RESUMEN
Hydrogen polysulfides (H2Sn) have emerged as critical physiological mediators that are closely associated with hydrogen sulfide (H2S) signaling. H2Sn exhibit greater nucleophilicity than H2S while also having electrophilic characteristics, enabling unique activities such as protein S-persulfidation. Despite their physiological importance, mechanisms and reactivities of H2Sn remain inadequately explored due to their inherent instability in aqueous environments. Consequently, there is a need to develop biocompatible methods for controlled H2Sn generation to elucidate their behaviors in biological contexts. Herein, we present a dual enzyme system (containing glucose oxidase (GOx) and chloroperoxidase (CPO)) with thioglucose as the substrate to facilitate the controlled release of H2Sn. Fluorescence measurements with SSP4 and the trapping studies allowed us to confirm the production of H2Sn. Such a method may be useful in elucidating the reactivity of hydrogen polysulfides in biological systems as well as provide a potential delivery of H2Sn to target sites for biological applications.
Asunto(s)
Cloruro Peroxidasa , Glucosa Oxidasa , Sulfuros , Glucosa Oxidasa/metabolismo , Glucosa Oxidasa/química , Cloruro Peroxidasa/metabolismo , Cloruro Peroxidasa/química , Sulfuros/química , Sulfuros/metabolismo , Sulfuro de Hidrógeno/metabolismo , Sulfuro de Hidrógeno/química , Aspergillus niger/enzimologíaRESUMEN
Halloysite nanotube (HNT) is a natural bio-compatible and stable nanomaterial available in abundance at low-cost. In this work, HNT was modified by two strategies to make it suitable for supporting immobilization of chloroperoxidase (CPO). Firstly, Fe3O4 nanoparticles were deposited on HNT, so magnetic separation can be used instead of centrifugation. Then, the magnetic HNT was modified by 3-aminopropyltriethoxysilane (APTES), which can provide amine group on surface of HNT and meanwhile inhibit the agglomeration of magnetic HNT. Then, HNT-Fe3O4 -APTES was linked with branched polyethyleneimine (PEI) to provide more amino for binding with enzyme. The so-prepared CPO@HNT-Fe3O4-APTES-PEI showed enhanced enzyme loading, reusability, improved thermal stability and tolerance to organic solvents than free CPO. For example, after 10 repeated uses, CPO@HNT- Fe3O4-APTES-PEI can maintain 92.20% of its original activity compared with 65.12% of activity of CPO@HNT-APTES-PEI and 45.69% of activity of CPO@HNT. The kinetic parameters indicated the affinity and specificity of immobilized enzyme to substrate was increased. CPO@HNT-Fe3O4-APTES-PEI was very efficient when it was applied in the degradation of pesticides mesotrione in wastewater. The degradation efficiency can reach 90% within 20 min at range of 5-40 µmol·L-1. These results ensure the potential practical application of this bio-materials in wastewater treatment.
Asunto(s)
Ascomicetos/enzimología , Cloruro Peroxidasa/química , Arcilla/química , Enzimas Inmovilizadas/química , Óxido Ferrosoférrico/química , Proteínas Fúngicas/química , Nanotubos/química , Plaguicidas/química , Aguas Residuales/químicaRESUMEN
Neutrophils can responsively release reactive oxygen species (ROS) to actively combat infections by exogenous stimulus and cascade enzyme catalyzed bio-oxidation. A supramolecular nanogel is now used as an artificial neutrophil by enzymatic interfacial self-assembly of peptides (Fmoc-Tyr(H2 PO3 )-OH) with magnetic nanoparticles (MNPs) and electrostatic loading of chloroperoxidase (CPO). The MNPs within the nanogel can elevate H2 O2 levels in cancer cells under programmed alternating magnetic field (AMF) similar to the neutrophil activator, and the loaded CPO within protective peptides nanolayer converts the H2 O2 into singlet oxygen (1 O2 ) in a sustained manner for neutrophil-inspired tumor therapy. As a proof of concept study, both the H2 O2 and 1 O2 in cancer cells increase stepwise under a programmed alternating magnetic field. An active enzyme dynamic therapy by magnetically stimulated oxygen stress and sustained enzyme bio-oxidation is thus shown with studies on both cells and animals.
