Recovery of Phenolic Acid and Enzyme Production from Corn Silage Biologically Treated by Trametes versicolor.

PubMed

Bucić-Kojić, Ana; Šelo, Gordana; Zelić, Bruno; Planinić, Mirela; Tišma, Marina

2017-03-01

Corn silage is used as high-energy forage for dairy cows and more recently for biogas production in a process of anaerobic co-digestion with cow manure. In this work, fresh corn silage after the harvest was used as a substrate in solid-state fermentations with T. versicolor with the aim of phenolic acid recovery and enzyme (laccase and manganese peroxidase) production. During 20 days of fermentation, 10.4-, 3.4-, 3.0-, and 1.8-fold increments in extraction yield of syringic acid, vanillic acid, p-hydroxybenzoic acid, and caffeic acid, respectively, were reached when compared to biologically untreated corn silage. Maximal laccase activity was gained on the 4th day of fermentation (V.A. = 180.2 U/dm 3 ), and manganese peroxidase activity was obtained after the 3rd day of fermentation (V.A. = 30.1 U/dm 3 ). The addition of copper(II) sulfate as inducer during solid state fermentation resulted in 8.5- and 7-fold enhancement of laccase and manganese peroxidase activities, respectively. Furthermore, the influence of pH and temperature on enzyme activities was investigated. Maximal activity of laccase was obtained at T = 50 °C and pH = 3.0, while manganese peroxidase is active at temperature range T = 45-70 °C with the maximal activity at pH = 4.5.

Description of the first fungal dye-decolorizing peroxidase oxidizing manganese(II)

DOE PAGES

Fernandez-Fueyo, Elena; Linde, Dolores; Almendral, David; ...

2015-05-13

Two phylogenetically divergent genes of the new family of dye-decolorizing peroxidases (DyPs) were found during comparison of the four DyP genes identified in the Pleurotus ostreatus genome with over 200 DyP genes from other basidiomycete genomes. The heterologously expressed enzymes ( Pleos-DyP1 and Pleos-DyP4, following the genome nomenclature) efficiently oxidize anthraquinoid dyes (such as Reactive Blue 19), which are characteristic DyP substrates, as well as low redox-potential dyes (such as 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and substituted phenols. However, only Pleos-DyP4 oxidizes the high redox-potential dye Reactive Black 5, at the same time that it displays high thermal and pH stability. Unexpectedly, bothmore » enzymes also oxidize Mn 2+ to Mn 3+, albeit with very different catalytic efficiencies. Pleos-DyP4 presents a Mn 2+ turnover (56 s –1) nearly in the same order of the two other Mn 2+-oxidizing peroxidase families identified in the P. ostreatus genome: manganese peroxidases (100 s–1 average turnover) and versatile peroxidases (145 s –1 average turnover), whose genes were also heterologously expressed. Oxidation of Mn 2+ has been reported for an Amycolatopsis DyP (24 s –1) and claimed for other bacterial DyPs, albeit with lower activities, but this is the first time that Mn 2+ oxidation is reported for a fungal DyP. Interestingly, Pleos-DyP4 (together with ligninolytic peroxidases) is detected in the secretome of P. ostreatus grown on different lignocellulosic substrates. In conclusion, it is suggested that generation of Mn 3+ oxidizers plays a role in the P. ostreatus white-rot lifestyle since three different families of Mn 2+-oxidizing peroxidase genes are present in its genome being expressed during lignocellulose degradation.« less

Description of the first fungal dye-decolorizing peroxidase oxidizing manganese(II)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fernandez-Fueyo, Elena; Linde, Dolores; Almendral, David

Two phylogenetically divergent genes of the new family of dye-decolorizing peroxidases (DyPs) were found during comparison of the four DyP genes identified in the Pleurotus ostreatus genome with over 200 DyP genes from other basidiomycete genomes. The heterologously expressed enzymes ( Pleos-DyP1 and Pleos-DyP4, following the genome nomenclature) efficiently oxidize anthraquinoid dyes (such as Reactive Blue 19), which are characteristic DyP substrates, as well as low redox-potential dyes (such as 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and substituted phenols. However, only Pleos-DyP4 oxidizes the high redox-potential dye Reactive Black 5, at the same time that it displays high thermal and pH stability. Unexpectedly, bothmore » enzymes also oxidize Mn 2+ to Mn 3+, albeit with very different catalytic efficiencies. Pleos-DyP4 presents a Mn 2+ turnover (56 s –1) nearly in the same order of the two other Mn 2+-oxidizing peroxidase families identified in the P. ostreatus genome: manganese peroxidases (100 s–1 average turnover) and versatile peroxidases (145 s –1 average turnover), whose genes were also heterologously expressed. Oxidation of Mn 2+ has been reported for an Amycolatopsis DyP (24 s –1) and claimed for other bacterial DyPs, albeit with lower activities, but this is the first time that Mn 2+ oxidation is reported for a fungal DyP. Interestingly, Pleos-DyP4 (together with ligninolytic peroxidases) is detected in the secretome of P. ostreatus grown on different lignocellulosic substrates. In conclusion, it is suggested that generation of Mn 3+ oxidizers plays a role in the P. ostreatus white-rot lifestyle since three different families of Mn 2+-oxidizing peroxidase genes are present in its genome being expressed during lignocellulose degradation.« less

Lignolytic enzymes produced by Trametes villosa ccb176 under different culture conditions

PubMed Central

Yamanaka, Renata; Soares, Clarissa F.; Matheus, Dácio R.; Machado, Kátia M.G.

2008-01-01

The expression of the enzymatic system produced by basidiomycetous fungi, which is involved in the degradation of xenobiotics, mainly depends on culture conditions, especially of the culture medium composition. Trametes villosa is a strain with a proven biotechnological potential for the degradation of organochlorine compounds and for the decolorization of textile dyes. The influence of glucose concentration, addition of a vegetable oil-surfactant emulsion, nature of the surfactant and the presence of manganese and copper on the growth, pH and production of laccase, total peroxidase and manganese-dependent peroxidase activities were evaluated. In general, acidification of the medium was observed, with the pH reaching a value close to 3.5 within the first days of growth. Laccase was the main activity detected under the different conditions and was produced throughout the culture period of the fungus, irrespective of the growth phase. Supplementation of the medium with vegetable oil emulsified with a surfactant induced manganese-dependent peroxidase activity in T. villosa. Higher specific yields of laccase activity were obtained with the addition of copper. PMID:24031184

The secretome of Trametes versicolor grown on tomato juice medium and purification of the secreted oxidoreductases including a versatile peroxidase.

PubMed

Carabajal, Maira; Kellner, Harald; Levin, Laura; Jehmlich, Nico; Hofrichter, Martin; Ullrich, René

2013-10-10

The present work was carried out with the aim to analyze the secretome of Trametes versicolor BAFC 2234 grown on tomato juice medium supplemented with copper and manganese. T. versicolor BAFC 2234 was selected among diverse wood dwelling agaricomycetes from Argentina by its ability to cause a strong white rot on hardwood and in addition to show high tolerance toward phenolic compounds. A considerable number of the identified proteins were related to the degradation/modification of lignocelluloses. Hydrolases, peroxidases and phenoloxidases were the most abundant enzymes produced under the above-mentioned culture conditions. The lignin-modifying oxidoreductases laccase, manganese peroxidase (MnP) and versatile peroxidase (VP) were successfully purified - the latter for the first time from T. versicolor. The native VP protein has a molecular mass of 45kDa and an isoelectric point of pH 3.7. The study clearly shows that complex plant-based media being rich in phenolics, such as tomato juice, can stimulate the secretion of a broad set of extracellular lignocellulolytic enzymes. Using such natural products as fungal culture media may give the opportunity to investigate plant biomass decomposition as well as the biodegradation of organic pollutants in an environment close to nature. Copyright © 2013 Elsevier B.V. All rights reserved.

Methylene blue as a lignin surrogate in manganese peroxidase reaction systems.

PubMed

Goby, Jeffrey D; Penner, Michael H; Lajoie, Curtis A; Kelly, Christine J

2017-11-15

Manganese peroxidase (MnP) is associated with lignin degradation and is thus relevant to lignocellulosic-utilization technologies. Technological applications require reaction mixture optimization. A surrogate substrate can facilitate this if its susceptibility to degradation is easily monitored and mirrors that of lignin. The dye methylene blue (MB) was evaluated in these respects as a surrogate substrate by testing its reactivity in reaction mixtures containing relevant redox mediators (dicarboxylic acids, fatty acids). Relative rates of MB degradation were compared to available literature reports of lignin degradation under similar conditions, and suggest that MB can be a useful lignin surrogate in MnP systems. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative analysis of lignin peroxidase and manganese peroxidase activity on coniferous and deciduous wood using ToF-SIMS.

PubMed

MacDonald, Jacqueline; Goacher, Robyn E; Abou-Zaid, Mamdouh; Master, Emma R

2016-09-01

White-rot fungi are distinguished by their ability to efficiently degrade lignin via lignin-modifying type II peroxidases, including manganese peroxidase (MnP) and lignin peroxidase (LiP). In the present study, time-of flight secondary ion mass spectrometry (ToF-SIMS) was used to evaluate lignin modification in three coniferous and three deciduous wood preparations following treatment with commercial preparations of LiP and MnP from two different white-rot fungi. Percent modification of lignin was calculated as a loss of intact methoxylated lignin over nonfunctionalized aromatic rings, which is consistent with oxidative cleavage of methoxy moieties within the lignin structure. Exposure to MnP resulted in greater modification of lignin in coniferous compared to deciduous wood (28 vs. 18 % modification of lignin); and greater modification of G-lignin compared to S-lignin within the deciduous wood samples (21 vs. 12 %). In contrast, exposure to LiP resulted in similar percent modification of lignin in all wood samples (21 vs 22 %), and of G- and S-lignin within the deciduous wood (22 vs. 23 %). These findings suggest that the selected MnP and LiP may particularly benefit delignification of coniferous and deciduous wood, respectively. Moreover, the current analysis further demonstrates the utility of ToF-SIMS for characterizing enzymatic modification of lignin in wood fibre along with potential advantages over UV and HPCL-MS detection of solubilized delignification products.

Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis.

PubMed

Min, K L; Kim, Y H; Kim, Y W; Jung, H S; Hah, Y C

2001-08-15

The white-rot fungus Phellinus ribis produced a single form of laccase, which was purified to apparent electrophoretic homogeneity from cultures induced with 2,5-xylidine. This protein was a dimer, consisting of two subunits of 76 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that the enzyme contained about 28% carbohydrate content. The laccase appeared to be different from other known laccases by the UV-visible absorption spectrum analysis. One enzyme molecule contained one copper, one manganese, and two zinc atoms. The laccase showed optimal activity at pH 4.0-6.0, 5.0, and 6.0 with 2,6-dimethoxyphenol, ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], and syringaldazine, respectively. The enzyme preferably oxidized dimethoxyphenol and aromatic amine compounds. The stability of the laccase was low at acidic pH, whereas it showed high stability at neutral pH and mild temperature. The N-terminal amino acid sequence revealed a very low homology with other microbial laccases. With some substrates, the addition of manganese and H2O2 resulted in a remarkable increase in the oxidation rate. Without an appropriate phenolic substrate, the enzyme could not oxidize Mn(II) in the presence of H2O2 or pyrophosphate. Copyright 2001 Academic Press.

Kinetics of Mn3+-oxalate formation and decay in reactions catalyzed by manganese peroxidase of Ceriporiopsis subvermispora

Treesearch

Ulises Urzua; Philip J. Kersten; Rafael Vicuna

1998-01-01

The kinetics of Mn3+- oxalate formation and decay were investigated in reactions catalyzed by manganese peroxidase (MnP) from the basiomycete Ceriporiopsis subvermispora in the absence of externally added hydrogen peroxide. A characteristic lag observed in the formation of this complex was shortened by glyoxylate or catalytic amounts of Mn3+ or hydrogen peroxide. MnP...

Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, P.; Whitwam, R.E.; Tien, Ming

1996-03-01

A manganese peroxidase (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3{prime} untranslated region of the glucoamylase gene of Asperigillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies. 34 refs., 7 figs., 1 tab.

A novel extracellular multicopper oxidase from Phanerochaete chrysosporium with ferroxidase activity

Treesearch

Luis F. Larrondo; Loreto Salas; Francisco Melo; Rafael Vicuna; Daniel Cullen

2003-01-01

Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified...

Oxidizability of unsaturated fatty acids and of a non-phenolic lignin structure in the manganese peroxidase-dependent lipid peroxidation system

Treesearch

Alexander N. Kapich; Tatyana V. Korneichik; Annele Hatakka; Kenneth E. Hammel

2010-01-01

Unsaturated fatty acids have been proposed to mediate the oxidation of recalcitrant, non-phenolic lignin structures by fungal manganese peroxidases (MnP), but their precise role remains unknown. We investigated the oxidizability of three fatty acids with varying degrees of polyunsaturation (linoleic, linolenic, and arachidonic acids) by measuring conjugated dienes...

Comparative evaluation of manganese peroxidase- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids by different methods

Treesearch

Alexander N. Kapich; Tatyana V. Korneichik; Kenneth E. Hammel; Annele Hatakka

2011-01-01

The peroxidation of C18 unsaturated fatty acids by fungal manganese peroxidase (MnP)/Mn(II) and by chelated Mn(III) was studied with application of three different methods: by monitoring oxygen consumption, by measuring conjugated dienes and by thiobarbituric acid-reactive substances (TBARS) formation. All tested polyunsaturated fatty acids (PUFAs) were oxidized by MnP...

Interactions of carbon nanotubes and/or graphene with manganese peroxidase during biodegradation of endocrine disruptors and triclosan.

PubMed

Chen, Ming; Zeng, Guangming; Lai, Cui; Zhang, Chang; Xu, Piao; Yan, Min; Xiong, Weiping

2017-10-01

Molecular-level biodegradation processes of bisphenol A (BPA), nonylphenol (NP) and triclosan (TCS) mediated by manganese peroxidase (MnP) were investigated with and without single-walled carbon nanotube (SWCNT) and/or graphene (GRA). Although the incorporation of SWCNT, GRA or their combination (SWCNT+GRA) did not break up the complexes composed of manganese peroxidase (MnP) and these substrates, they had different effects on the native contacts between the substrates and MnP. GRA tended to decrease the overall stability of the binding between MnP and its substrates. SWCNT or SWCNT+GRA generally had a minor impact on the mean binding energy between MnP and its substrates. We detected some sensitive residues from MnP that were dramatically disturbed by the GRA, SWCNT or SWCNT+GRA. Nanomaterials changed the number and behavior of water molecules adjacent to both MnP and its substrates, which was not due to the destruction of H-bond network formed by sensitive regions and water molecules. The present results are useful for understanding the molecular basis of pollutant biodegradation affected by the nanomaterials in the environment, and are also helpful in assessing the risks of these materials to the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Diversity of Ligninolytic Enzymes and Their Genes in Strains of the Genus Ganoderma: Applicable for Biodegradation of Xenobiotic Compounds?

PubMed Central

Torres-Farradá, Giselle; Manzano León, Ana M.; Rineau, François; Ledo Alonso, Lucía L.; Sánchez-López, María I.; Thijs, Sofie; Colpaert, Jan; Ramos-Leal, Miguel; Guerra, Gilda; Vangronsveld, Jaco

2017-01-01

White-rot fungi (WRF) and their ligninolytic enzymes (laccases and peroxidases) are considered promising biotechnological tools to remove lignin related Persistent Organic Pollutants from industrial wastewaters and contaminated ecosystems. A high diversity of the genus Ganoderma has been reported in Cuba; in spite of this, the diversity of ligninolytic enzymes and their genes remained unexplored. In this study, 13 native WRF strains were isolated from decayed wood in urban ecosystems in Havana (Cuba). All strains were identified as Ganoderma sp. using a multiplex polymerase chain reaction (PCR)-method based on ITS sequences. All Ganoderma sp. strains produced laccase enzymes at higher levels than non-specific peroxidases. Native-PAGE of extracellular enzymatic extracts revealed a high diversity of laccase isozymes patterns between the strains, suggesting the presence of different amino acid sequences in the laccase enzymes produced by these Ganoderma strains. We determined the diversity of genes encoding laccases and peroxidases using a PCR and cloning approach with basidiomycete-specific primers. Between two and five laccase genes were detected in each strain. In contrast, only one gene encoding manganese peroxidase or versatile peroxidase was detected in each strain. The translated laccases and peroxidases amino acid sequences have not been described before. Extracellular crude enzymatic extracts produced by the Ganoderma UH strains, were able to degrade model chromophoric compounds such as anthraquinone and azo dyes. These findings hold promises for the development of a practical application for the treatment of textile industry wastewaters and also for bioremediation of polluted ecosystems by well-adapted native WRF strains. PMID:28588565

Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J.

2014-12-01

The variable C-terminal tail of manganese peroxidases, a group of enzymes involved in lignin degradation, is implicated in their catalytic and stability properties, as shown by new crystal structures, molecular-simulation and directed-mutagenesis data. Based on this structural–functional evaluation, short and long/extralong manganese peroxidase subfamilies have been accepted; the latter are characterized by exceptional stability, while it is shown for the first time that the former are able to oxidize other substrates at the same site where manganese(II) is oxidized. The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, whichmore » share an Mn{sup 2+}-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn{sup 2+} oxidation by the internal propionate, but prevents the oxidation of 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd{sup 2+} binds at the Mn{sup 2+}-oxidation site and competitively inhibits oxidation of both Mn{sup 2+} and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not

Differential regulation of mnp2, a new manganese peroxidase-encoding gene from the ligninolytic fungus Trametes versicolor PRL 572.

PubMed

Johansson, Tomas; Nyman, Per Olof; Cullen, Daniel

2002-04-01

A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO(4) to cultures increased mnp2 transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of T. versicolor, mnp1, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in T. versicolor are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the mnp1 and mnp2 polypeptides to have internal homology.

Differential Regulation of mnp2, a New Manganese Peroxidase-Encoding Gene from the Ligninolytic Fungus Trametes versicolor PRL 572

PubMed Central

Johansson, Tomas; Nyman, Per Olof; Cullen, Daniel

2002-01-01

A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO4 to cultures increased mnp2 transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of T. versicolor, mnp1, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in T. versicolor are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the mnp1 and mnp2 polypeptides to have internal homology. PMID:11916737

Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent.

PubMed

Michel, F C; Dass, S B; Grulke, E A; Reddy, C A

1991-08-01

The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.

Biochemical and molecular characterization of an atypical manganese peroxidase of the litter-decomposing fungus Agrocybe praecox.

PubMed

Hildén, Kristiina; Mäkelä, Miia R; Steffen, Kari T; Hofrichter, Martin; Hatakka, Annele; Archer, David B; Lundell, Taina K

2014-11-01

Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of Tree Species on Enzyme Secretion by the Shiitake Medicinal Mushroom, Lentinus edodes (Agaricomycetes).

PubMed

Plotnikov, Evgeny V; Glukhova, Lubov B; Sokolyanskaya, Ludmila O; Karnachuk, Olga V; Solioz, Marc

2016-01-01

We compared cold and hot wood extracts of 3 endemic Siberian trees-namely, Prunus padus (bird cherry), Populus tremula (aspen), and Betula sp. (birch)-on biomass production and laccase and peroxidase secretion in submerged cultures by the medicinal mushroom Lentinus edodes. Of the conditions tested, only hot Prunus extracts stimulated biomass production, whereas all extracts stimulated laccase and peroxidase secretion, albeit to different extents. A large, differential stimulation of manganese peroxidase was observed by hot Prunus extracts. The results highlight important differences between tree species in the stimulation of biomass and enzyme production by L. edodes and point to potentially interesting stimulatory factors present in hot Prunus extracts. These findings are of relevance in the use of L. edodes for medicinal or biotechnological applications.

Role of fungal peroxidases in biological ligninolysis

Treesearch

Kenneth E. Hammel; Dan Cullen

2008-01-01

The degradation of lignin by filamentous fungi is a major route for the recycling of photosynthetically fixed carbon, and the oxidative mechanisms employed have potential biotechnological applications. The lignin peroxidases (LiPs), manganese peroxidases (MnPs), and closely related enzymes of white rot basidiomycetes are likely contributors to fungal ligninolysis. Many...

Biodegradation of Direct Blue 15 by free and immobilized Trametes versicolor.

PubMed

Pazarlioglu, Nurdan Kasikara; Akkaya, Alper; Akdogan, Hatice Ardag; Gungor, Burcin

2010-07-01

To investigate biodegradability by Trametes versicolor, five structurally different direct azo-dyes--Direct Black 38, Direct Blue 15 (DB 15), Direct Orange 26, Direct Green 6, and Direct Yellow 12--were studied. The DB 15 was determined as the best biodegradable dye by this white-rot fungus. Laccase and manganese peroxidase activities were monitored with the biodegradation process; it was observed that laccase played an important role in the dye degradation, while manganese peroxidase activity could not be detected. Possible degradation products also were examined by gas chromatography-mass spectrometry, but no metabolite was detected after the degradation and/or decolorization process. To enhance performance of the fungi during the degradation, Trametes versicolor cells were immobilized in alginate beads. Then, DB 15 decolorization by immobilized Trametes versicolor was studied in a small-scale packed-bed reactor. The color removal efficiency in repeated batches was found to be 98 and 93% for 50 mg/L DB 15.

Oxidation of a non-phenolic lignin model compound by two Irpex lacteus manganese peroxidases: evidence for implication of carboxylate and radicals.

PubMed

Qin, Xing; Sun, Xianhua; Huang, Huoqing; Bai, Yingguo; Wang, Yuan; Luo, Huiying; Yao, Bin; Zhang, Xiaoyu; Su, Xiaoyun

2017-01-01

Manganese peroxidase is one of the Class II fungal peroxidases that are able to oxidize the low redox potential phenolic lignin compounds. For high redox potential non-phenolic lignin degradation, mediators such as GSH and unsaturated fatty acids are required in the reaction. However, it is not known whether carboxylic acids are a mediator for non-phenolic lignin degradation. The white rot fungus Irpex lacteus is one of the most potent fungi in degradation of lignocellulose and xenobiotics. Two manganese peroxidases ( Il MnP1 and Il MnP2) from I. lacteus CD2 were over-expressed in Escherichia coli and successfully refolded from inclusion bodies. Both Il MnP1 and Il MnP2 oxidized the phenolic compounds efficiently. Surprisingly, they could degrade veratryl alcohol, a non-phenolic lignin compound, in a Mn 2+ -dependent fashion. Malonate or oxalate was found to be also essential in this degradation. The oxidation of non-phenolic lignin was further confirmed by analysis of the reaction products using LC-MS/MS. We proved that Mn 2+ and a certain carboxylate are indispensable in oxidation and that the radicals generated under this condition, specifically superoxide radical, are at least partially involved in lignin oxidative degradation. Il MnP1 and Il MnP2 can also efficiently decolorize dyes with different structures. We provide evidence that a carboxylic acid may mediate oxidation of non-phenolic lignin through the action of radicals. MnPs, but not LiP, VP, or DyP, are predominant peroxidases secreted by some white rot fungi such as I. lacteus and the selective lignocellulose degrader Ceriporiopsis subvermispora . Our finding will help understand how these fungi can utilize MnPs and an excreted organic acid, which is usually a normal metabolite, to efficiently degrade the non-phenolic lignin. The unique properties of Il MnP1 and Il MnP2 make them good candidates for exploring molecular mechanisms underlying non-phenolic lignin compounds oxidation by MnPs and for

Degradation of alkylphenols by white rot fungus Irpex lacteus and its manganese peroxidase.

