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1.
Int J Biol Macromol ; 271(Pt 1): 132627, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38797290

RESUMEN

Melanins are dark-brown to black-colored biomacromolecules which have been thoroughly studied in animals and microorganisms. However, the biochemical and molecular basis of plant melanins are poorly understood. We first characterized melanin from the black radish (Raphanus sativus var. niger) 'HLB' through spectroscopic techniques. p-Coumaric acid was identified as the main precursor of radish melanin. Moreover, a joint analysis of transcriptome and coexpression network was performed for the two radish accessions with black and white cortexes, 'HLB' and '55'. A set of R2R3-type RsMYBs and enzyme-coding genes exhibited a coexpression pattern, and were strongly correlated with melanin formation in radish. Transient overexpression of two phenol oxidases RsLAC7 (laccase 7) or RsPOD22-1 (peroxidase 22-1) resulted in a deeper brown color around the infiltration sites and a significant increase in the total phenol content. Furthermore, co-injection of the transcriptional activator RsMYB48/RsMYB97 with RsLAC7 and/or RsPOD22-1, markedly increased the yield of black extracts. Spectroscopic analyses revealed that these extracts are similar to the melanin found in 'HLB'. Our findings advance the understanding of structural information and the transcriptional regulatory mechanism underlying melanin formation in radish.


Asunto(s)
Regulación de la Expresión Génica de las Plantas , Melaninas , Monofenol Monooxigenasa , Raphanus , Raphanus/genética , Raphanus/metabolismo , Melaninas/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Transcriptoma , Perfilación de la Expresión Génica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Ácidos Cumáricos/metabolismo
2.
Acta Crystallogr D Struct Biol ; 80(Pt 2): 101-112, 2024 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-38265876

RESUMEN

Catalase is an antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into molecular oxygen and water. In all monofunctional catalases the pathway that H2O2 takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15 Šlonger and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H2O2 routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with kcat and kcat/Km values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.


Asunto(s)
Ácido Glutámico , Peróxido de Hidrógeno , Catalasa/química , Peróxido de Hidrógeno/química , Hemo/química , Treonina
3.
Angew Chem Int Ed Engl ; 62(1): e202211552, 2023 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-36334012

RESUMEN

De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di-copper site and mimicking the Type 3 (T3) copper-containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di-metal coordination spheres to engineer the di-copper site into a simple four-helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O2 -dependent oxidation of catechols to o-quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four-helix bundle protein.


Asunto(s)
Cobre , Metaloproteínas , Cobre/química , Catecoles/química , Metaloproteínas/química , Oxidación-Reducción
4.
Int. microbiol ; 25(3): 495-502, Ago. 2022. graf
Artículo en Español | IBECS | ID: ibc-216209

RESUMEN

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


Asunto(s)
Humanos , Péptido Hidrolasas , Lacasa , Pleurotus , Fenol , Microbiología
5.
Int Microbiol ; 25(3): 495-502, 2022 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-35113262

RESUMEN

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


Asunto(s)
Pleurotus , Ácido Edético/farmacología , Lacasa , Péptido Hidrolasas , Inhibidores de Proteasas/farmacología
6.
3 Biotech ; 11(5): 214, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33928002

RESUMEN

This study is to test the capacity of the white rot fungus Coriolopsis gallica for the biodegradation of Diesel Fuel hydrocarbons (DHs). Using the experimental face centered central composite design (FCCCD), culture conditions of the Diesel-mended medium were optimized to reach 110.43% of DHs removal rate, and l5267.35 U L-1 of laccase production by C. gallica, simultaneously. The optimal combination of the cultural parameters was: Diesel concentration range of 2.95-3.14%, inoculum size of 3%, incubation time of 15 days, Tween 80 concentration of 0.05%, and the ratio glucose/peptone (G/P) range of 10.15-10.27. Further, the degradation ability of C. gallica for Diesel Fuel was evaluated through mycelial pellets uptake and oxidative action of fungal enzymes in the optimized degrading-medium using gas chromatography-mass spectrometry (GC-MS). Cyclosiloxanes and C20 PAHs detected as the major compound in Diesel Fuel (46%) was completely bio-transformed to simple metabolites including, essentially benzoic acid ester (71%), alcohols (1.52%) epoxy alkane (1.07%), carboxylic acids (1.24%) and quinones (0.33%). Germination rate and root elongation, as a rapid phytotoxicity test demonstrated that toxicity of Diesel's PAHs is minimized by fungal treatment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02769-w.

