Characterization of the biosurfactant production and enzymatic potential of bacteria isolated from an oil-contaminated saline soil / Caracterización de la producción de biosurfactantes y potencial enzimático de bacterias aisladas de un suelo salino contaminado con aceite

Biosurfactants are amphiphilic compounds with extensive applications in oily contaminated environments to remove hydrocarbons. Moreover, enzymes such as laccase and manganese peroxidase are responsible for the oxidation of a variety of phenolic compounds and aromatic amines. Therefore, in the present study, bacteria with the potential to produce biosurfactants and enzymes (namely, laccase, manganese peroxidase, and endoglucanase carboxymethyl cellulose (CMCase)) were isolated from petroleum oil-contaminated soil. From 15 isolated bacteria, three isolates were selected as the best producers of biosurfactants according to the related tests, such as tests for surface tension reduction. These three bacteria indicated tolerance to a salinity test and were classified as resistant and very resistant. The isolates 3, 12, 13, and 14 showed positive results for the degradation of guaiacol, phenol red, and carboxymethylcellulose, as well as the decoloration of methylene blue by the creation of a clear halo around the bacterial colony. Upon the quantitation of the laccase and manganese peroxidase activities, 22.58 U/L and 21.81 U/L, respectively, were measured by isolate 13. Furthermore, CMCase activity was recorded with 0.057436 U/ml belonging to isolate 14. Bacterial strains with appreciable laccase, peroxidase, CMCase activity, and biosurfactant production potentials were identified through 16S rDNA sequence analysis as Bacillus sp. (isolate 3), Bacillus toyonensis (isolate 12), Bacillus cereus (isolate 13), and Bacillus tropicus (isolate 14), and their nucleotide sequences were deposited in the GenBank. The potentials for the industrial applicability of the biosurfactants and enzymes abound, and production needs to be optimized by the selected bacterial strains.(AU)

Extracellular proteases and laccases produced by Pleurotus ostreatus PoB: the effects of proteases on laccase activity / Proteasas y lacasas extracelulares producidas por Pleurotus ostreatus PoB: los efectos de las proteasas en la actividad de lacasa

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)

Modulation of laccase transcriptome during biodegradation of naphthalene by white rot fungus Pleurotus ostreatus

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) using Pleurotus ostreatus was investigated in the current study along with the expression levels of laccase genes involved in biodegradation under variable conditions. Biodegradation of PAHs (naphthalene, anthracene, and 1,10-phenanthroline) was detected spectrophotometrically. Recorded data revealed that biodegradation of the tested PAHs was time dependent. Elevated level of naphthalene biodegradation (86.47%) was observed compared to anthracene (27.87%) and 1,10-phenanthroline (24.51%) within 3 days post incubation. Naphthalene was completely degraded within 5 days. Further incubation enhanced the biodegradation of both anthracene and 1,10-phenanthroline until reaches 93.69% and 92.00% biodegradation of the initial concentration within an incubation period of 11 and 14 days, respectively. Naphthalene was selected as a PAH model. HPLC and thin layer chromatography of naphthalene biodegradation products at time intervals proposed that naphthalene was first degraded to alpha- and ß-naphthol which was further metabolized to salicylic and benzoic acid. The metabolic pathway of naphthalene degradation by this fungus was elucidated based on the detected metabolites. The expression profile of six laccase isomers was evaluated using real-time PCR. The transcriptome of the fungal laccase isomers recorded higher levels of transcription under optimized fermentation conditions especially in presence of both naphthalene and Tween 80. The accumulation of such useful metabolites from the biodegradation of PAH pollutants recommended white rot fungus as a potential candidate for production of platform chemicals from PAH wastes

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Aislamiento y evaluación de la actividad enzimática de hongos descomponedores de madera (Quindío, Colombia) / Isolation of wood-decaying fungi and evaluation of their enzymatic activity (Quindío, Colombia)

