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1.
Bioelectrochemistry ; 132: 107399, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31835110

RESUMO

Pyranose dehydrogenase is a flavin-dependent carbohydrate oxidoreductase classified among Auxiliary Activities Family 3, along with structurally and catalytically related enzymes like pyranose oxidase and cellobiose dehydrogenase, and probably fulfils biological functions in lignocellulose breakdown. It is limited to a rather small group of litter-decomposing basidiomycetes adapted to humic-rich habitats, and shows an equally rare combination of structural and biochemical properties. It displays broader substrate specificity and regioselectivity compared to similar enzymes, catalyzing monooxidations at C1, C2, C3 or dioxidations at C2, 3 or C3, 4, depending on the pyranose sugar form (mono-/di-/oligo-saccharide or glycoside) and the enzyme source. It is unable to utilize oxygen as electron acceptor, using substituted benzoquinones and (organo)metallic ions instead, which suggests a role in redox cycling of (hydro)quinones and complexed metal ions. Pyranose dehydrogenase is a promising candidate for enzymatic sensors of various sugars, for the anodic reaction in enzymatic biofuel cells powered by carbohydrate mixtures, and as a versatile biocatalyst for the production of di- and tri-carbonyl sugar derivatives as chiral intermediates for the synthesis of rare sugars, novel drugs and fine chemicals.


Assuntos
Biocatálise , Desidrogenases de Carboidrato/metabolismo , Técnicas Eletroquímicas/métodos , Agaricus/enzimologia , Fontes de Energia Bioelétrica , Desidrogenases de Carboidrato/química , Elétrons , Glicosilação , Oxirredução , Especificidade por Substrato
2.
Food Chem ; 293: 285-290, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151613

RESUMO

Exogenous adenosine triphosphate (ATP) treatment at 0, 250, 500, 750, and 1000 µM retarded cap browning in mushrooms by 0, 34, 26, 51 and 32 %, respectively, during storage at 4 °C for 18 days. Triggering signaling H2O2 accumulation arising from elevating NADPH oxidase enzyme activity during 6 days of storage at 4 °C may be pivotal for promoting shikimate dehydrogenase enzyme activity in mushrooms treated with ATP during 18 days of storage at 4 °C. Promoting melatonin accumulation (390 µg kg-1 FW vs. 160 µg kg-1 FW) in mushrooms treated with ATP during cold storage may attribute to signaling H2O2 accumulation. Higher DPPH scavenging capacity (72 % vs. 65 %) in mushrooms treated with ATP may attribute to higher phenols accumulation arising from higher phenylalanine ammonialyase/polyphenol oxidase enzymes activity concomitant with higher alternative oxidase gene expression during 18 days of storage at 4 °C.


Assuntos
Trifosfato de Adenosina/farmacologia , Agaricus/efeitos dos fármacos , Temperatura Baixa , Armazenamento de Alimentos , Reação de Maillard , Trifosfato de Adenosina/administração & dosagem , Agaricus/enzimologia , Agaricus/fisiologia , Oxirredutases do Álcool/metabolismo , Compostos de Bifenilo/química , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/metabolismo , Melatonina/metabolismo , Proteínas Mitocondriais/genética , NADPH Oxidases/metabolismo , Oxirredutases/genética , Fenóis/metabolismo , Picratos/química , Proteínas de Plantas/genética , Transdução de Sinais
3.
J Enzyme Inhib Med Chem ; 34(1): 927-936, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31039625

RESUMO

Skin ageing results from enhanced activation of intracellular enzymes such as collagenases, elastases and tyrosinase, stimulated by intrinsic ageing and photoageing factors. Recently, caffeine-based cosmetics are introduced that demonstrates to slow down skin photoageing process. However, no attempts have been done so for to understand caffeine functional inhibitory activity against photoageing related enzymes. Hence, this study established the caffeine molecular interaction and inhibition activity profiles against respective enzymes using in silico and in vitro methods, respectively. Results from in silico study indicates that caffeine has comparatively good affinity with collagenase (-4.6 kcal/mol), elastase (-3.36 kcal/mol) and tyrosinase (-2.86 kcal/mol) and formed the stable protein-ligand complex as validated by molecular dynamics simulation (protein-ligand contacts, RMSD, RMSF and secondary structure changes analysis). Moreover, in vitro data showed that caffeine (1000 µg/mL) has statistically significant maximum inhibition activity of 41.86, 36.44 and 13.72% for collagenase, elastase and tyrosinase, respectively.


