investigated by response surface methodology as factor variables. At optimal conditions, i.e., 8.8 mg/mL CMC and 10 mg/mL Br, hyperactivation of the enzyme was achieved, leading to the specific proteolytic activity of 2.3 U/mg and immobilization efficiency of 39.1 %. The antimicrobial activity was observed against Gram-positive bacteria (S. epidermidis, S. aureus and E. faecalis) for membranes with immobilized N and was superior when in situ modified BnC membranes were used. N immobilized on the BnC or BnC-CMC membranes was cytocompatible and did not cause changes in normal human dermal fibroblast cell morphology. BnC membranes perform as an efficient carrier for Br or N immobilization, holding promise in wound debridement and providing antimicrobial action against Gram-positive bacteria, respectively.
Asunto(s)
Acetobacteraceae , Bromelaínas , Celulosa , Nisina , Nisina/farmacología , Nisina/química , Bromelaínas/química , Bromelaínas/farmacología , Celulosa/química , Celulosa/farmacología , Acetobacteraceae/química , Humanos , Antibacterianos/farmacología , Antibacterianos/química , Cicatrización de Heridas/efectos de los fármacos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Nanoestructuras/química , Pruebas de Sensibilidad MicrobianaRESUMEN
This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.
Asunto(s)
Fibrinolíticos/química , Nanopartículas de Magnetita/química , Mucor/enzimología , Péptido Hidrolasas/química , Péptido Hidrolasas/aislamiento & purificación , Cromatografía por Intercambio Iónico , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Enzimas Inmovilizadas/farmacología , Fibrinógeno/química , Fibrinógeno/metabolismo , Fibrinolíticos/aislamiento & purificación , Fibrinolíticos/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Mucor/química , Mucor/genética , Péptido Hidrolasas/farmacología , TemperaturaRESUMEN
It has been planned to minimize the yield and quality impairment of the seed corn, which is strategically important in the world, by pests under storage conditions with a biological product produced with a biotechnological approach. In this context, the present study aimed to control the maize weevil Sitophilus zeamais, known as a warehouse pest, using a nanoformulation. In the study, the chitinase enzyme from Lactobacillus coryniformis was purified first using ammonium sulfate precipitation and then by using the HiTrap Capto DEAE column, and the molecular mass of the purified enzyme was determined to be ~ 33 kDa, and the optimum pH and the values as pH 6.0 and 65-75 °C, respectively. Five different doses of nanoformulation (2, 4, 6, 8 and 10 mg/L) were applied to corn grains by the spraying method with three repetitions so that the insect can ingest the formulation through feeding. The effects of the applications on the death rate and mean time of death of Sitophilus zeamais were determined. According to these findings, it was concluded that the best practice was nanoformulation with 6 mg/L, considering both the mortality rate (100%) and the average death time (2.4 days). Chitinase from L. coryniformis is a promising candidate for corn lice control and management.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Quitinasas/química , Quitinasas/farmacología , Insecticidas/química , Insecticidas/farmacología , Lactobacillus/enzimología , Gorgojos/efectos de los fármacos , Animales , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Lactobacillus/química , Nanopartículas/química , Gorgojos/crecimiento & desarrollo , Óxido de Zinc/químicaRESUMEN
Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.
Asunto(s)
Quitosano , Enzimas Inmovilizadas , Ensayo de Materiales , Nanopartículas/química , Fenilalanina Hidroxilasa , Quitosano/química , Quitosano/farmacología , Evaluación Preclínica de Medicamentos , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Células HEK293 , Células Hep G2 , Humanos , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/farmacologíaRESUMEN
The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.
Asunto(s)
Biocatálisis , Carbono/química , Carbono/farmacología , Nitrógeno/química , Nitrógeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Corona de Proteínas/metabolismo , Puntos Cuánticos/química , Puntos Cuánticos/metabolismo , Células A549 , Animales , Apirasa/química , Apirasa/farmacología , Catalasa/química , Catalasa/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Células HeLa , Humanos , Ratas , Especies Reactivas de Oxígeno/metabolismo , beta-Galactosidasa/química , beta-Galactosidasa/farmacologíaRESUMEN
Herein, our suggestion is to immobilize enzymes in-situ on absorbable shape-memory stents instead of injecting therapeutic enzymes into the blood. Chitosan (CHI)-based stents were tailored as novel support and the enzyme-immobilizing ability was elucidated using L-asparaginase (L-ASNase). For developing shape-memory stents, CHI-glycerol (GLY) solution was prepared and further blended with different ratios of polyethylene glycol (PEG), and polyvinyl alcohol (PVA). Afterward, the blends were modified by ionic crosslinking with sodium tripolyphosphate to obtain a shape-memory character. L-ASNase was included in the blends by using in-situ method before ionic crosslinking. The prepared stents, with or without L-ASNase, were comprehensively characterized by using several techniques. Collectively, immobilized L-ASNase exhibited much better performance in immobilization parameters than free one, thanks to its improved stability and reusability. For instance, CHI/GLY/PEG-3@L-ASNase retained about 70% of the initial activity after storage at 30 °C for 2 weeks, whereas the free form lost half of its initial activity. Besides, it retained 73.4% residual activity after 15 consecutive cycles. Most importantly, stent formulations exhibited ~60% activity in the bioreactor system after 4 weeks of incubation. Given the above results, shape-memory stents can be a promising candidate as a new platform for immobilization, especially in the blood circulation system.