Se realizó la colecta de hongos (rdas.) en troncos caídos con diferentes estados de descomposición en un bosque subandino (la reserva natural La Montaña del Ocaso) y se evaluó su actividad ligninolítica. Se cultivaron en Agar extracto de malta y se realizaron pruebas semicuantitativas de actividad lacasa utilizando como inductor enzimático el ácido 2,2’azino-bis-[3-etilbenzotiazolin-6-sulfónico] y el 2,6-diclorofenolindofenol para la celobiosa deshidrogenasa (CDH). Se seleccionaron los hongos con mayor actividad enzimática de troncos con diferente grado de descomposición: Cookeina sulcipes (de estado 1), un hongo de la familia Corticiaceae (de estado 2), Xylaria polymorpha (de estado 3) y Earliella sp. (de estado 4). La fermentación se realizó a 28°C durante 11 días, a 150r.p.m., con mediciones diarias para biomasa, glucosa, actividad lacasa, actividad CDH y proteínas. Los hongos de los troncos con estados de descomposición 1 a 3 presentaron mayor actividad lacasa, a medida que aumentaba el estado de descomposición. Hubo un aumento en la actividad CDH a medida que se incrementó el estado de descomposición de los troncos. Hubo una relación positiva entre la producción de las 2 enzimas. Earliella sp. fue el hongo con mayor producción de biomasa (1.140,19g/l), actividad lacasa (157Ul−1) y CDH (43,50Ul−1). Este trabajo es el primer reporte de actividad lacasa y CDH en C. sulcipes y Earliella sp. Además, sienta las bases para la utilización de estos hongos nativos en aplicaciones biotecnológicas y se adentra en el conocimiento de su función dentro del proceso de descomposición de la madera en bosques(AU)

White rotfungi(AscomycotaandBasidiomycota)werecollectedonfallentrunkswithdifferentdecaystages, inasubandeanforest(LaMontan˜a delOcasonaturereserve),anditwasevaluatedtheirligninoliticactivity.Theywereculturedonmaltextractagar.Thenitwasperformedsemiquantitativetestsforlaccaseand cellobiosedehydrogenase(CDH)activityusingABTSandDCPIPasenzymaticinducers.Basedontheresults ofthesetests,thefungiwithhigheractivitiesfromtrunkswithdifferentdecaystageswereselected:Cookeina sulcipes (for stage1),afungusfromthefamilyCorticiaceae(forstage2), Xylariapolymorpha (forstage 3)and Earliella sp. (forstage4).Afermentationwasperformedat28 1C, during11days,inarotatoryshaker at150rpm.Biomass,glucose,proteinsandenzymeactivitiesmeasurementswereperformeddaily.The fungithatwereinthetrunkswithdecaystatesfrom1to3,showedhigherlaccaseactivityasthestateof decayincreased.AhigherDCHactivitywasalsoassociatedwithahigher.Also,therewasapositiverelationship betweenbothenzymes’activities.Erliellawasthefunguswhichpresentedthehighestbiomass production(1140,19g/l),laccaseactivity(157UL 1) andCDHactivity(43,50UL 1). Thisworkisthefirst reportoflaccaseandCDHactivityfor Cookeina sulcipes and Earliella sp. Moreover,itgivesbasisforthe useofthesenativefungiinbiotechnologicalapplicationsandtheacknowledgmentoftheirfunctioninthe wooddecayprocessinnativeforest

Detoxification of azo dyes by a novel pH-versatile, salt-resistant laccase from Streptomyces ipomoea

A newly identified extracellular laccase produced by Streptomyces ipomoea CECT 3341 (SilA) was cloned and overexpressed, and its physicochemical characteristics assessed together with its capability to decolorize and detoxify an azotype dye. Molecular analysis of the deduced sequence revealed that SilA contains a TAT-type signal peptide at the N-terminus and only two cupredoxine domains; this is consistent with reports describing two other Streptomyces laccases but contrasts with most laccases, which contain three cupredoxine domains. The heterologous expression and purification of SilA revealed that the homodimer is the only active form of the enzyme. Its stability at high pH and temperature, together with its resistance to high concentrations of NaCl and to typical laccase inhibitors such as sodium azide confirmed the unique properties of this novel laccase. The range of substrates that SilA is able to oxidize was found to be pH-dependent; at alkaline pH, SilA oxidized a wide range of phenolic compounds, including the syringyl and guayacil moieties derived from lignin. The oxidative potential of this enzyme to use phenolic compounds as natural redox mediators was shown through the coordinated action of SilA and acetosyringone (as mediator), which resulted in the complete detoxification of the azo-type dye Orange II (AU)