Assuntos
Cafeína/farmacologia , Colagenases/metabolismo , Simulação por Computador , Inibidores Enzimáticos/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Elastase Pancreática/antagonistas & inibidores , Agaricus/enzimologia , Animais , Cafeína/química , Clostridium histolyticum/enzimologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Técnicas In Vitro , Ligantes , Simulação de Dinâmica Molecular , Monofenol Mono-Oxigenase/metabolismo , Pâncreas/enzimologia , Elastase Pancreática/metabolismo , Relação Estrutura-Atividade , Suínos
4.
Chem Biodivers ; 16(7): e1900167, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31145516

RESUMO

A dozen of phosphonic and phosphinic acid derivatives containing pyridine moiety were synthesized and its inhibitory activity toward mushroom tyrosinase was investigated. Moreover, molecular docking of these compounds to the active site of the enzyme was performed. All the compounds (1-10) demonstrated the inhibitory effect with the IC50 and inhibition constants ranging millimolar concentrations. The obtained results indicate that the compounds show different types of inhibition (competitive, noncompetitive, mixed), but all of them are reversible inhibitors. The obtained outcomes allowed to make the structure-activity relationship (SAR) analysis. Compound 4 ([(benzylamino)(pyridin-2-yl)methyl]phenylphosphinic acid) revealed the lowest IC50 value of 0.3 mm and inhibitory constant of Ki 0.076 mm, with noncompetitive type and reversible mechanism of inhibition. According to SAR analysis, introducing bulky phenyl moieties to phosphonic and amino groups plays an important role in the inhibitory potency on activity of mushroom tyrosinase and could be useful in design and development of a new class of potent organophosphorus inhibitors of tyrosinase. Combined results of molecular docking and SAR analysis can be helpful in designing novel tyrosinase inhibitors of desired properties. They may have broad application in food industry and cosmetology.


Assuntos
Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidores , Ácidos Fosfínicos/farmacologia , Ácidos Fosforosos/farmacologia , Agaricus/enzimologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Cinética , Estrutura Molecular , Monofenol Mono-Oxigenase/metabolismo , Ácidos Fosfínicos/química , Ácidos Fosfínicos/isolamento & purificação , Ácidos Fosforosos/química , Ácidos Fosforosos/isolamento & purificação , Relação Estrutura-Atividade
5.
Anal Bioanal Chem ; 411(11): 2415-2424, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30880350

RESUMO

An amperometric biosensor compatible with a flow injection analysis (FIA) for highly selective determination of acetaminophen (APAP) in a sample of human urine was developed. This biosensor is also suitable for use in the routine pharmaceutical practice. To prove this statement, two different commercially available pharmaceutical formulations were analyzed. This nano-(bio)electroanalytical device was made from a commercially available screen-printed carbon electrode covered by a thin layer of non-functionalized graphene (NFG) as amperometric transducer. A biorecognition layer was prepared from mushroom (Agaricus bisporus) tyrosinase (EC 1.14.18.1) cross-linked using glutaraldehyde, where resulting aggregates were covered by Nafion®, a known ion exchange membrane. Owing to the use of tyrosinase and presence of NFG, the developed analytical instrument is able to measure even at potentials of 0 V. Linear ranges differ according to choice of detection potential, namely up to 130 µmol L-1 at 0 V, up to 90 µmol L-1 at -0.1 V, and up to 70 µmol L-1 at -0.15 V. The first mentioned linear range is described by the equation Ip [µA] = 0.236 - 0.1984c [µmol L-1] and correlation coefficient r = 0.9987; this equation was used to quantify the content of APAP in each sample. The limit of detection of APAP was estimated to be 1.1 µmol L-1. A recovery of 96.8% (c = 25 µmol L-1, n = 5 measurements) was calculated. The obtained results show that FIA is a very selective method for APAP determination, being comparable to the chosen reference method of reversed-phase high-performance liquid chromatography.