Asunto(s)
Cloruro Peroxidasa/metabolismo , Nanopartículas de Magnetita/química , Nanogeles/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cloruro Peroxidasa/química , Humanos , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Campos Magnéticos , Ratones , Nanogeles/uso terapéutico , Nanogeles/toxicidad , Neoplasias/tratamiento farmacológico , Neoplasias/mortalidad , Neoplasias/patología , Neutrófilos/química , Neutrófilos/inmunología , Tamaño de la Partícula , Péptidos/química , Oxígeno Singlete/química , Oxígeno Singlete/metabolismo , Electricidad Estática , Tasa de Supervivencia , Trasplante HeterólogoRESUMEN
Chloroperoxidase (CPO) is a hybrid of two different families of enzymes, peroxidases and P450s. However, it is poorly understood on CPO's multiple catalytic functions. Herein, phenol was selected as a model substrate to investigate the multiple catalytic roles of CPO. Results showed that phenol was readily transformed into a variety of brominated organic compounds (BOCs) via the CPO-mediated oxidative process. A total of 16 BOCs were identified using gas and liquid chromatography coupled with mass spectrometry. Possible reaction pathways could be attributable to four CPO-mediated processes, including bromination, radical coupling, intramolecular cyclization and debromination. Higher bromide concentrations and lower pH conditions both facilitated the formation of brominated products. While a higher bromination capacity was observed in pH 3.0 solutions, CPO-mediated radical couplings were more favorable at pH 5.0 and 6.0. Although CPO might catalyze chlorination when chloride and bromide coexisted in the solution, BOCs were the dominant products of CPO-mediated phenol oxidation. Results of this study suggest that various catalytic roles of CPO may contribute to the biotic formation of BOCs in the natural environment.
Asunto(s)
Bromuros/química , Cloruro Peroxidasa/química , Hidrocarburos Bromados/química , Fenol/química , Catálisis , Cromatografía Liquida , Halogenación , Estructura Molecular , Oxidación-Reducción , Espectrometría de Masas en TándemRESUMEN
A multitude of industrial processes are catalyzed by two or more enzymes working together in a cascade way. However, designing efficient enzymatic cascade reactions is still a challenge. In this work, a TiO2 thin film with mesoporous pores was prepared and used as carrier for co-immobilization of chloroperoxidase (CPO) and glucose peroxidase (GOx). By adjusting the dosage of hexadecyltrimethylammonium bromide (CTAB) and the ratio of the two enzymes, CPO and GOx were well distributed and positional orientated to their own appropriate pores to form an ordered "occupation" based on a "feet in right shoes" effect. Moreover, when the pore size was controlled around 12 nm, the enzymes aggregation was inhibited so as to avoid the decrease of activity of enzyme; The catalytic performance of TiO2-GOx and CPO composites was evaluated by the application of decolorization of Orange G dye in a cascaded manner. The oxidant H2O2 needed by CPO is generated in situ through glucose oxidation by GOx. Upon co-immobilization of CPO and GOx on the same carrier, a large increase in the initial catalytic efficiency was detected when compared to an equimolar mixture of the free enzymes, which was four times greater. Moreover, the affinity of the enzyme toward substrate binding was improved according to the kinetic assay. The thermal stability of TiO2-GOx and CPO composites were greatly improved than free enzymes. The TiO2-GOx and CPO composites can be easily separated from the reaction media which facilitate its recycle use.
Asunto(s)
Compuestos Azo/química , Cloruro Peroxidasa/química , Enzimas Inmovilizadas/química , Membranas Artificiales , Peroxidasas/química , Titanio/química , Oxidación-Reducción , PorosidadRESUMEN
Chloroperoxidase (CPO), a heme-thiolate protein, from Caldariomyces fumago catalyzes a plethora of reactions including halogenation, dismutation, epoxidation, and oxidation. Although all CPO-catalyzed reactions go through a common intermediate, compound I, different mechanisms are followed in subsequent transformations. To understand the mechanism of CPO-catalyzed halide-dependent degradation of orange G, the role of halide and pH was systematically investigated. It is revealed that formation and protonation of compound X, a long-sought after hypochlorite heme adduct intermediate existed during CPO-catalyzed halide-dependent reactions, significantly lowers the reaction barrier and increases the efficiency of CPO-catalyzed orange G degradation. The extremely acidic optimal reaction pH suggests the protonation of a residue, presumably, Glu 183 in CPO catalysis. Halide dependent studies showed that Kcat is higher in the presence of Br(-) than in the presence of Cl(-). The degradation products of orange G indicate the cleavage at a single position of orange G, demonstrating a high regioselectivity of CPO-catalyzed degradation. Based on our kinetic, NMR and QM/MM studies, the mechanism of CPO-catalyzed orange G degradation was proposed.