7.
Mar Biotechnol (NY) ; 22(5): 673-682, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32833111

RESUMEN

In this study, we report our contribution to the application of whole cells of Brazilian marine-derived fungi in the biotransformation of ethinylestradiol 1. A preliminary screening with twelve marine-derived fungi strains revealed that the fungus Penicillium oxalicum CBMAI 1996 promoted the biotransformation of ethinylestradiol 1. Then, P. oxalicum CBMAI 1996 was employed in the reactions in decaplicate in order to purify and characterize the main biotransformation products of ethinylestradiol 1. Compounds 1b and 1c were characterized by NMR, HRMS, [α]D and mp. Compound 1b was also characterized by single crystal X-ray diffraction. In addition, kinetic monitoring of the biotransformation of ethinylestradiol 1 by P. oxalicum CBMAI 1996 was evaluated in this study in order to obtain high yields of compounds 1b and 1c with a reduction of the reaction time. In this work, we proposed a biotransformation pathway of ethinylestradiol 1, which suggests the presence of several enzymes such as phenol oxidases, monooxygenases, and ene-reductases in the fungus P. oxalicum CBMAI 1996. In summary, the rapid biodegradation of ethinylestradiol 1 and compounds 1b and 1c also has an environmental relevance, since ethinylestradiol 1 and other steroidal compounds are improperly discarded in the environment, and part of these compounds are displaced into the oceans.


Asunto(s)
Biotransformación , Etinilestradiol/metabolismo , Penicillium/metabolismo , Biodegradación Ambiental , Contaminantes Químicos del Agua/metabolismo
8.
Braz. arch. biol. technol ; 63: e20190015, 2020. tab, graf
Artículo en Inglés | LILACS | ID: biblio-1132271

RESUMEN

Abstract (1) Background: Oxygen supply is an important parameter to be considered in submerged cultures. This study evaluated the influence of different conditions for dissolved oxygen (DO) concentration on laccases activities and growth of Pleurotus sajor-caju PS-2001 in submerged process in stirred-tank bioreactor. (2) Methods: Initially, three different conditions were tested: uncontrolled DO and minimum levels of 30% and 80% of saturation, with the pH controlled between 4.5 and 7.0. (3) Results: Best results were observed at 30% DO (26 U mL-1 of laccases at 96 h), whereas higher mycelial biomass was observed at 30% and 80% DO (above 4.5 g L-1). Four different conditions of DO (uncontrolled, 10%, 30% and 50% of saturation) were tested at pH 6.5, with higher laccases activity (80 U mL-1 at 66 h) and lower mycelial growth (1.36 g L-1 at 90 h) being achieved with DO of 30%. In this test, the highest values for volumetric productivity and specific yield factor were determined. Under the different pH conditions tested, the production of laccases is favoured at DO concentration of 30% of saturation, while superior DO levels favours fungal growth. (4) Conclusion: The results indicate that dissolved oxygen concentration is a critical factor for the culture of P. sajor-caju PS-2001 and has important effects not only on laccases production but also on fungal growth.


Asunto(s)
Oxígeno Disuelto , Biomasa , Reactores Biológicos , Pleurotus/crecimiento & desarrollo , Pleurotus/enzimología , Lacasa/biosíntesis
9.
J Environ Manage ; 236: 581-590, 2019 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-30771677

RESUMEN

In this work, phenol removal from aqueous solutions by Pleurotus sajor-caju PS-2001 phenol oxidases was assessed under different conditions. In stirred-tank reactor (STR), 77, 82, 92 and 36% of removal were attained when initial concentrations of 1.0, 2.0, 3.0 and 4.0 mmol L-1 phenol, respectively, were used. Among the different enzymes produced by this fungus, phenol removal seems to be related to the activity of laccases that attained maximum values between 33 and 91 U mL-1 in STR. With an internal-loop airlift reactor (ILAR), phenol concentrations of 1.0, 2.0, 3.0, 4.0 and 5.0 mmol L-1 were evaluated, and removal of 70, 76, 82, 77 and 82%, respectively, were observed. In ILAR, however, superior maximum titres of laccases were quantified (80-285 U mL-1). Crude enzyme broths have also been tested for phenol removal from 3.0 mmol L-1 aqueous solutions, the best result (55% of removal) being obtained at pH 3.2 and 30 °C, without agitation, using 60 U mL-1 laccases. According to the data presented, phenol can be efficiently removed from liquid media in submerged cultures of P. sajor-caju PS-2001 even when carried out in a simple pneumatic reactor, whereas significantly less amount of the pollutant is degraded when a crude enzyme broth is used.


Asunto(s)
Pleurotus , Lacasa , Monofenol Monooxigenasa , Fenol , Fenoles
10.
Parasit Vectors ; 10(1): 171, 2017 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-28376914

RESUMEN

BACKGROUND: Phenol oxidases (POs) catalyze the oxidation of dopa and dopamine to melanin, which is crucial for cuticle formation and innate immune maintenance in insects. Although, Laccase 2, a member of the PO family, has been reported to be a requirement for melanin-mediated cuticle tanning in the development stages of some insects, whether it participates in cuticle construction and other physiological processes during the metamorphosis of mosquito pupae is unclear. METHODS: The association between the phenotype and the expression profile of Anopheles sinensis Laccase 2 (AsLac2) was assessed from pupation to adult eclosion. Individuals showing an expression deficiency of AsLac2 that was produced by RNAi and their phenotypic defects and physiological characterizations were compared in detail with the controls. RESULTS: During the dominant expression period, knockdown of AsLac2 in pupae caused the cuticle to be unpigmented, and produced thin and very soft cuticles, which further impeded the eclosion rate of adults as well as their fitness. Moreover, melanization immune responses in the pupae were sharply decreased, leading to poor resistance to microorganism infection. Both the high conservation among Laccase 2 homologs and a very similar genomic synteny of the neighborhood in Anopheles genus implies a conservative function in the pupal stage. CONCLUSIONS: To our knowledge, this is the first study to report the serious phenotypic defects in mosquito pupae caused by the dysfunction of Laccase 2. Our findings strongly suggest that Laccase 2 is crucial for Anopheles cuticle construction and melanization immune responses to pathogen infections during pupal metamorphosis. This irreplaceability provides valuable information on the application of Lacccase 2 and/or other key genes in the melanin metabolism pathway for developing mosquito control strategies.