Assuntos
Acetaminofen/urina , Agaricus/enzimologia , Analgésicos não Entorpecentes/urina , Técnicas Biossensoriais/métodos , Análise de Injeção de Fluxo/métodos , Monofenol Mono-Oxigenase/química , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Análise de Injeção de Fluxo/instrumentação , Humanos , Limite de Detecção , Urinálise/instrumentação , Urinálise/métodos
6.
J Sci Food Agric ; 99(2): 790-796, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29998459

RESUMO

BACKGROUND: In the present study, we investigated the role of ornithine decarboxylase (ODC) in the methyl jasmonate (MeJA)-regulated postharvest quality maintenance of Agaricus bisporus (J. E. Kange) Imbach button mushrooms by pretreating mushrooms with a specific irreversible inhibitor called α-difluoromethylornithine (DFMO) before exposure to MeJA vapor. RESULTS: Mushrooms were treated with 0 or 100 µmol L-1 MeJA or a combination of 120 µmol L-1 DFMO and 100 µmol L-1 MeJA, respectively, before storage at 4 °C for 21 days. Treatment with MeJA alone induced the increase in ODC activity whereas this effect was greatly suppressed by pretreatment with DFMO. α-Difluoromethylornithine strongly attenuated the effect of MeJA on decreasing cap opening, slowing the decline rate of soluble protein and total sugar, and accumulating total phenolics and flavonoids. α-Difluoromethylornithine pretreatment also counteracted the ability of MeJA to inhibit polyphenol oxidase and lipoxygenase activities, and malondialdehyde production, and to stimulate superoxide dismutase and catalase activities. It also largely downregulated MeJA-induced accumulation of free putrescine (Put). CONCLUSION: These results reveal that ODC is involved in MeJA-regulated postharvest quality retention of button mushrooms, and this involvement is likely to be associated with Put levels. © 2018 Society of Chemical Industry.


Assuntos
Acetatos/farmacologia , Agaricus/química , Agaricus/efeitos dos fármacos , Ciclopentanos/farmacologia , Proteínas Fúngicas/metabolismo , Ornitina Descarboxilase/metabolismo , Oxilipinas/farmacologia , Agaricus/enzimologia , Agaricus/crescimento & desenvolvimento , Catecol Oxidase/metabolismo , Flavonoides/análise , Flavonoides/metabolismo , Malondialdeído/metabolismo , Fenóis/análise , Fenóis/metabolismo , Putrescina/análise , Putrescina/metabolismo , Controle de Qualidade , Superóxido Dismutase/metabolismo
7.
Bioorg Chem ; 82: 129-138, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30312868

RESUMO

Pyrimidine-fused compounds are of great interest for the discovery of potent bioactive agents. This study describes the synthesis of novel pyranopyrimidines 3a-f and pyranotriazolopyrimidines 4a-d derivatives via the cyclocondensation reaction of α-functionalized iminoether 2, which was obtained from 2-amino-3-cyanopyrane 1, with a series of primary aromatic amines and hydrazides, respectively. Structures of all synthesized compounds were established on the basis of spectroscopic methods including 1H NMR, 13C NMR and ES-HRMS. They were finally tested for their anticoagulant and anti-tyrosinase activities. Significant results have been obtained and the structure-activity relationship (SAR) was discussed with the help of molecular docking analysis.


