Identification and characterization of laccase-type multicopper oxidases involved in dye-decolorization by the fungus Leptosphaerulina sp.

13 p.-4 fig.-4 tab.[Background] Fungal laccases are multicopper oxidases (MCOs) with high biotechnological potential due to their capability to oxidize a wide range of aromatic contaminants using oxygen from the air. Albeit the numerous laccase-like genes described in ascomycete fungi, ascomycete laccases have been less thoroughly studied than white-rot basidiomycetous laccases. A variety of MCO genes has recently been discovered in plant pathogenic ascomycete fungi, however little is known about the presence and function of laccases in these fungi or their potential use as biocatalysts. We aim here to identify the laccase-type oxidoreductases that might be involved in the decolorization of dyes by Leptosphaerulina sp. and to characterize them as potential biotechnological tools.[Results] A Leptosphaerulina fungal strain, isolated from lignocellulosic material in Colombia, produces laccase as the main ligninolytic oxidoreductase activity during decolorization of synthetic organic dyes. Four laccase-type MCO genes were partially amplified from the genomic DNA using degenerate primers based on laccase-specific signature sequences. The phylogenetic analysis showed the clustering of Lac1, Lac4 and Lac3 with ascomycete laccases, whereas Lac2 grouped with fungal ferroxidases (together with other hypothetical laccases). Lac3, the main laccase produced by Leptosphaerulina sp. in dye decolorizing and laccase-induced cultures (according to the shotgun analysis of both secretomes) was purified and characterized in this study. It is a sensu-stricto laccase able to decolorize synthetic organic dyes with high efficiency particularly in the presence of natural mediator compounds.[Conclusions] The searching for laccase-type MCOs in ascomycetous families where their presence is poorly known, might provide a source of biocatalysts with potential biotechnological interest and shed light on their role in the fungus. The information provided by the use of genomic and proteomic tools must be combined with the biochemical evaluation of the enzyme to prove its catalytic activity and applicability potential.This research was supported by the Program for Interuniversity Cooperation and Scientific Reasearch (PCI) from the Spanish Agency for International Cooperation and Development (AECID), Project AP/033932/11, and the Spanish Project NOESIS BIO2014-56388-R.Peer reviewe

Elimination of Isoxazolyl-Penicillins antibiotics in waters by the ligninolytic native Colombian strain Leptosphaerulina sp. considerations on biodegradation process and antimicrobial activity removal

In this work, Leptosphaerulina sp. (a Colombian native fungus) significantly removed three Isoxazolyl-Penicillin antibiotics (IP): oxacillin (OXA, 16000 µg L-1), cloxacillin (CLX, 17500 µg L-1) and dicloxacillin (DCX, 19000 µg L-1) from water. The biological treatment was performed at pH 5.6, 28 °C, and 160 rpm for 15 days. The biotransformation proccess and lack of toxicity of the final solutions (antibacterial activity (AA) and cytotoxicity) were tested. The role of enzymes in IP removal was analysed through in vitro studies with enzymatic extracts (crude and pre-purified) from Leptosphaerulina sp., commercial enzymes and enzymatic inhibitors. Futhermore, the applicabililty of mycoremediation process to a complex matrix (simulated hospital wastewater) was evaluated. IP were considerably abated by the fungus, OXA was the fastest degraded (day 6), followed by CLX (day 7) and DCX (day 8). Antibiotics biodegradation was associated to laccase and versatile peroxidase action. Assays using commercial enzymes (i.e. laccase from Trametes versicolor and horseradish peroxidase) and inhibitors (EDTA, NaCl, sodium acetate, manganese (II) ions) confirmed the significant role of enzymatic transformation. Whereas, biomass sorption was not an important process in the antibiotics elimination. Evaluation of AA against Staphylococcus aureus ATCC 6538 revealed that Leptosphaerulina sp. also eliminated the AA. In addition, the cytotoxicity assay (MTT) on the HepG2 cell line demonstrated that the IP final solutions were non-toxic. Finally, Leptosphaerulina sp. eliminated OXA and its AA from synthetic hospital wastewater at 6 days. All these results evidenced the potential of Leptosphaerulina sp. mycoremediation as a novel environmentally friendly process for the removal of IP from aqueous systems

Decolorization of Reactive Black 5 Dye by Heterogeneous Photocatalysis with TiO2/UV