Asunto(s)
Ascomicetos/enzimología , Compuestos Azo/química , Cloruro Peroxidasa/química , Proteínas Fúngicas/química , Modelos Químicos , Catálisis , Cinética , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains "spectroscopically silent" V(V), which does not change oxidation state during the reaction. We employed an NMR crystallography approach based on (51)V magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO). The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5-100-fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained due to the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated at pH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and mutant VCPO and their pH-dependence. This study demonstrates that (51)V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.
Asunto(s)
Ascomicetos/enzimología , Cloruro Peroxidasa/química , Ascomicetos/genética , Dominio Catalítico , Cloruro Peroxidasa/genética , Cloruro Peroxidasa/metabolismo , Cristalografía , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Mutación Puntual , Conformación Proteica , Vanadatos/metabolismoRESUMEN
The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.
Asunto(s)
Cianuros/química , Hemo/química , Animales , Dominio Catalítico , Bovinos , Cloruro Peroxidasa/química , Electrones , Enzimas/química , Radicales Libres , Hongos/metabolismo , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Ligandos , Oxidación-Reducción , Oxígeno/química , Peróxidos/química , Plantas/metabolismo , Unión ProteicaRESUMEN
Halogenated phenazine meroterpenoids are a structurally unusual family of marine actinobacterial natural products that exhibit antibiotic, antibiofilm, and cytotoxic bioactivities. Despite a lack of established phenazine halogenation biochemistry, genomic analysis of Streptomyces sp. CNZ-289, a prolific lavanducyanin and C2-halogenated derivative producer, suggested the involvement of vanadium-dependent haloperoxidases. We subsequently discovered lavanducyanin halogenase (LvcH), characterized it in vitro as a regioselective vanadium-dependent chloroperoxidase, and applied it in late-stage chemoenzymatic synthesis.
Asunto(s)
Cloruro Peroxidasa , Halogenación , Vanadio , Cloruro Peroxidasa/metabolismo , Cloruro Peroxidasa/química , Vanadio/química , Estructura Molecular , Streptomyces/química , Estereoisomerismo , Fenazinas/química , Fenazinas/farmacología , Fenazinas/síntesis química , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis químicaRESUMEN
To unravel the mechanism of chloroperoxidase (CPO)-catalyzed regioselective oxidation of indole, we studied the structure of the CPO-indole complex using nuclear magnetic resonance (NMR) relaxation measurements and computational techniques. The dissociation constant (KD) of the CPO-indole complex was calculated to be approximately 21 mM. The distances (r) between protons of indole and the heme iron calculated via NMR relaxation measurements and molecular docking revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. Both KD and r values are independent of pH in the range of 3.0-6.5. The stability and structure of the CPO-indole complex are also independent of the concentration of chloride or iodide ion. Molecular docking suggests the formation of a hydrogen bond between the NH group of indole and the carboxyl O of Glu 183 in the binding of indole to CPO. Simulated annealing of the CPO-indole complex using r values from NMR experiments as distance restraints reveals that the van der Waals interactions were much stronger than the Coulomb interactions in the binding of indole to CPO, indicating that the association of indole with CPO is primarily governed by hydrophobic rather than electrostatic interactions. This work provides the first experimental and theoretical evidence of the long-sought mechanism that leads to the "unexpected" regioselectivity of the CPO-catalyzed oxidation of indole. The structure of the CPO-indole complex will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications.