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Production of two novel laccase isoforms by a thermotolerant strain of Pycnoporus sanguineus isolated from an oil-polluted tropical habitat

A thermotolerant and halotolerant strain of Pycnoporus sanguineus was isolated from an oil-polluted site in a tropical area located in Veracruz, Mexico. This strain was able to grow at 47 degrees C and in culture medium containing 500 mM NaCl. The strain was also tolerant to the presence of 30,000 ppm of crude Maya oil. A 68-kDa protein purified from submerged cultures exhibited laccase activity towards 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), guaiacol, syringaldazine, and o-dianisidine, for which it presented the highest affinity (Km = 43 microM). Two-dimensional gel electrophoresis analysis showed that, unusual for laccases, the enzyme has two active isoforms, with isoelectric points of 7.00 and 7.08. The purified enzyme showed high thermostability, retaining 40% of its original activity after 3 h at 60 degrees C. This property seems to correlate with a long «shelf-life», given that at 40 degrees C enzyme activity was only gradually lost over a 5-day period incubation. Both the fungus and its laccase are likely to have high potential for biotechnological applications (AU)

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Laccase activity in Cryptococcus gattii strains isolated from goats

Cryptococcus neoformans y Cryptococcus gattii son los principales agentesde criptococosis, una grave micosis del hombre y los animales. Entre losfactores de patogenicidad de estas especies cabe destacar la lacasa(fenoloxidasa), enzima producida por éstas y otras especies fúngicas, queinduce la síntesis de melanina a partir de compuestos di-hidroxifenólicos.La gran mayoría de los numerosos estudios sobre la lacasa se han efectuadocon C. neoformans, y la información específica en C. gattii es muy escasa.El objetivo de este estudio ha sido evaluar la actividad de la lacasa enaislamientos de C. gattii serotipo B procedentes de cabrasinmunocompetentes muertas por criptococosis durante varios brotesepidémicos desarrollados en Cáceres (Extremadura, España).La producción de lacasa de estos aislamientos se ha comparado con la deotros de la misma especie y también con cepas de C. neoformans.Se procedió a la ruptura de las levaduras por métodos físicos, y elsobrenadante de cada aislamiento se añadió a una solución 20 mM de L-dopa. La actividad enzimática se midió a través de la absorbancia como unidadesenzimáticas (EU) a 450 nm. Los valores máximos de EU se observaron en trescepas de C. gattii aisladas de cabras (EU >12), mientras que el valor mas bajose observó en una cepa ambiental de C. gattii serotipo C (EU = 0,7).Para C. neoformans la mayor actividad lacasa se obtuvo en una cepa delserotipo A aislada en un paciente con meningitis criptocócica.Todos los aislamientos de C. gattii procedentes de los animales muertos enbrotes epidémicos mostraron diferentes grados de actividad lacasa.Esta enzima parece representar un factor de patogenicidad importante,aunque no exclusivo, en esta especie(au)

Cryptococcosis is a life-threatening infection in humans and animals causedby encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformansand Cryptococcus gattii are the main agents of this mycosis Until 2002C. gattii was classified as a variety of C. neoformans but now is accepted asan independent species. The laccase (phenoloxydase) enzyme produced bythese yeasts is considered one of the main pathogenic factors for its ability toinduce melanin from dihydroxyphenolic compounds. The vast majority of thestudies in laccase and melanin synthesis have been developed using isolatesof C. neoformans. The main objective of this study was to evaluate laccaseactivity in strains of C. gattii, serotype B isolated from immunocompetentgoats that died of lung and disseminated cryptococcosis, in several outbreaksoccurring in Spain. The laccase activities of these isolates were compared withthose of other strains of C. gattii and C. neoformans. After fungal cell rupture,the supernatant of each isolate was analyzed for its laccase activity using assubstrate an L-dopa 20 mM solution. The degree of enzymatic activity wasassessed according to its absorbance at 450 nm and scored using EnzymaticUnits (EU). The maximum values were observed in three strains of C. gattiifrom goats (EU > 12). The smallest values were observed in one environmentalisolate of C. gattii serotype C (EU = 0.7). The highest recorded value forC. neoformans was 6.3 EU in a serotype A isolate from one human case ofmeningitis. C. gattii serotype B obtained from goats showed different degreesof laccase activity, being the highest in those isolated from severe outbreaksof cryptococcosis. This enzyme appears to represent a major, thoughnonexclusive, pathogenic factor for Cryptococcus gattii(AU)