Reactive Black 5 (RB5) is an azo dye widely used in the textile industry because of its high chemical stability. Since it does not entirely fix on the fabrics, it pollutes water sources. In this work, the decolorization of aqueous solutions with RB5 was performed by heterogeneous photocatalysis with TiO2/UV. The reaction was carried out in an aluminum photoreactor equipped with five lamps. The effect of TiO2 (0.1, 0.175, and 0.25 g L-1), RB5 concentration (50, 75, and 100 mg L-1), and pH (3, 7, and 11) was evaluated for 14 h, using a Box–Behnken experimental statistical design. Complete decolorization of RB5 was obtained at 14 h, employing 0.175 g L-1 TiO2, 50 mg L-1 RB5, and pH 3. A 98.44% of decolorization was achieved in 10 h (0.25 g L-1 of TiO2, 50 mg L-1 of RB5, and pH 7). The highest decolorization percentage of RB5 (99.51%) was obtained at 10 h of exposure to UV light, using 0.5 g L-1 of TiO2, 50 mg L-1 of the dye, and a pH of 3. Cytotoxicity tests on the HepG2 cell line indicated that photocatalytic degradation of RB5 did not generate cytotoxic byproducts.El Negro Remazol B (NRB) es un colorante azoico, usado en la industria textil por su estabilidad química. Este tinte no se fija al 100%, ocasionando contaminación en el agua. En este trabajo se evaluó la decoloración de soluciones acuosas con NRB mediante fotocatálisis heterogénea con TiO2/UV. La reacción se realizó en un fotorreactor de aluminio equipado con cinco lámparas. El efecto de la concentración de TiO2 (0,1; 0,175 y 0,25 g L-1), y NRB (50, 75 y 100 mg L-1) y el pH (3, 7 y 11) fue evaluado durante 14 h, tomando un diseño estadístico experimental Box-Behnken. La decoloración completa del NRB se obtuvo después de 14 h (0,175 g L-1 de TiO2, 50 mg L-1 de NRB, y pH 3). Se alcanzó un 98,44% de decoloración después de 10 h (0,25 g L-1 de TiO2, 50 mg L-1 de NRB, y pH 7). Se encontró que el mejor porcentaje de decoloración del NRB (99,51%) se obtuvo a las 10 h de exposición a luz UV, utilizando 0,5 g L-1 de TiO2, 50 mg L-1 del colorante y un pH de 3. Los ensayos de citotoxicidad sobre la línea celular HepG2 indicaron que la degradación fotocatalítica del NRB no generó subproductos citotóxicos.O Negro Remazol B (RNB) é um corante azoico usado na indústria têxtil pela sua estabilidade química. O corante não é fixo no tecido em 100%, então contamina as fontes de água. Neste trabalho, avaliou-se a descoloração de soluções aquosas com RNB por meio de fotocatálise heterogênea com TiO2/UV. A reação foi feita no fotoreactor de alumínio com cinco lâmpadas. O efeito da concentração de TiO2 (0,1; 0,175 e 0,25 g L-1), RNB (50, 75 e 100 mg L-1) e pH (3, 7 e 11) foi avaliado durante 14 h utilizando o modelo estatístico Box-Behnken. Ás 14 h, foi atingida a descoloração completa do RNB (pH 3; 0,175 g L-1 de TiO2 e 50 mg L-1 de RNB). Utilizando 0,25 g L-1 de TiO2, 50 mg L-1 de RNB e pH 7, obteve-se descoloração de 98,44% em 10 h. Encontrou-se que a melhor porcentagem de descoloração do RNB (91,51%) foi obtida com 10 h de exposição à luz UV usando 0,5 g L-1 de TiO2, 50 mg L-1 do corante e pH de 3. Adicionalmente, os testes de citotoxicidade na linha celular HepG2 mostraram que a degradação fotocatalítica do RNB não gerou subprodutos citotóxicos

Decolorization of Reactive Black 5 Dye by Heterogeneous Photocatalysis with TiO2/UV

Reactive Black 5 (RB5) is an azo dye widely used in the textile industry because of its high chemical stability. Since it does not entirely fix on the fabrics, it pollutes water sources. In this work, the decolorization of aqueous solutions with RB5 was performed by heterogeneous photocatalysis with TiO2/UV. The reaction was carried out in an aluminum photoreactor equipped with five lamps. The effect of TiO2 (0.1, 0.175, and 0.25 g L-1), RB5 concentration (50, 75, and 100 mg L-1), and pH (3, 7, and 11) was evaluated for 14 h, using a Box–Behnken experimental statistical design. Complete decolorization of RB5 was obtained at 14 h, employing 0.175 g L-1 TiO2, 50 mg L-1 RB5, and pH 3. A 98.44% of decolorization was achieved in 10 h (0.25 g L-1 of TiO2, 50 mg L-1 of RB5, and pH 7). The highest decolorization percentage of RB5 (99.51%) was obtained at 10 h of exposure to UV light, using 0.5 g L-1 of TiO2, 50 mg L-1 of the dye, and a pH of 3. Cytotoxicity tests on the HepG2 cell line indicated that photocatalytic degradation of RB5 did not generate cytotoxic byproducts