PubMed

Moon, Dong-Soo; Song, Hong-Gyu

2012-10-01

Alkylphenols are common endocrine disrupters that are produced from the degradation of widely used surfactants. Since they cause various harmful effects on aquatic life and in humans, they should be removed from the environments being contaminated. White rot fungus Irpex lacteus can completely degrade 100 mg/L of octylphenol, nonylphenol, and phenylphenol during 1 day of incubation in the complex YMG medium, which was the highest degrading capability among nine strains of white rot fungi tested. In the N-limited Kirk's basal salts medium, I. lacteus could degrade almost 100 % of 100 mg/L octylphenol and nonylphenol in 1 h, and exhibited a high activity of manganese peroxidase (MnP; 1,790 U/L). MnP of I. lacteus was purified by ion exchange chromatography, and this degraded 99 % of 50 mg/L octylphenol and removed 80 % of estrogenic activity in 2 hours. In addition, the purified MnP (10 U/mL) degraded over 90 % of 50 mg/L nonylphenol in 1 h.

Differential Regulation by Organic Compounds and Heavy Metals of Multiple Laccase Genes in the Aquatic Hyphomycete Clavariopsis aquatica

PubMed Central

Solé, Magali; Müller, Ines; Pecyna, Marek J.; Fetzer, Ingo; Harms, Hauke

2012-01-01

To advance the understanding of the molecular mechanisms controlling microbial activities involved in carbon cycling and mitigation of environmental pollution in freshwaters, the influence of heavy metals and natural as well as xenobiotic organic compounds on laccase gene expression was quantified using quantitative real-time PCR (qRT-PCR) in an exclusively aquatic fungus (the aquatic hyphomycete Clavariopsis aquatica) for the first time. Five putative laccase genes (lcc1 to lcc5) identified in C. aquatica were differentially expressed in response to the fungal growth stage and potential laccase inducers, with certain genes being upregulated by, e.g., the lignocellulose breakdown product vanillic acid, the endocrine disruptor technical nonylphenol, manganese, and zinc. lcc4 is inducible by vanillic acid and most likely encodes an extracellular laccase already excreted during the trophophase of the organism, suggesting a function during fungal substrate colonization. Surprisingly, unlike many laccases of terrestrial fungi, none of the C. aquatica laccase genes was found to be upregulated by copper. However, copper strongly increases extracellular laccase activity in C. aquatica, possibly due to stabilization of the copper-containing catalytic center of the enzyme. Copper was found to half-saturate laccase activity already at about 1.8 μM, in favor of a fungal adaptation to low copper concentrations of aquatic habitats. PMID:22544244

Characterisation of manganese peroxidase and laccase producing bacteria capable for degradation of sucrose glutamic acid-Maillard reaction products at different nutritional and environmental conditions.

PubMed

Kumar, Vineet; Chandra, Ram

2018-02-02

Maillard reactions products (MRPs) are a major colorant of distillery effluent. It is major source of environmental pollution due to its complex structure and recalcitrant nature. This study has revealed that sucrose glutamic acid-Maillard reaction products (SGA-MRPs) showed many absorption peaks between 200 and 450 nm. The absorption maximum peak was noted at 250 nm in spectrophotometric detection. This indicated the formation of variable molecular weight Maillard products during the SGA-MRPs formation at high temperature. The identified aerobic bacterial consortium consisting Klebsiella pneumoniae (KU726953), Salmonella enterica (KU726954), Enterobacter aerogenes (KU726955), Enterobacter cloaceae (KU726957) showed optimum production of MnP and laccase at 120 and 144 h of growth, respectively. The potential bacterial consortium showed decolourisation of Maillard product up to 70% in presence of glucose (1%), peptone (0.1%) at optimum pH (8.1), temperature (37 °C) and shaking speed (180 rpm) within 192 h of incubation. The reduction of colour of Maillard product correlated with shifting of absorption peaks in UV-Vis spectrophotometry analysis. Further, the changing of functional group in FT-IR data showed appearance of new peaks and GC-MS analysis of degraded sample revealed the depolymerisation of complex MRPs. The toxicity evaluation using seed of Phaseolus mungo L. showed reduction of toxicity of MRPs after bacterial treatment. Hence, this study concluded that developed bacterial consortium have capability for decolourisation of MRPs due to high content of MnP and laccase.

Heterologous Expression of Phanerochaete chrysoporium Glyoxal Oxidase and its Application for the Coupled Reaction with Manganese Peroxidase to Decolorize Malachite Green

PubMed Central

Son, Yu-Lim; Kim, Hyoun-Young; Thiyagarajan, Saravanakumar; Xu, Jing Jing

2012-01-01

cDNA of the glx1 gene encoding glyoxal oxidase (GLX) from Phanerochaete chrysosporium was isolated and expressed in Pichia pastoris. The recombinant GLX (rGLX) produces H2O2 over 7.0 nmol/min/mL using methyl glyoxal as a substrate. Use of rGLX as a generator of H2O2 improved the coupled reaction with recombinant manganese peroxidase resulting in decolorization of malachite green up to 150 µM within 90 min. PMID:23323052

Patterns of lignin degradation and oxidative enzyme secretion by different wood- and litter-colonizing basidiomycetes and ascomycetes grown on beech-wood.

PubMed

Liers, Christiane; Arnstadt, Tobias; Ullrich, René; Hofrichter, Martin

2011-10-01

The degradation of lignocellulose and the secretion of extracellular oxidoreductases were investigated in beech-wood (Fagus sylvatica) microcosms using 11 representative fungi of four different ecophysiological and taxonomic groups causing: (1) classic white rot of wood (e.g. Phlebia radiata), (2) 'nonspecific' wood rot (e.g. Agrocybe aegerita), (3) white rot of leaf litter (Stropharia rugosoannulata) or (4) soft rot of wood (e.g. Xylaria polymorpha). All strong white rotters produced manganese-oxidizing peroxidases as the key enzymes of ligninolysis (75-2200 mU g(-1)), whereas lignin peroxidase activity was not detectable in the wood extracts. Interestingly, activities of two recently discovered peroxidases - aromatic peroxygenase and a manganese-independent peroxidase of the DyP-type - were detected in the culture extracts of A. aegerita (up to 125 mU g(-1)) and Auricularia auricula-judae (up to 400 mU g(-1)), respectively. The activity of classic peroxidases correlated to some extent with the removal of wood components (e.g. Klason lignin) and the release of small water-soluble fragments (0.5-1.0 kDa) characterized by aromatic constituents. In contrast, laccase activity correlated with the formation of high-molecular mass fragments (30-200 kDa). The differences observed in the degradation patterns allow to distinguish the rot types caused by basidiomycetes and ascomycetes and may be suitable for following the effects of oxidative key enzymes (ligninolytic peroxidases vs. laccases, role of novel peroxidases) during wood decay. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed.

PubMed

Clough, Richard C; Pappu, Kameshwari; Thompson, Kevin; Beifuss, Katherine; Lane, Jeff; Delaney, Donna E; Harkey, Robin; Drees, Carol; Howard, John A; Hood, Elizabeth E

2006-01-01

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.

Insights into lignin degradation and its potential industrial applications.

PubMed

Abdel-Hamid, Ahmed M; Solbiati, Jose O; Cann, Isaac K O

2013-01-01

Lignocellulose is an abundant biomass that provides an alternative source for the production of renewable fuels and chemicals. The depolymerization of the carbohydrate polymers in lignocellulosic biomass is hindered by lignin, which is recalcitrant to chemical and biological degradation due to its complex chemical structure and linkage heterogeneity. The role of fungi in delignification due to the production of extracellular oxidative enzymes has been studied more extensively than that of bacteria. The two major groups of enzymes that are involved in lignin degradation are heme peroxidases and laccases. Lignin-degrading peroxidases include lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP), and dye-decolorizing peroxidase (DyP). LiP, MnP, and VP are class II extracellular fungal peroxidases that belong to the plant and microbial peroxidases superfamily. LiPs are strong oxidants with high-redox potential that oxidize the major non-phenolic structures of lignin. MnP is an Mn-dependent enzyme that catalyzes the oxidation of various phenolic substrates but is not capable of oxidizing the more recalcitrant non-phenolic lignin. VP enzymes combine the catalytic activities of both MnP and LiP and are able to oxidize Mn(2+) like MnP, and non-phenolic compounds like LiP. DyPs occur in both fungi and bacteria and are members of a new superfamily of heme peroxidases called DyPs. DyP enzymes oxidize high-redox potential anthraquinone dyes and were recently reported to oxidize lignin model compounds. The second major group of lignin-degrading enzymes, laccases, are found in plants, fungi, and bacteria and belong to the multicopper oxidase superfamily. They catalyze a one-electron oxidation with the concomitant four-electron reduction of molecular oxygen to water. Fungal laccases can oxidize phenolic lignin model compounds and have higher redox potential than bacterial laccases. In the presence of redox mediators, fungal laccases can oxidize non

Degradation of polyethylene by Trichoderma harzianum--SEM, FTIR, and NMR analyses.

PubMed

Sowmya, H V; Ramalingappa; Krishnappa, M; Thippeswamy, B

2014-10-01

Trichoderma harzianum was isolated from local dumpsites of Shivamogga District for use in the biodegradation of polyethylene. Soil sample of that dumpsite was used for isolation of T. harzianum. Degradation was carried out using autoclaved, UV-treated, and surface-sterilized polyethylene. Degradation was monitored by observing weight loss and changes in physical structure by scanning electron microscopy, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. T. harzianum was able to degrade treated polyethylene (40%) more efficiently than autoclaved (23%) and surface-sterilized polyethylene (13%). Enzymes responsible for polyethylene degradation were screened from T. harzianum and were identified as laccase and manganese peroxidase. These enzymes were produced in large amount, and their activity was calculated using spectrophotometric method and crude extraction of enzymes was carried out. Molecular weight of laccase was determined as 88 kDa and that of manganese peroxidase was 55 kDa. The capacity of crude enzymes to degrade polyethylene was also determined. By observing these results, we can conclude that this organism may act as solution for the problem caused by polyethylene in nature.

The effect of iron to manganese substitution on microperoxidase 8 catalysed peroxidase and cytochrome P450 type of catalysis.

PubMed

Primus, J L; Boersma, M G; Mandon, D; Boeren, S; Veeger, C; Weiss, R; Rietjens, I M

1999-06-01

This study describes the catalytic properties of manganese microperoxidase 8 [Mn(III)MP8] compared to iron microperoxidase 8 [Fe(III)MP8]. The mini-enzymes were tested for pH-dependent activity and operational stability in peroxidase-type conversions, using 2-methoxyphenol and 3,3'-dimethoxybenzidine, and in a cytochrome P450-like oxygen transfer reaction converting aniline to para-aminophenol. For the peroxidase type of conversions the Fe to Mn replacement resulted in a less than 10-fold decrease in the activity at optimal pH, whereas the aniline para-hydroxylation is reduced at least 30-fold. In addition it was observed that the peroxidase type of conversions are all fully blocked by ascorbate and that aniline para-hydroxylation by Fe(III)MP8 is increased by ascorbate whereas aniline para-hydroxylation by Mn(III)MP8 is inhibited by ascorbate. Altogether these results indicate that different types of reactive metal oxygen intermediates are involved in the various conversions. Compound I/II, scavenged by ascorbate, may be the reactive species responsible for the peroxidase reactions, the polymerization of aniline and (part of) the oxygen transfer to aniline in the absence of ascorbate. The para-hydroxylation of aniline by Fe(III)MP8, in the presence of ascorbate, must be mediated by another reactive iron-oxo species which could be the electrophilic metal(III) hydroperoxide anion of microperoxidase 8 [M(III)OOH MP8]. The lower oxidative potential of Mn, compared to Fe, may affect the reactivity of both compound I/II and the metal(III) hydroperoxide anion intermediate, explaining the differential effect of the Fe to Mn substitution on the pH-dependent behavior, the rate of catalysis and the operational stability of MP8.

Size exclusion chromatography for the removal of pigments from extracellular ligninolytic enzyme extracts from decayed wheat straw.

PubMed

Shukla, Dharmendra; Patel, Bhavesh; Modi, Hasmukh; Vyas, Bharat Rajiv Manuel

2011-11-01

Solid-state fermentation of wheat straw was carried out by a native white rot basidiomycete Daedaleopsis flavida strain 5A. Extract prepared from the 12-day decayed wheat straw contained extracellular ligninolytic enzymes like manganese peroxidase (MnP), manganese-independent peroxidase (MIP), lignin peroxidase (LiP) and laccase along with straw-degraded products and pigments. Sephacryl S-200 size exclusion chromatography in 16/100 column was used for the separation of these ligninolytic enzymes and straw-degraded products and pigments. Recovery of pigment-free ligninolytic enzyme activities as protein was 40% of the total proteins loaded and specific LiP activity increased 34 fold after size exclusion chromatography. Thus accurate estimation of LiP by veratryl alcohol oxidation assay was possible only after the removal of interfering pigments. The reproducibility of size exclusion chromatography is adjudged satisfactory from the consistent results obtained after seven repetitive uses of matrices.

Laccase enzyme detoxifies hydrolysates and improves biogas production from hemp straw and miscanthus.

PubMed

Schroyen, Michel; Van Hulle, Stijn W H; Holemans, Sander; Vervaeren, Han; Raes, Katleen

2017-11-01

The impact of various phenolic compounds, vanillic acid, ferulic acid, p-coumaric acid and 4-hydroxybenzoic acid on anaerobic digestion of lignocellulosic biomass (hemp straw and miscanthus) was studied. Such phenolic compounds have been known to inhibit biogas production during anaerobic digestion. The different phenolic compounds were added in various concentrations: 0, 100, 500, 1000 and 2000mg/L. A difference in inhibition of biomethane production between the phenolic compounds was noted. Hydrolysis rate, during anaerobic digestion of miscanthus was inhibited up to 50% by vanillic acid, while vanillic acid had no influence on the initial rate of biogas production during the anaerobic digestion of hemp straw. Miscanthus has a higher lignin concentration (12-30g/100gDM) making it less accessible for degradation, and in combination with phenolic compounds released after harsh pretreatments, it can cause severe inhibition levels during the anaerobic digestion, lowering biogas production. To counter the inhibition, lignin degrading enzymes can be used to remove or degrade the inhibitory phenolic compounds. The interaction of laccase and versatile peroxidase individually with the different phenolic compounds was studied to have insight in the polymerization of inhibitory compounds or breakdown of lignocellulose. Hemp straw and miscanthus were incubated with 0, 100 and 500mg/L of the different phenolic compounds for 0, 6 and 24h and pretreated with the lignin degrading enzymes. A laccase pretreatment successfully detoxified the substrate, while versatile peroxidase however was inhibited by 100mg/L of each of the individual phenolic compounds. Finally a combination of enzymatic detoxification and subsequent biogas production showed that a decrease in phenolic compounds by laccase treatment can considerably lower the inhibition levels of the biogas production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cloning and characterization of a cDNA encoding a novel extracellular peroxidase from Trametes versicolor.

PubMed

Collins, P J; O'Brien, M M; Dobson, A D

1999-03-01

The white rot basidiomycete Trametes versicolor secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from T. versicolor CU1, the transcript levels of which are repressed by low concentrations of Mn2+ and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression.

Cloning and Characterization of a cDNA Encoding a Novel Extracellular Peroxidase from Trametes versicolor

PubMed Central

Collins, Patrick J.; O’Brien, Margaret M.; Dobson, Alan D. W.

1999-01-01

The white rot basidiomycete Trametes versicolor secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from T. versicolor CU1, the transcript levels of which are repressed by low concentrations of Mn2+ and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression. PMID:10049906

A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

PubMed Central

Larrondo, Luis F.; Salas, Loreto; Melo, Francisco; Vicuña, Rafael; Cullen, Daniel

2003-01-01

Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe2+, with a Km close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity. PMID:14532088

Isolation of Thermophilic Lignin Degrading Bacteria from Oil-Palm Empty Fruit Bunch (EFB) Compost

NASA Astrophysics Data System (ADS)

Lai, C. M. T.; Chua, H. B.; Danquah, M. K.; Saptoro, A.

2017-06-01

Empty Fruit Bunch (EFB) is a potential and sustainable feedstock for bioethanol production due to its high cellulosic content and availability in Malaysia. Due to high lignin content of EFB and the lack of effective delignification process, commercial bioethanol production from EFB is presently not viable. Enzymatic delignification has been identified as one of the key steps in utilising EFB as a feedstock for bioethanol conversion. To date, limited work has been reported on the isolation of lignin degrading bacteria. Hence, there is a growing interest to search for new lignin degrading bacteria with greater tolerance to temperature and high level of ligninolytic enzymes for more effective lignin degradation. This study aimed to isolate and screen thermophilic ligninolytic microorganisms from EFB compost. Ten isolates were successfully isolated from EFB compost. Although they are not capable of decolorizing Methylene Blue (MB) dye under agar plate assay method, they are able to utilize lignin mimicked compound - guaiacol as a sole carbon on the agar plate assay. This infers that there is no correlation of ligninolytic enzymes with dye decolourization for all the isolates that have been isolated. However, they are able to produce ligninolytic enzymes (Lignin peroxidase, Manganese peroxidase, Laccase) in Minimal Salt Medium with Kraft Lignin (MSM-KL) with Lignin Peroxidase (LiP) as the predominant enzyme followed by Manganese Peroxidase (MnP) and Laccase (Lac). Among all the tested isolates, CLMT 29 has the highest LiP production up to 8.7673 U/mL following 24 h of growth.

Effect of soya lecithin on the enzymatic system of the white-rot fungi Anthracophyllum discolor.

PubMed

Bustamante, M; González, M E; Cartes, A; Diez, M C

2011-01-01

The present work optimized the initial pH of the medium and the incubation temperature for ligninolytic enzymes produced by the white-rot fungus Anthracophyllum discolor. Additionally, the effect of soya lecithin on mycelial growth and the production of ligninolytic enzymes in static batch cultures were evaluated. The critical micelle concentration of soya lecithin was also studied by conductivity. The effects of the initial pH (3, 4, and 5) and incubation temperature (20, 25, and 30°C) on different enzymatic activities revealed that the optimum conditions to maximize ligninolytic activity were 26°C and pH 5.5 for laccase and manganese peroxidase (MnP) and 30°C and pH 5.5 for manganese-independent peroxidase (MiP). Under these culture conditions, the maximum enzyme production was 10.16, 484.46, and 112.50 U L(-1) for laccase, MnP, and manganese-independent peroxidase MiP, respectively. During the study of the effect of soya lecithin on A. discolor, we found that the increase in soya lecithin concentration from 0 to 10 g L(-1) caused an increase in mycelial growth. On the other hand, in the presence of soya lecithin, A. discolor produced mainly MnP, which reached a maximum concentration of 30.64 ± 4.61 U L(-1) after 25 days of incubation with 1 g L(-1) of the surfactant. The other enzymes were produced but to a lesser extent. The enzymatic activity of A. discolor was decreased when Tween 80 was used as a surfactant. The critical micelle concentration of soya lecithin calculated in our study was 0.61 g L(-1).

Induction, Purification and Characterization of a Novel Manganese Peroxidase from Irpex lacteus CD2 and Its Application in the Decolorization of Different Types of Dye

PubMed Central

Qin, Xing; Zhang, Jie; Zhang, Xiaoyu; Yang, Yang

2014-01-01

Manganese peroxidase (MnP) is the one of the important ligninolytic enzymes produced by lignin-degrading fungi which has the great application value in the field of environmental biotechnology. Searching for new MnP with stronger tolerance to metal ions and organic solvents is important for the maximization of potential of MnP in the biodegradation of recalcitrant xenobiotics. In this study, it was found that oxalic acid, veratryl alcohol and 2,6-Dimehoxyphenol could stimulate the synthesis of MnP in the white-rot fungus Irpex lacteus CD2. A novel manganese peroxidase named as CD2-MnP was purified and characterized from this fungus. CD2-MnP had a strong capability for tolerating different metal ions such as Ca2+, Cd2+, Co2+, Mg2+, Ni2+ and Zn2+ as well as organic solvents such as methanol, ethanol, DMSO, ethylene glycol, isopropyl alcohol, butanediol and glycerin. The different types of dyes including the azo dye (Remazol Brilliant Violet 5R, Direct Red 5B), anthraquinone dye (Remazol Brilliant Blue R), indigo dye (Indigo Carmine) and triphenylmethane dye (Methyl Green) as well as simulated textile wastewater could be efficiently decolorized by CD2-MnP. CD2-MnP also had a strong ability of decolorizing different dyes with the coexistence of metal ions and organic solvents. In summary, CD2-MnP from Irpex lacteus CD2 could effectively degrade a broad range of synthetic dyes and exhibit a great potential for environmental biotechnology. PMID:25412169

Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum

DOE Office of Scientific and Technical Information (OSTI.GOV)

D /Souza, T.M.; Merritt, C.S.; Reddy, C.A.

1999-12-01

Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen shaken cultures were much greater than those seen in low-nitrogen, malt extract, or wool-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units aremore » structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HM cultures showed two laccase activity bands, where as isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, and 5.1. Low levels of MnP activity were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.« less

Optimization of manganese peroxidase production from Schizophyllum sp. F17 in solid-state fermentation of agro-industrial residues.

PubMed

Zhou, Yue; Yang, Bing; Yang, Yang; Jia, Rong

2014-03-01

Manganese peroxidase (MnP), a crucial enzyme in lignin degradation, has wide potential applications in environmental protection. However, large-scale industrial application of this enzyme is limited due to several factors primarily related to cost and availability. Special attention has been paid to the production of MnP from inexpensive sources, such as lignocellulosic residues, using solid-state fermentation (SSF) systems. In the present study, a suitable SSF medium for the production of MnP by Schizophyllum sp. F17 from agro-industrial residues has been optimized. The mixed solid medium, comprising pine sawdust, rice straw, and soybean powder at a ratio of 0.52:0.15:0.33, conferred a maximum enzyme activity of 11.18 U/g on the sixth day of SSF. The results show that the use of wastes such as pine sawdust and rice straw makes the enzyme production more economical as well as helps solve environmental problems.

A Novel Lentinula edodes Laccase and Its Comparative Enzymology Suggest Guaiacol-Based Laccase Engineering for Bioremediation

PubMed Central

Wong, Kin-Sing; Cheung, Man-Kit; Au, Chun-Hang; Kwan, Hoi-Shan

2013-01-01

Laccases are versatile biocatalysts for the bioremediation of various xenobiotics, including dyes and polyaromatic hydrocarbons. However, current sources of new enzymes, simple heterologous expression hosts and enzymatic information (such as the appropriateness of common screening substrates on laccase engineering) remain scarce to support efficient engineering of laccase for better “green” applications. To address the issue, this study began with cloning the laccase family of Lentinula edodes. Three laccases perfectio sensu stricto (Lcc4A, Lcc5, and Lcc7) were then expressed from Pichia pastoris, characterized and compared with the previously reported Lcc1A and Lcc1B in terms of kinetics, stability, and degradation of dyes and polyaromatic hydrocarbons. Lcc7 represented a novel laccase, and it exhibited both the highest catalytic efficiency (assayed with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS]) and thermostability. However, its performance on “green” applications surprisingly did not match the activity on the common screening substrates, namely, ABTS and 2,6-dimethoxyphenol. On the other hand, correlation analyses revealed that guaiacol is much better associated with the decolorization of multiple structurally different dyes than are the two common screening substrates. Comparison of the oxidation chemistry of guaiacol and phenolic dyes, such as azo dyes, further showed that they both involve generation of phenoxyl radicals in laccase-catalyzed oxidation. In summary, this study concluded a robust expression platform of L. edodes laccases, novel laccases, and an indicative screening substrate, guaiacol, which are all essential fundamentals for appropriately driving the engineering of laccases towards more efficient “green” applications. PMID:23799101

Induction of fungal laccase production under solid state bioprocessing of new agroindustrial waste and its application on dye decolorization.