Influence of nutrients on enhancing laccase production by Botryosphaeria rhodina MAMB-05

The physiological requirements needed to enhance the production of laccases by the ascomycete Botryosphaeria rhodina MAMB-05 in submerged cultivation were examined under non-induced and induced (veratryl alcohol, VA) conditions. Under non-induced conditions (-VA), the initial pH, C:N ratio, and inorganic N source did not influence laccase production, in contrast to Tween 80, soybean oil, and copper, which significantly increased laccase production, and proline and urea, which suppressed laccase formation. In addition, Tween 60 could serve as the sole carbon source for the production of these enzymes. Under VA-induced conditions of fungal growth, factors such as inoculum type, time-point of addition of inducer, initial pH, C:N ratio, and type of N source, influenced the production of laccases; however, unlike the non-induced conditions, proline and urea did not act as suppressors. Each of these physiological conditions exerted different effects on biomass production. The nutritional conditions examined for B. rhodina MAMB-05 are discussed in relation to their influence on fungal growth and laccase production (AU)

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Acción de Stereum hirsutum (Wild) Pers. en la degradación de colorantes / Stereum hirsutum (Wild) Pers. action in dye degradation

Stereum hirsutum, un basidiomicete responsable de la pudrición blanca de lamadera, mostró un buen desarrollo durante un proceso de fermentación enestado sólido. Esta se realizó en salvado de trigo, salvado de soja o en unamezcla de ambos. En salvado de soja presentó la mayor actividad decolorantesobre xilidina (Ponceau 2R ), índigo-carmín y verde de malaquita. Seobtuvieron valores óptimos de decoloración y detoxificación con una relaciónde 30 g de peso fresco (sustrato+hongo) en 500 ml de solución de verde demalaquita, con una actividad lacasa de 42 U/l. El proceso de decoloraciónse llevó a cabo sin el agregado de nutrientes ni de mediadores. Los máximosde decoloración no fueron coincidentes con las máximas actividadesenzimáticas, pero la acción oxidante de la lacasa producida por S. hirsutumsobre los colorantes quedó confirmada con el análisis electroforético, quepermitió relacionar la actividad de dicha enzima y su acción decolorante,utilizando geles teñidos con índigo-carmín y con verde de malaquita, conABTS (2,2’-azino-bis-3-etil-benzotiazolin-6-ácido sulfónico) como mediador.La detoxificación se estableció en base al crecimiento de Phanerochaetechrysosporium, un hongo sensible al verde de malaquita(AU)

Stereum hirsutum, a white rot fungus, has a good growth in solid statefermentation. This was carried on with wheat bran, soy bran and a mixture ofboth. Mycelia grown on soy bran showed the highest decolorization activity onPonceau 2R (xylidine), indigo carmine and malachite green. Optimalrelationship between decolorization and detoxification of malachite green was30 g of fresh weight (mycelia plus substrate) in 500 ml malachite greensolution, 42 U/l of laccase was measured in this solution. Decolorization wascarried on without the addition either of nutrients or mediators. Conditions formaximal decolorization did not agree with those for maximal ligninolyticenzyme production, but effectiveness of laccase activity on decolorization wasevidenced by electrophoretic analysis, that allowed laccase identification andits decolorization activity in gels stained with indigo carmine and malachitegreen, with ABTS as mediator. Detoxification was assayed using the sensiblefungus Phanerochaete chrysosporium(AU)