Assuntos
Anticoagulantes/farmacologia , Inibidores Enzimáticos/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Piranos/farmacologia , Pirimidinas/farmacologia , Triazóis/farmacologia , Agaricus/enzimologia , Anticoagulantes/sangue , Anticoagulantes/síntese química , Anticoagulantes/química , Sítios de Ligação , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Monofenol Mono-Oxigenase/química , Tempo de Tromboplastina Parcial , Piranos/sangue , Piranos/síntese química , Piranos/química , Pirimidinas/sangue , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Triazóis/sangue , Triazóis/síntese química , Triazóis/química
8.
J Agric Food Chem ; 66(51): 13464-13472, 2018 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-30482011

RESUMO

Theaflavins, the orange-red pigments contained in black tea, have attracted attention as a result of their health-promoting effects. However, their synthetic preparation, in which the enzymatic oxidation of catechol-type catechin is followed by the quinone-induced oxidative dimerization of selectively combined catechol- and pyrogallol-type catechins, provides only a low yield. In the present study, we found that a 1-octanol/buffer biphasic system improved the yield of theaflavin 3-gallate in a tyrosinase-catalyzed synthetic reaction with (-)-epicatechin and (-)-epigallocatechin gallate. When the enzymatic reaction proceeded in a buffer solution, oxidized (-)-epigallocatechin gallate was preferentially used for self-dimerization. However, self-dimerization was suppressed in the octanol phase, allowing oxidized (-)-epigallocatechin gallate to participate in coupling with (-)-epicatechin quinone, leading to effective production of theaflavin 3-gallate. Furthermore, the preferential localization of theaflavin 3-gallate in the octanol phase prevented (-)-epicatechin-quinone-induced degradation.


Assuntos
1-Octanol/química , Agaricus/enzimologia , Biflavonoides/química , Catequina/análogos & derivados , Catequina/química , Proteínas Fúngicas/química , Ácido Gálico/análogos & derivados , Biocatálise , Dimerização , Ácido Gálico/química , Estrutura Molecular , Monofenol Mono-Oxigenase , Oxirredução
9.
Biomed Res Int ; 2018: 5314320, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30320135

RESUMO

Torreya grandis Fort. ex Lindl. is a plant belonging to the Taxaceae family and Torreya grandis cv. Merrillii is the only grafted and thoroughbred species belonging to this species. In this study, we extracted five different seed oils, including T. grandis seed oil (TGSO), T. grandis "Xiangyafei" seed oil (XYSO), T. grandis "Zhimafei" seed oil (ZMSO), T. grandis "Majus"seed oil (TGMSO), and T. grandis "cunguangfei" seed oil (CGSO) using physical pressure. The resulting extracts were analyzed to determine their fatty acid composition, antioxidant activity, and inhibitory activity towards tyrosinase. The results of the antioxidant activity assays revealed that XYSO and ZMSO exhibited much greater DPPH radical scavenging activity and ferric reducing power than TGSO. Notably, all five of the seed oils showed dose-dependent inhibitory activity towards tyrosinase. XYSO and TGSO gave the highest activities of all of the seed oils tested in the current study against monophenolase and diphenolase, with IC50 values of 227.0 and 817.5µg/mL, respectively. The results of this study show that wild TGSOs exhibit strong antioxidant and tyrosinase inhibition activities. These results therefore suggest that wild TGSOs could be used as a potential source of natural antioxidant agents and tyrosinase inhibitors.


Assuntos
Agaricus/enzimologia , Antioxidantes/química , Inibidores Enzimáticos/química , Proteínas Fúngicas/antagonistas & inibidores , Monofenol Mono-Oxigenase/antagonistas & inibidores , Óleos Vegetais/química , Sementes/química , Taxaceae/química , Proteínas Fúngicas/química , Monofenol Mono-Oxigenase/química
10.
Anal Chim Acta ; 1040: 128-135, 2018 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-30327102

