Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest ...due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.
Fungal laccases Rivera-Hoyos, Claudia M.; Morales-Álvarez, Edwin David; Poutou-Piñales, Raúl A. ...
Fungal biology reviews,
December 2013, 2013-12-00, 20131201, Letnik:
27, Številka:
3-4
Journal Article
Recenzirano
Laccases are enzymes widely distributed in plants, fungi, bacteria, and insects. They are multicopper oxidases that catalyze the transformation of aromatic and non-aromatic compounds with reduction ...of molecular oxygen to water. These enzymes participate in processes such as biosynthesis and lignin degradation, morphogenesis, and pigment biosynthesis, among others. In this review we discuss relevant aspects of fungal laccases regarding the existence of fungal laccases gene families, the growing interest in investigating mechanisms of their molecular regulation, and factors that influence the production of laccases, due to their potential biotechnological applications. In addition we comparatively analyzed some structural similarities and differences depicting general features of laccases' active site, demonstrating their frequency as monomeric proteins with highly conserved cupredoxine type domains. Although inter- and intra-specific differences have been determined, structural differences encountered between fungal laccases remain unclear based on Crystallography and X-ray diffraction.
•Exist several lacasses genes as a consequence of physiological function diversity.•Crystallography or X-ray resolutions influence the structural differences encountered.•Not all recombinant lacasses combines attributes to promote production processes.•There are intra-specific differences, among laccases of the same fungus.•We discuss the diversity of fungal laccases with crystallography structures.
An efficient sequential, biological and photocatalytic treatment to reduce the pollutant levels in wastewater due to the bleaching process during paper production is reported. For a biological ...pre-treatment, 800 ml of non-sterilized effluent was inoculated with Trametes versicolor immobilized in polyurethane foam, with 25 g l(-1) glucose, 6.75 mM CuSO(4), and 0.22 mM MnSO(4) added, and cultured at 25 degrees C with an air flow of 800 ml min(-1) for 8d. The fungus did not inhibit growth of the heterotropic populations of the effluent. After 4d of culture, the chemical oxygen demand (COD) reduction and colour removal (CR) were 82% and 80%, respectively, with laccase (LAC) and manganese peroxidase (MnP) activities of 345 U l(-1) and 78 U l(-1), respectively. The COD reduction and CR correlated positively (p<0.0001) with LAC and MnP activities. Chlorophenol removal was 99% of pentachlorophenol, 99% of 2,3,4,6-tetrachlorophenol (2,3,4,6-TCP), 98% of 3,4-dichlorophenol (3,4-DCP) and 77% of 4-chlorophenol (4-CP), while 2,4,5-trichlorophenol (2,4,5-TCP) increased to 0.2 mg l(-1). The pre-treated effluent was then exposed to a photocatalytic treatment. The treatment with photolysis resulted in 9% CR and 46% COD reduction, 42% CR and 60% COD reduction by photocatalysis, and 62% CR and 85% COD reduction by heterogeneous photocatalysis with the system TiO(2)/Ru(x)Se(y) (Fig. 4). With this treatment the bacterial and fungal populations also decreased by 5 logarithmic units with respect to the biological treatment alone (Fig. 5). The total sequential treatment resulted in a 92% CR (from 5800 UC), 97% COD reduction (from 59 g l(-1)) and 99% chlorophenol removal at 96 h and 20 min.
An efficient sequential, biological and photocatalytic treatment to reduce the pollutant levels in wastewater due to the bleaching process during paper production is reported. For a biological ...pre-treatment, 800
ml of non-sterilized effluent was inoculated with
Trametes versicolor immobilized in polyurethane foam, with 25
g
l
−1 glucose, 6.75
mM CuSO
4, and 0.22
mM MnSO
4 added, and cultured at 25
°C with an air flow of 800
ml
min
−1 for 8
d. The fungus did not inhibit growth of the heterotropic populations of the effluent. After 4
d of culture, the chemical oxygen demand (COD) reduction and colour removal (CR) were 82% and 80%, respectively, with laccase (LAC) and manganese peroxidase (MnP) activities of 345
U
l
−1 and 78
U
l
−1, respectively. The COD reduction and CR correlated positively (
p
<
0.0001) with LAC and MnP activities. Chlorophenol removal was 99% of pentachlorophenol, 99% of 2,3,4,6-tetrachlorophenol (2,3,4,6-TCP), 98% of 3,4-dichlorophenol (3,4-DCP) and 77% of 4-chlorophenol (4-CP), while 2,4,5-trichlorophenol (2,4,5-TCP) increased to 0.2
mg
l
−1. The pre-treated effluent was then exposed to a photocatalytic treatment. The treatment with photolysis resulted in 9% CR and 46% COD reduction, 42% CR and 60% COD reduction by photocatalysis, and 62% CR and 85% COD reduction by heterogeneous photocatalysis with the system TiO
2/Ru
x
Se
y
(Fig. 4). With this treatment the bacterial and fungal populations also decreased by 5 logarithmic units with respect to the biological treatment alone (Fig. 5). The total sequential treatment resulted in a 92% CR (from 5800 UC), 97% COD reduction (from 59
g
l
−1) and 99% chlorophenol removal at 96
h and 20
min.