Asunto(s)
Asparaginasa/farmacología , Quitosano/química , Polietilenglicoles/química , Alcohol Polivinílico/química , Asparaginasa/química , Estabilidad de Medicamentos , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Concentración de Iones de Hidrógeno , Cinética , Polietilenglicoles/farmacología , Stents , Temperatura , HumectabilidadRESUMEN
In the present study different enzymes (α- amylase, trypsin, cellulase, horse-radish peroxidase and pectinex ultra clear) were studied for bacterial biofilm inhibition and Pectinex ultra clear showed best inhibition. So, m-combi-CLEA of Pectinex ultra clear was developed by cross linked enzyme aggregate (CLEA) formation on APTES (3-aminopropyltriethoxysilane) modified iron oxide nanoparticles. Different parameters were optimized and it was observed that 0.4 mg/ml of protein (containing 25 U/mg cellulase activity), 0.5 mg/ml BSA and 10 mM glutaraldehyde when incubated for 3 h gives 100% enzyme activity using ethanol as the precipitant. The CLEA formed were thermally more stable as compared to free enzyme. m-combi-CLEA of Pectinex ultra clear shows 75-78% biofilm inhibition of E. coli and S. aureus. Furthermore, m-combi-CLEA can be reused till 4 cycles with same efficiency. The carbohydrate contents of E. coli biofilm decreased from 64.629 µg to 6.23 µg and for S. aureus biofilm, it decreased from 58.46 µg to 5.52 µg when treated with m-combi CLEA in comparison to untreated biofilms. FTIR, darkfield illumination Fluorescence Microscopy, and Scanning Electron Microscopy was further used for characterization.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Enzimas Inmovilizadas/farmacología , Escherichia coli/efectos de los fármacos , Tecnología Química Verde , Magnetismo , Complejos Multienzimáticos/farmacología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Antibacterianos/metabolismo , Biopelículas/crecimiento & desarrollo , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Matriz Extracelular de Sustancias Poliméricas/metabolismo , Hidrólisis , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/metabolismoRESUMEN
The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N-acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta. Using directed enzyme evolution, we were able to increase the enzyme's temperature resistance (T50, the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom's infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.
Asunto(s)
Hidrolasas de Éster Carboxílico/farmacología , Erwinia amylovora/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Nanosferas/química , Enfermedades de las Plantas/prevención & control , Percepción de Quorum/efectos de los fármacos , Acil-Butirolactonas/metabolismo , Hidrolasas de Éster Carboxílico/administración & dosificación , Hidrolasas de Éster Carboxílico/genética , Evolución Molecular Dirigida/métodos , Enzimas Inmovilizadas/administración & dosificación , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/farmacología , Erwinia amylovora/fisiología , Modelos Moleculares , Péptidos/química , Enfermedades de las Plantas/microbiología , Pyrus/microbiologíaRESUMEN
Bacterial adhesion infection caused by medical materials in clinical application has become a serious threat, and it urgently needs new strategies to deal with these clinical challenges. In this work, LED209, a highly selective histidine sensor kinase inhibitor of Gram-negative bacteria, was covalently attached on cellulose membrane (CM) via click reaction. The data of contact angle measurements, ATR-FTIR and X-ray photoelectron spectroscopy confirmed the successful synthesis of LED-CM. In addition, the results of antibacterial activity of the membranes shown that LED-CM exhibited excellent anti-adhesion ability to Enterohemorrhagic Escherichia coli (EHEC), and significantly reduced the formation of bacterial biofilm. Importantly, LED-CM was able to repress the expression of virulence genes in EHEC. Furthermore, LED209-functionalized cellulose membrane indicated no cytotoxicity to mammalian cells. Hence, our present work demonstrated that CM modified with LED209 possessed markedly anti-adhesion activity against EHEC, which offered a potent antimicrobial material for combating bacterial infections.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Enzimas Inmovilizadas/farmacología , Infecciones por Escherichia coli/tratamiento farmacológico , Escherichia coli O157/efectos de los fármacos , Proteínas de Escherichia coli/antagonistas & inhibidores , Complejos Multienzimáticos/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/efectos de los fármacos , Celulosa/química , Membranas Artificiales , Ratones , Células 3T3 NIHRESUMEN