RESUMO

The presence of phenol in industrial wastewater is an issue of great relevance for petrochemical and energy companies, among others. The high toxicity level of this substance requires polluting industries to continuously monitor the concentration of phenol in their wastewaters so as to comply with environmental regulations and to minimize environmental impact. This research work proposes the experimental development of an analytical method for "in situ" measurement of the concentration of phenol diluted in water, with application in Oil & Gas production wastewater monitoring. The method is based on the principle of selectivity exhibited by the oxidoreductase enzymes in the presence of phenolic compounds. The differences in the performance found when using organic tissues and microorganisms, as natural alternative sources of the enzyme, are also highlighted. These alternative sources of oxidoreductase enzyme work as the recognition element of the biosensor. A dissolved oxygen sensor is used as the transducer of the chemical signal produced by the reaction between the analyte and the enzyme. The bioencapsulation technique is very adequate in this case, because it offers a nutrient medium to the microorganisms and is reusable, making it ideal for repetitive measurement applications. The results show that the biosensor exhibits an approximately linear behavior when measuring phenol concentrations from 0.2 to 2 ppm.


Assuntos
Técnicas Biossensoriais , Oxirredutases/química , Fenóis/análise , Água/química , Agaricus/enzimologia , Estrutura Molecular , Oxirredutases/metabolismo , Fenóis/metabolismo , Água/metabolismo
11.
Bioorg Chem ; 81: 577-586, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30248509

RESUMO

The group of 19 thiosemicarbazones (TSCs) were synthesized and its inhibitory activity toward mushroom tyrosinase and ability to inhibition of melanogenesis in B16 cells were investigated. Moreover, molecular docking of these compounds to the active site of the enzyme was performed. The obtained results allowed to make the structure-activity relationship (SAR) analysis. Kinetic studies revealed that TSCs 1, 2, 11 and 18 have better inhibitory properties than kojic acid, a reference compound, with the best inhibitory constant (Ki) value of 0.38 µM for TSC 2. According to SAR analysis, the smaller and less branched molecules exhibit higher affinity to the enzyme. Melanin production in B16 cells was inhibited by all investigated compounds at micromolar level. Most of compounds studied in this work can be considered as potent inhibitors of tyrosinase and melanogenesis. They may have broad application in food preservatives and cosmetics. Combined results of molecular docking and SAR analysis can be helpful in designing novel tyrosinase inhibitors of desired properties.


Assuntos
Agaricales/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Melaninas/antagonistas & inibidores , Monofenol Mono-Oxigenase/antagonistas & inibidores , Tiossemicarbazonas/química , Tiossemicarbazonas/farmacologia , Agaricus/enzimologia , Animais , Vias Biossintéticas/efeitos dos fármacos , Linhagem Celular Tumoral , Melaninas/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/metabolismo
12.
PLoS One ; 13(7): e0201090, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30040824

RESUMO

Agaricus bisporus consumes carbohydrates contained in wheat straw based compost used for commercial mushroom production. Double substituted arabinoxylan is part of the ~40% of the compost polysaccharides that are not degraded by A. bisporus during its growth and development. Genes encoding α-1,3-l-arabinofuranosidase (AXHd3) enzymes that act on xylosyl residues doubly substituted with arabinosyl residues are absent in this mushroom forming fungus. Here, the AXHd3 encoding hgh43 gene of Humicola insolens was expressed in A. bisporus with the aim to improve its substrate utilization and mushroom yield. Transformants secreted active AXHd3 in compost as shown by the degradation of double substituted arabinoxylan oligomers in an in vitro assay. However, carbohydrate composition and degree of arabinosyl substitution of arabinoxylans were not affected in compost possibly due to inaccessibility of the doubly substituted xylosyl residues.


Assuntos
Agaricus/enzimologia , Compostagem , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Xilanos/metabolismo , Agaricus/classificação , Agaricus/genética , Agaricus/crescimento & desenvolvimento , Metabolismo dos Carboidratos , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Organismos Geneticamente Modificados , Sordariales/enzimologia , Sordariales/genética , Transformação Genética
13.
Int J Biol Macromol ; 117: 538-545, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29803745