PubMed

Akpinar, Merve; Ozturk Urek, Raziye

2017-06-01

Lignocellulosic wastes are generally produced in huge amounts worldwide. Peach waste of these obtained from fruit juice industry was utilized as the substrate for laccase production by Pleurotus eryngii under solid state bioprocessing (SSB). Its chemical composition was determined and this bioprocess was carried out under stationary conditions at 28 °C. The effects of different compounds; copper, iron, Tween 80, ammonium nitrate and manganese, and their variable concentrations on laccase production were investigated in detail. The optimum production of laccase (43,761.33 ± 3845 U L -1 ) was achieved on the day of 20 by employing peach waste of 5.0 g and 70 µM Cu 2+ , 18 µM Fe 2+ , 0.025% (v/v) Tween 80, 4.0 g L -1 ammonium nitrate, 750 µM Mn 2+ as the inducers. The dye decolorization also researched to determine the degrading capability of laccase produced from peach culture under the above-mentioned conditions. Within this scope of the study, methyl orange, tartrazine, reactive red 2 and reactive black dyes were treated with this enzyme. The highest decolorization was performed with methyl orange as 43 ± 2.8% after 5 min of treatment when compared to other dyes. Up to now, this is the first report on the induction of laccase production by P. eryngii under SSB using peach waste as the substrate.

Grouping of multicopper oxidases in Lentinula edodes by sequence similarities and expression patterns.

PubMed

Sakamoto, Yuichi; Nakade, Keiko; Yoshida, Kentaro; Natsume, Satoshi; Miyazaki, Kazuhiro; Sato, Shiho; van Peer, Arend F; Konno, Naotake

2015-12-01

The edible white rot fungus Lentinula edodes possesses a variety of lignin degrading enzymes such as manganese peroxidases and laccases. Laccases belong to the multicopper oxidases, which have a wide range of catalytic activities including polyphenol degradation and synthesis, lignin degradation, and melanin formation. The exact number of laccases in L. edodes is unknown, as are their complete properties and biological functions. We analyzed the draft genome sequence of L. edodes D703PP-9 and identified 13 multicopper oxidase-encoding genes; 11 laccases in sensu stricto, of which three are new, and two ferroxidases. lcc8, a laccase previously reported in L. edodes, was not identified in D703PP-9 genome. Phylogenetic analysis showed that the 13 multicopper oxidases can be classified into laccase sensu stricto subfamily 1, laccase sensu stricto subfamily 2 and ferroxidases. From sequence similarities and expression patterns, laccase sensu stricto subfamily 1 can be divided into two subgroups. Laccase sensu stricto subfamily 1 group A members are mainly secreted from mycelia, while laccase sensu stricto subfamily 1 group B members are expressed mainly in fruiting bodies during growth or after harvesting but are lowly expressed in mycelia. Laccase sensu stricto subfamily 2 members are mainly expressed in mycelia, and two ferroxidases are mainly expressed in the fruiting body during growth or after harvesting, and are expressed at very low levels in mycelium. Our data suggests that L. edodes laccases in same group share expression patterns and would have common biological functions.

Isolation, characterization and transcriptome analysis of a novel Antarctic Aspergillus sydowii strain MS-19 as a potential lignocellulosic enzyme source.

PubMed

Cong, Bailin; Wang, Nengfei; Liu, Shenghao; Liu, Feng; Yin, Xiaofei; Shen, Jihong

2017-05-30

With the growing demand for fossil fuels and the severe energy crisis, lignocellulose is widely regarded as a promising cost-effective renewable resource for ethanol production, and the use of lignocellulose residues as raw material is remarkable. Polar organisms have important value in scientific research and development for their novelty, uniqueness and diversity. In this study, a fungus Aspergillus sydowii MS-19, with the potential for lignocellulose degradation was screened out and isolated from an Antarctic region. The growth profile of Aspergillus sydowii MS-19 was measured, revealing that Aspergillus sydowii MS-19 could utilize lignin as a sole carbon source. Its ability to synthesize low-temperature lignin peroxidase (Lip) and manganese peroxidase (Mnp) enzymes was verified, and the properties of these enzymes were also investigated. High-throughput sequencing was employed to identify and characterize the transcriptome of Aspergillus sydowii MS-19. Carbohydrate-Active Enzymes (CAZyme)-annotated genes in Aspergillus sydowii MS-19 were compared with those in the brown-rot fungus representative species, Postia placenta and Penicillium decumbens. There were 701CAZymes annotated in Aspergillus sydowii MS-19, including 17 cellulases and 19 feruloyl esterases related to lignocellulose-degradation. Remarkably, one sequence annotated as laccase was obtained, which can degrade lignin. Three peroxidase sequences sharing a similar structure with typical lignin peroxidase and manganese peroxidase were also found and annotated as haem-binding peroxidase, glutathione peroxidase and catalase-peroxidase. In this study, the fungus Aspergillus sydowii MS-19 was isolated and shown to synthesize low-temperature lignin-degrading enzymes: lignin peroxidase (Lip) and manganese peroxidase (Mnp). These findings provide useful information to improve our understanding of low-temperature lignocellulosic enzyme production by polar microorganisms and to facilitate research and

Bioprospecting and biotechnological applications of fungal laccase.

PubMed

Upadhyay, Pooja; Shrivastava, Rahul; Agrawal, Pavan Kumar

2016-06-01

Laccase belongs to a small group of enzymes called the blue multicopper oxidases, having the potential ability of oxidation. It belongs to enzymes, which have innate properties of reactive radical production, but its utilization in many fields has been ignored because of its unavailability in the commercial field. There are diverse sources of laccase producing organisms like bacteria, fungi and plants. In fungi, laccase is present in Ascomycetes, Deuteromycetes, Basidiomycetes and is particularly abundant in many white-rot fungi that degrade lignin. Laccases can degrade both phenolic and non-phenolic compounds. They also have the ability to detoxify a range of environmental pollutants. Due to their property to detoxify a range of pollutants, they have been used for several purposes in many industries including paper, pulp, textile and petrochemical industries. Some other application of laccase includes in food processing industry, medical and health care. Recently, laccase has found applications in other fields such as in the design of biosensors and nanotechnology. The present review provides an overview of biological functions of laccase, its mechanism of action, laccase mediator system, and various biotechnological applications of laccase obtained from endophytic fungi.

Blood tolerant laccase by directed evolution.

PubMed

Mate, Diana M; Gonzalez-Perez, David; Falk, Magnus; Kittl, Roman; Pita, Marcos; De Lacey, Antonio L; Ludwig, Roland; Shleev, Sergey; Alcalde, Miguel

2013-02-21

High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnology. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific composition of human blood (above neutral pH, high chloride concentration) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chemical syntheses, and detoxification. Copyright © 2013 Elsevier Ltd. All rights reserved.

Laccase/HBT and laccase-CBM/HBT treatment of softwood kraft pulp: impact on pulp bleachability and physical properties.

PubMed

Ravalason, Holy; Bertaud, Frédérique; Herpoël-Gimbert, Isabelle; Meyer, Valérie; Ruel, Katia; Joseleau, Jean-Paul; Grisel, Sacha; Olivé, Caroline; Sigoillot, Jean-Claude; Petit-Conil, Michel

2012-10-01

Pycnoporus cinnabarinus laccase and a chimeric laccase-CBM were applied in softwood kraft pulp biobleaching in the presence of 1-hydroxybenzotriazole (HBT). The presence of CBM could enhance the laccase biobleaching potential as a decrease in the enzymatic charge and chlorine dioxide consumption, as well as an increase in pulp brightness were observed. Laccase/HBT treatment could be improved by increasing oxygen pressure from 1 to 3bar and pulp consistency from 5% to 10%. Conversely, under the same conditions, no improvement of laccase-CBM/HBT treatment was observed, indicating a different behavior of both systems. However, laccase-CBM/HBT treatment led to a better preservation of pulp properties. This effect was probably due to fiber surface modifications involving the action of the CBM. Transmission electron microscopy examination of pulp fibers indicated a retention of laccase-CBM inside the pulp fibers due to CBM binding and an increased external microfibrillation of the fibers due to enzymatic treatments. Copyright © 2012 Elsevier Ltd. All rights reserved.

Lignin-degrading Peroxidases from Genome of Selective Ligninolytic Fungus Ceriporiopsis subvermispora*

PubMed Central

Fernández-Fueyo, Elena; Ruiz-Dueñas, Francisco J.; Miki, Yuta; Martínez, María Jesús; Hammel, Kenneth E.; Martínez, Angel T.

2012-01-01

The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora. PMID:22437835

Ligninases production by Basidiomycetes strains on lignocellulosic agricultural residues and their application in the decolorization of synthetic dyes

PubMed Central

Gomes, Eleni; Aguiar, Ana Paula; Carvalho, Caio César; Bonfá, Maricy Raquel B.; da Silva, Roberto; Boscolo, Mauricio

2009-01-01

Wood rotting Basidiomycetes collected in the “Estação Ecológica do Noroeste Paulista”, São José do Rio Preto, São Paulo State, Brazil, concerning Aphyllophorales order and identified as Coriolopsis byrsina SXS16, Lentinus strigellus SXS355, Lentinus sp SXS48, Picnoporus sanguineus SXS 43 and Phellinus rimosus SXS47 were tested for ligninases production by solid state fermentation (SSF) using wheat bran or rice straw as culture media. C. byrsina produced the highest laccase (200 U mL-1) and Lentinus sp produced the highest activities of manganese peroxidase (MnP) and lignin peroxidase (LiP) (7 and 8 U mL-1, respectively), when cultivated on wheat bran. The effect of N addition on enzyme production was studied in medium containing rice straw and the data showed an increase of 3 up to 4-fold in the laccase production compared to that obtained in SSF on wheat bran. The laccases presented optimum pH at 3.0-3.5 and were stable at neutral pH values. Optimum pH for MnP and LiP activities was at 3.5 and between 4.5 and 6.0, respectively. All the strains produced laccase with optimum activities between 55-60ºC while the peroxidases presented maximum activity at temperatures of 30 to 55ºC. The crude enzymes promoted decolorization of chemically different dyes with around 70% of decolorization of RBBR and cybacron blue 3GA in 6h of treatment. The data indicated that enzymes from these basidiomycetes strains are able to decolorize synthetic dyes. PMID:24031314

Construction and direct electrochemistry of orientation controlled laccase electrode

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ying; Zhang, Jiwei; Huang, Xirong, E-mail: xrhuang@sdu.edu.cn

2014-03-28

Highlights: • A recombinant laccase with Cys-6×His tag at the N or C terminus was generated. • Orientation controlled laccase electrodes were constructed via self assembly. • The electrochemical behavior of laccase electrodes was orientation dependent. • The C terminus tagged laccase was better for bioelectrocatalytic reduction of O{sub 2}. - Abstract: A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, usingmore » genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O{sub 2} reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells.« less

Laccases from Aureobasidium pullulans

USDA-ARS?s Scientific Manuscript database

Laccases are polyphenol oxidases (EC 1.10.3.2) that have numerous industrial and bioremediation applications. Laccases are well known as lignin-degrading enzymes, but these enzymes can play numerous other roles in fungi. In this study, 41 strains of the fungus Aureobasidium pullulans were examined f...

Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase.

PubMed

Garg, Neha; Bieler, Nora; Kenzom, Tenzin; Chhabra, Meenu; Ansorge-Schumacher, Marion; Mishra, Saroj

2012-10-23

Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase. In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg(-1) protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac. Laccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200

Secretome-based Manganese(II) Oxidation by Filamentous Ascomycete Fungi

NASA Astrophysics Data System (ADS)

Zeiner, C. A.; Purvine, S.; Zink, E.; Paša-Tolić, L.; Chaput, D.; Wu, S.; Santelli, C. M.; Hansel, C. M.

2017-12-01

Manganese (Mn) oxides are among the strongest oxidants in the environment, and Mn(II) oxidation to Mn(III/IV) (hydr)oxides includes both abiotic and microbially-mediated processes. While white-rot Basidiomycete fungi oxidize Mn(II) using laccases and Mn peroxidases in association with lignocellulose degradation, the mechanisms by which filamentous Ascomycete fungi oxidize Mn(II) and a physiological role for Mn(II) oxidation in these organisms remain poorly understood. Through a combination of chemical and in-gel assays, bulk mass spectrometry, and iTRAQ proteomics, we demonstrate enzymatic Mn(II) oxidation in the secretomes of three phylogenetically diverse Ascomycetes that were isolated from Mn-laden sediments. Candidate Mn(II)-oxidizing enzymes were species-specific and included bilirubin oxidase and tyrosinase in Stagonospora sp. SRC1lsM3a, GMC oxidoreductase in Paraconiothyrium sporulosum AP3s5-JAC2a, and FAD-binding oxidoreductases in Pyrenochaeta sp. DS3sAY3a. These findings were supported by full proteomic characterization of the secretomes, which revealed a lack of Mn, lignin, and versatile peroxidases in these Ascomycetes but a substantially higher proportion of LMCOs and GMC oxidoreductases compared to wood-rot Basidiomycetes. We also identified the potential for indirect enzymatic Mn(II) oxidation by hydroxyl radical, as the secretomes were rich in diverse lignocellulose-degrading enzymes that could participate in Fenton chemistry. A link between Mn(II) oxidation and carbon oxidation analogous to white-rot Basidiomycetes remains unknown in these Ascomycetes. Interestingly, growth rates on rich medium were unaffected by the presence of Mn(II), and the production of Mn(II)-oxidizing proteins in the secretome was constitutive and not inducible by Mn(II). Thus, no physiological benefit of Mn(II) oxidation in these Ascomycetes has yet been identified, and Mn(II) oxidation appears to be a side reaction. Future work will explore the lignin-degrading capacity of

Improved recovery of active recombinant laccase from maize seed.

PubMed

Bailey, M R; Woodard, S L; Callaway, E; Beifuss, K; Magallanes-Lundback, M; Lane, J R; Horn, M E; Mallubhotla, H; Delaney, D D; Ward, M; Van Gastel, F; Howard, J A; Hood, E E

2004-01-01

Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.

Application of ligninolytic potentials of a white-rot fungus Ganoderma lucidum for degradation of lindane.

PubMed

Kaur, Harsimran; Kapoor, Shammi; Kaur, Gaganjyot

2016-10-01

Lindane, a broad-spectrum organochlorine pesticide, has caused a widespread environmental contamination along with other pesticides due to wrong agricultural practices. The high efficiency, sustainability and eco-friendly nature of the bioremediation process provide an edge over traditional physico-chemical remediation for managing pesticide pollution. In the present study, lindane degradation was studied by using a white-rot fungus, Ganoderma lucidum GL-2 strain, grown on rice bran substrate for ligninolytic enzyme induction at 30 °C and pH 5.6 after incorporation of 4 and 40 ppm lindane in liquid as well as solid-state fermentation. The estimation of lindane residue was carried out by gas chromatography coupled to mass spectrometry (GC-MS) in the selected ion monitoring mode. In liquid-state fermentation, 100.13 U/ml laccase, 50.96 U/ml manganese peroxidase and 17.43 U/ml lignin peroxidase enzymes were obtained with a maximum of 75.50 % lindane degradation on the 28th day of incubation period, whereas under the solid-state fermentation system, 156.82 U/g laccase, 80.11 U/g manganese peroxidase and 18.61 U/g lignin peroxidase enzyme activities with 37.50 % lindane degradation were obtained. The lindane incorporation was inhibitory to the production of ligninolytic enzymes and its own degradation but was stimulatory for extracellular protein production. The dialysed crude enzyme extracts of ligninolytic enzymes were though efficient in lindane degradation during in vitro studies, but their efficiencies tend to decrease with an increase in the incubation period. Hence, lindane-degrading capabilities of G. lucidum GL-2 strain make it a potential candidate for managing lindane bioremediation at contaminated sites.

Chlorophenol degradation in soil columns inoculated with Anthracophyllum discolor immobilized on wheat grains.

PubMed

Diez, M C; Gallardo, F; Tortella, G; Rubilar, O; Navia, R; Bornhardt, C

2012-03-01

The white-rot fungus Anthracophyllum discolor immobilized on wheat grains was evaluated for chlorophenol (2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol) degradation in allophanic soil columns activated by acidification. Columns without inoculation were used as the control to evaluate the adsorption capacity of the soil columns. The chlorophenols were removed efficiently in soil columns by both adsorption and degradation processes. In inoculated soil columns, 2,4-dichlorophenol was highly degraded and this degradation is associated with a high production of manganese peroxidase. 2,4,6-trichlorophenol was degraded to a lesser extent compared with 2,4-dichlorophenol. Pentachlorophenol was first removed by adsorption and then through degradation by the fungus. Manganese peroxidase activity was lowest when the column was fed with pentachlorophenol and highest when the column was fed with 2,4-dichlorophenol. Laccase was also produced by the fungus but to a lesser degree. Copyright © 2010 Elsevier Ltd. All rights reserved.

Fungal Laccases: Production, Function, and Applications in Food Processing

PubMed Central

Brijwani, Khushal; Rigdon, Anne; Vadlani, Praveen V.

2010-01-01

Laccases are increasingly being used in food industry for production of cost-effective and healthy foods. To sustain this trend widespread availability of laccase and efficient production systems have to be developed. The present paper delineate the recent developments that have taken place in understanding the role of laccase action, efforts in overexpression of laccase in heterologous systems, and various cultivation techniques that have been developed to efficiently produce laccase at the industrial scale. The role of laccase in different food industries, particularly the recent developments in laccase application for food processing, is discussed. PMID:21048859

Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase

PubMed Central

2012-01-01

Background Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase. Results In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg-1 protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac. Conclusion Laccase of C. bulleri was successfully produced extra

Construction and direct electrochemistry of orientation controlled laccase electrode.

PubMed

Li, Ying; Zhang, Jiwei; Huang, Xirong; Wang, Tianhong

2014-03-28

A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, using genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O2 reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells. Copyright © 2014 Elsevier Inc. All rights reserved.

[Degradation of anthraquinone blue by Trametes trogii].

PubMed

Levin, L; Jordan, A; Forchiassin, F; Viale, A

2001-01-01

The ability of the white rot fungus Trametes trogii BAFC 463 (high producer of ligninolytic enzymes, especially laccase and manganese peroxidase) to degrade the dye anthraquinone blue, refractory to bacterial attack, was evaluated. Both tropho- and idiophasic T. trogii cultures in synthetic medium (glucose/asparagine) and complex medium (malt extract/glucose) were able to transform up to 88% dye in 4 hours. The activity of laccase, an oxygen-dependent phenoloxidase which was present at high levels in all the conditions assayed, might be related to the ability of the fungus to degrade the colorant. This is supported by the fact that in bioreactor experiences carried out at pH 4.5 the addition of anthraquinone blue caused a decrease in the levels of soluble oxygen. However, although high levels of laccase were produced at pH 7.5, the enzyme was not active, and neither dye transformation nor loss in the levels of soluble oxygen were quantified.

Decolourisation of mushroom farm wastewater by Pleurotus ostreatus.

PubMed

Rodríguez Pérez, Suyén; García Oduardo, Nora; Bermúdez Savón, Rosa C; Fernández Boizán, Maikel; Augur, Christopher

2008-07-01

Mushroom production on coffee pulp as substrate generates an intense black residual liquid, which requires suitable treatment. In the present study, Pleurotus ostreatus growth in wastewater from mushroom farm was evaluated as a potential biological treatment process for decolourisation as well as to obtain biomass (liquid inoculum). Culture medium components affecting mycelial growth were determined, evaluating colour removal. Laccase activity was monitored during the process. P. ostreatus was able to grow in non diluted WCP. Highest biomass yield was obtained when glucose (10 g/l) was added. The addition of this carbon source was necessary for efficient decolourisation. Agitation of the culture improved biodegradation of WCP as well as fungal biomass production. Laccase and manganese-independent peroxidase activities were detected during fungal treatment of the WCP by P. ostreatus CCEBI 3024. The laccase enzyme showed good correlation with colour loss. Both wastewater colour and pollution load (as chemical oxygen demand) decreased more than 50% after 10 days of culture. Phenols were reduced by 92%.

Laccase/Mediator Systems: Their Reactivity toward Phenolic Lignin Structures.

PubMed

Hilgers, Roelant; Vincken, Jean-Paul; Gruppen, Harry; Kabel, Mirjam A

2018-02-05

Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize the nonphenolic subunits of lignin (70-90% of the polymer). The phenolic subunits (10-30% of the polymer), which can also be oxidized without mediators, have received considerably less attention. Consequently, it remains unclear to what extent the presence of a mediator influences the reactions of the phenolic subunits of lignin. To get more insight in this, UHPLC-MS was used to study the reactions of a phenolic lignin dimer (GBG), initiated by a laccase from Trametes versicolor , alone or in combination with the mediators HBT and ABTS. The role of HBT was negligible, as its oxidation by laccase occurred slowly in comparison to that of GBG. Laccase and laccase/HBT oxidized GBG at a comparable rate, resulting in extensive polymerization of GBG. In contrast, laccase/ABTS converted GBG at a higher rate, as GBG was oxidized both directly by laccase but also by ABTS radical cations, which were rapidly formed by laccase. The laccase/ABTS system resulted in Cα oxidation of GBG and coupling of ABTS to GBG, rather than polymerization of GBG. Based on these results, we propose reaction pathways of phenolic lignin model compounds with laccase/HBT and laccase/ABTS.

Manganese availability is negatively associated with carbon storage in northern coniferous forest humus layers.

PubMed

Stendahl, Johan; Berg, Björn; Lindahl, Björn D

2017-11-14

Carbon sequestration below ground depends on organic matter input and decomposition, but regulatory bottlenecks remain unclear. The relative importance of plant production, climate and edaphic factors has to be elucidated to better predict carbon storage in forests. In Swedish forest soil inventory data from across the entire boreal latitudinal range (n = 2378), the concentration of exchangeable manganese was singled out as the strongest predictor (R 2  = 0.26) of carbon storage in the extensive organic horizon (mor layer), which accounts for one third of the total below ground carbon. In comparison, established ecosystem models applied on the same data have failed to predict carbon stocks (R 2  < 0.05), and in our study manganese availability overshadowed both litter production and climatic factors. We also identified exchangeable potassium as an additional strong predictor, however strongly correlated with manganese. The negative correlation between manganese and carbon highlights the importance of Mn-peroxidases in oxidative decomposition of recalcitrant organic matter. The results support the idea that the fungus-driven decomposition could be a critical factor regulating humus carbon accumulation in boreal forests, as Mn-peroxidases are specifically produced by basidiomycetes.

Expression of manganese peroxidase by Lentinula edodes and Lentinula boryana in solid state and submerged system fermentation.

