Effects of Bromelia pinguin (Bromeliaceae) on soil ecosystem function and fungal diversity in the lowland forests of Costa Rica.

BACKGROUND: Bromelia pinguin (Bromeliaceae) is a terrestrial bromeliad commonly found under forest stands throughout the Neotropics that has been shown to have antifungal activity in vitro. We have hypothesized that this bromeliad would also have an effect on the fungal populations in nearby soil by decreasing fungaldiversity and negatively impacting C and N cycle-related activities. A previous study in the lowland forest of Costa Rica showed the soil beneath these bromeliads had decreased fungal ITS DNA and differences in C and N levels compared to adjacent primary forest soils. RESULTS: In this follow-up study, we found that the bromeliad soils had lower rates of C and N biomass development and lower phenol oxidase activity (suggesting less decreased fungal decomposition activity). The results of T-RFLP and cloning-based taxonomic analyses showed the community level diversity and abundance of fungal ITS DNA was less in bromeliad soils. Sequence analysis of fungal ITS DNA clones showed marked differences in fungal community structure between habitats of Basidiomycota (Tremellales, Agricales, Thelephorales), Ascomycota (Helotiales), and Zycomycota populations. CONCLUSIONS: The data show there to be differences in the soil nutrient dynamics and fungal community structure and activity associated with these bromeliads, as compared to the adjacent primary forest. This suggests the possibility that the anti-fungal activity of the bromeliad extends into the soil. The bromeliad-dense regions of these primary forest habitats provide a unique natural micro-habitat within the forests and the opportunity to better identify the role of fungal communities in the C and N cycles in tropical soils.

Estudo da aplicação da tirosinase vegetal no tratamento de efluentes e verificação da genotoxicidade do efluente tratado em células vegetais / Study on tyrosinase application in the treatment of plant effluent and verification of genotoxicity of the treated effluent into plant cells

Os problemas ambientais relacionados à crescente atividade industrial têm gerado preocupações aos órgãos governamentais e entidades de proteção ambientais, sendo necessários estudos de base que busquem novas alternativas para a recuperação de áreas poluídas e a solução de problemas operacionais relacionados com as técnicas empregadas. Um dos compostos mais encontrados em diversos efluentes industriais, principalmente de indústrias bioquímico-farmacêuticas, é o fenol que provoca um impacto danoso no ambiente devido ao fato de ser um poluente tóxico. O presente trabalho propõe, portanto, avaliar a oxidação e destruição do fenol através da utilização da enzima tirosinase extraída de vegetais, cujos resultados podem ser úteis para o tratamento de outros compostos fenólicos como o hormônio 17β-estradiol ou os que se encontram nos efluentes procedentes da produção de azeite ("águas de vegetação") após a recuperação dos polifenóis importantes como antioxidantes. A tirosinase tem a capacidade de transformar fenóis em produtos menos solúveis em água e menos danosos, permitindo assim uma agressão menor ao ambiente. Outro método de remoção do fenol também foi avaliado utilizando queratina extraída de penas de galinha, quitina e quitosana como bioadsorventes. A atividade enzimática foi determinada espectrofotometricamente com soluções de fosfato de potássio e L-tirosina. Para determinar a concentração de fenol aps a oxidação foi utilizada a Cromatografia Líquida de Alta Eficiência (HPLC). Para estudar a adsorção do fenol aplicou-se o método colorimétrico a partir das soluções de tampão borato, 4 aminoantipirina e ferricianeto de potássio e as absorbâncias foram lidas em espectrofotômetro UV-Vis a 546nm, enquanto a determinação de polifenis presentes na "água de vegetação" foi realizada pelo método Folin-Ciocalteu. A quantidade de tirosinase nas batatas das variedades Ágata e Galette di Bologna apresentou-se muito baixa a ponto de modificarmos a matéria prima para...