We combined the chemical and physical methods of papain immobilization through the aldehyde groups available on oxidized bacterial cellulose (OxBC) to provide high proteolytic activity for future applications as bioactive dressing. Bacterial cellulose (BC) was obtained by the fermentation of Komagataeibacter hansenii in Hestrin-Schramm medium for 5 days, followed by purification and oxidation using NaIO4. Surface response methodology was used to optimize papain immobilization (2%, w/v) for 24 h. The independent variables: pH (3-7) and temperature (5 to 45 °C) were investigated. The mathematically validated optimal conditions of 45 °C and pH 7 had a statistical effect on the immobilization yield (IY) of papain in OxBC (52.9%). These ideal conditions were also used for papain immobilization in BC (unoxidized). The IY of 9.1% was lower than that of OxBC. OxBC-Papain and BC-Papain were investigated using thermal analysis, confocal microscopy, and diffusion testing. The OxBC support exhibited a more interactive chemical structure than the BC support, and was capable of immobilizing papain by covalent bonds (-C-NHR) and adsorption (ion exchange), with 93.3% recovered activity, 49.4% immobilization efficiency, and better thermal stability. Papain immobilized to OxBC by adsorption displayed 53% widespread papain activity. The results indicate the potential of prolonged bioactivity in debrided chronic wounds.
Asunto(s)
Celulosa Oxidada/química , Papaína/química , Péptido Hidrolasas/química , Piel/efectos de los fármacos , Acetobacteraceae/enzimología , Adsorción/efectos de los fármacos , Celulosa Oxidada/farmacología , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Papaína/biosíntesis , Papaína/farmacología , Péptido Hidrolasas/farmacología , Piel/lesionesRESUMEN
Bacterial cellulose (BC) is recognized as a wound dressing material well-suited for chronic wounds; however, it has no intrinsic antimicrobial activity. Further, the formation of biofilms can limit the effectiveness of the pre-saturation of BC with antimicrobial agents. Here, to hinder biofilm formation by P. aeruginosa, we immobilized the hydrolytic domain of PelA (a glycohydrolase involved in the synthesis of biofilm polysaccharide Pel) on the surface of BC. The immobilization of 32.35⯱â¯1.05â¯mg PelAh per g BC membrane resulted in an eight-fold higher P. aeruginosa cell detachment from BC membrane, indicating reduced biofilm matrix stability. Further, 1D and 2D infrared spectroscopy analysis indicated systematic reduction of polysaccharide biofilm elements, confirming the specificity of immobilized PelAh. Importantly, BC-PelAh was not cytotoxic towards L929 fibroblast cells. Thus, we conclude that PelAh can be used in BC wound dressings for safe and specific protection against biofilm formation by P. aeruginosa.
Asunto(s)
Acetobacteraceae/química , Vendajes , Biopelículas/efectos de los fármacos , Celulosa/química , Glicósido Hidrolasas/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Acetobacteraceae/fisiología , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Biopelículas/crecimiento & desarrollo , Línea Celular , Celulosa/biosíntesis , Celulosa/aislamiento & purificación , Clonación Molecular , Enzimas Inmovilizadas/biosíntesis , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/genética , Ratones , Dominios Proteicos , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
The prolonged use of enzymes under oxidative stress is a major challenge in enabling effective enzymatic reaction pathways. Herein, we report a biomimetic antioxidant defensive strategy capable of providing adequate protection of enzymes against superoxide-mediated oxidation. Superoxide dismutase (SOD) and catalase (CAT) were chosen as scavengers and covalently encapsulated into silica nanoreactors, together with glucose dehydrogenase (GDH), which simultaneously should produce the coenzyme nicotinamide adenine dinucleotide (NADH, reduced form). By the enzymatic reactions of SOD and CAT, the interior of silica nanoreactors becomes a "ROS safe zone" to protect the glucose-dependent NADH production of coencapsulated GDH. We further combined this protected NADH-producing module with photocatalytic nanoparticles that enable the light-triggered oxidation of NADH back to NAD+ (oxidized form). In combination, these two modules allow interconversion between NAD+ and NADH by the addition of glucose or by light irradiation (LED lamp or sunlight). This protection and regeneration strategy is a versatile tool for enzyme applications for biological reactors, catalysis, or prototypes of artificial organelles or building blocks that contains fragile biomolecules and rely on the coenzyme NAD+/NADH.