PubMed

Hermann, Katia L; Costa, Alessandra; Helm, Cristiane V; De Lima, Edson A; Tavares, Lorena B B

2013-09-01

The production of ethanol from lignocellulosic biomass is referred as a second generation biofuel, whose processing is one of the most promising technologies under development. There are few available studies on the use of enzymes produced by fungi as active for the biodegradation of lignocellulosic biomass. However, the manganese peroxidase (MnP) enzyme presents high potential to degrade lignin and the basidiomycetes are the major producers of this oxidase. Thus, this study aimed at evaluating the ability of fungi Lentinula edodes and Lentinula boryana to produce this enzyme when cultivated in submerged fermentation system (SS) and also in solid-state fermentation system (SSF) containing Eucalyptus benthamii sawdust with or without corn cob meal. In the SS the greatest MnP expression occurred on the 25th day, being of 70 UI.L-1 for L. boryana and of 20 UI.L-1 for L. edodes. In the SSF, the best results were obtained on the 10th day for L. edodes, while for L. boryana it happened between the 20th and the 25th days, despite both species presented values close to 110 UI.L-1. Therefore, the results indicated that the studied fungi express the enzyme of interest and that its production is enhanced when cultivated in solid system.

Automated chromatographic laccase-mediator-system activity assay.

PubMed

Anders, Nico; Schelden, Maximilian; Roth, Simon; Spiess, Antje C

2017-08-01

To study the interaction of laccases, mediators, and substrates in laccase-mediator systems (LMS), an on-line measurement was developed using high performance anion exchange chromatography equipped with a CarboPac™ PA 100 column coupled to pulsed amperometric detection (HPAEC-PAD). The developed method was optimized for overall chromatographic run time (45 to 120 min) and automated sample drawing. As an example, the Trametes versicolor laccase induced oxidation of 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane (adlerol) using 1-hydroxybenzotriazole (HBT) as mediator was measured and analyzed on-line. Since the Au electrode of the PAD detects only hydroxyl group containing substances with a limit of detection being in the milligram/liter range, not all products are measureable. Therefore, this method was applied for the quantification of adlerol, and-based on adlerol conversion-for the quantification of the LMS activity at a specific T. versicolor laccase/HBT ratio. The automated chromatographic activity assay allowed for a defined reaction start of all laccase-mediator-system reactions mixtures, and the LMS reaction progress was automatically monitored for 48 h. The automatization enabled an integrated monitoring overnight and over-weekend and minimized all manual errors such as pipetting of solutions accordingly. The activity of the LMS based on adlerol consumption was determined to 0.47 U/mg protein for a laccase/mediator ratio of 1.75 U laccase/g HBT. In the future, the automated method will allow for a fast screening of combinations of laccases, mediators, and substrates which are efficient for lignin modification. In particular, it allows for a fast and easy quantification of the oxidizing activity of an LMS on a lignin-related substrate which is not covered by typical colorimetric laccase assays. ᅟ.

Structural insights into 2,2'-azino-Bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)-mediated degradation of reactive blue 21 by engineered Cyathus bulleri Laccase and characterization of degradation products.

PubMed

Kenzom, T; Srivastava, P; Mishra, S

2014-12-01

Advanced oxidation processes are currently used for the treatment of different reactive dyes which involve use of toxic catalysts. Peroxidases are reported to be effective on such dyes and require hydrogen peroxide and/or metal ions. Cyathus bulleri laccase, expressed in Pichia pastoris, catalyzes efficient degradation (78 to 85%) of reactive azo dyes (reactive black 5, reactive orange 16, and reactive red 198) in the presence of synthetic mediator ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]. This laccase was engineered to degrade effectively reactive blue 21 (RB21), a phthalocyanine dye reported to be decolorized only by peroxidases. The 816-bp segment (toward the C terminus) of the lcc gene was subjected to random mutagenesis and enzyme variants (Lcc35, Lcc61, and Lcc62) were selected based on increased ABTS oxidizing ability. Around 78 to 95% decolorization of RB21 was observed with the ABTS-supplemented Lcc variants in 30 min. Analysis of the degradation products by mass spectrometry indicated the formation of several low-molecular-weight compounds. Mapping the mutations on the modeled structure implicated residues both near and far from the T1 Cu site that affected the catalytic efficiency of the mutant enzymes on ABTS and, in turn, the rate of oxidation of RB21. Several inactive clones were also mapped. The importance of geometry as well as electronic changes on the reactivity of laccases was indicated. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Fungal biodegradation and enzymatic modification of lignin

PubMed Central

Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng

2010-01-01

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed. PMID:21968746

Structural Insights into 2,2′-Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS)-Mediated Degradation of Reactive Blue 21 by Engineered Cyathus bulleri Laccase and Characterization of Degradation Products

PubMed Central

Kenzom, T.; Srivastava, P.

2014-01-01

Advanced oxidation processes are currently used for the treatment of different reactive dyes which involve use of toxic catalysts. Peroxidases are reported to be effective on such dyes and require hydrogen peroxide and/or metal ions. Cyathus bulleri laccase, expressed in Pichia pastoris, catalyzes efficient degradation (78 to 85%) of reactive azo dyes (reactive black 5, reactive orange 16, and reactive red 198) in the presence of synthetic mediator ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]. This laccase was engineered to degrade effectively reactive blue 21 (RB21), a phthalocyanine dye reported to be decolorized only by peroxidases. The 816-bp segment (toward the C terminus) of the lcc gene was subjected to random mutagenesis and enzyme variants (Lcc35, Lcc61, and Lcc62) were selected based on increased ABTS oxidizing ability. Around 78 to 95% decolorization of RB21 was observed with the ABTS-supplemented Lcc variants in 30 min. Analysis of the degradation products by mass spectrometry indicated the formation of several low-molecular-weight compounds. Mapping the mutations on the modeled structure implicated residues both near and far from the T1 Cu site that affected the catalytic efficiency of the mutant enzymes on ABTS and, in turn, the rate of oxidation of RB21. Several inactive clones were also mapped. The importance of geometry as well as electronic changes on the reactivity of laccases was indicated. PMID:25261507

Laccases: Production, Expression Regulation, and Applications in Pharmaceutical Biodegradation

PubMed Central

Yang, Jie; Li, Wenjuan; Ng, Tzi Bun; Deng, Xiangzhen; Lin, Juan; Ye, Xiuyun

2017-01-01

Laccases are a family of copper-containing oxidases with important applications in bioremediation and other various industrial and biotechnological areas. There have been over two dozen reviews on laccases since 2010 covering various aspects of this group of versatile enzymes, from their occurrence, biochemical properties, and expression to immobilization and applications. This review is not intended to be all-encompassing; instead, we highlighted some of the latest developments in basic and applied laccase research with an emphasis on laccase-mediated bioremediation of pharmaceuticals, especially antibiotics. Pharmaceuticals are a broad class of emerging organic contaminants that are recalcitrant and prevalent. The recent surge in the relevant literature justifies a short review on the topic. Since low laccase yields in natural and genetically modified hosts constitute a bottleneck to industrial-scale applications, we also accentuated a genus of laccase-producing white-rot fungi, Cerrena, and included a discussion with regards to regulation of laccase expression. PMID:28559880

Engineering and Applications of fungal laccases for organic synthesis

PubMed Central

Kunamneni, Adinarayana; Camarero, Susana; García-Burgos, Carlos; Plou, Francisco J; Ballesteros, Antonio; Alcalde, Miguel

2008-01-01

Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis. Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed. PMID:19019256

Laccase versus Laccase-Like Multi-Copper Oxidase: A Comparative Study of Similar Enzymes with Diverse Substrate Spectra

PubMed Central

Reiss, Renate; Ihssen, Julian; Richter, Michael; Eichhorn, Eric; Schilling, Boris; Thöny-Meyer, Linda

2013-01-01

Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Owing to their broad substrate range, copper-containing laccases are versatile biocatalysts, capable of oxidizing numerous natural and non-natural industry-relevant compounds, with water as the sole by-product. In the present study, 10 of the 11 multi-copper oxidases, hitherto considered to be laccases, from fungi, plant and bacterial origin were compared. A substrate screen of 91 natural and non-natural compounds was recorded and revealed a fairly broad but distinctive substrate spectrum amongst the enzymes. Even though the enzymes share conserved active site residues we found that the substrate ranges of the individual enzymes varied considerably. The EC classification is based on the type of chemical reaction performed and the actual name of the enzyme often refers to the physiological substrate. However, for the enzymes studied in this work such classification is not feasible, even more so as their prime substrates or natural functions are mainly unknown. The classification of multi-copper oxidases assigned as laccases remains a challenge. For the sake of simplicity we propose to introduce the term “laccase-like multi-copper oxidase” (LMCO) in addition to the term laccase that we use exclusively for the enzyme originally identified from the sap of the lacquer tree Rhus vernicifera. PMID:23755261

[Advance of heterologous expression study of eukaryote-origin laccases].

PubMed

Ning, Na; Tan, Huijun; Sun, Xinxin; Ni, Jinfeng

2017-04-25

Laccases are enzymes belonging to the group of multi-copper oxidases. These enzymes are widely distributed in insects, plants, fungi and bacteria. In general, laccases can oxidize an exceptionally high number of substrates, so they have broad applications in textile, pulp, food and the degradation of lignin. However, low yield, low activity and thermo-instability of laccase in nature limit the application of laccase. High efficient heterologous expression of the protein is an effective way for solving this problem. Here, we summarize the research advances of heterologous expression of eukaryote-origin laccases. We focus on the overexpression of eukaryote-origin laccases using different expression system and the method for improving the production yield and enzyme activity in yeast cells. Information provided in this review would be helpful for researchers in the field.

Expression of the Laccase Gene from a White Rot Fungus in Pichia pastoris Can Enhance the Resistance of This Yeast to H2O2-Mediated Oxidative Stress by Stimulating the Glutathione-Based Antioxidative System

PubMed Central

Fan, Fangfang; Zhuo, Rui; Ma, Fuying; Gong, Yangmin; Wan, Xia; Jiang, Mulan

2012-01-01

Laccase is a copper-containing polyphenol oxidase that has great potential in industrial and biotechnological applications. Previous research has suggested that fungal laccase may be involved in the defense against oxidative stress, but there is little direct evidence supporting this hypothesis, and the mechanism by which laccase protects cells from oxidative stress also remains unclear. Here, we report that the expression of the laccase gene from white rot fungus in Pichia pastoris can significantly enhance the resistance of yeast to H2O2-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H2O2 and to protect cells from lipid oxidative damage. The mechanism by which laccase gene expression increases resistance to oxidative stress was then investigated further. We found that laccase gene expression in Pichia pastoris could increase the level of glutathione-based antioxidative activity, including the intracellular glutathione levels and the enzymatic activity of glutathione peroxidase, glutathione reductase, and γ-glutamylcysteine synthetase. The transcription of the laccase gene in Pichia pastoris was found to be enhanced by the oxidative stress caused by exogenous H2O2. The stimulation of laccase gene expression in response to exogenous H2O2 stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system, including PpYAP1, PpGPX1, PpPMP20, PpGLR1, and PpGSH1. Taken together, these results suggest that the expression of the laccase gene in Pichia pastoris can enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage. PMID:22706050

Characterization and decolorization applicability of xerogel matrix immobilized manganese peroxidase produced from Trametes versicolor IBL-04.

PubMed

Iqbal, Hafiz Muhammad Nasir; Asgher, Muhammad

2013-05-01

A novel manganese peroxidase (MnP) isolated from solid state culture of Trametes versicolor IBL-04 was immobilized using xerogel matrix composed of trimethoxysilane (TMOS) and propyltetramethoxysilane (PTMS). FTIR spectroscopy confirmed the successful entrapment of MnP into the xerogel matrix. An immobilization efficiency of 92.2% was achieved with a purified active fraction containing 2 mg/mL MnP. After 24 h incubation at varying pH and temperatures, the immobilized MnP retained 82 and 75% activity at pH 4 and 80°C, respectively. Xerogel matrix immobilization enhanced the catalytic efficiency of entrapped MnP. Metal ions including Cu2+, Mn2+ and Fe2+ stimulated enzyme activity while cysteine, EDTA and Ag+ inhibited the activity. MnP preserved 82% of its initial activity during oxidation of MnSO4 in 10 consecutive cycles, demonstrating the reusability of xerogel entrapped MnP. The immobilized MnP could be stored for up to 75 days at 4°C without significant activity loss. To explore the industrial applicability of MnP, the immobilized MnP was tested for decolorization of textile industry effluent in a Packed Bed Reactor System (PBRS). After five consecutive cycles, 98.8% decolorization of effluent was achieved within 5 h. The kinetic properties, storage stability and reusability of entrapped MnP from T. versicolor IBL-04 reflect its prospects as biocatalyst for bioremediation and other industrial applications.

Synthesis of novel laccase-biotitania biocatalysts for malachite green decolorization.

PubMed

Zhang, Xinying; Wang, Meiyin; Lin, Linlin; Xiao, Gao; Tang, Zhenping; Zhu, Xuefeng

2018-07-01

Biomimetic mineralization has emerged as a novel tool for generating excellent supports for enzyme stabilization. In this work, protamine was used to induce titanium (IV) bis(ammonium lactato) dihydroxide (Ti-BALDH) into titania nanoparticles. This biomimetic titanification process was adopted for laccase immobilization. Laccase-biotitania biocatalyst was prepared and the effect of different parameters (buffer solution, titania precursor concentration, protamine concentration, and enzyme loading) on the encapsulation efficiency and recovery of laccase were evaluated. Compared with free laccase, the thermal and pH stability of immobilized laccase were improved significantly. In addition, laccase loaded on titania was effective at enhancing its storage stability. After seven consecutive cycles, the immobilized laccase still retained 51% of its original activity. Finally, laccase-biotitania biocatalysts showed good performance on decolorization of malachite green (MG), which can be attributed to an adsorption and degradation effect. The intermediates of the MG degradation were identified by gas chromatography-mass spectrometry (GC-MS) analysis, and the most probable degradation pathway was proposed. This study provides deeper understanding of the laccase-biotitania particles as a fast biocatalyst for MG decolorization. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Production of laccase by Coriolus versicolor and its application in decolorization of dyestuffs: (I). Production of laccase by batch and repeated-batch processes.

PubMed

Lin, Jian-Ping; Wei, Lian; Xia, Li-Ming; Cen, Pei-Lin

2003-01-01

The production of laccase by Coriolus versicolor was studied. The effect of cultivation conditions on laccase production by Coriolus versicolor was examined to obtain optimal medium and cultivation conditions. Both batch and repeated-batch processes were performed for laccase production. In repeated-batch fermentation with self-immobilized mycelia, total of 14 cycles were performed with laccase activity in the range between 3.4 and 14.8 U/ml.

Engineering laccases: in search for novel catalysts.

PubMed

Robert, Viviane; Mekmouche, Yasmina; Pailley, Pierre R; Tron, Thierry

2011-04-01

Laccases (p-diphenol oxidase, EC 1.10.3.2) are blue multicopper oxidases that catalyze the reduction of dioxygen to water, with a concomitant oxidation of small organic substrates. Since the description at the end of the nineteenth century of a factor catalyzing the rapid hardening of the latex of the Japanese lacquer trees (Rhus sp.) exposed to air laccases from different origins (plants, fungi bacteria) have been continuously discovered and extensively studied. Nowadays, molecular evolution and other powerful protein modification techniques offer possibilities to develop tailored laccases for a wide array of applications including drug synthesis, biosensors or biofuel cells. Here, we give an overview on strategies and results of our laboratory in the design of new biocatalysts based on laccases.

Engineering Laccases: In Search for Novel Catalysts

PubMed Central

Robert, Viviane; Mekmouche, Yasmina; Pailley, Pierre R; Tron, Thierry

2011-01-01

Laccases (p-diphenol oxidase, EC 1.10.3.2) are blue multicopper oxidases that catalyze the reduction of dioxygen to water, with a concomitant oxidation of small organic substrates. Since the description at the end of the nineteenth century of a factor catalyzing the rapid hardening of the latex of the Japanese lacquer trees (Rhus sp.) exposed to air laccases from different origins (plants, fungi bacteria) have been continuously discovered and extensively studied. Nowadays, molecular evolution and other powerful protein modification techniques offer possibilities to develop tailored laccases for a wide array of applications including drug synthesis, biosensors or biofuel cells. Here, we give an overview on strategies and results of our laboratory in the design of new biocatalysts based on laccases. PMID:21966250

Fungal Laccases and Their Applications in Bioremediation

PubMed Central

Viswanath, Buddolla; Rajesh, Bandi; Janardhan, Avilala; Kumar, Arthala Praveen; Narasimha, Golla

2014-01-01

Laccases are blue multicopper oxidases, which catalyze the monoelectronic oxidation of a broad spectrum of substrates, for example, ortho- and para-diphenols, polyphenols, aminophenols, and aromatic or aliphatic amines, coupled with a full, four-electron reduction of O2 to H2O. Hence, they are capable of degrading lignin and are present abundantly in many white-rot fungi. Laccases decolorize and detoxify the industrial effluents and help in wastewater treatment. They act on both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and they can be effectively used in paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation and act as biosensors. Recently, laccase has been applied to nanobiotechnology, which is an increasing research field, and catalyzes electron transfer reactions without additional cofactors. Several techniques have been developed for the immobilization of biomolecule such as micropatterning, self-assembled monolayer, and layer-by-layer techniques, which immobilize laccase and preserve their enzymatic activity. In this review, we describe the fungal source of laccases and their application in environment protection. PMID:24959348

Heterologous expression of Trametes versicolor laccase in Saccharomyces cerevisiae.

PubMed

Iimura, Yosuke; Sonoki, Tomonori; Habe, Hiroshi

2018-01-01

Laccase is used in various industrial fields, and it has been the subject of numerous studies. Trametes versicolor laccase has one of the highest redox potentials among the various forms of this enzyme. In this study, we optimized the expression of laccase in Saccharomyces cerevisiae. Optimizing the culture conditions resulted in an improvement in the expression level, and approximately 45 U/L of laccase was functionally secreted in the culture. The recombinant laccase was found to be a heavily hypermannosylated glycoprotein, and the molecular weight of the carbohydrate chain was approximately 60 kDa. These hypermannosylated glycans lowered the substrate affinity, but the optimum pH and thermo-stability were not changed by these hypermannosylated glycans. This functional expression system described here will aid in molecular evolutionary studies conducted to generate new variants of laccase. Copyright © 2017 Elsevier Inc. All rights reserved.

Enhancing the laccase production and laccase gene expression in the white-rot fungus Trametes velutina 5930 with great potential for biotechnological applications by different metal ions and aromatic compounds.

PubMed

Yang, Yang; Wei, Fuxiang; Zhuo, Rui; Fan, Fangfang; Liu, Huahua; Zhang, Chen; Ma, Li; Jiang, Mulan; Zhang, Xiaoyu

2013-01-01

Laccase is useful for various biotechnological and industrial applications. The white-rot fungus Trametes velutina 5930 and its laccase, isolated from the Shennongjia Nature Reserve in China by our laboratory, has great potential for practical application in environmental biotechnology. However, the original level of laccase produced by Trametes velutina 5930 was relatively low in the absence of any inducer. Therefore, in order to enhance the laccase production by Trametes velutina 5930 and make better use of this fungus in the field of environmental biotechnology, the regulation of laccase production and laccase gene expression in Trametes velutina 5930 were investigated in this study. Different metal ions such as Cu(2+) and Fe(2+) could stimulate the laccase synthesis and laccase gene transcription in Trametes velutina 5930. Some aromatic compounds structurally related to lignin, such as tannic acid, syringic acid, cinnamic acid, gallic acid and guaiacol, could also enhance the level of laccase activity and laccase gene transcription. We also found that there existed a positive synergistic effect of aromatic compound and metal ion on the laccase production and laccase gene transcription in Trametes velutina 5930. Taken together, our study may contribute to the improvement of laccase productivity by Trametes velutina 5930.

Conditions Optimizing and Application of Laccase-mediator System (LMS) for the Laccase-catalyzed Pesticide Degradation

PubMed Central

Jin, Xiaoting; Yu, Xiangyang; Zhu, Guangyan; Zheng, Zuntao; Feng, Fayun; Zhang, Zhiyong

2016-01-01

A high capacity of laccase from Trametes versicolor capable of degrading pesticides has been revealed. The conditions for degrading of five selected pesticides including chlorpyrifos, chlorothalonil, pyrimethanil, atrazine and isoproturon with the purified laccases from Trametes versicolor were optimized. The results showed that the optimum conditions for the highest activity were pH at 5.0 and temperature at 25 °C. The best mediators were violuric acid for pyrimethanil and isoproturon, vanillin for chlorpyrifos, and acetosyringone and HBT for chlorothalonil and atrazine, respectively. The laccase was found to be stable at a pH range from 5.0 to 7.0 and temperature from 25 to 30 °C. It was observed that each pesticide required a different laccase mediator concentration typically between 4.0–6.0 mmol/L. In the experiment, the degradation rates of pyrimethanil and isoproturon were significantly faster than those of chlorpyrifos, chlorothalonil and atrazine. For example, it was observed that pyrimethanil and isoproturon degraded up to nearly 100% after 24 hours while the other three pesticides just reached up 90% of degradation after 8 days of incubation. PMID:27775052

Conditions Optimizing and Application of Laccase-mediator System (LMS) for the Laccase-catalyzed Pesticide Degradation.

PubMed

Jin, Xiaoting; Yu, Xiangyang; Zhu, Guangyan; Zheng, Zuntao; Feng, Fayun; Zhang, Zhiyong

2016-10-24

A high capacity of laccase from Trametes versicolor capable of degrading pesticides has been revealed. The conditions for degrading of five selected pesticides including chlorpyrifos, chlorothalonil, pyrimethanil, atrazine and isoproturon with the purified laccases from Trametes versicolor were optimized. The results showed that the optimum conditions for the highest activity were pH at 5.0 and temperature at 25 °C. The best mediators were violuric acid for pyrimethanil and isoproturon, vanillin for chlorpyrifos, and acetosyringone and HBT for chlorothalonil and atrazine, respectively. The laccase was found to be stable at a pH range from 5.0 to 7.0 and temperature from 25 to 30 °C. It was observed that each pesticide required a different laccase mediator concentration typically between 4.0-6.0 mmol/L. In the experiment, the degradation rates of pyrimethanil and isoproturon were significantly faster than those of chlorpyrifos, chlorothalonil and atrazine. For example, it was observed that pyrimethanil and isoproturon degraded up to nearly 100% after 24 hours while the other three pesticides just reached up 90% of degradation after 8 days of incubation.

Conditions Optimizing and Application of Laccase-mediator System (LMS) for the Laccase-catalyzed Pesticide Degradation

NASA Astrophysics Data System (ADS)

Jin, Xiaoting; Yu, Xiangyang; Zhu, Guangyan; Zheng, Zuntao; Feng, Fayun; Zhang, Zhiyong

2016-10-01

A high capacity of laccase from Trametes versicolor capable of degrading pesticides has been revealed. The conditions for degrading of five selected pesticides including chlorpyrifos, chlorothalonil, pyrimethanil, atrazine and isoproturon with the purified laccases from Trametes versicolor were optimized. The results showed that the optimum conditions for the highest activity were pH at 5.0 and temperature at 25 °C. The best mediators were violuric acid for pyrimethanil and isoproturon, vanillin for chlorpyrifos, and acetosyringone and HBT for chlorothalonil and atrazine, respectively. The laccase was found to be stable at a pH range from 5.0 to 7.0 and temperature from 25 to 30 °C. It was observed that each pesticide required a different laccase mediator concentration typically between 4.0-6.0 mmol/L. In the experiment, the degradation rates of pyrimethanil and isoproturon were significantly faster than those of chlorpyrifos, chlorothalonil and atrazine. For example, it was observed that pyrimethanil and isoproturon degraded up to nearly 100% after 24 hours while the other three pesticides just reached up 90% of degradation after 8 days of incubation.

