Trametes versicolor (L.) Lloyd as a source of thermostable serine protease: production and characterization

Proteases are ubiquitously present and are among the largest groups of commercially important enzymes. Here, we investigated a wood-rot basidiomycete Trametes versicolor (L.) Lloyd [Syn. Coriolus versicolor (L.) Quél.; Polyporus versicolor (L.) Fr.] as a source of the enzyme serine protease, its production, and optimized to obtain a higher yield of the enzyme.. The significant variables with optimized values for maximum production of the enzyme were temperature (30?C), incubation time (120 h) and wheat bran (10 g). The yield increased by 30.76% by statistically optimizing the media. The optimized temperature and pH for the maximum protease activity was 50?C and pH 7.0, respectively. The enzyme was purified through ion exchange (using DEAE cellulose 52 resin) and gel filtration chromatography (using Superdex 200 column). The purified enzyme had a retention time of 7 min in RP-HPLC. The enzyme was stable at a broad range of temperature (30-60?C) and pH (5.0-8.0) with a half-life of 58.72 min, Vmax of 37.17 ?M min/mL and Km of 0.657 mg/mL. Its activity was enhanced by Na+, Ca2+, Mg2+ ions and SDS surfactant. These properties make this enzyme a valuable candidate for industrial applications

Expression and characterization of NADPH-cytochrome P450 reductase from Trametes versicolor in Escherichia coli / 生物工程学报

Trametes versicolor has strong ability to degrade environmental organic pollutants. NADPH-cytochrome P450 reductase (CPR) of T. versicolor transfers electron to cytochrome P450s (CYPs) and participates in the degradation process of organic pollutants. Sequence analysis showed that the genome of T. versicolor contains 1 potential CPR and multiple potential CYP sequences. To further study the molecular mechanism for the involvement of T. versicolor CPR in the cellular degradation of organic pollutants, a CPR gene from T. versicolor was cloned and heterologously expressed in Escherichia coli. Subsequently, the main properties of the recombinant enzyme were investigated. A truncated CPR protein lacking the predicted membrane anchor region (residues 1-24), named CPRΔ24, was overexpressed as a soluble form in E. coli. The recombinant CPRΔ24 protein showed a molecular weight consistent with the theoretical value of 78 kDa. Recombinant CPRΔ24 was purified using a Ni²⁺-chelating column followed by size exclusion chromatography. The specific activity of the purified CPRΔ24 was 5.82 U/mg. The CPRΔ24 enzyme displayed the maximum activity at 35 ℃ and pH 8.0. It has different degrees of tolerance against several types of metal ions and organic solvents. The apparent Km and kcat values of recombinant CPRΔ24 for NADPH were 19.7 μmol/L and 3.31/s, respectively, and those for the substrate cytochrome c were 25.9 μmol/L and 10.2/s, respectively, under conditions of 35 ℃ and pH 8.0. The above research provides the basis for exploring the functional mechanism of T. versicolor CPR in the degradation pathway of environmental organic pollutants.

Natural decomposition of hornbeam wood decayed by the white rot fungus Trametes versicolor

ABSTRACT The impacts of white-rot fungi on altering wood chemistry have been studied mostly in vitro. However, in vivo approaches may enable better assessment of the nature of interactions between saprotrophic fungi and host tree in nature. Hence, decayed and sound wood samples were collected from a naturally infected tree (Carpinus betulus L.). Fruiting bodies of the white rot fungus Trametes versicolor grown on the same tree were identified using rDNA ITS sequencing. Chemical compositions (cellulose and lignin) of both sound and infected wood were studied. FT-IR spectroscopy was used to collect spectra of decayed and un-decayed wood samples. The results of chemical compositions indicated that T. versicolor reduced cellulose and lignin in similar quantities. Fungal activities in decayed wood causes serious decline in pH content. The amount of alcohol-benzene soluble extractives was severely decreased, while a remarkable increase was found in 1% sodium hydroxide soluble and hot water extractive contents in the decayed wood samples, respectively. FT-IR analyses demonstrated that T. versicolor causes simultaneous white rot in the hornbeam tree in vivo which is in line with in vitro experiments.

Estudo da descoloração do corante FD&C azul no 2 Indigotina pelo tratamento combinado do fungo Trametes versicolor e processo de filtração lenta / Study of decolorization of FD&C blue # 2 indigotine by fungus Trametes versicolor combined with slow sand filtration

O uso de fungos na descoloração de corantes com métodos economicamente viáveis de produção de água bacteriologicamente segura há muito vem sendo descrito por diversos autores. Este trabalho teve por objetivo investigar a eficiência da remoção de corante artificial FD&C azul no 2 Indigotina, com uso do fungo de degradação branca Trametes versicolor em combinação com a filtração lenta. Para a realização dos trabalhos, foram instalados dois protótipos de filtros lentos denominados FL-A e FL-B - no sobrenadante do filtro FL-A foi inoculado o referido fungo, e o filtro FL-B foi utilizado como controle (sem inoculação do microrganismo). O melhor percentual de remoção do corante pelo fungo Trametes versicolor em combinação com a filtração lenta foi de 44,74% 24 horas após a atividade máxima registrada de lacase. Os resultados mostraram que a filtração lenta combinada com o tratamento com o fungo T. versicolor não apresenta grande potencial para remoção de cor em 21 dias de tratamento, visto que os produtos microbianos gerados interferem no processo de filtração, diminuindo a eficiência do processo físico. Entretanto, restringindo o tempo de tratamento a 24 horas após a atividade enzimática máxima, o tratamento combinado apresentou boa eficiência.