Asunto(s)
Anopheles/enzimología , Anopheles/inmunología , Resistencia a la Enfermedad , Integumento Común/fisiología , Lacasa/deficiencia , Animales , Perfilación de la Expresión Génica , Silenciador del Gen , Pupa/enzimología , Pupa/inmunología , Interferencia de ARN , Análisis de Secuencia de ADN , Curtiembre
11.
J Microbiol ; 55(4): 280-288, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28124773

RESUMEN

The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological-biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was activated. These genes encode enzymes such as tyrosinase, chitinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Grifola/crecimiento & desarrollo , Hidrolasas/biosíntesis , Hifa/crecimiento & desarrollo , Reishi/crecimiento & desarrollo , Hongos Shiitake/crecimiento & desarrollo , Transcripción Genética , Grifola/enzimología , Grifola/ultraestructura , Hidrolasas/genética , Hifa/enzimología , Hifa/ultraestructura , Microscopía , Morfogénesis , Reishi/enzimología , Reishi/ultraestructura , Hongos Shiitake/enzimología , Hongos Shiitake/ultraestructura
12.
FEMS Microbiol Lett ; 362(11)2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25926529

RESUMEN

In order to explore the abundance and potential environmental functions of green algal laccases, we screened various algae for extracellular laccase-like activities, characterized basic features of these activities in selected species and exemplarily studied the transformation of environmental pollutants and complex natural compounds by the laccase of Tetracystis aeria. Oxidation of the classical laccase substrate ABTS was found to be widespread in chlorophycean algae. The oxidation activity detected in members of the 'Scenedesmus' clade was caused by an unknown thermostable low-molecular-mass compound. In contrast, species of the Moewusinia, including Chlamydomonas moewusii and T. aeria, excreted putative 'true' laccases. Phenolic substrates were oxidized by these enzymes optimally at neutral to alkaline pH. The Tetracystis laccase efficiently transformed bisphenol A, 17α-ethinylestradiol, nonylphenol and triclosan in the presence of ABTS as redox mediator, while anthracene, veratrylalcohol and adlerol were not attacked. Lignosulfonate and humic acid underwent slight (de)polymerization reactions in the presence of the laccase and mediator(s), probably involving the oxidation of phenolic constituents. Possible natural functions of the enzymes, such as the synthesis of complex polymers or detoxification processes, may assist the survival of the algae in adverse environments. In contaminated surface waters, laccase-producing green algae might contribute to the environmental breakdown of phenolic pollutants.


Asunto(s)
Chlorophyta/enzimología , Contaminantes Ambientales/metabolismo , Lacasa/metabolismo , Fenoles/metabolismo , Antracenos/metabolismo , Compuestos de Bencidrilo/metabolismo , Chlorophyta/clasificación , Chlorophyta/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Filogenia , Especificidad por Sustrato , Triclosán/metabolismo
13.
J Biotechnol ; 182-183: 37-45, 2014 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-24800960

RESUMEN

We report for the first time that the medicinal basidiomycete Lentinus edodes can reduce Au(III) from chloroauric acid (HAuCl4) to elemental Au [Au(0)], forming nanoparticles. Several methods, including transmission electron microscopy, electron energy loss spectroscopy, X-ray fluorescence, and dynamic light scattering, were used to show that when the fungus was grown submerged, colloidal gold accumulated on the surface of and inside the mycelial hyphae as electron-dense particles mostly spherical in shape, with sizes ranging from 5 to 50nm. Homogeneous proteins (the fungal enzymes laccase, tyrosinase, and Mn-peroxidase) were found for the first time to be involved in the reduction of Au(III) to Au(0) from HAuCl4. A possible mechanism forming Au nanoparticles is discussed.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Oro/química , Oro/metabolismo , Nanopartículas del Metal/química , Hongos Shiitake/metabolismo , Cloruros/química , Cloruros/metabolismo , Proteínas Fúngicas/metabolismo , Compuestos de Oro/química , Compuestos de Oro/metabolismo , Microscopía Electrónica de Transmisión , Monofenol Monooxigenasa/metabolismo , Tamaño de la Partícula , Hongos Shiitake/química , Hongos Shiitake/citología , Hongos Shiitake/enzimología
14.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 3): 398-408, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23519415

RESUMEN

Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide.


Asunto(s)
Ascomicetos/enzimología , Ascomicetos/genética , Catalasa/química , Regulación Fúngica de la Expresión Génica , Monofenol Monooxigenasa/química , Catalasa/genética , Catalasa/metabolismo , Cristalografía por Rayos X , Activación Enzimática/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética
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