Lignin-degrading peroxidases in white-rot fungus Trametes hirsuta 072. Absolute expression quantification of full multigene family

PubMed Central

Vasina, Daria V.; Moiseenko, Konstantin V.; Fedorova, Tatiana V.; Tyazhelova, Tatiana V.

2017-01-01

Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle. PMID:28301519

Applications and Prospective of Peroxidase Biocatalysis in the Environmental Field

NASA Astrophysics Data System (ADS)

Torres-Duarte, Cristina; Vazquez-Duhalt, Rafael

Environmental protection is, doubtless, one of the most important challenges for the human kind. The huge amount of pollutants derived from industrial activities represents a threat for the environment and ecologic equilibrium. Phenols and halogenated phenols, polycyclic aromatic hydrocarbons, endocrine disruptive chemicals, pesticides, dioxins, polychlorinated biphenyls, industrial dyes, and other xenobiotics are among the most important pollutants. A large variety of these xenobiotics are substrates for peroxidases and thus susceptible to enzymatic transformation. The literature reports mainly the use of horseradish peroxidase, manganese peroxidase, lignin peroxidase, and chloroperoxidase on the transformation of these pollutants. Peroxidases are enzymes able to transform a variety of compounds following a free radical mechanism, giving oxidized or polymerized products. The peroxidase transformation of these pollutants is accompanied by a reduction in their toxicity, due to a biological activity loss, a reduction in the bioavailability or due to the removal from aqueous phase, especially when the pollutant is found in water. In addition, when the pollutants are present in soil, peroxidases catalyze a covalent binding to soil organic matter. In most of cases, oxidized products are less toxic and easily biodegradable than the parent compounds. In spite of their versatility and potential use in environmental processes, peroxidases are not applied at large scale yet. Diverse challenges, such as stability, redox potential, and the production of large amounts, should be solved in order to apply peroxidases in the pollutant transformation. In this chapter, we critically review the transformation of different xenobiotics by peroxidases, with special attention on the identified transformation products, the probable reaction mechanisms, and the toxicity reports. Finally, the design and development of an environmental biocatalyst is discussed. The design challenges are

Dye decolorization and detoxification potential of Ca-alginate beads immobilized manganese peroxidase.

PubMed

Bilal, Muhammad; Asgher, Muhammad

2015-12-10

In view of compliance with increasingly stringent environmental legislation, an eco-friendly treatment technology of industrial dyes and effluents is a major environmental challenge in the color industry. In present study, a promising and eco-friendly entrapment approach was adopted to immobilize purified manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum IBL-05 on Ca-alginate beads. The immobilized MnP was subsequently used for enhanced decolorization and detoxification of textile reactive dyes). MnP isolated from solid-state culture of G. lucidum IBL-05, presented highest immobilization yield (83.9 %) using alginate beads prepared at optimized conditions of 4 % (w/v) sodium alginate, 2 % (w/v) Calcium chloride (CaCl2) and 0.5 mg/ml enzyme concentration. Immobilization of MnP enhanced optimum temperature but caused acidic shift in optimum pH of the enzyme. The immobilized MnP showed optimum activity at pH 4.0 and 60 °C as compared to pH 5.0 and 35 °C for free enzyme. The kinetic parameters K(m) and V(max) of MnP were significantly improved by immobilization. The enhanced catalytic potential of immobilized MnP led to 87.5 %, 82.1 %, 89.4 %, 95.7 % and 83 % decolorization of Sandal-fix Red C4BLN, Sandal-fix Turq Blue GWF, Sandal-fix Foron Blue E2BLN, Sandal-fix Black CKF and Sandal-fix Golden Yellow CRL dyes, respectively. The insolubilized MnP was reusable for 7 repeated cycles in dye color removal. Furthermore, immobilized MnP also caused a significant reduction in biochemical oxygen demand (BOD) (94.61-95.47 %), chemical oxygen demand (COD) (91.18-94.85 %), and total organic carbon (TOC) (89.58-95 %) of aqueous dye solutions. G. lucidum MnP was immobilized in Ca-alginate beads by entrapment method to improve its practical effectiveness. Ca-alginate bound MnP was catalytically more vigorous, thermo-stable, reusable and worked over wider ranges of pH and temperature as compared to its free counterpart. Results of cytotoxicity like

Heterologous laccase production and its role in industrial applications

PubMed Central

Pezzella, Cinzia; Giardina, Paola; Faraco, Vincenza; Sannia, Giovanni

2010-01-01

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry. PMID:21327057

Lignin oxidation and pulp delignification by laccase and mediators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bourbonnais, R.; Paice, M.G.; Reid, I.D.

1996-10-01

The phenol oxidizing enzyme laccase is produced abundantly by the lignin-degrading fungus Trametes versicolor. We found previously that laccase can oxidize veratryl alcohol and other non-phenolic lignin model compounds when a mediator such as 2,2{prime}-azinobis(3-ethylbenzthiazoline-5-sulphonate) (ABTS) was present. The laccase/mediator couple was also shown to be effective for delignification of kraft pulps. Two different isozymes of laccase produced by this fungus were purified and their reactivities towards lignins and kraft pulps were studied. The mediator ABTS was shown to be essential for pulp delignification and to reverse the polymerization of kraft lignin by either laccase. Pulp delignification with laccase andmore » ABTS was also optimized. resulting in up to 55% lignin removal from kraft pulp following sequential enzyme treatments and alkaline extractions. Several variables were surveyed including enzyme and mediator dosage, oxygen pressure, temperature, reaction time, and pH.« less

Roles of small laccases from Streptomyces in lignin degradation.

PubMed

Majumdar, Sudipta; Lukk, Tiit; Solbiati, Jose O; Bauer, Stefan; Nair, Satish K; Cronan, John E; Gerlt, John A

2014-06-24

Laccases (EC 1.10.3.2) are multicopper oxidases that can oxidize a range of substrates, including phenols, aromatic amines, and nonphenolic substrates. To investigate the involvement of the small Streptomyces laccases in lignin degradation, we generated acid-precipitable polymeric lignin obtained in the presence of wild-type Streptomyces coelicolor A3(2) (SCWT) and its laccase-less mutant (SCΔLAC) in the presence of Miscanthus x giganteus lignocellulose. The results showed that strain SCΔLAC was inefficient in degrading lignin compared to strain SCWT, thereby supporting the importance of laccase for lignin degradation by S. coelicolor A3(2). We also studied the lignin degradation activity of laccases from S. coelicolor A3(2), Streptomyces lividans TK24, Streptomyces viridosporus T7A, and Amycolatopsis sp. 75iv2 using both lignin model compounds and ethanosolv lignin. All four laccases degraded a phenolic model compound (LM-OH) but were able to oxidize a nonphenolic model compound only in the presence of redox mediators. Their activities are highest at pH 8.0 with a low krel/Kapp for LM-OH, suggesting that the enzymes’ natural substrates must be different in shape or chemical nature. Crystal structures of the laccases from S. viridosporus T7A (SVLAC) and Amycolatopsis sp. 75iv2 were determined both with and without bound substrate. This is the first report of a crystal structure for any laccase bound to a nonphenolic β-O-4 lignin model compound. An additional zinc metal binding site in SVLAC was also identified. The ability to oxidize and/or rearrange ethanosolv lignin provides further evidence of the utility of laccase activity for lignin degradation and/or modification.

Trametes meyenii possesses elevated dye degradation abilities under normal nutritional conditions compared to other white rot fungi

PubMed Central

2014-01-01

Several species of white-rot fungi were investigated for their utility in prolonged decolouration of the recalcitrant sulfonated azo dye, amaranth. Trametes pubescens, T. multicolor, T. meyenii and T. versicolor decoloured amaranth azo-dye best on low-nitrogen agar-solidified media whereas Bjerkandera adusta and Phlebia radiata were most effective in low nitrogen medium supplemented with manganese. Trametes cotonea did not decolour effectively under any condition. The decolouring Trametes species were also effective in liquid culture whereas B. adusta and P. radiata were not. Trametes meyenii, T. pubescens and T. multicolor were equal to or better than commonly employed T. versicolor at decolouring amaranth. This is the first study to show the dye decolouration potential of T. meyenii, T. pubescens, and T. multicolor. Supplementing with Mn(II) increased assayable manganese peroxidase activity, but not long-term decolouration, indicating that laccase is the main decolourizing enzyme in these Trametes species. This appears to be because of inadequate Mn3+ chelation required by manganese peroxidase because adding relatively low amounts of malonate enhanced decolouration rates. The ability of Trametes meyenii to simultaneously decolour dye over prolonged periods of time while growing in relatively nutrient-rich medium appears to be unique amongst white-rot fungi, indicating its potential in wastewater bioremediation. PMID:25401075

Screening for ligninolytic enzymes from autochthonous fungi and applications for decolorization of Remazole Marine Blue

PubMed Central

Erden, Emre; Ucar, M. Cigdem; Gezer, Tekin; Pazarlioglu, Nurdan Kasikara

2009-01-01

This study presents new and alternative fungal strains for the production of ligninolytic enzymes which have great potential to use in industrial and biotechnological processes. Thirty autochthonous fungal strains were harvested from Bornova-Izmir in Turkiye. In the fresh fruitbody extracts laccase, manganese peroxidase and lignin peroxidase activities, which are the principal enzymes responsible for ligninocellulose degradation by Basidiomycetes, were screened. Spores of some of the basidiomycetes species such as Cortinarius sp., Trametes versicolor, Pleurotus ostreatus, Abortiporus biennis, Lyophyllum subglobisporium, Ramaria stricta, Ganoderma carnosum, Lactarius delicious ve Lepista nuda were isolated and investigated optimum cultivation conditions in submerged fermentation for high yields of ligninolytic enzyme production. In addition, isolated fungal strains were monitored on agar plates whether having the capability of decolorization of a textile dye Remazol Marine Blue. PMID:24031371

Uses of Laccases in the Food Industry

PubMed Central

Osma, Johann F.; Toca-Herrera, José L.; Rodríguez-Couto, Susana

2010-01-01

Laccases are an interesting group of multi copper enzymes, which have received much attention of researchers in the last decades due to their ability to oxidise both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. This makes these biocatalysts very useful for their application in several biotechnological processes, including the food industry. Thus, laccases hold great potential as food additives in food and beverage processing. Being energy-saving and biodegradable, laccase-based biocatalysts fit well with the development of highly efficient, sustainable, and eco-friendly industries. PMID:21048873

Heat shock treatment improves Trametes versicolor laccase production.

PubMed

Wang, Feng; Guo, Chen; Wei, Tao; Zhang, Tian; Liu, Chun-Zhao

2012-09-01

An efficient heat shock strategy has been developed to improve laccase production in submerged Trametes versicolor cultures. The optimized heat shock strategy consists of subjecting T. versicolor mycelial pellets to three heat shock treatments at 45 °C for 45 min, starting at culture day 0, with a 24-h interval between treatments. Laccase production increased by more than 1.6-fold relative to the control in both flasks and a 5-L bioreactor because the expression of the laccase gene was enhanced by heat shock induction. The present work demonstrates that heat shock induction is a promising method because it both improves fungal laccase production and has a good potential in industrial application.

A novel Laccase Biosensor based on Laccase immobilized Graphene-Cellulose Microfiber Composite modified Screen-Printed Carbon Electrode for Sensitive Determination of Catechol

PubMed Central

Palanisamy, Selvakumar; Ramaraj, Sayee Kannan; Chen, Shen-Ming; Yang, Thomas C. K.; Yi-Fan, Pan; Chen, Tse-Wei; Velusamy, Vijayalakshmi; Selvam, Sonadevi

2017-01-01

In the present work, we demonstrate the fabrication of laccase biosensor to detect the catechol (CC) using laccase immobilized on graphene-cellulose microfibers (GR-CMF) composite modified screen printed carbon electrode (SPCE). The direct electrochemical behavior of laccase was investigated using laccase immobilized different modified SPCEs, such as GR/SPCE, CMF/SPCE and GR-CMF/SPCE. Compared with laccase immobilized GR and CMF modified SPCEs, a well-defined redox couple of CuI/CuII for laccase was observed at laccase immobilized GR-CMF composite modified SPCE. Cyclic voltammetry results show that the as-prepared biosensor has 7 folds higher catalytic activity with lower oxidation potential towards CC than SPCE modified with GR-CMF composite. Under optimized conditions, amperometric i-t method was used for the quantification of CC, and the amperometric response of the biosensor was linear over the concertation of CC ranging from 0.2 to 209.7 μM. The sensitivity, response time and the detection limit of the biosensor for CC is 0.932 μMμA−1 cm−2, 2 s and 0.085 μM, respectively. The biosensor has high selectivity towards CC in the presence of potentially active biomolecules and phenolic compounds. The biosensor also accessed for the detection of CC in different water samples and shows good practicality with an appropriate repea. PMID:28117357

Effect of growth substrate, method of fermentation, and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes.

PubMed

Elisashvili, Vladimir; Kachlishvili, Eva; Penninckx, Michel

2008-11-01

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml(-1)) and xylanase (135 U ml(-1)) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l(-1)). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.

[Enhancement of laccase activity by combining white rot fungal strains].

PubMed

He, Rong-yu; Liu, Xiao-feng; Yan, Zhi-ying; Yuan, Yue-xiang; Liao, Yin-zhang; Li, Xu-dong

2010-02-01

The method of combining white rot fungal strains was used to enhance laccase activity, and the interaction mechanism between strains was also studied. The laccase activity of combined fungi of strain 55 (Trametes trogii) and strain m-6 (Trametes versicolor) were 24.13 and 4.07-fold higher than that of strain 55 and strain m-6, respectively. No inhibitory effect was observed when the two strains were co-cultivated. On plate cultivation, there was hyphal interference in the contact area, where laccase activity was the highest followed by brown pigmentation. In liquid cultivation, strain m-6 played much more important role on enhancement of laccase activity, and the laccase activity of strain 55 by adding strain m-6 was 7.03-fold higher than that of strain m-6 by adding strain 55, furthermore, filter sterilized- and high temperature autoclaved-extracellular substances of strain m-6 could also stimulate strain 55 to excrete more laccase, which led to 6.79-fold and 4. 60-fold increase in laccase activity by adding 20 mL, respectively. The native staining results of Native-PAGE showed that the types of laccase isozymes were not changed when strains were co-cultured, but the concentration of three types increased.

Fungal pretreatment of sweet sorghum bagasse with supplements: improvement in lignin degradation, selectivity and enzymatic saccharification.

PubMed

Mishra, Vartika; Jana, Asim K; Jana, Mithu Maiti; Gupta, Antriksh

2017-06-01

Sweet sorghum bagasse (SSB) from food processing and agricultural industry has attracted the attention for uses in production of biofuel, enzymes and other products. The alteration in lignocellulolytic enzymes by use of supplements in fungal pretreatment of SSB to achieve higher lignin degradation, selectivity value and enzymatic hydrolysis to fermentable sugar was studied. Fungal strain Coriolus versicolor was selected for pretreatment due to high ligninolytic and low cellulolytic enzyme production resulting in high lignin degradation and selectivity value. SSB was pretreated with supplements of veratryl alcohol, syringic acid, catechol, gallic acid, vanillin, guaiacol, CuSO 4 and MnSO 4 . The best results were obtained with CuSO 4 , gallic acid and syringic acid supplements. CuSO 4 increased the activities of laccase (4.9-fold) and polyphenol oxidase (1.9-fold); gallic acid increased laccase (3.5-fold) and manganese peroxidase (2.5-fold); and syringic acid increased laccase (5.6-fold), lignin peroxidase (13-fold) and arylalcohol oxidase (2.8-fold) resulting in enhanced lignin degradations and selectivity values than the control. Reduced cellulolytic enzyme activities resulted in high cellulose recovery. Enzymatic hydrolysis of pretreated SSB yielded higher sugar due to degradation of lignin and reduced the crystallinity of cellulose. The study showed that supplements could be used to improve the pretreatment process. The results were confirmed by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and thermogravimetric/differential thermogravimetric analysis of SSB.

Light-induced inhibition of laccase in Pycnoporus sanguineus.

PubMed

Hernández, Christian A; Perroni, Yareni; Pérez, José Antonio García; Rivera, Beatriz Gutiérrez; Alarcón, Enrique

2016-03-01

The aim was to determine which specific regions of the visible light spectrum were responsible for the induction or inhibition of laccase in Pycnoporus sanguineus. Cultures were exposed to various bandwidth lights: blue (460 nm), green (525 nm), white (a combination of 460 and 560 nm), red (660 nm), and darkness. The results indicate that short wavelengths strongly inhibit the production of laccase: green (3.76 ± 1.12 U/L), blue (1.94 ± 0.36 U/L), and white (1.05 ± 0.21 U/L) in proportions of 85.8, 92.6, and 96.0%, respectively; whereas long wavelengths inhibit laccase production only partially i.e., red light (14.05 ± 4.79 U/L) in a proportion of 46.8%. Maximum activity was induced in absence of visible light (30 °C, darkness), i.e., 30.76 ± 4.0 U/L. It is concluded that the production of laccase in P. sanguineus responds to light stimuli [measured as wavelengths and lx] and that it does so inversely. This can be explained as an ecological mechanism of environmental recognition, given that P. sanguineus develops inside lignocellulose structures in conditions of darkness. The presence of short wavelength light (460-510 nm) would indicate that the organism finds itself in an external environment, unprovided of lignin, and that it is therefore unnecessary to secrete laccase. This possible new regulation in the laccase production in P. sanguineus has important biotechnological implications, for it would be possible to control the production of laccase using light stimuli.

Removal of acetaminophen in water by laccase immobilized in barium alginate.

PubMed

Ratanapongleka, Karnika; Punbut, Supot

2018-02-01

This research has focused on the optimization of immobilized laccase condition and utilization in degradation of acetaminophen contaminated in aqueous solution. Laccase from Lentinus polychrous was immobilized in barium alginate. The effects of laccase immobilization such as sodium alginate concentration, barium chloride concentration and gelation time were studied. The optimal conditions for immobilization were sodium alginate 5% (w/v), barium chloride 5% (w/v) and gelation time of 60 min. Immobilized laccase was then used for acetaminophen removal. Acetaminophen was removed quickly in the first 50 min. The degradation rate and percentage of removal increased when the enzyme concentration increased. Immobilized laccase at 0.57 U/g-alginate showed the maximum removal at 94% in 240 min. The removal efficiency decreased with increasing initial acetaminophen concentration. The K m value for immobilized laccase (98.86 µM) was lower than that of free laccase (203.56 µM), indicating that substrate affinity was probably enhanced by immobilization. The immobilized enzyme exhibited high activity and good acetaminophen removal at pH 7 and temperature of 35°C. The activation energies of free and immobilized laccase for degradation of acetaminophen were 8.08 and 17.70 kJ/mol, respectively. It was also found that laccase stability to pH and temperature increased after immobilization. Furthermore, immobilized laccase could be reused for five cycles. The capability of removal and enzyme activity were retained above 70%.

Laccase of Cyathus bulleri: structural, catalytic characterization and expression in Escherichia coli.

PubMed

Salony; Garg, N; Baranwal, R; Chhabra, M; Mishra, S; Chaudhuri, T K; Bisaria, V S

2008-02-01

Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% alpha-helical, 26% beta-sheet and 38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher beta-sheet structures. The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.

Exploiting the oxidizing capabilities of laccases exploiting the oxidizing capabilities of laccases for sustainable chemistry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cannatelli, Mark D.

Part one of this dissertation research has focused on harnessing the ability of laccases to generate reactive para-quinones in situ from the corresponding hydroquinones, followed by reaction with a variety of nucleophiles to perform novel carbon-carbon, carbon-nitrogen, and carbon-sulfur bond forming reactions for the synthesis of new and existing compounds. In part two of this dissertation, the fundamental laccase-catalyzed coupling chemistry developed in part one was applied to functionalize the surface of kraft lignin.

Bacterial versus fungal laccase: potential for micropollutant degradation

PubMed Central

2013-01-01

Relatively high concentrations of micropollutants in municipal wastewater treatment plant (WWTP) effluents underscore the necessity to develop additional treatment steps prior to discharge of treated wastewater. Microorganisms that produce unspecific oxidative enzymes such as laccases are a potential means to improve biodegradation of these compounds. Four strains of the bacterial genus Streptomyces (S. cyaneus, S. ipomoea, S. griseus and S. psammoticus) and the white-rot fungus Trametes versicolor were studied for their ability to produce active extracellular laccase in biologically treated wastewater with different carbon sources. Among the Streptomyces strains evaluated, only S. cyaneus produced extracellular laccase with sufficient activity to envisage its potential use in WWTPs. Laccase activity produced by T. versicolor was more than 20 times greater, the highest activity being observed with ash branches as the sole carbon source. The laccase preparation of S. cyaneus (abbreviated LSc) and commercial laccase from T. versicolor (LTv) were further compared in terms of their activity at different pH and temperatures, their stability, their substrate range, and their micropollutant oxidation efficiency. LSc and LTv showed highest activities under acidic conditions (around pH 3 to 5), but LTv was active over wider pH and temperature ranges than LSc, especially at near-neutral pH and between 10 and 25°C (typical conditions found in WWTPs). LTv was also less affected by pH inactivation. Both laccase preparations oxidized the three micropollutants tested, bisphenol A, diclofenac and mefenamic acid, with faster degradation kinetics observed for LTv. Overall, T. versicolor appeared to be the better candidate to remove micropollutants from wastewater in a dedicated post-treatment step. PMID:24152339

Chitosan multiple addition enhances laccase production from Trametes versicolor.

PubMed

Adekunle, Abiodun Emmanuel; Wang, Feng; Hu, Jianhua; Ma, Anzhou; Guo, Chen; Zhuang, Guoqiang; Liu, Chun-Zhao

2015-10-01

Chitosan multiple addition strategy was developed to improve laccase production from Trametes versicolor cultures. The optimized multiple addition strategy was carried out by two-time addition of 0.1 g L(-1) chitosan to a 2-day-old culture media, with 24-h interval between the treatments. Under these conditions, laccase activity of 644.9 U l(-1) was achieved on the seventh day and laccase production was improved by 93.5 % higher than the control. Chitosan treatment increased reactive oxygen species generation and extracellular protein concentration in the treated mycelia. In contrast, the inducer inhibited the mycelia growth. The result of the quantitative reverse transcription polymerase chain reaction showed that the copy number of the laccase gene transcript increased by 16.7-fold in the treated mycelia relative to the control. This study provides insight into some of the intrinsic metabolic processes involved in the upregulation of laccase production in the presence of chitosan inducer in fungal culture.

Reactivity of bacterial and fungal laccases with lignin under alkaline conditions.

PubMed

Moya, Raquel; Saastamoinen, Päivi; Hernández, Manuel; Suurnäkki, Anna; Arias, Enriqueta; Mattinen, Maija-Liisa

2011-11-01

The ability of Streptomyces ipomoea laccase to polymerize secoisolariciresinol lignan and technical lignins was assessed. The reactivity of S. ipomoea laccase was also compared to that of low redox fungal laccase from Melanocarpus albomyces using low molecular mass p-coumaric, ferulic and sinapic acid as well as natural (acetosyringone) and synthetic 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) mediators as substrates. Oxygen consumption measurement, MALDI-TOF MS and SEC were used to follow the enzymatic reactions at pH 7, 8, 9 and 10 at 30°C and 50°C. Polymerization of lignins and lignan by S. ipomoea laccase under alkaline reaction conditions was observed, and was enhanced in the presence of acetosyringone almost to the level obtained with M. albomyces laccase without mediator. Reactivities of the enzymes towards acetosyringone and TEMPO were similar, suggesting exploitation of the compounds and low redox laccase in lignin valorization under alkaline conditions. The results have scientific impact on basic research of laccases. Copyright © 2011 Elsevier Ltd. All rights reserved.