RESUMO

Omeprazole was first evaluated for its antityrosinase activity and preservation of fresh-cut apples. The results obtained from enzymic analyses showed that the omeprazole inhibited tyrosinase activity (IC50 = 40 ±â€¯1.2 µM) with a reversible and competitive mechanism. Fluorescence quenching assays demonstrated that the interaction between omeprazole and tyrosinase was driven by hydrophobic forces and hydrogen bonds in a static procedure. Molecular docking further revealed that hydrogen bonds and hydrophobic forces were generated by omeprazole with the amino acid residues located in the A chain of tyrosinase. Moreover, the results from preservation assays showed that omeprazole could inhibit the activities of polyphenol oxidase (PPO) and peroxidase (POD), prevent the oxidation of total phenolics and flavonoid, thereby delay the browning of fresh-cut apples. Hence, this work identified a novel tyrosinase inhibitor and expands its feasible application as a food preservative.


Assuntos
Conservação de Alimentos/métodos , Malus/enzimologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Omeprazol/farmacologia , Agaricus/enzimologia , Catecol Oxidase/metabolismo , Di-Hidroxifenilalanina/metabolismo , Flavonoides/análise , Cinética , Malus/efeitos dos fármacos , Simulação de Acoplamento Molecular , Omeprazol/química , Peroxidase/metabolismo , Fenóis/análise , Soluções , Espectrometria de Fluorescência , Especificidade por Substrato
14.
J Med Chem ; 61(9): 3908-3917, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29634898

RESUMO

The inhibition of tyrosinase (Ty, EC 1.14.18.1) represents an efficient strategy of decreasing melanogenesis and skin hyperpigmentation. A combination of crystallographic and docking studies on two different tyrosinases, that from Bacillus megaterium (TyBm) and that from a mushroom (TyM), has contributed to increasing our knowledge about their structural information and translating that information to the most druggable human Ty (TyH) isozyme. In particular, we designed and synthesized a series of 1-(4-fluorobenzyl)piperazine and 1-(4-fluorobenzyl)piperidine derivatives showing inhibitory activities on TyM at micromolar ranges and more potency than that of the reference compound, kojic acid. The crystal structures of TyBm with inhibitor 3 (IC50 value of 25.11 µM) and 16 (IC50 value of 5.25 µM) were solved, confirming the binding poses hypothesized by in silico studies and revealing the main molecular determinants for the binding recognition of the inhibitors.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Piperazinas/química , Piperazinas/farmacologia , Agaricus/enzimologia , Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Cinética , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Piperazinas/metabolismo , Relação Estrutura-Atividade
15.
Int J Biol Macromol ; 113: 1142-1148, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29545062

RESUMO

Agaricus bisporus CU13 laccase was purified using ammonium sulfate precipitation (40-80%), Sephadex G100, and DEAE Sephadex A50 anion exchange column chromatography, respectively. Two laccase isoenzymes (Lacc1 & Lacc2) with purification folds of 1.40 and 5.81 respectively, were obtained from DEAE Sephadex A50 column. Optimal temperature and pH were recorded at 55 °C and pH 5.0 for both laccase isoenzymes using ABTS as substrate. Lacc1 was more thermostable than Lacc2 with residual activity of 95, 80 and 6%, while Lacc2 only retained 72, 25 and 0.4% of its activity after incubation for 90 min. at 50, 60 and 70 °C, respectively. Lacc2 retained about 93 and 86% of the initial activity at pH 9.0 and 7.0, whereas Lacc1 was stable at pH 7.0 and 5.0 followed by pH 9.0 and retained about 87, 76, and 36% of its activity respectively, after 4 h of incubation. Lacc1 was activated by 40% in the presence of Cu2+ (10 mM). Km and Vmax values found to be 0.394 and 0.158 µM, and 0.1351 and 0.4755 µmol min-1 for Lacc1 and Lacc2, respectively. The efficiency of both isoenzymes to decolorize Acid blue dye, make the enzyme seems to be a prospective for further biotechnological applications.