Asunto(s)
Cloruro Peroxidasa/química , Indoles/química , Espectroscopía de Resonancia Magnética/métodos , Catálisis , Concentración de Iones de Hidrógeno , Simulación del Acoplamiento Molecular , Oxidación-ReducciónRESUMEN
The use of stable chlorine isotopic signatures (δ(37)Cl) of organochlorine compounds has been suggested as a tool to determine both their origins and transformations in the environment. Here we investigated the δ(37)Cl fractionation of two important pathways for enzymatic natural halogenation: chlorination by chloroperoxidase (CPO) and flavin-dependent halogenases (FDH). Phenolic products of CPO were highly (37)Cl depleted (δ(37)Cl = -12.6 ± 0.9); significantly more depleted than all known industrially produced organochlorine compounds (δ(37)Cl = -7 to +6). In contrast, four FDH products did not exhibit any observable isotopic shifts (δ(37)Cl = -0.3 ± 0.6). We attributed the different isotopic effect to the distinctly different chlorination mechanisms employed by the two enzymes. Furthermore, the δ(37)Cl in bulk organochlorines extracted from boreal forest soils were only slightly depleted in (37)Cl relative to inorganic Cl. In contrast to previous suggestions that CPO plays a key role in production of soil organochlorines, this observation points to the additional involvement of either other chlorination pathways, or that dechlorination of naturally produced organochlorines can neutralize δ(37)Cl shifts caused by CPO chlorination. Overall, this study demonstrates that chlorine isotopic signatures are highly useful to understand sources and cycling of organochlorines in nature. Furthermore, this study presents δ(37)Cl values of FDH products as well of bulk organochlorines extracted from pristine forest soil for the first time.
Asunto(s)
Cloruro Peroxidasa/química , Cloro/química , Hidrocarburos Clorados/química , Oxidorreductasas/química , Flavina-Adenina Dinucleótido/química , Isótopos , Suelo/química , ÁrbolesRESUMEN
The initiation of this study relies on a targeted genome-mining approach to highlight the presence of a putative vanadium-dependent haloperoxidase-encoding gene in the deep-sea hydrothermal vent fungus Hortaea werneckii UBOCC-A-208029. To date, only three fungal vanadium-dependent haloperoxidases have been described, one from the terrestrial species Curvularia inaequalis, one from the fungal plant pathogen Botrytis cinerea, and one from a marine derived isolate identified as Alternaria didymospora. In this study, we describe a new vanadium chloroperoxidase from the black yeast H. werneckii, successfully cloned and overexpressed in a bacterial host, which possesses higher affinity for bromide (Km = 26 µM) than chloride (Km = 237 mM). The enzyme was biochemically characterized, and we have evaluated its potential for biocatalysis by determining its stability and tolerance in organic solvents. We also describe its potential three-dimensional structure by building a model using the AlphaFold 2 artificial intelligence tool. This model shows some conservation of the 3D structure of the active site compared to the vanadium chloroperoxidase from C. inaequalis but it also highlights some differences in the active site entrance and the volume of the active site pocket, underlining its originality.
Asunto(s)
Ascomicetos , Cloruro Peroxidasa , Exophiala , Respiraderos Hidrotermales , Cloruro Peroxidasa/genética , Cloruro Peroxidasa/química , Cloruro Peroxidasa/metabolismo , Exophiala/metabolismo , Saccharomyces cerevisiae/metabolismo , Vanadio/metabolismo , Inteligencia Artificial , Ascomicetos/genéticaRESUMEN
We report that incorporation of very low concentrations of redox protein cytochrome c and redox active small molecule vitamin C impacted the outcome of one-electron oxidations mediated by structurally distinct plant/fungal heme peroxidases. Evidence suggests that cytochrome c and vitamin C function as a redox relay for diffusible reduced oxygen species in the reaction system, without invoking specific or affinity-based molecular interactions for electron transfers. The findings provide novel perspectives to understanding - (1) the promiscuous role of cytochrome b(5) in the metabolism mediated by liver microsomal xenobiotic metabolizing systems and (2) the roles of antioxidant molecules in affording relief from oxidative stress.
Asunto(s)
Antioxidantes/química , Ácido Ascórbico/química , Cloruro Peroxidasa/química , Citocromos c/química , Hemo/química , Peroxidasa de Rábano Silvestre/química , Oxidación-ReducciónRESUMEN
Quercetin, rutin, naringin, hesperidin and chrysin were tested as substrates for chloroperoxidase to produce reactive quinones to graft onto chitosan. Quercetin and rutin quinones were successfully chemically attached to low molecular weight chitosan. The quercetin-modified chitosan showed an enhancement of plastic, antioxidant and antimicrobial properties as well as of thermal degradability. Finally, chitosan-quercetin films visibly decreased enzymatic oxidation when applied to Opuntia ficus indica cladodes.