The use of fungi in the decolorization of dyes with economically viable methods of producing bacteriologically safe water has long been described by several authors. This study aimed to investigate the removal efficiency of artificial coloring FD&C Blue no 2 Indigo, using the degradation white fungus Trametes versicolor in combination with slow sand filtration. Two prototype filters slowly termed FL-A and FL-B were installed - the supernatant water of filter FL-A was inoculated with the fungus, while filter FL-B was used as control. The best percentage of dye removal by the fungus Trametes versicolor in combination with the slow sand filtration was 44.74% achieved 24 hours after the maximum laccase activity. The results show that the combination of the fungus T. versicolor with slow sand filtration treatment presents no great potential for color removal at 21 days of treatment, whereas microbial products generated interfere with the filtration process, lowering the efficiency of the physical process. However, with the restriction of the handling time into 24 hours after the maximum enzymatic activity, combined treatment showed good efficiency.

Biosorption kinetics of a direct azo dye Sirius Blue K-CFN by Trametes versicolor

In this study, lyophilized Trametes versicolor biomass is used as a sorbent for biosorption of a textile dye, Sirius Blue K-CFN, from an aqueous solution. The batch sorption was studied with respect to dye concentration, adsorbent dose and equilibrium time. The effect of pH and temperature on dye uptake was also investigated and kinetic parameters were determined. Optimal initial pH (3.0), equilibrium time (2 hrs), initial dye concentration ( 100 mg l-1) and biomass concentration (1.2 mg l-1) were determined at 26ºC. The maximum biosorption capacity (q max) of Sirius Blue K-CFN dye on lyophilized T. versicolor biomass is 62.62 mg/g. The kinetic and isotherm studies indicated that the biosorption process obeys to a pseudo-second order model and Langmuir isotherm model. In addition, the biosorption capacities of fungal biomass compared to other well known adsorbents such as activated carbon and Amberlite, fungal biomass biosorptions capacities were found to be more efficient.

Inmovilización de hongos ligninolíticos para la remoción del colorante negro reactivo 5 / Immobilising lignilolytic fungus for removing reactive black 5 dye

La presencia de colorantes azoicos en aguas residuales de la industria textil es un problema ambiental y sanitario, porque muchos de estos compuestos son cancerígenos. Los tratamientos biológicos son una alternativa para la remoción de ese tipo de colorantes. En el presente trabajo se evaluó el efecto de tres hongos de podredumbre blanca, Trametes versicolor, Pleurotus ostreatus y Phanerochaete chrysosporium sobre la decoloración de un agua que contiene colorante negro reactivo 5 (NR5), ampliamente usado en la industria textil. Se estudió la inmovilización de estos hongos en dos soportes, espuma de poliuretano y estropajo (L. cylíndrica) para seleccionar el mejor soporte y el hongo con mayor capacidad para la decoloración. Ambos soportes fueron igualmente efectivos, pero se seleccionó estropajo por ser un producto natural. El hongo que generó los mayores porcentajes de decoloración en 4 días fue Trametes versicolor, con 96%, 98% y 98% para agua con concentración de NR5 300 ppm, 150 ppm y 75 ppm, respectivamente. La actividad lacasa para cada concentración de NR5 fue 8 U L-1, 7 U L-1 y 5 U L-1.

Waste water from the textile industry represents a major environmental and health problem because it contains azo dyes whose carcirogenic effect has been tested in research. Biological treatment represents a valuable alternative for removing these dyes. The effect of Trametes versicolor, Pleurotus ostreatus and Phanerochaete chrysosporium rot fungi on decoloration of water containing reactive black five (NR5) textile dye was evaluated in this work. Immobilising the fungi on polyurethane foam and luffa sponge (Luffa cylindrica) supports was studied in order to select the best support and the fungi having the best decolorisation. Both supports were equally effective; however, the luffa sponge was selected as being a natural product. Trametes versicolor produced the highest decolorisation percentages in four days (96%, 98% and 98% for 300 ppm, 150 ppm and 75 ppm NR5 concentrations, respectively) while lacase enzyme activity was 8 UL-1, 7 UL-1 and 5 UL-1 for each of them.

Trametes versicolor growth and laccase induction with by-products of pulp and paper industry

The cultivation of Trametes versicolor for laccase production and cell growth were strongly dependent on experimental conditions namely physical and chemical parameters as well as nutrient availability and inducer stimulation. Biomass growth was compared for a rich medium and for a defined medium in two different temperatures. The best temperature was 28ºC and the maximum specific growth rates were µmax = 0.083 h-1 for the rich medium and µmax = 0.043 h-1 for the defined medium. It was clearly shown that laccase production is not associated with cell growth, indicating that this ligninolytic enzyme must be produced in the defined medium by a secondary metabolism. In order to obtain laccase induction, addition of solid lignin, lignosulphonates, veratryl alcohol, xylidine and ethanol was tested at different concentrations. To optimise laccase activity, the combined effect of inducer addition and simultaneously glucose suppression was studied. The best result for laccase induction (1240 U/L) was obtained with solid lignin, a by-product of pulp and paper industry and the higher laccase activity attained (1583 U/L) was obtained with the combined effect of xylidine addition and glucose suppression.