Stabilized Laccases as Heterogeneous Bioelectrocatalysts (Postprint)

DTIC Science & Technology

2012-10-01

nature, with examples that range from bacterial species to fungi and plants.19·101 However, certain properties of laccases, such as their protein...identified, includ- ing examples of extremophilic enzymes,l11·121 but the extracellu- lar laccases from wood-rotting fungi , such as Trametes spp...biomolecules not only affects the stability and selectivity of an enzyme towards different substrates but also the charge trans- fer at the electrode

Induction of laccases in Trametes versicolor by aqueous wood extracts.

PubMed

Bertrand, Brandt; Martínez-Morales, Fernando; Tinoco, Raunel; Rojas-Trejo, Sonia; Serrano-Carreón, Leobardo; Trejo-Hernández, María R

2014-01-01

The induction of laccase isoforms in Trametes versicolor HEMIM-9 by aqueous extracts (AE) from softwood and hardwood was studied. Samples of sawdust of Pinus sp., Cedrela sp., and Quercus sp. were boiled in water to obtain AE. Different volumes of each AE were added to fungal cultures to determine the amount of AE needed for the induction experiments. Laccase activity was assayed every 24 h for 15 days. The addition of each AE (50 to 150 μl) to the fungal cultures increased laccase production compared to the control (0.42 ± 0.01 U ml(-1)). The highest laccase activities detected were 1.92 ± 0.15 U ml(-1) (pine), 1.87 ± 0.26 U ml(-1) (cedar), and 1.56 ± 0.34 U ml(-1) (oak); laccase productivities were also significantly increased. Larger volumes of any AE inhibited mycelial growth. Electrophoretic analysis revealed two laccase bands (lcc1 and lcc2) for all the treatments. However, when lcc2 was analyzed by isoelectric focusing, inducer-dependent isoform patterns composed of three (pine AE), four (oak AE), and six laccase bands (cedar AE) were observed. Thus, AE from softwood and hardwood had induction effects in T. versicolor HEMIM-9, as indicated by the increase in laccase activity and different isoform patterns. All of the enzymatic extracts were able to decolorize the dye Orange II. Dye decolorization was mainly influenced by pH. The optimum pH for decolorization was pH 5 (85%), followed by pH 7 (50%) and pH 3 (15%). No significant differences in the dye decolorizing capacity were detected between the control and the differentially induced laccase extracts (oak, pine and cedar). This could be due to the catalytic activities of isoforms with pI 5.4 and 5.8, which were detected under all induction conditions.

Biological decolourisation of wastewater from molasses fermentation by Trametes versicolor in an airlift reactor.

PubMed

Rioja, R; García, M T; Peña, M; González, G

2008-06-01

Continuous decolourisation of wastewater from molasses fermentation using mycelium of Trametes versicolor in pellets shape was performed in an airlift bioreactor (semi-pilot scale) with the aim of operating steadily for a long period, maintaining the colour removal activity. The influences of influent flow and glucose feed rate were tested. Induction of peroxidases secretion by Mn(2+) addition was also studied. The efficiency of the decolourisation process was followed by monitoring colour and enzymatic activities. The experimental results showed that continuous decolourisation in an airlift bioreactor can be considered a suitable alternative for treating molasses fermentation wastewater. A colour removal yield around 60% remained practically constant during 23 days under continuous operation. Laccase was found to be the main enzyme secreted by the strain, being responsible for the decolourisation process. Mn(2+) addition was not likely to induct manganese-dependent peroxidase secretion.

Effects of Kraft Pulp and Lignin on Trametes versicolor Carbon Metabolism

PubMed Central

Roy, Brian P.; Archibald, Frederick

1993-01-01

The white rot basidiomycete Trametes (Coriolus) versicolor can substantially increase the brightness and decrease the lignin content of washed, unbleached hardwood kraft pulp (HWKP). Monokaryotic strain 52J was used to study how HWKP and the lignin in HWKP affect the carbon metabolism and secretions of T. versicolor. Earlier work indicated that a biobleaching culture supernatant contained all components necessary for HWKP biobleaching and delignification, but the supernatant needed frequent contact with the fungus to maintain these activities. Thus, labile small fungal metabolites may be the vital biobleaching system components renewed or replaced by the fungus. Nearly all of the CO2 evolved by HWKP-containing cultures came from the added glucose, indicating that HWKP is not an important source of carbon or energy during biobleaching. Carbon dioxide appeared somewhat earlier in the absence of HWKP, but the culture partial O2 pressure was little affected by the presence of pulp. The presence of HWKP in a culture markedly increased the culture's production of a number of acidic metabolites, including 2-phenyllactate, oxalate, adipate, glyoxylate, fumarate, mandelate, and glycolate. Although the total concentration of these pulp-induced metabolites was only 4.3 mM, these compounds functioned as effective manganese-complexing agents for the manganese peroxidase-mediated oxidation of phenol red, propelling the reaction at 2.4 times the rate of 50 mM sodium malonate, the standard chelator-buffer. The presence of HWKP in a culture also markedly stimulated fungal secretion of the enzymes manganese peroxidase, cellulase, and cellobiose-quinone oxidoreductase, but not laccase (phenol oxidase) or lignin peroxidase. PMID:16348963

Use of Laccase as a Novel, Versatile Reporter System in Filamentous Fungi

PubMed Central

Mander, Gerd J.; Wang, Huaming; Bodie, Elizabeth; Wagner, Jens; Vienken, Kay; Vinuesa, Claudia; Foster, Caroline; Leeder, Abigail C.; Allen, Gethin; Hamill, Valerie; Janssen, Giselle G.; Dunn-Coleman, Nigel; Karos, Marvin; Lemaire, Hans Georg; Subkowski, Thomas; Bollschweiler, Claus; Turner, Geoffrey; Nüsslein, Bernhard; Fischer, Reinhard

2006-01-01

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2′-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain. PMID:16820501

Use of laccase as a novel, versatile reporter system in filamentous fungi.

PubMed

Mander, Gerd J; Wang, Huaming; Bodie, Elizabeth; Wagner, Jens; Vienken, Kay; Vinuesa, Claudia; Foster, Caroline; Leeder, Abigail C; Allen, Gethin; Hamill, Valerie; Janssen, Giselle G; Dunn-Coleman, Nigel; Karos, Marvin; Lemaire, Hans Georg; Subkowski, Thomas; Bollschweiler, Claus; Turner, Geoffrey; Nüsslein, Bernhard; Fischer, Reinhard

2006-07-01

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.

Recent developments and applications of immobilized laccase.

PubMed

Fernández-Fernández, María; Sanromán, M Ángeles; Moldes, Diego

2013-12-01

Laccase is a promising biocatalyst with many possible applications, including bioremediation, chemical synthesis, biobleaching of paper pulp, biosensing, textile finishing and wine stabilization. The immobilization of enzymes offers several improvements for enzyme applications because the storage and operational stabilities are frequently enhanced. Moreover, the reusability of immobilized enzymes represents a great advantage compared with free enzymes. In this work, we discuss the different methodologies of enzyme immobilization that have been reported for laccases, such as adsorption, entrapment, encapsulation, covalent binding and self-immobilization. The applications of laccase immobilized by the aforementioned methodologies are presented, paying special attention to recent approaches regarding environmental applications and electrobiochemistry. Copyright © 2012 Elsevier Inc. All rights reserved.

Ultrasound-assisted extraction and characterization of hydrolytic and oxidative enzymes produced by solid state fermentation.

PubMed

Szabo, Orsolya Erzsebet; Csiszar, Emilia; Toth, Karolina; Szakacs, George; Koczka, Bela

2015-01-01

Ligninolytic and hydrolytic enzymes were produced with six selected fungi on flax substrate by solid state fermentation (SSF). The extracellular enzyme production of the organisms in two SSF media was evaluated by measuring the soluble protein concentration and the filter paper, endoxylanase, 1,4-β-d-glucosidase, 1,4-β-d-endoglucanase, polygalacturonase, lignin peroxidase, manganese peroxidase and laccase activities of the clear culture solutions produced by conventional extraction from the SSF materials. The SSF material of the best enzyme producer (Trichoderma virens TUB F-498) was further investigated to enhance the enzyme recovery by low frequency ultrasound treatment. Performance of both the original and ultrasound macerated crude enzyme mixtures was evaluated in degradation of the colored lignin-containing and waxy materials of raw linen fabric. Results proved that sonication (at 40%, 60% and 80% amplitudes, for 60min) did not result in reduction in the filter paper, lignin peroxidase and laccase activities of the crude enzyme solution, but has a significant positive effect on the efficiency of enzyme extraction from the SSF material. Depending on the parameters of sonication, the enzyme activities in the extracts obtained can be increased up to 129-413% of the original activities measured in the control extracts recovered by a common magnetic stirrer. Sonication also has an effect on both the enzymatic removal of the lignin-containing color materials and hydrophobic surface layer from the raw linen. Copyright © 2014 Elsevier B.V. All rights reserved.

Laccase-mediator catalyzed conversion of model lignin compounds

USDA-ARS?s Scientific Manuscript database

Identifying suitable reaction conditions remains an important task in the development of practical enzyme catalysts. Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both comm...

The ability of white-rot fungi to degrade the endocrine-disrupting compound nonylphenol.

PubMed

Soares, Ana; Jonasson, Karin; Terrazas, Enrique; Guieysse, Benoit; Mattiasson, Bo

2005-03-01

Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l-1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l-1 and 99 mg l-1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14 degrees C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l-1 and 79 mg l-1 of nonylphenol, respectively. No pollutant removal was recorded at 4 degrees C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg-1 of nonylphenol. Only 1.3+/-0.1% of nonylphenol remained in the soil after 5 weeks of incubation.

Laccase production by Monotospora sp., an endophytic fungus in Cynodon dactylon.

PubMed

Wang, J W; Wu, J H; Huang, W Y; Tan, R X

2006-03-01

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by the endophytic fungus Monotospora sp. were evaluated. The optimal temperature and initial pH for laccase production by Monotospora sp. in submerged culture were found to be 30 degrees C and 8.5, respectively. Maltose (2 g l(-1)) and ammonium tartrate (10 g l(-1)) were the most suitable carbon and nitrogen source for laccase production. Under optimal culture medium, the maximum laccase activity was determined to be 13.55 U ml(-1), which was approximately four times higher than that in basal medium. This is the first report on laccase production by an endophytic fungus.

Laccase-mediator catalyzed conversion of model lignin compounds

USDA-ARS?s Scientific Manuscript database

Laccases play an important role in the biological breakdown of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined a variety of laccases, both commercially prepared and crude extracts, for their ability to oxidize three model lignol compounds (p-coumaryl...

Laccase engineering: from rational design to directed evolution.

PubMed

Mate, Diana M; Alcalde, Miguel

2015-01-01

Laccases are multicopper oxidoreductases considered by many in the biotechonology field as the ultimate "green catalysts". This is mainly due to their broad substrate specificity and relative autonomy (they use molecular oxygen from air as an electron acceptor and they only produce water as by-product), making them suitable for a wide array of applications: biofuel production, bioremediation, organic synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced production cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (organic solvents, ionic liquids, physiological fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts. Copyright © 2014 Elsevier Inc. All rights reserved.

Combined sequence and structure analysis of the fungal laccase family.

PubMed

Kumar, S V Suresh; Phale, Prashant S; Durani, S; Wangikar, Pramod P

2003-08-20

Plant and fungal laccases belong to the family of multi-copper oxidases and show much broader substrate specificity than other members of the family. Laccases have consequently been of interest for potential industrial applications. We have analyzed the essential sequence features of fungal laccases based on multiple sequence alignments of more than 100 laccases. This has resulted in identification of a set of four ungapped sequence regions, L1-L4, as the overall signature sequences that can be used to identify the laccases, distinguishing them within the broader class of multi-copper oxidases. The 12 amino acid residues in the enzymes serving as the copper ligands are housed within these four identified conserved regions, of which L2 and L4 conform to the earlier reported copper signature sequences of multi-copper oxidases while L1 and L3 are distinctive to the laccases. The mapping of regions L1-L4 on to the three-dimensional structure of the Coprinus cinerius laccase indicates that many of the non-copper-ligating residues of the conserved regions could be critical in maintaining a specific, more or less C-2 symmetric, protein conformational motif characterizing the active site apparatus of the enzymes. The observed intraprotein homologies between L1 and L3 and between L2 and L4 at both the structure and the sequence levels suggest that the quasi C-2 symmetric active site conformational motif may have arisen from a structural duplication event that neither the sequence homology analysis nor the structure homology analysis alone would have unraveled. Although the sequence and structure homology is not detectable in the rest of the protein, the relative orientation of region L1 with L2 is similar to that of L3 with L4. The structure duplication of first-shell and second-shell residues has become cryptic because the intraprotein sequence homology noticeable for a given laccase becomes significant only after comparing the conservation pattern in several fungal

Enhanced delignification of lignocellulosic substrates by Pichia GS115 expressed recombinant laccase.

PubMed

Kumar, Vidya Pradeep; Kolte, Atul P; Dhali, Arindam; Naik, Chandrashekar; Sridhar, Manpal

2018-04-25

Utilization of energy-rich crop residues by ruminants is restricted by the presence of lignin, which is recalcitrant to digestion. Application of lignin degrading enzymes on the lignocellulosic biomass exposes the cellulose for easy digestion by ruminants. Laccases have been found to be considerably effective in improving the digestibility by way of delignification. However, laccase yields from natural hosts are not sufficient for industrial scale applications, which restricts their use. A viable option would be to express the laccase gene in compatible hosts to achieve higher production yields. A codon-optimized synthetic variant of Schizophyllum commune laccase gene was cloned into a pPIC9K vector and expressed in P. pastoris GS115 (his4) under the control of an alcohol oxidase promoter. Colonies were screened for G418 resistance and the methanol utilization phenotype was established. The transformant yielded a laccase activity of 344 U·mL -1 after 5 days of growth at 30°C (0.019 g·mL -1 wet cell weight). The laccase protein produced by the recombinant Pichia clone was detected as two bands with apparent molecular weights of 55 kDa and 70 kDa on SDS-PAGE. Activity staining on native PAGE confirmed the presence of bioactive laccase. Treatment of five common crop residues with recombinant laccase recorded a lignin loss ranging between 1.64% in sorghum stover, to 4.83% in finger millet, with an enhancement in digestibility ranging between 8.71% in maize straw to 24.61% in finger millet straw. Treatment with recombinant laccase was effective in enhancing the digestibility of lignocellulosic biomass for ruminant feeding through delignification. To date, a number of hosts have been adventured to produce laccase in large quantities, but, to our knowledge, there are no reports of the expression of laccase protein from Schizophyllum commune in Pichia pastoris, and also on the treatment of crop residues using recombinant laccase for ruminant feeding.

Effect of different compounds on the induction of laccase production by Agaricus blazei.

PubMed

Valle, J S; Vandenberghe, L P S; Oliveira, A C C; Tavares, M F; Linde, G A; Colauto, N B; Soccol, C R

2015-12-03

Laccases are polyphenol oxidases produced by many fungi and have many applications in textile, food and beverage, and pulp and paper industries. Laccase production can be induced using aromatic or phenolic compounds that mostly affect the transcription of laccase-encoding genes. In this study, we analyzed laccase and biomass production by Agaricus blazei in the presence of different concentrations of nitrogen, copper, and inducers such as pyrogallol, veratryl alcohol, xylidine, vanillin, guaiacol, and ethanol. Laccase production by A. blazei U2-4 reached 43.8 U/mL in the presence of 2.8 g/L nitrogen and 150 μM copper. However, addition of copper to the cultivation medium decreased biomass production. Different compounds differentially induced laccase production by A. blazei. Moreover, different concentrations of these inducers exerted different effects on laccase activity. Ethanol (1.0 mM), guaiacol (0.5 mM), and vanillin (0.5 mM) were the best inducers and increased laccase activity by 120% (A. blazei U2-2), 30% (A. blazei U2-3), and 9% (A. blazei U2-4), respectively. In contrast, pyrogallol and xylidine decreased laccase activity but increased biomass production.

Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

PubMed Central

Addleman, Katherine; Archibald, Frederick

1993-01-01

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching. Images PMID:16348851

Characterization of C-terminally engineered laccases.

PubMed

Liu, Yingli; Cusano, Angela Maria; Wallace, Erin C; Mekmouche, Yasmina; Ullah, Sana; Robert, Viviane; Tron, Thierry

2014-08-01

Extremities of proteins are potent sites for functionalization. Carboxy terminus variants of the Trametes sp. strain C30 LAC3 laccase were generated and produced in Saccharomyces cerevisiae. A variant deleted of the last 13 residues (CΔ) and its 6 His tagged counterpart (CΔ6H) were found active enzymes. The production of CΔ6H resulted in the synthesis of a unusually high proportion of highly glycosylated forms of the enzyme therefore allowing the additional purification of a hyper-glycosylated form of CΔ6H noted CΔ6Hh. Properties of CΔ, CΔ6H and CΔ6Hh were compared. Globally, LAC3 catalytic efficiency was moderately affected by terminal modifications except in CΔ for which the kcat/KM ratio decreased 4 fold (with syringaldazine as substrate) and 10 fold (with ABTS as substrate) respectively. The catalytic parameters kcat and KM of CΔ6H and CΔ6Hh were found to be strictly comparable revealing that over glycosylation does not affect the enzyme catalytic efficiency. To the contrary, in vitro deglycosylation of laccase drastically reduced its activity. So, despite a complex glycosylated pattern observed for some of the variant enzymes, terminal sequences of laccases appear to be appropriate sites for the functionalization/immobilization of laccase. Copyright © 2014 Elsevier B.V. All rights reserved.

FTIR Spectroscopy Applied in Remazol Blue Dye Oxidation by Laccases

NASA Astrophysics Data System (ADS)

Juárez-Hernández, J.; Zavala-Soto, M. E.; Bibbins-Martínez, M.; Delgado-Macuil, R.; Díaz-Godinez, G.; Rojas-López, M.

2008-04-01

We have used FTIR with attenuated total reflectance (ATR) technique to analyze the decolourization process of Remazol Blue dye (RB19) caused by the oxidative activity of laccase enzyme. It is known that laccases catalyze the oxidation of a large range of phenolic compounds and aromatic amines carrying out one-electron oxidations, although also radicals could be formed which undergo subsequent nonenzymatic reactions. The enzyme laccase is a copper-containing polyphenol oxidase (EC 1.10.3.2) which has been tested as a potential alternative in detoxification of environmental pollutants such as dyes present in wastewaters generated for the textile industry. In order to ensure degradation or avoid formation of toxic compounds it is important to establish the mechanism by which laccase oxidizes dyes. In this research individual ATR-FTIR spectra have been recorded for several reaction times between 0 to 236 hours, and the temporal dependence of the reaction was analyzed through the relative diminution of the intensity of the infrared band at 1127 cm-1 (associated to C-N vibration), with respect to the intensity of the band at 1104 cm-1 (associated to S = O) from sulphoxide group. Decolourization process of this dye by laccase could be attributed to its accessibility on the secondary amino group, which is a potential point of attack of laccases, abstracting the hydrogen atom. This decolourization process of remazol blue dye by laccase enzyme might in a future replace the traditionally high chemical, energy and water consuming textile operations.

Ultrasound-intensified laccase production from Trametes versicolor.

PubMed

Wang, Feng; Ma, An-Zhou; Guo, Chen; Zhuang, Guo-Qiang; Liu, Chun-Zhao

2013-01-01

An efficient intermittent ultrasonic treatment strategy was developed to improve laccase production from Trametes versicolor mycelia cultures. The optimized strategy consisted of exposing 2-day-old mycelia cultures to 5-min ultrasonic treatments for two times with a 12-h interval at the fixed ultrasonic power and frequency (120 W, 40 kHz). After 5 days of culture, this strategy produced the highest extracellular laccase activity of 588.9 U/L among all treatments tested which was 1.8-fold greater than the control without ultrasound treatment. The ultrasonic treatment resulted in a higher pellet porosity that facilitated the mass transfer of nutrients and metabolites from the pellets to the surrounding liquid. Furthermore, the ultrasonic treatment induced the expression of the laccase gene (lcc), which correlated with a sharp increase in both extracellular and intracellular laccase activity. This is the first study to find positive effects of ultrasound on gene expression in fungal cells. These results provide a basis for understanding the stimulation of metabolite production and process intensification by ultrasonic treatment in filamentous fungal culture. Copyright © 2012 Elsevier B.V. All rights reserved.

A New Laccase Based Biosensor for Tartrazine.

PubMed

Mazlan, Siti Zulaikha; Lee, Yook Heng; Hanifah, Sharina Abu

2017-12-09

Laccase enzyme, a commonly used enzyme for the construction of biosensors for phenolic compounds was used for the first time to develop a new biosensor for the determination of the azo-dye tartrazine. The electrochemical biosensor was based on the immobilization of laccase on functionalized methacrylate-acrylate microspheres. The biosensor membrane is a composite of the laccase conjugated microspheres and gold nanoparticles (AuNPs) coated on a carbon-paste screen-printed electrode. The reaction involving tartrazine can be catalyzed by laccase enzyme, where the current change was measured by differential pulse voltammetry (DPV) at 1.1 V. The anodic peak current was linear within the tartrazine concentration range of 0.2 to 14 μM ( R ² = 0.979) and the detection limit was 0.04 μM. Common food ingredients or additives such as glucose, sucrose, ascorbic acid, phenol and sunset yellow did not interfere with the biosensor response. Furthermore, the biosensor response was stable up to 30 days of storage period at 4 °C. Foods and beverage were used as real samples for the biosensor validation. The biosensor response to tartrazine showed no significant difference with a standard HPLC method for tartrazine analysis.

A New Laccase Based Biosensor for Tartrazine

PubMed Central

Mazlan, Siti Zulaikha; Lee, Yook Heng; Hanifah, Sharina Abu

2017-01-01

Laccase enzyme, a commonly used enzyme for the construction of biosensors for phenolic compounds was used for the first time to develop a new biosensor for the determination of the azo-dye tartrazine. The electrochemical biosensor was based on the immobilization of laccase on functionalized methacrylate-acrylate microspheres. The biosensor membrane is a composite of the laccase conjugated microspheres and gold nanoparticles (AuNPs) coated on a carbon-paste screen-printed electrode. The reaction involving tartrazine can be catalyzed by laccase enzyme, where the current change was measured by differential pulse voltammetry (DPV) at 1.1 V. The anodic peak current was linear within the tartrazine concentration range of 0.2 to 14 μM (R2 = 0.979) and the detection limit was 0.04 μM. Common food ingredients or additives such as glucose, sucrose, ascorbic acid, phenol and sunset yellow did not interfere with the biosensor response. Furthermore, the biosensor response was stable up to 30 days of storage period at 4 °C. Foods and beverage were used as real samples for the biosensor validation. The biosensor response to tartrazine showed no significant difference with a standard HPLC method for tartrazine analysis. PMID:29232842

Overproduction of recombinant laccase using a homologous expression system in Coriolus versicolor.

PubMed

Kajita, Shinya; Sugawara, Shinsuke; Miyazaki, Yasumasa; Nakamura, Masaya; Katayama, Yoshihiro; Shishido, Kazuo; Iimura, Yosuke

2004-12-01

One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants microg(-1) of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.