Assuntos
Agaricus/enzimologia , Corantes/metabolismo , Lacase/isolamento & purificação , Lacase/metabolismo , Agaricus/citologia , Biocatálise , Cor , Estabilidade Enzimática , Espaço Extracelular/enzimologia , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Lacase/química , Metais/farmacologia , Oxirredução , Temperatura
16.
Protein Expr Purif ; 145: 64-70, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29326063

RESUMO

A highly-active tyrosinase (H subunit) isoform has been purified from a commercial crude extract of Agaricus bisporus by a specific, two step-hydrophobic chromatography cascade process based on the differential adsorption of the proteins from the extract to hydrophobic-functionalized supports. At first, commercial, crude tyrosinase from Agaricus bisporus (AbTyr) dissolved in aqueous media was added to octadecyl-Sepabeads matrix at 25 °C. Under these conditions, the support specifically adsorbed a protein with a molecular weight of 47 kDa which showed no tyrosinase activity. The known H subunit of tyrosinase from Agaricus bisporus (45 kDa, H-AbTyr) and another protein of 50 kDa were present in the supernatant. Sodium phosphate buffer was added to adjust the ionic strength of the solution up to 100 mM and Triton X-100 was added (final concentration of 0.07% v/v) to control the hydrophobicity effect for both proteins. This solution was offered again to fresh octadecyl-Sepabeads support, immobilizing selectively the H-AbTyr and leaving exclusively the 50 kDa protein as a pure sample in the supernatant. This tyrosinase isoform of 50 kDa was almost 4-fold more active than the known H-TyrAb, with a specific tyrosinase activity of more than 38,000 U/mg.


Assuntos
Agaricus/enzimologia , Cromatografia/métodos , Monofenol Mono-Oxigenase/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação
17.
Anal Bioanal Chem ; 410(1): 27-32, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29150808

RESUMO

Up to now, knowledge of enzymes capable of degrading various contaminants of emerging concern (CEC) is limited, which is especially due to the lack of rapid screening methods. Thus, a miniaturized high-throughput setup using a chip-based robotic nanoelectrospray ionization system coupled to mass spectrometry has been developed to rapidly screen enzymatic reactions with environmentally relevant CECs. Three laccases, two tyrosinases, and two peroxidases were studied for their ability to transform ten pharmaceuticals and benzotriazole. Acetaminophen was most susceptible to enzymatic conversion by horseradish peroxidase (HRP), laccase from Trametes versicolor (LccTV), and a tyrosinase from Agaricus bisporus (TyrAB). Diclofenac and mefenamic acid were converted by HRP and LccTV, whereas sotalol was solely amenable to HRP conversion. Benzotriazole, carbamazepine, gabapentin, metoprolol, primidone, sulfamethoxazole, and venlafaxine remained persistent in this study. The results obtained here emphasize that enzymes are highly selective catalysts and more effort is required in the use of fast monitoring technologies to find suitable enzyme systems. Despite the methodological limitations discussed in detail, the automated tool provides a routine on-line screening of various enzymatic reactions to identify potential enzymes that degrade CECs. Graphical abstract A chip-based robotic nano-ESI-MS tool to rapidly monitor enzymatic degradation of environmentally relevant emerging contaminants.


Assuntos
Monitoramento Ambiental/instrumentação , Poluentes Ambientais/metabolismo , Ensaios de Triagem em Larga Escala/instrumentação , Preparações Farmacêuticas/metabolismo , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Agaricus/enzimologia , Armoracia/enzimologia , Biocatálise , Monitoramento Ambiental/economia , Monitoramento Ambiental/métodos , Poluentes Ambientais/isolamento & purificação , Recuperação e Remediação Ambiental , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Peroxidase do Rábano Silvestre/metabolismo , Dispositivos Lab-On-A-Chip , Lacase/metabolismo , Miniaturização/instrumentação , Miniaturização/métodos , Monofenol Mono-Oxigenase/metabolismo , Preparações Farmacêuticas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/economia , Espectrometria de Massas por Ionização por Electrospray/métodos , Fatores de Tempo , Trametes/enzimologia
18.
Biotechnol Prog ; 34(1): 42-50, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28726354