Environmental problems related to growing industrial activity have generated concerns among government entities and environmental protection, being necessary more baseline studies that seek new alternatives for the recovery of polluted areas and solution of problems related to the operational techniques employed. One of the compounds most commonly found in many industrial effluents, mainly from biochemical and pharmaceutical industries, is phenol, which causes a detrimental impact on the environment due to its toxicity. Therefore, this work proposes the oxidation and destruction of phenol using the enzyme tyrosinase, extracted from plants, whose results could be useful in the future for the treatment of other phenolic compounds such as 17β-estradiol hormone or those found in the effluent coming from the production for olive oil ("vegetation water") after polyphenols recovery. Such an enzyme has the ability of transforming phenols into products less soluble in water and less dangerous, thereby allowing for a minor impact on the environment. Another method of phenol removal was also evaluated using keratin extracted from chicken feathers, chitin and chitosan as phenol biosorbents. Potassium phosphate buffer and L-tyrosine solutions were used for the determination of enzymatic activity, the high performance liquid chromatography (HPLC) for the determination of phenol concentration after oxidation, and a colorimetric method making use of solutions of borate buffer, 4-aminoantipyrine and potassium ferricyanide as well as reading of the absorbance at 546nm to investigate phenol biosorption, while the presence of polyphenols in "vegetation water" was determined by the Folin-Ciocalteu method. The presence of the tyrosinase in potato varieties Agata and Galette di Bologna was shown to be very low, thus suggesting to change the biosorbent material. So, additional tests were done on apples, kiwi, banana and mushroom, but only the last showed a considerable activity...

Production and characterization of tyrosinase activity in Pycnoporus sanguineus CCT-4518 crude extract

Tyrosinase is an enzyme of industrial interest. The production and characterization of tyrosinase from P. sanguineus CCT-4518 were investigated. The selection of inductors, luminosity influence, inoculum size and type of culture medium on the production of tyrosinase and the effect of inhibitors on enzyme activity were performed. Optimum conditions for intracellular tyrosinase production was observed after 2 days using 0.15% L-tyrosine as inducer, in the presence of light, with inoculum size of 10 mycelium discs, using 2% malt extract broth medium, incubated at 30°C, and constant agitation of 150 rpm. Tyrosinase activity was completely inhibited by the addition of 6 mM salicylhydroxamic acid or phenylthiourea, however an inhibition of 4.15% was recorded by the addition of 0.1 mM sodium azide. No inhibition could be detected in case of 0.1 mM phenyl methanesulfonyl fluoride addition. Optimal conditions for intracellular tyrosinase activity using L-dopa as substrate were observed at pH 6.6 and 45°C. Thermal stability studies indicated that the enzyme is stable at 45°C for 15 minutes. Higher temperatures decreased tyrosinase activity. Enzyme production was confirmed by non-denaturing polyacrylamide gel electrophoresis and the protein profile was investigated by denaturing polyacrylamide gel electrophoresis.

Partial purification and characterization of polyphenoloxidase from culinary-medicinal Royal Sun mushroom (the Himematsutake), Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae).

The Royal Sun mushroom, the Himematsutake culinary-medicinal mushroom, Agaricus brasiliensis has several polyphenoloxidase activities in a broad sense. Here we report the partial purification of tyrosinase-type polyphenoloxidase (PPO). PPO is purified from A. brasiliensis without browning using a two-phase partitioning with Triton X-114 and ammonium sulfate fractionation. Partially denaturing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis) staining with L-3,4-dihydroxyphenylalanine was performed and the indicated molecular sizes were approximately 70 kDa and 45 kDa. The purified enzyme is in its latent state and can be activated maximally in the presence of 1.6 mM sodium dodecyl sulfate (SDS). This enzyme catalyzes two distinct reactions, monophenolase and diphenolase activity, and the monophenolase activity showed a lag time typical of polyphenoloxidase. The K(m) value for 4-tert-butylcatechol was quite similar in the presence and absence of SDS, but the apparent V(max) value was increased 2.0-fold by SDS. Mimosine was a typical competitive inhibitor with K(i) values of 138.2 microM and 281.0 microM n the presence and absence of SDS, respectively.

Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus.

Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N-terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18-residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68,993 Da. ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o-diphenols and to exhibit temperature-dependency of o-diphenolase activity like a psychrophilic enzyme.