Objective. To isolate biosurfactant-producing microorganisms from the rhizosphere of fique and to select the best genus to evaluate the effect of nutritional and fermentation conditions on the ...production of rhamnolipids. Materials and methods. Rhizospheric soil was sampled in three areas of Cauca. The best genus was selected for the experimental designs (Plackett Burman and 22 factorial) and to find the production conditions for the growth kinetics at an Erlenmeyer flask scale. Results. Isolates from the rhizosphere of fique plants were from groups (or genera) of Bacillus, Pseudomonas and Streptomyces, being Pseudomonas the more responsive in preliminary testing for emulsification. From the results of the experimental designs and the kinetics of production, we found that rhamnose synthesis associated with rhamnolipids (3.2 g/l) and emulsification (68% EC24) was significantly favored (p <0.0001) by cultivating an inoculum of 10% v/v of Pseudomonas fluorescens in a medium composed of: soybean oil 2% (v/v), K2HPO4 0.2% (w/v), yeast extract 0.4 g/l, NH4NO3 3.7 g/l, 1 ml trace elements (CoCl3 20 mg/l, H3BO3 30 mg/l, ZnSO4 10 mg/l, Cu2SO4 1 mg/l, Na2MoO4 3 mg/l, FeSO4 10 mg/l MnSO4 2,6 mg/l) and pH 7.2. Conclusion. Of all the microbial genera isolated from the rhizosphere of fique, Pseudomonas fluorescens had the greatest potential in the production of biosurfactants of the rhamnolipids family. Key words: Pseudomonas fluorescens, biosurfactant, rhamnose, emulsification index, soybean oil.
An efficient sequential, biological and photocatalytic treatment to reduce the pollutant levels in wastewater due to the bleaching process during paper production is reported. For a biological ...pre-treatment, 800 ml of non-sterilized effluent is inoculated with Trametes versicolor immobilized in polyurethane foam, with 25 g L super(-1) glucose, 6.75 mM CuSO sub(4), and 0.22 mM MnSO sub(4) is added, and cultured at 25 degree C with an air flow of 800 ml min super(-1) for 8 d. It is observed that the fungus does not inhibit growth of the heterotrophic populations of the effluent. The total sequential treatment results in a 92% CR, 97% COD reduction and 99% chlorophenol removal at 96 h and 20 min.
The adsorption performance of a Cu-TiO2 composite for removing acid blue 80 (AB80) dye from aqueous solutions was investigated in terms of kinetics, equilibrium, and thermodynamics. The effect of ...operating variables, such as solution pH, initial dye concentration, contact time, and temperature, on AB80 adsorption was studied in batch experiments. AB80 adsorption increased with increasing contact time, initial dye concentration, and temperature and with decreasing solution pH. Modeling of adsorption kinetics showed good agreement of experimental data with the pseudo-second-order kinetics model. The experimental equilibrium data for AB80 adsorption were evaluated for compliance with different two-parameter, three-parameter, and four-parameter isotherm models. The Langmuir isotherm model best described the AB80 adsorption equilibrium data. The thermodynamic data revealed that the AB80 adsorption process was endothermic and nonspontaneous. Kinetics, equilibrium, and thermodynamic results indicate that Cu-TiO2 adsorbs AB80 by a chemical sorption reaction.
Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest ...due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZ alpha A-LaccGluc-Stop and pGAPZ alpha A-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 plus or minus 6.46 UL-1 compared to activities of 0.13 plus or minus 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and alpha -factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.
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