Redox Chemistry in Laccase-Catalyzed Oxidation of N-Hydroxy Compounds

PubMed Central

Xu, Feng; Kulys, Juozas J.; Duke, Kyle; Li, Kaichang; Krikstopaitis, Kastis; Deussen, Heinz-Josef W.; Abbate, Eric; Galinyte, Vilija; Schneider, Palle

2000-01-01

1-Hydroxybenzotriazole, violuric acid, and N-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an “outer-sphere” type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation. PMID:10788380

Laccase-catalyzed synthesis of 2,3-ethylenedithio-1,4-quinones

DOE PAGES

Cannatelli, Mark D.; Ragauskas, Arthur J.

2015-06-05

Laccases (benzenediol:oxygen oxidoreductase EC 1.10.3.2) are part of a family of multicopper oxidases. These environmentally friendly enzymes require O 2 as their only co-substrate and produce H 2O as their sole by-product. As a result, they have acquired increasing use in biotechnological applications, particularly in the field of organic synthesis. In the current study, laccases have been employed to successfully couple 1,2-ethanedithiol to various substituted hydroquinones to produce novel 2,3-ethylenedithio-1,4-quinones in good yields via an oxidation–addition–oxidation–addition–oxidation mechanism. The reactions proceeded in one-pot under mild conditions (room temperature, pH 5.0). This study further supports the use of laccases as green toolsmore » in organic chemistry. Furthermore, it provides evidence that laccase-catalyzed cross-coupling reactions involving small thiols are possible, in spite of research that suggests small thiols are potent inhibitors of laccases.« less

2.0 Angstrom Structure of Prostaglandin H2 Synthase-1 Reconstituted with a Manganese Porphyrin Cofactor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta,K.; Selinsky, B.; Loll, P.

2006-01-01

Prostaglandin H{sub 2} synthase (EC 1.14.99.1) is a clinically important drug target that catalyzes two key steps in the biosynthesis of the eicosanoid hormones. The enzyme contains spatially distinct cyclooxygenase and peroxidase active sites, both of which require a heme cofactor. Substitution of ferric heme by Mn{sup III} protoporphyrin IX greatly diminishes the peroxidase activity, but has little effect on the cyclooxygenase activity. Here, the 2.0 Angstrom resolution crystal structure of the Mn{sup III} form of ovine prostaglandin H{sub 2} synthase-1 is described (R = 21.8%, R{sub free} = 23.7%). Substitution of Mn{sup III} for Fe{sup III} causes no structuralmore » perturbations in the protein. However, the out-of-plane displacement of the manganese ion with respect to the porphyrin is greater than that of the iron by approximately 0.2 Angstroms. This perturbation may help to explain the altered catalytic properties of the manganese enzyme.« less

Laccase pretreatment for agrofood wastes valorization.

PubMed

Giacobbe, Simona; Pezzella, Cinzia; Lettera, Vincenzo; Sannia, Giovanni; Piscitelli, Alessandra

2018-06-01

Apple pomace, potato peels, and coffee silverskin are attractive agrofood wastes for the production of biofuels and chemicals, due to their abundance and carbohydrate content. As lignocellulosic biomasses, their conversion is challenged by the presence of lignin that prevents hydrolysis of polysaccharides, hence demanding a pretreatment step. In this work, the effectiveness of Pleurotus ostreatus laccases (with and without mediator) to remove lignin, improving the subsequent saccharification, was assessed. Optimized conditions for sequential protocol were set up for all agrofood wastes reaching delignification and detoxification yields correlated with high saccharification. Especially noteworthy were results for apple pomace and coffee silverskin for which 83% of and 73% saccharification yields were observed, by using laccase and laccase mediator system, respectively. The herein developed sequential protocol, saving soluble sugars and reducing the amount of wastewater, can improve the overall process for obtaining chemicals or fuels from agrofood wastes. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Copper induction and differential expression of laccase in Aspergillus flavus

PubMed Central

Gomaa, Ola M.; Momtaz, Osama A.

2015-01-01

Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus . Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent. PMID:26221119

Transcriptional response of lignin-degrading enzymes to 17α-ethinyloestradiol in two white rots

PubMed Central

Přenosilová, L; Křesinová, Z; Amemori, A Slavíková; Cajthaml, T; Svobodová, K

2013-01-01

Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g−1 of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l−1 EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation. PMID:23170978

Zinc tetraaminophthalocyanine-Fe3O4 nanoparticle composite for laccase immobilization

PubMed Central

Huang, Jun; Liu, Cheng; Xiao, Haiyan; Wang, Juntao; Jiang, Desheng; GU, Erdan

2007-01-01

Zinc tetraaminophthalocyanine-Fe3O4 nanoparticle composites were prepared by organic-inorganic complex technology and characterized. It has been proved that the ZnTAPc dispersed randomly onto the surface of Fe3O4 nanoparticles to form molecular dispersion layer and there was a relatively strong bond between central zinc cation and oxygen. The nanoparticle composite took the shape of roundish spheres with the mean diameter of about 15 nm. Active amino groups of magnetic carriers could be used to bind laccase via glutaraldehyde. The optimal pH for the activity of the immobilized laccases and free laccase were the same at pH 3.0 and the optimal temperature for laccase immobilization on ZnTAPc-Fe3O4 nanoparticle composite was 45°. The immobilization yields and Km value of the laccase immobilized on ZnTAPc-Fe3O4 nanoparticle composite were 25% and 20.1 μM, respectively. This kind of immobilized laccase has good thermal, storage and operation stability, and could be used as the sensing biocomponent for the fiber optic biosensor based on enzyme catalysis. PMID:18203444

Optimization of laccase production by Trametes versicolor cultivated on industrial waste.

PubMed

Tišma, Marina; Znidaršič-Plazl, Polona; Vasić-Rački, Durđa; Zelić, Bruno

2012-01-01

Laccases are very interesting biocatalysts for several industrial applications. Its production by different white-rot fungi can be stimulated by a variety of inducing substrates, and the use of lignocellulosic wastes or industrial by-products is one of the possible approaches to reduce production costs. In this work, various industrial wastes were tested for laccase production by Trametes versicolor MZKI G-99. Solid waste from chemomechanical treatment facility of a paper manufacturing plant showed the highest potential for laccase production. Enzyme production during submerged cultivation of T. versicolor on the chosen industrial waste has been further improved by medium optimization using genetic algorithm. Concentrations of five components in the medium were optimized within 60 shake-flasks experiments, where the highest laccase activity of 2,378 U dm(-3) was achieved. Waste from the paper industry containing microparticles of CaCO(3) was found to stimulate the formation of freely dispersed mycelium and laccase production during submerged cultivation of T. versicolor. It was proven to be a safe and inexpensive substrate for commercial production of laccase and might be more widely applicable for metabolite production by filamentous fungi.

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

PubMed Central

Rodríguez-Escribano, David; de Salas, Felipe; Camarero, Susana

2017-01-01

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin–Ciocalteau reagent (FCR), to detect the decrease in phenolic content produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). The method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates. PMID:28820431

Laccase: Microbial Sources, Production, Purification, and Potential Biotechnological Applications

PubMed Central

Shraddha; Shekher, Ravi; Sehgal, Simran; Kamthania, Mohit; Kumar, Ajay

2011-01-01

Laccase belongs to the blue multicopper oxidases and participates in cross-linking of monomers, degradation of polymers, and ring cleavage of aromatic compounds. It is widely distributed in higher plants and fungi. It is present in Ascomycetes, Deuteromycetes and Basidiomycetes and abundant in lignin-degrading white-rot fungi. It is also used in the synthesis of organic substance, where typical substrates are amines and phenols, the reaction products are dimers and oligomers derived from the coupling of reactive radical intermediates. In the recent years, these enzymes have gained application in the field of textile, pulp and paper, and food industry. Recently, it is also used in the design of biosensors, biofuel cells, as a medical diagnostics tool and bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases have received attention of researchers in the last few decades due to their ability to oxidize both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. It has been identified as the principal enzyme associated with cuticular hardening in insects. Two main forms have been found: laccase-1 and laccase-2. This paper reviews the occurrence, mode of action, general properties, production, applications, and immobilization of laccases within different industrial fields. PMID:21755038

Laccase: microbial sources, production, purification, and potential biotechnological applications.

PubMed

Shraddha; Shekher, Ravi; Sehgal, Simran; Kamthania, Mohit; Kumar, Ajay

2011-01-01

Laccase belongs to the blue multicopper oxidases and participates in cross-linking of monomers, degradation of polymers, and ring cleavage of aromatic compounds. It is widely distributed in higher plants and fungi. It is present in Ascomycetes, Deuteromycetes and Basidiomycetes and abundant in lignin-degrading white-rot fungi. It is also used in the synthesis of organic substance, where typical substrates are amines and phenols, the reaction products are dimers and oligomers derived from the coupling of reactive radical intermediates. In the recent years, these enzymes have gained application in the field of textile, pulp and paper, and food industry. Recently, it is also used in the design of biosensors, biofuel cells, as a medical diagnostics tool and bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases have received attention of researchers in the last few decades due to their ability to oxidize both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. It has been identified as the principal enzyme associated with cuticular hardening in insects. Two main forms have been found: laccase-1 and laccase-2. This paper reviews the occurrence, mode of action, general properties, production, applications, and immobilization of laccases within different industrial fields.

The ligninolytic peroxidases in the genus Pleurotus: divergence in activities, expression, and potential applications.

PubMed

Knop, Doriv; Yarden, Oded; Hadar, Yitzhak

2015-02-01

Mushrooms of the genus Pleurotus are comprised of cultivated edible ligninolytic fungi with medicinal properties and a wide array of biotechnological and environmental applications. Like other white-rot fungi (WRF), they are able to grow on a variety of lignocellulosic biomass substrates and degrade both natural and anthropogenic aromatic compounds. This is due to the presence of the non-specific oxidative enzymatic systems, which are mainly consisted of lacasses, versatile peroxidases (VPs), and short manganese peroxidases (short-MnPs). Additional, less studied, peroxidase are dye-decolorizing peroxidases (DyPs) and heme-thiolate peroxidases (HTPs). During the past two decades, substantial information has accumulated concerning the biochemistry, structure and function of the Pleurotus ligninolytic peroxidases, which are considered to play a key role in many biodegradation processes. The production of these enzymes is dependent on growth media composition, pH, and temperature as well as the growth phase of the fungus. Mn(2+) concentration differentially affects the expression of the different genes. It also severs as a preferred substrate for these preoxidases. Recently, sequencing of the Pleurotus ostreatus genome was completed, and a comprehensive picture of the ligninolytic peroxidase gene family, consisting of three VPs and six short-MnPs, has been established. Similar enzymes were also discovered and studied in other Pleurotus species. In addition, progress has been made in the development of molecular tools for targeted gene replacement, RNAi-based gene silencing and overexpression of genes of interest. These advances increase the fundamental understanding of the ligninolytic system and provide the opportunity for harnessing the unique attributes of these WRF for applied purposes.

Ethidium bromide stimulated hyper laccase production from bird's nest fungus Cyathus bulleri.

PubMed

Dhawan, S; Lal, R; Kuhad, R C

2003-01-01

Effect of ethidium bromide, a DNA intercalating agent, on laccase production from Cyathus bulleri was studied. The bird's nest fungus, Cyathus bulleri was grown on 2% (w/v) malt extract agar (MEA) supplemented with 1.5 microg ml(-1) of the phenanthridine dye ethidium bromide (EtBr) for 7 d and when grown subsequently in malt extract broth (MEB), produced a 4.2-fold increase in laccase production as compared to the untreated fungus. The fungal cultures following a single EtBr treatment, when regrown on MEA devoid of EtBr, produced a sixfold increase in laccase in MEB. However, on subsequent culturing on MEA in the absence of EtBr, only a 2.5-fold increase in laccase production could be maintained. In another attempt, the initial EtBr-treated cultures, when subjected to a second EtBr treatment (1.5 microg ml(-1)) on MEA for 7 d, produced a 1.4-fold increase in laccase production in MEB. The white-rot fungus Cyathus bulleri, when treated with EtBr at a concentration of 1.5 microg ml(-1) and regrown on MEA devoid of EtBr, produced a sixfold increase in laccase production in MEB. The variable form of C. bulleri capable of hyper laccase production can improve the economic feasibility of environmentally benign processes involving use of fungal laccases in cosmetics (including hair dyes), food and beverages, clinical diagnostics, pulp and paper industry, industrial effluent treatment, animal biotechnology and biotransformations.

Manganese

MedlinePlus

... de Manganèse, Dioxyde de Manganèse, Gluconate de Manganèse, Glycérophosphate de Manganèse, Manganèse, Manganese Amino Acid Chelate, Manganese ... Chloridetetrahydrate, Manganese Citrate, Manganese Dioxide, Manganese ... Sulfate, Manganese Sulfate Monohydrate, Manganese Sulfate Tetrahydrate, ...

Laccases as palladium oxidases.

PubMed

Mekmouche, Yasmina; Schneider, Ludovic; Rousselot-Pailley, Pierre; Faure, Bruno; Simaan, A Jalila; Bochot, Constance; Réglier, Marius; Tron, Thierry

2015-02-01

The first example of a coupled catalytic system involving an enzyme and a palladium(ii) catalyst competent for the aerobic oxidation of alcohol in mild conditions is described. In the absence of dioxygen, the fungal laccase LAC3 is reduced by a palladium(0) species as evidenced by the UV/VIS and ESR spectra of the enzyme. During the oxidation of veratryl alcohol performed in water, at room temperature and atmospheric pressure, LAC3 regenerates the palladium catalyst, is reduced and catalyzes the four-electron reduction of dioxygen into water with no loss of enzyme activity. The association of a laccase with a water-soluble palladium complex results in a 7-fold increase in the catalytic efficiency of the complex. This is the first step in the design of a family of renewable palladium catalysts for aerobic oxidation.

Adsorption of Trametes versicolor laccase to soil iron and aluminum minerals: enzyme activity, kinetics and stability studies.

PubMed

Wu, Yue; Jiang, Ying; Jiao, Jiaguo; Liu, Manqiang; Hu, Feng; Griffiths, Bryan S; Li, Huixin

2014-02-01

Laccases play an important role in the degradation of soil phenol or phenol-like substance and can be potentially used in soil remediation through immobilization. Iron and aluminum minerals can adsorb extracellular enzymes in soil environment. In the present study, we investigated the adsorptive interaction of laccase, from the white-rot fungus Trametes versicolor, with soil iron and aluminum minerals and characterized the properties of the enzyme after adsorption to minerals. Results showed that both soil iron and aluminum minerals adsorbed great amount of laccase, independent of the mineral specific surface areas. Adsorbed laccases retained 26-64% of the activity of the free enzyme. Compared to the free laccase, all adsorbed laccases showed higher Km values and lower Vmax values, indicating a reduced enzyme-substrate affinity and a lower rate of substrate conversion in reactions catalyzed by the adsorbed laccase. Adsorbed laccases exhibited increased catalytic activities compared to the free laccase at low pH, implying the suitable application of iron and aluminum mineral-adsorbed T. versicolor laccase in soil bioremediation, especially in acid soils. In terms of the thermal profiles, adsorbed laccases showed decreased thermal stability and higher temperature sensitivity relative to the free laccase. Moreover, adsorption improved the resistance of laccase to proteolysis and extended the lifespan of laccase. Our results implied that adsorbed T. versicolor laccase on soil iron and aluminum minerals had promising potential in soil remediation. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Inactivation of Laccase by the Attack of As (III) Reaction in Water.

PubMed

Hu, Jinyuan; Lu, Kun; Dong, Shipeng; Huang, Qingguo; Mao, Liang

2018-03-06

Laccase is a multicopper oxidase containing four coppers as reaction sites, including one type 1, one type 2, and two type 3. We here provide the first experimental data showing that As (III) can be effectively removed from water and transformed to As (V) through reactions mediated by laccase with the presence of oxygen. To this end, the As (III) removal, As (V) yields, total protein, active laccase, and copper concentrations in the aqueous phase were determined, respectively. Additionally, electron paramagnetic resonance spectra and UV-vis spectra were applied to probe possible structural changes of the laccase during the reaction. The data offer the first evidence that laccase can be inactivated by As (III) attack thus leading to the release of type 2 copper. The released copper has no reactivity with the As (III). These findings provide new ideas into a significant pathway likely to master the environmental transformation of arsenite, and advance the understanding of laccase inactivation mechanisms, thus providing a foundation for optimization of enzyme-based processes and potential development for removal and remediation of arsenite contamination in the environment.

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

PubMed

Dekker, Robert F H; Barbosa, Aneli M; Giese, Ellen C; Godoy, Saulo D S; Covizzi, Luiz G

2007-09-01

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.

Immobilization of laccase of Pycnoporus sanguineus CS43.

PubMed

Gonzalez-Coronel, Luis A; Cobas, Marta; Rostro-Alanis, Magdalena de J; Parra-Saldívar, Roberto; Hernandez-Luna, Carlos; Pazos, Marta; Sanromán, M Ángeles

2017-10-25

Laccase from Pycnoporus sanguineus CS43 was successfully immobilized onto Immobead-150 and Eupergit-C by covalent binding and by entrapment in LentiKats. The highest immobilization was onto Immobead-150 (97.1±1.2%) compared to the other supports, LentiKats (89±1.1%) and Eupergit-C (83.2±1.4%). All three immobilized enzyme systems showed increased thermostability and better mechanical properties than free laccase. Moreover, after 5 cycles of reuse of these systems, 90% of initial laccase activity was retained. Immobead-150 and LentiKats systems exhibited the highest efficiencies in removal of m-cresol under the combined actions of biodegradation and adsorption, while laccase entrapped in LentiKats showed a high ability for degradation of m-cresol within 24h. In addition, the typical Michaelis-Menten enzymatic model effectively described the kinetic profile of m-cresol degradation by the enzyme entrapped in LentiKats. Based on the results obtained in the present study, it can be established that the immobilized biocatalysts developed here possess significant potential for wastewater treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

Potential of acetylacetone as a mediator for Trametes versicolor laccase in enzymatic transformation of organic pollutants.

PubMed

Yang, Hua; Sun, Hongfei; Zhang, Shujuan; Wu, Bingdang; Pan, Bingcai

2015-07-01

Low-cost and environmentally friendly mediators could facilitate the application of laccase (EC 1.10.3.2) in variant biotechnological processes. Acetylacetone (AA) represents an inexpensive and low toxic small molecular diketone that has been proven as an effective mediator for laccase in free radical polymerization. However, the potential of AA as a mediator for laccase in pollutant detoxification and/or degradation is still unknown. In this work, the roles of AA in laccase-induced polymerization and transformation were investigated. AA was demonstrated to be a highly efficient mediator in the laccase-induced grafting copolymerization of acrylamide and chitosan. The efficacy of AA in the laccase-induced decoloration of malachite green (MG) was compared with that of the widely used 1-hydroxybenzotriazole (HBT). The laccase-AA system had the highest turnover number (TON, 39.1 μmol/U), followed by the laccase-only system (28.5 μmol/U), while the TON of the laccase-HBT system was the lowest (14.9 μmol/U). The pseudo-first-order transformation rate constant (k 1) of MG in the laccase-AA system was up to 0.283 h(-1) under the given conditions, while the k 1 of AA caused by laccase was only 0.008 h(-1). In the five-cycle run, the concentration of AA remained stable. The larger TON of the laccase-AA system and the stability of AA in the cycling runs demonstrate that AA was more recyclable than HBT in the LMS, leading to a prolonged serving life of laccase. These results suggest that AA might be a potential redox mediator for laccase.

Laccase-catalyzed oxidation of oxybenzone in municipal wastewater primary effluent.

PubMed

Garcia, Hector A; Hoffman, Catherine M; Kinney, Kerry A; Lawler, Desmond F

2011-02-01

Pharmaceuticals and personal care products (PPCPs) are now routinely detected in raw and treated municipal wastewater. Since conventional wastewater treatment processes are not particularly effective for PPCP removal, treated wastewater discharges are the main entry points for PPCPs into the environment, and eventually into our drinking water. This study investigates the use of laccase-catalyzed oxidation for removing low concentrations of PPCPs from municipal wastewater primary effluent. Oxybenzone was selected as a representative PPCP. Like many other PPCPs, it is not recognized directly by the laccase enzyme. Therefore, mediators were used to expand the oxidative range of laccase, and the efficacy of this laccase-mediator system in primary effluent was evaluated. Eight potential mediators were investigated, and 2,2'-Azino-bis(3-ethylbenzthiazoline-6sulphonic acid) diammonium salt (ABTS), a synthetic mediator, and acetosyringone (ACE), a natural mediator, provided the greatest oxybenzone removal efficiencies. An environmentally relevant concentration of oxybenzone (43.8 nM, 10 μg/L) in primary effluent was completely removed (below the detection limit) after two hours of treatment with ABTS, and 95% was removed after two hours of treatment with ACE. Several mediator/oxybenzone molar ratios were investigated at two different initial oxybenzone concentrations. Higher mediator/oxybenzone molar ratios were required at the lower (environmentally relevant) oxybenzone concentration, and ACE required higher molar ratios than ABTS to achieve comparable oxybenzone removal. Oxybenzone oxidation byproducts generated by the laccase-mediator system were characterized and compared to those generated during ozonation. Enzymatic treatment generated byproducts with higher mass to charge (m/z) ratios, likely due to oxidative coupling reactions. The results of this study suggest that, with further development, the laccase-mediator system has the potential to extend the treatment

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez-Escribano, David; de Salas, Felipe; Pardo, Isabel

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin-Ciocalteau reagent (FCR), to detect the decrease in phenolic contentmore » produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). As a result, the method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.« less

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

DOE PAGES

Rodriguez-Escribano, David; de Salas, Felipe; Pardo, Isabel; ...

2017-08-18

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin-Ciocalteau reagent (FCR), to detect the decrease in phenolic contentmore » produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). As a result, the method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.« less

Dehalogenation of Chlorinated Hydroxybiphenyls by Fungal Laccase

PubMed Central

Schultz, Asgard; Jonas, Ulrike; Hammer, Elke; Schauer, Frieder

2001-01-01

We have investigated the transformation of chlorinated hydroxybiphenyls by laccase produced by Pycnoporus cinnabarinus. The compounds used were transformed to sparingly water-soluble colored precipitates which were identified by gas chromatography-mass spectrometry as oligomerization products of the chlorinated hydroxybiphenyls. During oligomerization of 2-hydroxy-5-chlorobiphenyl and 3-chloro-4-hydroxybiphenyl, dechlorinated C—C-linked dimers were formed, demonstrating the dehalogenation ability of laccase. In addition to these nonhalogenated dimers, both monohalogenated and dihalogenated dimers were identified. PMID:11526052

Location of laccase in ordered mesoporous materials

NASA Astrophysics Data System (ADS)

Mayoral, Álvaro; Gascón, Victoria; Blanco, Rosa M.; Márquez-Álvarez, Carlos; Díaz, Isabel

2014-11-01

The functionalization with amine groups was developed on the SBA-15, and its effect in the laccase immobilization was compared with that of a Periodic Mesoporous Aminosilica. A method to encapsulate the laccase in situ has now been developed. In this work, spherical aberration (Cs) corrected scanning transmission electron microscopy combined with high angle annular dark field detector and electron energy loss spectroscopy were applied to identify the exact location of the enzyme in the matrix formed by the ordered mesoporous solids.

Overexpression and characterization of laccase from Trametes versicolor in Pichia pastoris.