Asunto(s)
Catalasa/farmacología , Enzimas Inmovilizadas/farmacología , Glucosa 1-Deshidrogenasa/farmacología , NAD/metabolismo , Nanopartículas/química , Superóxido Dismutasa/farmacología , Biomimética/métodos , Catalasa/química , Línea Celular Tumoral , Enzimas Inmovilizadas/química , Glucosa/química , Glucosa/metabolismo , Glucosa 1-Deshidrogenasa/química , Humanos , Luz , NAD/química , Nanopartículas/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Polímeros/química , Polímeros/efectos de la radiación , Dióxido de Silicio/química , Superóxido Dismutasa/química , Superóxidos/química , Superóxidos/metabolismoRESUMEN
We developed a polymer-encapsulated DNase, n(DNase), which can efficiently accumulate in biofilm and expose the DNase to cleave the eDNA of the biofilm. CLSM and crystal violet staining results demonstrated effective biofilm disintegration (92.2%) when treated with n(DNase). This work demonstrated a general approach for coating matrix-dispersion enzymes to achieve biofilm disintegration and provided a promising strategy for treating biofilm-associated infections.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Desoxirribonucleasas/farmacología , Enzimas Inmovilizadas/farmacología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/administración & dosificación , Desoxirribonucleasas/administración & dosificación , Portadores de Fármacos/química , Sinergismo Farmacológico , Enzimas Inmovilizadas/administración & dosificación , Humanos , Polímeros/química , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/fisiologíaRESUMEN
We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme-functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.
Asunto(s)
Antibacterianos/farmacología , ADN/química , Portadores de Fármacos/química , Muramidasa/farmacología , Nanoestructuras/química , Animales , Antibacterianos/química , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/toxicidad , Bacillus subtilis/química , Bacillus subtilis/efectos de los fármacos , Células COS , Chlorocebus aethiops , ADN/toxicidad , Portadores de Fármacos/toxicidad , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Escherichia coli/química , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Muramidasa/química , Nanoestructuras/toxicidad , Conformación de Ácido NucleicoRESUMEN
The recent emergence of antibiotic-resistant bacteria requires the development of new antibiotics or new agents capable of enhancing antibiotic activity. Lysozyme degrades bacterial cell wall without involving antibiotic resistance and has become a new antibacterial strategy. However, direct use of native, active proteins in clinical settings is not practical as it is fragile under various conditions. In this study, lysozyme was integrated into chitosan nanoparticles (CS-NPs) by the ionic gelation technique to obtain lysozyme immobilized chitosan nanoparticles (Lys-CS-NPs) and then characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), which showed a small particle size (243.1 ± 2.1 nm) and positive zeta potential (22.8 ± 0.2 mV). The immobilization significantly enhanced the thermal stability and reusability of lysozyme. In addition, compared with free lysozyme, Lys-CS-NPs exhibited superb antibacterial properties according to the results of killing kinetics in vitro and measurement of the minimum inhibitory concentration (MIC) of CS-NPs and Lys-CS-NPs against Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus). These results suggest that the integration of lysozyme into CS-NPs will create opportunities for the further potential applications of lysozyme as an anti-bacterium agent.
Asunto(s)
Antibacterianos/farmacología , Quitosano/química , Portadores de Fármacos/química , Muramidasa/farmacología , Nanopartículas/química , Dispersión Dinámica de Luz , Estabilidad de Enzimas , Enzimas Inmovilizadas/farmacología , Escherichia coli/efectos de los fármacos , Klebsiella pneumoniae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , TemperaturaRESUMEN
Direct immobilization of functional proteins on gold nanoparticles (AuNPs) affects their structure and function. Changes may vary widely and range from strong inhibition to the enhancement of protein function. More often though the outcome of direct protein immobilization results in protein misfolding and the loss of protein activity. Additional complications arise when the protein being immobilized is a zymogen which requires and relies on additional protein-protein interactions to exert its function. Here we describe molecular design of a glutathione-S-transferase-Staphylokinase fusion protein (GST-SAK) and its conjugation to AuNPs. The multivalent AuNP-(GST-SAK)n complexes generated show plasminogen activation activity in vitro. The methods described are transferable and could be adapted for conjugation and functional analysis of other plasminogen activators, thrombolytic preparations or other functional enzymes.