Asunto(s)
Antiinfecciosos/química , Antioxidantes/química , Quitosano/química , Cloruro Peroxidasa/química , Aditivos Alimentarios/química , Quercetina/química , Antiinfecciosos/farmacología , Antioxidantes/farmacología , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Calorimetría , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Cloruro Peroxidasa/metabolismo , Color , Flavanonas/química , Flavanonas/farmacología , Flavonoides/química , Flavonoides/farmacología , Aditivos Alimentarios/farmacología , Hesperidina/química , Hesperidina/farmacología , Reacción de Maillard/efectos de los fármacos , Opuntia/efectos de los fármacos , Opuntia/metabolismo , Oxidación-Reducción , Tallos de la Planta/efectos de los fármacos , Tallos de la Planta/metabolismo , Quercetina/farmacología , Rutina/química , Rutina/farmacología , EspectrofotometríaRESUMEN
Chloroperoxidase (CPO) is a haeme-thiolate enzyme able to catalyse the halogenation and oxidation of a wide range of organic substrates. In this work, the CPO-catalysed chlorination and bromination reaction of natural estrogens was characterised. Estradiol, estrone and equiline were efficiently converted to halogenated compounds in the presence of chloride or bromide and hydrogen peroxide. The catalytic efficiency of CPO in this reaction is similar to that measured for other aromatic substrates; as expected the bromination reaction proceeds more efficiently than the chlorination reaction. Three major products were detected for chlorination of estradiol; two of them were monohalogenated compounds while a third product was a dihalogenated compound at positions 2 and 4 of the aromatic ring A. Chlorinated compounds are not substrates for tyrosinase, suggesting that the halogenated form of estrogens is less susceptible to form o-quinones.
Asunto(s)
Cloruro Peroxidasa , Bromuros , Catálisis , Cloruro Peroxidasa/química , Cloruro Peroxidasa/metabolismo , Cloruros , Estradiol , Estrógenos , Estrona , Halogenación , Peróxido de Hidrógeno , Monofenol Monooxigenasa , QuinonasRESUMEN
Halogenases catalyze reactions that introduce halogen atoms into electron-rich organic molecules. Vanadium-dependent haloperoxidases are generally considered to be promiscuous halogenating enzymes that have thus far been derived exclusively from eukaryotes, where their cellular function is often disputed. We now report the first biochemical characterization of a bacterial vanadium-dependent chloroperoxidase, NapH1 from Streptomyces sp. CNQ-525, which catalyzes a highly stereoselective chlorination-cyclization reaction in napyradiomycin antibiotic biosynthesis. This finding biochemically links a vanadium chloroperoxidase to microbial natural product biosynthesis.
Asunto(s)
Antibacterianos/biosíntesis , Cloruro Peroxidasa/metabolismo , Naftoquinonas/metabolismo , Streptomyces/enzimología , Vanadio/metabolismo , Antibacterianos/química , Biocatálisis , Cloruro Peroxidasa/química , Ciclización , Estructura Molecular , Naftoquinonas/química , Estereoisomerismo , Streptomyces/genéticaRESUMEN
Heme peroxidases are subject to a mechanism-based oxidative inactivation. During the catalytic cycle, the heme group is activated to form highly oxidizing species, which may extract electrons from the protein itself. In this work, we analyze changes in residues prone to oxidation owing to their low redox potential during the peroxide-mediated inactivation of chloroperoxidase from Caldariomyces fumago under peroxidasic catalytic conditions. Surprisingly, we found only minor changes in the amino acid content of the fully inactivated enzyme. Our results show that tyrosine residues are not oxidized, whereas all tryptophan residues are partially oxidized in the inactive protein. The data suggest that the main process leading to enzyme inactivation is heme destruction. The molecular characterization of the peroxide-mediated inactivation process could provide specific targets for the protein engineering of this versatile peroxidase.