Aspectos imunologicos do sistema enzimatico fenoloxidase de Schistosoma mansoni / Immunological aspects of the phenol oxidase enzymatic system of Schistosoma mansoni

O sistema enzimatico fenoloxidase (EC1.10.3.1, EC1.10.3.2) esta amplamente distribuido entre os seres vivos, tendo sido descrito em diferentes especies do reino animal e vegetal. Apesar de desempenhar um papel fundamental na formacao da capsula ou parede dos ovos de trematodeos, o sistema enzimatico fenoloxidase (PO) tem sido pouco estudado nesses organismos. No presente trabalho sao apresentados os resultados iniciais de imunizacoes de coelhos contra PO de femeas adultas de S. mansoni e tirosinase de cogumelo (Sigma). As analises imunologicas, realizadas atraves de imunodifusao dupla (teste de Ouchterlony) e imunoeletroforese, revelaram identidade imunitaria parcial entre PO de machos e femeas....

[Isolation and partial characterization of phenoloxidase from apples (Malus domestica, var. Anna)]. / Aislamiento y caracterización parcial de la enzima fenoloxidasa de manzana (Malus domestica, var. Anna).

This study pursued the isolation and partial characterization of the enzyme polyphenoloxidase from apple (Malus domestica Anna variety), grown in the Hermosillo Coast (State of Sonora, Mexico). The effects of pH and temperature as well as its specificity towards substrates, and its behavior under conditions of hydrophobic chromatography, were studied. The enzyme was isolated from a residual powder obtained from ripe apples homogenized with cold acetone. The extract thus prepared was used to characterize the enzyme, and it showed an optimum pH of 5.36 and an optimum temperature of 35 degrees C. The substrate specificity proved to decrease from 4-methyl catechol, chlorogenic acid, catechol, and caffeic acid, to 3,4-dihydroxiphenyl alanine (DOPA). The enzyme resulted to be more thermostable (temperature range: 35 degrees C to 60 degrees C) than the rest of oxidases of plant origin. When the extract was eluted under conditions of hydrophobic chromatography separation, it appeared as a single peak resulting in a 300 fold purification. The phenolase activity characteristics found in the present study were similar to those observed in other apples from temperate climates; however, this particular polyphenoloxidase is more thermostable under natural conditions. This explains why apples of the Anna variety, at the high harvesting temperature, show a very fast formation of brown spots even when there is a minor damage. The content of compounds with phenolic group was high (1.16 g/100 g fresh weight). Further increase of the velocity of fruit enzymatic browning was due to this reason.

Aislamiento y caracterización parcial de la enzima fenoloxidasa de manzana (Malus domestica, var. Anna) / Isolation and partial characterization of phenoloxidase from apple (Malus domestica, var. Anna)

El objetivo de este trabajo fue el aislamiento y la caracterización parcial de la enzima polifenoloxidasa de manzana (Malus domestica Var. Anna), cosechada en la región semidesértica de la Costa de Hermosillo, Sonora, México. Se estudió el efecto que tienen el pH, temperatura, especificidad hacia sustratos y separación bajo condiciones de cromatografía hidrofóbica. La enzima se aisló a partir de manzanas maduras tratadas con acetona fría. Del polvo residual obtenido se extrajo la enzima con regulador de fosfatos, y el extracto se utilizó para realizar caracterización, encontrándose que el pH y temperatura óptimos eran 5.36 y 35§C, respectivamente. La especificidad hacia sustratos mostró ser decreciente desde 4-metil catecol, ácido clorogénico, catecol y ácido cafeico hasta 3.4-dihidroxifenilalanina (DOPA). La enzima resultó ser más termoestable que la generalidad de las oxidasas en el intervalo de temperatura de 35§C a 60§C. El comportamiento del extracto a través de cromatografía hidrofóbica produjo un solo pico con actividad polifenolásica, lográndose una purificación de aproximadamente 300 veces. El contenido de compuestos con grupo fenólico fue de 1.16 g/100 g de fruta fresca. Las características polifenolásicas encontradas se asemejan a las de manzanas de regiones templadas, aunque éstas presentan una mayor termoestabilidad, lo que explica hasta cierto grado la gran influencia que la temperatura ejerce sobre el fenómeno del oscurecimiento enzimático en las condiciones tan cálidas...