Asunto(s)
Glutatión Transferasa/genética , Oro/química , Metaloendopeptidasas/genética , Activadores Plasminogénicos/farmacología , Proteínas Recombinantes/farmacología , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Fibrinógeno/metabolismo , Glutatión Transferasa/química , Humanos , Nanopartículas del Metal , Metaloendopeptidasas/química , Modelos Moleculares , Activadores Plasminogénicos/química , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/químicaRESUMEN
Bacterial pathogens are responsible for millions of cases of illnesses and deaths each year throughout the world. The development of novel surfaces and coatings that effectively inhibit and prevent bacterial attachment, proliferation, and growth is one of the crucial steps for tackling this global challenge. Herein, we report a dual-functional coating for aluminum surfaces that relies on the controlled immobilization of lysozyme enzyme (muramidase) into interstitial spaces of presintered, nanostructured thin film based on â¼200 nm silica nanoparticles and the sequential chemisorption of an organofluorosilane to the available interfacial areas. The mean diameter of the resultant lysozyme microdomains was 3.1 ± 2.5 µm with an average spacing of 8.01 ± 6.8 µm, leading to a surface coverage of 15.32%. The coating had an overall root-mean-square (rms) roughness of 539 ± 137 nm and roughness factor of 1.50 ± 0.1, and demonstrated static, advancing, and receding water contact angles of 159.0 ± 1.0°, 155.4 ± 0.6°, and 154.4 ± 0.6°, respectively. Compared to the planar aluminum, the coated surfaces produced a 6.5 ± 0.1 (>99.99997%) and 4.0 ± 0.1 (>99.99%) log-cycle reductions in bacterial surfaces colonization against Gram-negative Salmonella Typhimurium LT2 and Gram-positive Listeria innocua, respectively. We anticipate that the implementation of such a coating strategy on healthcare environments and surfaces and food-contact surfaces can significantly reduce or eliminate potential risks associated with various contamination and cross-contamination scenarios.
Asunto(s)
Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Desinfectantes/farmacología , Enzimas Inmovilizadas/farmacología , Muramidasa/farmacología , Aluminio/química , Antibacterianos/química , Desinfectantes/química , Enzimas Inmovilizadas/química , Interacciones Hidrofóbicas e Hidrofílicas , Listeria/efectos de los fármacos , Muramidasa/química , Nanopartículas/química , Salmonella typhimurium/efectos de los fármacos , Dióxido de Silicio/química , HumectabilidadRESUMEN
BACKGROUND: L-asparaginase (L-AsnA) enzyme has gained significant attention in the food, biocatalysts and pharmaceutics industry. It (L-AsnA) has been widely used in food processing industries as a promising acrylamide mitigating agent and as a therapeutic agent in the treatment of certain human cancers. OBJECTIVE: Based on US Patent (4,433,054; 1984), L-asparaginase (L-AsnA) enzyme is immobilized by admixing the active enzyme on the polysaccharide to be in a gel form. The storage stability of immobilized L-AsnA enzyme and its anti-proliferation and antiviral activity were determined. METHODS: In the present study, S. maxima was cultured at large scales (300 liter) for the production of enough extracellular L-asparaginase (L-AsnA) using modified (high N concentration) Zarrouk medium as we reported in a previous study. L-AsnA was immobilized on natural polymers, as agar cake beads, agarose pieces and gelatin blocks, in order to evaluate the efficiency of physical entrapment techniques. Anti-proliferation properties of L-AsnA against lung carcinoma A549, hepatocellular carcinoma Hep-G2 and prostate carcinoma PC3 human cancer cell lines were assessed by the MTT cell viability method. In addition, the antiviral activity against Coxsackie B3 (CSB3) Virus was assessed. RESULTS: The highest L-AsnA immobilized activity and immobilization yield were achieved with agar cakes bead. The purified S. maxima L-AsnA showed good antiviral activity against Coxsackie B3 (CSB3) Virus in a dose-dependent manner with an IC50 value 17.03 µg/ml. The antiviral mode of action is presumably due to their capability of inhibiting attachment, blocking the adsorption and penetration event of the viral replication cycle with 89.24%, 72.78% and 72.78%, respectively. Also, S. maxima L-AsnA showed anti-proliferation effect against lung carcinoma A549, hepatocellular carcinoma Hep-G2 and prostate carcinoma PC3 human cancer cell lines, with an IC50 of 22.54, 24.65 and 56.61 µg/ml, respectively. CONCLUSION: It is interesting to favor L-asparaginase of S. maxima which showed antiviral activity and anti-proliferation effect against different types of human cell lines. Thus, S. maxima microalgae might be a good source for L-AsnA enzymes and can be immobilized on natural polymers.