RESUMO

Biological pre-treatment seems to be promising being an eco-friendly process, with no inhibitor generated during the process. The potential for elephant grass pre-treatment with white degradation fungi Pleurotus ostreatus, Agaricus blazei, Lentinula edodes, Pleurotus citrinopileatus, and Pleurotus djamor, in isolated or mixed cultures of these strains, was evaluated. The highest activities of enzymes involved in the degradation of lignocellulosic biomass (laccases, endoglucanases, xylanases, and ß-glucosidases) were observed for A. blazei, L. edodes and the combination of P. ostreatus and A. blazei. In the enzymatic hydrolysis, there was greater release of reducing sugars in the pre-treated elephant grass samples by A. blazei during 10 days (338.91 ± 7.39 mg g-1 of biomass). For this sample, higher lignin reductions, 24.81 and 57.45%, after 15 and 35 days of incubation, respectively, were also verified. These data indicate the potential of macromycetes such as A. blazei to perform biological pre-treatments. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:42-50, 2018.


Assuntos
Agaricus/enzimologia , Cenchrus/química , Lignina/química , Pleurotus/enzimologia , Agaricus/química , Biomassa , Celulase/química , Glucosidases/química , Hidrólise , Lacase/química , Pleurotus/química
19.
Molecules ; 22(11)2017 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-29143758

RESUMO

Tyrosinase is a type-3 copper enzyme that is widely distributed in plants, fungi, insects, and mammals. Developing high potent inhibitors against tyrosinase is of great interest in diverse fields including tobacco curing, food processing, bio-insecticides development, cosmetic development, and human healthcare-related research. In the crystal structure of Agaricus bisporus mushroom tyrosinase, there is an oxygen atom bridging the two copper ions in the active site. It is unclear whether the identity of this bridging oxygen is a water molecule or a hydroxide anion. In the present study, we theoretically determine the identity of this critical bridging oxygen by performing first-principles hybrid quantum mechanics/molecular mechanics/Poisson-Boltzmann-surface area (QM/MM-PBSA) calculations along with a thermodynamic cycle that aim to improve the accuracy. Our results show that the binding with water molecule is energy favored and the QM/MM-optimized structure is very close to the crystal structure, whereas the binding with hydroxide anions causes the increase of energy and significant structural changes of the active site, indicating that the identity of the bridging oxygen must be a water molecule rather than a hydroxide anion. The different binding behavior between water and hydroxide anions may explain why molecules with a carboxyl group or too many negative charges have lower inhibitory activity. In light of this, the design of high potent active inhibitors against tyrosinase should satisfy both the affinity to the copper ions and the charge neutrality of the entire molecule.


Assuntos
Agaricus/enzimologia , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Agaricus/química , Domínio Catalítico , Cobre/química , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Oxigênio/química , Conformação Proteica , Teoria Quântica , Termodinâmica
20.
Biotech Histochem ; 92(6): 411-416, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28800260

RESUMO

The light subunit of mushroom, Agaricus bisporus, tyrosinase (LSMT), has been identified as an extrinsic component of the enzyme. Its function is unknown, but it can cross an epithelial cell layer, which suggests that it can be absorbed by the intestine. A similar capability has been demonstrated for the HA-33 component of the progenitor toxin from Clostridium botulinum, which is the closest structural homolog of LSMT. Unlike HA-33, LSMT appears to be non-immunogenic as shown by preliminary tests in Swiss Webster mice. We investigated the immunogenicity and histopathology of LSMT in mice to determine its safety in vivo. LSMT did not evoke generation of antibodies after prolonged periods of intraperitoneal administration. Histopathological observations confirmed the absence of responses in organs after twelve weekly administrations of LSMT. We found that LSMT is not toxic and is less immunogenic than the C. botulinum HA-33 protein, which supports further research and development for pharmaceutical application.


Assuntos
Agaricus/enzimologia , Monofenol Mono-Oxigenase/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Imunogenicidade da Vacina , Masculino , Camundongos , Monofenol Mono-Oxigenase/genética , Tamanho do Órgão/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade
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