Asunto(s)
Ascomicetos/enzimología , Cloruro Peroxidasa/antagonistas & inhibidores , Hemo/química , Peróxido de Hidrógeno/farmacología , Cloruro Peroxidasa/química , Cloruro Peroxidasa/metabolismo , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Activación Enzimática/efectos de los fármacos , Peróxido de Hidrógeno/química , Modelos Moleculares , Oxidación-Reducción , Ingeniería de ProteínasAsunto(s)
Enzimas/química , Hemo/química , Biocatálisis , Dominio Catalítico , Cloruro Peroxidasa/química , Cloruro Peroxidasa/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Enzimas/metabolismo , Hemo Oxigenasa (Desciclizante)/química , Hemo Oxigenasa (Desciclizante)/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/metabolismo , Peroxidasas/química , Peroxidasas/metabolismo , Protoporfirinas/química , Triptófano Oxigenasa/química , Triptófano Oxigenasa/metabolismoRESUMEN
The catalytic performance of chloroperoxidase (CPO) in peroxidation of 2, 2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) and oxidation of indole in a reverse micelle composed of surfactant-water-isooctane-pentanol was investigated and optimized in this work. Some positive results were obtained as follows: the peroxidation activity of CPO was enhanced 248% and 263%, while oxidation activity was enhanced 215% and 222% in cetyltrimethylammonium bromide (CTABr) reverse micelle medium and dodecyltrimethylammonium bromide (DTABr) medium, respectively. Thermostability was also greatly improved in reverse micelle: at 40 °C, CPO essentially lost all its activity after 5 h incubation, while 58-76% catalytic activity was retained for both reactions in the two reverse micelle media. At 50 °C, about 44-75% catalytic activity remained for both reactions in reverse micelle after 2 h compared with no observed activity in pure buffer under the same conditions. The enhancement of CPO activity was dependent mainly on the surfactant concentration and structure, organic solvent ratio (V(pentanol)/V(isooctane)), and water content in the reverse micelle. The obtained kinetic parameters showed that the catalytic turnover frequency (k(cat)) was increased in reverse micelle. Moreover, the lower K(m) and higher k(cat)/K(m) demonstrated that both the affinity and specificity of CPO to substrates were improved in reverse micelle media. Fluorescence, circular dichroism (CD) and UV-vis spectra assays indicated that a catalytically favorable conformation of enzyme was achieved in reverse micelle, including the strengthening of the protein α-helix structure, and greater exposure of the heme prosthetic group for easy access of the substrate in bulk solution. These results are promising in view of the industrial applications of this versatile biological catalyst.
Asunto(s)
Cloruro Peroxidasa/química , Benzotiazoles/metabolismo , Biocatálisis , Cloruro Peroxidasa/metabolismo , Dicroismo Circular , Estabilidad de Enzimas , Micelas , Octanos/química , Oxidación-Reducción , Conformación Proteica , Compuestos de Amonio Cuaternario/química , Ácidos Sulfónicos/metabolismo , Tensoactivos/química , TemperaturaRESUMEN
Recently, enzyme dynamic therapy (EDT) has drawn much attention as a new type of dynamic therapy. However, the selection of suitable nanocarriers to deliver chloroperoxidase (CPO) and enhancement of the level of hydrogen peroxide (H2 O2 ) in the tumor microenvironment (TME) are critical factors for improving the efficiency of EDT. In this study, a rapidly decomposing nanocomposite is designed using tetra-sulfide-bond-incorporating dendritic mesoporous organosilica (DMOS) as a nanocarrier, followed by loading CPO and sodium-hyaluronate-modified calcium peroxide nanoparticles (CaO2 -HA NPs). The nanocomposite can effectively generate singlet oxygen (1 O2 ) for tumor therapy without any exogenous stimulus via trimodal-enhanced EDT, including DMOS-induced depletion of glutathione (GSH), H2 O2 compensation from CaO2 -HA NPs in mildly acidic TME, and oxidative stress caused by overloading of Ca2+ . As tetra-sulfide bonds are sensitive to GSH, DMOS can generate hydrogen sulfide (H2 S) gas as a new kind of H2 S gas nanoreactor. Additionally, the overloading of Ca2+ can cause tumor calcification to accelerate in vivo tumor necrosis and promote computed tomography imaging efficacy. Therefore, a novel H2 S gas, EDT, and Ca2+ -interference combined therapy strategy is developed.