PubMed

Li, Q; Pei, J; Zhao, L; Xie, J; Cao, F; Wang, G

2014-01-01

A laccase-encoding gene of Trametes versicolor, lccA, was cloned and expressed in Pichia pastoris X33. The lccA gene consists ofa 1560 bp open reading frame encoding 519 amino acids, which was classified into family copper blue oxidase. To improve the expression level of recombinant laccase in P. pastoris, conditions of the fermentation were optimized by the single factor experiments. The optimal fermentation conditions for the laccase production in shake flask cultivation using BMGY medium were obtained: the optimal initial pH 7.0, the presence of 0.5 mM Cu2+, 0.6% methanol added into the culture every 24 h. The laccase activity was up to 11.972 U/L under optimal conditions after 16 days of induction in a medium with 4% peptone. After 100 h of large scale production in 5 L fermenter the enzyme activity reached 18.123 U/L. The recombinant laccase was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimum pH and temperature for the laccase were pH 2.0 and 50 degrees C with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. The recombinant laccase was stable over a pH range of 2.0-7.0. The K(m) and the V(max) value of LccA were 0.43 mM and 82.3 U/mg for ABTS, respectively.

Screening of white-rot fungi manganese peroxidases: a comparison between the specific activities of the enzyme from different native producers

PubMed Central

2012-01-01

In this study manganese peroxidase (MnP) enzymes from selected white-rot fungi were isolated and compared for potential future recombinant production. White-rot fungi were cultivated in small-scale in liquid media and a simplified process was established for the purification of extracellular enzymes. Five lignin degrading organisms were selected (Bjerkandera sp., Phanerochaete (P.) chrysosporium, Physisporinus (P.) rivulosus, Phlebia (P.) radiata and Phlebia sp. Nf b19) and studied for MnP production in small-scale. Extracellular MnP activity was followed and cultivations were harvested at proximity of the peak activity. The production of MnPs varied in different organisms but was clearly regulated by inducing liquid media components (Mn2+, veratryl alcohol and malonate). In total 8 different MnP isoforms were purified. Results of this study reinforce the conception that MnPs from distinct organisms differ substantially in their properties. Production of the extracellular enzyme in general did not reach a substantial level. This further suggests that these native producers are not suitable for industrial scale production of the enzyme. The highest specific activities were observed with MnPs from P. chrysosporium (200 U mg-1), Phlebia sp. Nf b19 (55 U mg-1) and P. rivulosus (89 U mg-1) and these MnPs are considered as the most potential candidates for further studies. The molecular weight of the purified MnPs was estimated to be between 45–50 kDa. PMID:23190610

Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

DOE PAGES

Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J.; ...

2014-11-22

The genome ofCeriporiopsis subvermisporaincludes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn 2+-oxidation site and have varying lengths of the C-terminal tail. We expressed short, long and extralong MnPs heterologously and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn 2+oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. Furthermore, the tail, which is anchored by numerous contacts, notmore » only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd 2+binds at the Mn 2+-oxidation site and competitively inhibits oxidation of both Mn 2+and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of anin silicoshortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.« less

Degradation and detoxification of the triphenylmethane dye malachite green catalyzed by crude manganese peroxidase from Irpex lacteus F17.

PubMed

Yang, Xueting; Zheng, Jinzhao; Lu, Yongming; Jia, Rong

2016-05-01

Malachite green (MG), a recalcitrant, carcinogenic, and mutagenic triphenylmethane dye, was decolorized and detoxified using crude manganese peroxidase (MnP) prepared from the white rot fungus Irpex lacteus F17. In this study, the key factors (pH, temperature, MG, Mn(2+), H2O2, MnP) in these processes were investigated. Under optimal conditions, 96 % of 200 mg L(-1) of MG was decolorized when 66.32 U L(-1) of MnP was added for 1 h. The K m, V max, and k cat values were 109.9 μmol L(-1), 152.8 μmol L(-1) min(-1), and 44.5 s(-1), respectively. The decolorization of MG by MnP followed first-order reaction kinetics with a kinetic rate constant of 0.0129 h(-1). UV-vis and UPLC analysis revealed degradation of MG. Furthermore, seven different intermediates formed during the MnP treatment of 0.5 h were identified by LC-TOF-MS. These degradation products were generated via two different routes by either N-demethylation of MG or the oxidative cleavage of the C-C double bond in MG. Based on ecotoxicity analyses performed on bacteria and algae, it was confirmed that MG metabolites produced by the MnP-catalyzed system were appreciably less toxic than the parent compound. These studies indicate the potential use of this enzyme system in the clean-up of aquatic and terrestrial environments.

Comparative analysis of spatial organization of laccases from Cerrena maxima and Coriolus zonatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhukova, Yu. N.; Zhukhlistova, N. E.; Lyashenko, A. V.

2007-09-15

Laccase (oxygen oxidoreductase, EC 1.10.3.2) belongs to the multicopper oxidase family. The main function of this enzyme is to perform electron transfer from the oxidized substrate through the mononuclear copper-containing site T1 to the oxygen molecule bound to the site T3 in the trinuclear T2/T3 cluster. The structures of two new fungal laccases from C. maxima and C. zonatus were solved on the basis of synchrotron X-ray diffraction data. Both laccases show high structural homology with laccases from other sources. The role of the carbohydrate component of laccases in structure stabilization and formation of ordered protein crystals was demonstrated. Inmore » the structures of C. maxima and C. zonatus laccases, two water channels of functional importance were found and characterized. The structural results reported in the present study characterize one of the functional states of the enzyme fixed in the crystal structure.« less

Investigating the degradation process of kraft lignin by β-proteobacterium, Pandoraea sp. ISTKB.

PubMed

Kumar, Madan; Singh, Jyoti; Singh, Manoj Kumar; Singhal, Anjali; Thakur, Indu Shekhar

2015-10-01

The present study investigates the kraft lignin (KL) degrading potential of novel alkalotolerant Pandoraea sp. ISTKB utilizing KL as sole carbon source. The results displayed 50.2 % reduction in chemical oxygen demand (COD) and 41.1 % decolorization after bacterial treatment. The maximum lignin peroxidase (LiP) and manganese peroxidase (MnP) activity detected was 2.73 and 4.33 U ml(-1), respectively, on day 3. The maximum extracellular and intracellular laccase activities observed were 1.32 U ml(-1) on day 5 and 4.53 U ml(-1) on day 4, respectively. The decolorization and degradation was maximum on day 2. Further, it registered an increase with the production of extracellular laccase. This unusual trend of decolorization and degradation was studied using various aromatic compounds and dyes. SEM and FTIR results indicated significant change in surface morphology and functional group composition during the course of degradation. Gas chromatography and mass spectroscopy (GC-MS) analysis confirmed KL degradation by emergence of new peaks and the identification of low molecular weight aromatic intermediates in treated sample. The degradation of KL progressed through the generation of phenolic intermediates. The identified intermediates implied the degradation of hydroxyphenyl, ferulic acid, guaiacyl, syringyl, phenylcoumarane, and pinoresinol components commonly found in lignin. The degradation, decolorization, and GC-MS analysis indicated potential application of the isolate Pandoraea sp. ISTKB in treatment of lignin-containing pollutants and KL valorization.

Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

NASA Astrophysics Data System (ADS)

Gottschalk, Leda M. F.; Bon, Elba P. S.; Nobrega, Ronaldo

It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.

Role of laccase from Coriolus versicolor MTCC-138 in selective oxidation of aromatic methyl group.

PubMed

Chaurasia, Pankaj Kumar; Singh, Sunil Kumar; Bharati, Shashi Lata

2014-01-01

Now a day, laccases are the most promising enzymes in the area of biotechnology and synthesis. One of the best applications of laccases is the selective oxidation of aromatic methyl group to aldehyde group. Such transformations are valuable because it is difficult to stop the reaction at aldehyde stage. Chemical methods used for such biotransformations areexpensive and give poor yields. But, the laccase-catalyzed biotransformations of such type are non-expensive and yield is excellent. Authors have used crude laccase obtained from the liquid culture growth medium of fungal strain Coriolus versicolor MTCC-138 for the biotransformations of toluene, 3-nitrotoluene, and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde, and 4-chlorobenzaldehyde, respectively, instead of purified laccase because purification process requires much time and cost. This communication reports that crude laccase can also be used in the place of purified laccase as effective biocatalyst.

Mycoremediation of manganese and phenanthrene by Pleurotus eryngii mycelium enhanced by Tween 80 and saponin.

PubMed

Wu, Minghui; Xu, Yongan; Ding, Wenbo; Li, Yuanyuan; Xu, Heng

2016-08-01

Bioremediation of areas co-contaminated with metals and polycyclic aromatic hydrocarbons (PAHs) by mushrooms has attracted considerable attention in recent years. In this study, Pleurotus eryngii was introduced for the removal of Mn and phenanthrene (Phe) from potato liquid medium (PDL) simultaneously. Effects of Tween 80 and saponin on P. eryngii growth together with Mn uptake as well as Phe removal were investigated. Although pollutants had a negative effect on mycelial morphology and growth, P. eryngii could still tolerate and remove Mn and Phe. Tween 80 increased removal of Mn and Phe through increase of P. eryngii growth, Phe solubility, pollutants bioavailability, and specific surface area of mycelium pellets, moreover, the activities of manganese peroxidase (MnP) and laccase, which played an important role on PAHs biodegradation. The maximal removal of Mn and Phe was achieved (92.17 and 93.85 % after 15 days incubation, respectively) with 0.6 g L(-1) Tween 80. Treatments with saponin markedly inhibited P. eryngii growth (50.17-66.32 % lower relative to control) due to its fungistatic activity. Nevertheless, saponin could slightly enhance Phe removal through increasing solubility of Phe, and Phe removal rate varied from 80.53 to 87.06 % in saponin treatments. Joint stress of Mn and Phe induced a strong antioxidative response, and superoxide dismutase (SOD) activity decreased in surfactants-treated mycelium compared with control. Generally, Tween 80 was more suitable for strengthening mycoremediation by P. eryngii than saponin, and could be a promising alternative for the remediation of heavy metals and PAHs co-contaminated sites by mushrooms.

Purification and characterization of a melanin biodegradation enzyme from Geotrichum sp.

PubMed

Kim, B S; Blaghen, M; Hong, H-S; Lee, K-M

2016-12-01

Melanin is a black or brown phenolic polymer present mainly in skin and hair. Although melanin can be degraded by some microbial species, the melanin degradation capacity of Geotrichum sp. is unknown. The aim of this study was to characterize a melanin biodegradation enzyme from Geotrichum sp. In this study, we assessed the melanin degradation activity of Geotrichum sp. in comparison with the major melanin-degrading enzymes, manganese-dependent peroxidase (MnP), manganese-independent peroxidase, lignin peroxidase and laccase. Furthermore, the effect of several carbohydrates on melanin degradation by Geotrichum sp. was determined. The MnP enzyme was purified using ammonium sulphate precipitation and Sephadex G-200 column chromatography, and then the conditions for optimal enzymatic activity were determined by adjusting the pH, temperature and Tween-80 concentration. Compared with extracellular ligninolytic enzymes of Geotrichum sp., MnP had the highest ligninolytic enzyme activity; and the highest enzymatic activity was observed in the presence of glucose. The final purified MnP enzyme exhibited 6 U mL -1 activity and had a molecular weight of 54.2 kDa. The enzymatic activity was highest at pH 4.5 and 25-35°C in the absence of Tween-80. These results indicate the potential of MnP purified from Geotrichum sp. as a skin-lightening agent in the cosmetic industry. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Upgrading Laccase Production and Biochemical Properties: Strategies and Challenges.

PubMed

Bertrand, Brandt; Martínez-Morales, Fernando; Trejo-Hernández, María R

2017-07-01

Improving laccases continues to be crucial in novel biotechnological developments and industrial applications, where they are concerned. This review breaks down and explores the potential of the strategies (conventional and modern) that can be used for laccase enhancement (increased production and upgraded biochemical properties such as stability and catalytic efficiency). The challenges faced with these approaches are briefly discussed. We also shed light on how these strategies merge and give rise to new options and advances in this field of work. Additionally, this article seeks to serve as a guide for students and academic researchers interested in laccases. This document not only gives basic information on laccases, but also provides updated information on the state of the art of various technologies that are used in this line of investigation. It also gives the readers an idea of the areas extensively studied and the areas where there is still much left to be done. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1015-1034, 2017. © 2017 American Institute of Chemical Engineers.

Laccase/mediator assisted degradation of triarylmethane dyes in a continuous membrane reactor.

PubMed

Chhabra, Meenu; Mishra, Saroj; Sreekrishnan, Trichur Ramaswamy

2009-08-10

Laccase/mediator systems are important bioremediation agents as the rates of reactions can be enhanced in the presence of the mediators. The decolorization mechanism of two triarylmethane dyes, namely, Basic Green 4 and Acid Violet 17 is reported using Cyathus bulleri laccase. Basic Green 4 was decolorized through N-demethylation by laccase alone, while in mediator assisted reactions, dye breakdown was initiated from oxidation of carbinol form of the dye. Benzaldehyde and N,N-dimethyl aniline were the major end products. With Acid Violet 17, laccase carried out N-deethylation and in mediator assisted reactions, oxidation of the carbinol form of the dye occurred resulting in formation of formyl benzene sulfonic acid, carboxy benzene sulfonic acid and benzene sulfonic acid. Toxicity analysis revealed that Basic Green 4 was toxic and treatment with laccase/mediators resulted in 80-100% detoxification. The treatment of the textile dye solution using laccase and 2,2'-azino-di-(-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was demonstrated in an enzyme membrane reactor. At a hydraulic retention time of 6h, the process was operated for a period of 15 days with nearly 95% decolorization, 10% reduction in flux and 70% recovery of active ABTS.

Decolorization and Detoxification of Textile Dyes with a Laccase from Trametes hirsuta

PubMed Central

Abadulla, Elias; Tzanov, Tzanko; Costa, Silgia; Robra, Karl-Heinz; Cavaco-Paulo, Artur; Gübitz, Georg M.

2000-01-01

Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC50) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (ΔE*) below 1.1 were measured for most dyes. PMID:10919791

Pycnoporus cinnabarinus laccases: an interesting tool for food or non-food applications.

PubMed

Georis, J; Lomascolo, A; Camarero, S; Dorgeo, V; Herpoël, I; Asther, M; Martinez, A T; Dauvrin, T

2003-01-01

The effects of the addition of ferulic acid and ethanol in P. cinnabarinus ss3 culture medium in fermentor were compared in 15-L fermentor. In the presence of 30 g l(-1) ethanol, laccase activity (270,000 U/L1) was 3-fold higher as compared with ferulic acid-induced cultures, and 150-fold higher as compared with non-induced cultures, respectively. High-quality flax pulp was bleached in a totally-chlorine free (TCF) sequence using a laccase-mediator system constituted by laccase from Pycnoporus cinnabarinus and 1-hydroxybenzotriazole (HBT) as mediator. Up to 90% delignification and strong brightness increase were attained after the laccase-mediator treatment followed by H2O2 bleaching. This TCF sequence was further improved by applying H2O2 under pressurized O2. In this way, up to 82% ISO brightness was obtained (compared with 37% in the initial pulp and 60% in the peroxide-bleached control) as well as very low kappa number. A positive evaluation of the laccase has been also performed in a food application. The colour of a tea-based beverage was significantly improved by incubating an infusion of green tea with the Pycnoporus laccase.

Location of laccase in ordered mesoporous materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mayoral, Álvaro; Gascón, Victoria; Blanco, Rosa M.

2014-11-01

The functionalization with amine groups was developed on the SBA-15, and its effect in the laccase immobilization was compared with that of a Periodic Mesoporous Aminosilica. A method to encapsulate the laccase in situ has now been developed. In this work, spherical aberration (C{sub s}) corrected scanning transmission electron microscopy combined with high angle annular dark field detector and electron energy loss spectroscopy were applied to identify the exact location of the enzyme in the matrix formed by the ordered mesoporous solids.

Thermokinetic comparison of trypan blue decolorization by free laccase and fungal biomass.

PubMed

Razak, N N A; Annuar, M S M

2014-03-01

Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k1', value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ∼ 22 kJ mol(-1), while energy for laccase inactivation was 18 kJ mol(-1). Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (ΔT = 40 K) as opposed to fungal biomass (ΔT = 15 K). Comparison of entropy change (ΔS) values indicated metabolism of the dye by the biomass.

Media optimization for laccase production by Trichoderma harzianum ZF-2 using response surface methodology.

PubMed

Gao, Huiju; Chu, Xiang; Wang, Yanwen; Zhou, Fei; Zhao, Kai; Mu, Zhimei; Liu, Qingxin

2013-12-01

Trichoderma harzianum ZF-2 producing laccase was isolated from decaying samples from Shandong, China, and showed dye decolorization activities. The objective of this study was to optimize its culture conditions using a statistical analysis of its laccase production. The interactions between different fermentation parameters for laccase production were characterized using a Plackett-Burman design and the response surface methodology. The different media components were initially optimized using the conventional one-factor-at-a-time method and an orthogonal test design, and a Plackett-Burman experiment was then performed to evaluate the effects on laccase production. Wheat straw powder, soybean meal, and CuSO4 were all found to have a significant influence on laccase production, and the optimal concentrations of these three factors were then sequentially investigated using the response surface methodology with a central composite design. The resulting optimal medium components for laccase production were determined as follows: wheat straw powder 7.63 g/l, soybean meal 23.07 g/l, (NH4)2SO4 1 g/l, CuSO4 0.51 g/l, Tween-20 1 g/l, MgSO4 1 g/l, and KH2PO4 0.6 g/l. Using this optimized fermentation method, the yield of laccase was increased 59.68 times to 67.258 U/ml compared with the laccase production with an unoptimized medium. This is the first report on the statistical optimization of laccase production by Trichoderma harzianum ZF-2.

Scale-up laccase production from Trametes versicolor stimulated by vanillic acid.

PubMed

Wang, Ke-Feng; Hu, Jian-Hua; Guo, Chen; Liu, Chun-Zhao

2016-07-01

An efficient strategy for laccase production in Trametes versicolor cultures was developed using vanillic acid as the inducer. The optimized vanillic acid treatment strategy consisted of exposing 2-day-old mycelia cultures to 80 mg/L vanillic acid. After 4 days, laccase activity of 588.84 U/L was achieved in flasks which represented a 1.79-fold increase compared to the control. In 200-L airlift bioreactor, the maximal laccase activity reached up to 785.12 U/L using the optimized vanillic acid treatment strategy. The zymograms of culture supernatants revealed three bands with laccase activity, among which Lac1 and Lac2 were abundant laccase isoforms constitutively expressed, and Lac3 was an inducible isozyme by vanillic acid. The results of real-time quantitative PCR showed that the transcription level of lcc in T. versicolor cultures grown with vanillic acid for 7 days was about 5.64-fold greater than that without vanillic acid in flasks. In 200-L airlift bioreactor cultures of T. versicolor with addition of vanillic acid, the transcript level of lcc at day 7 was 2.62-fold higher than that in flasks with vanillic acid due to the good mass transfer and oxygen supply in the bioreactor system. This study provides a basis for understanding the induction mechanism of vanillic acid for laccase production and has good potential for industrial applications.

Disruption of LACCASE4 and 17 Results in Tissue-Specific Alterations to Lignification of Arabidopsis thaliana Stems[W

PubMed Central

Berthet, Serge; Demont-Caulet, Nathalie; Pollet, Brigitte; Bidzinski, Przemyslaw; Cézard, Laurent; Le Bris, Phillipe; Borrega, Nero; Hervé, Jonathan; Blondet, Eddy; Balzergue, Sandrine; Lapierre, Catherine; Jouanin, Lise

2011-01-01

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers. PMID:21447792

Laccase-catalyzed oxidation of iodide and formation of organically bound iodine in soils.

PubMed

Seki, Miharu; Oikawa, Jun-ichi; Taguchi, Taro; Ohnuki, Toshihiko; Muramatsu, Yasuyuki; Sakamoto, Kazunori; Amachi, Seigo

2013-01-02

Laccase oxidizes iodide to molecular iodine or hypoiodous acid, both of which are easily incorporated into natural soil organic matter. In this study, iodide sorption and laccase activity in 2 types of Japanese soil were determined under various experimental conditions to evaluate possible involvement of this enzyme in the sorption of iodide. Batch sorption experiment using radioactive iodide tracer ((125)I(-)) revealed that the sorption was significantly inhibited by autoclaving (121 °C, 40 min), heat treatment (80 and 100 °C, 10 min), γ-irradiation (30 kGy), N(2) gas flushing, and addition of reducing agents and general laccase inhibitors (KCN and NaN(3)). Interestingly, very similar tendency of inhibition was observed in soil laccase activity, which was determined using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as a substrate. The partition coefficient (K(d): mL g(-1)) for iodide and specific activity of laccase in soils (Unit g(-1)) showed significant positive correlation in both soil samples. Addition of a bacterial laccase with an iodide-oxidizing activity to the soils strongly enhanced the sorption of iodide. Furthermore, the enzyme addition partially restored iodide sorption capacity of the autoclaved soil samples. These results suggest that microbial laccase is involved in iodide sorption on soils through the oxidation of iodide.

One-pot synthesis of active copper-containing carbon dots with laccase-like activities

NASA Astrophysics Data System (ADS)

Ren, Xiangling; Liu, Jing; Ren, Jun; Tang, Fangqiong; Meng, Xianwei

2015-11-01

Herein, an effective strategy for designing a new type of nanozyme, blue fluorescent laccase mimics, is reported. Active copper-containing carbon dots (Cu-CDs) were synthesized through a simple, nontoxic and one-pot hydrothermal method, which showed favorable photoluminescence properties and good photostability under high-salt conditions or in a broad pH range (3.0-13.5). The Cu-CDs possessed intrinsic laccase-like activities and could catalyze the oxidation of the laccase substrate p-phenylenediamine (PPD) to produce a typical color change from colorless to brown. Poly(methacrylic acid sodium salt) (PMAA) not only was used as the carbon source and reducing agent, but also provided carboxyl groups to assist flocculation between Cu-CDs and polyacrylamide, which facilitated the removal of PPD. Importantly, the intrinsic fluorescence of the as-prepared Cu-CDs could indicate the presence of hydroquinone, one of the substrates of laccases, based on laccase mimics and fluorescence quenching.Herein, an effective strategy for designing a new type of nanozyme, blue fluorescent laccase mimics, is reported. Active copper-containing carbon dots (Cu-CDs) were synthesized through a simple, nontoxic and one-pot hydrothermal method, which showed favorable photoluminescence properties and good photostability under high-salt conditions or in a broad pH range (3.0-13.5). The Cu-CDs possessed intrinsic laccase-like activities and could catalyze the oxidation of the laccase substrate p-phenylenediamine (PPD) to produce a typical color change from colorless to brown. Poly(methacrylic acid sodium salt) (PMAA) not only was used as the carbon source and reducing agent, but also provided carboxyl groups to assist flocculation between Cu-CDs and polyacrylamide, which facilitated the removal of PPD. Importantly, the intrinsic fluorescence of the as-prepared Cu-CDs could indicate the presence of hydroquinone, one of the substrates of laccases, based on laccase mimics and fluorescence quenching

Use of bacteria for improving the lignocellulose biorefinery process: importance of pre-erosion.

PubMed

Zhuo, Shengnan; Yan, Xu; Liu, Dan; Si, Mengying; Zhang, Kejing; Liu, Mingren; Peng, Bing; Shi, Yan

2018-01-01

Biological pretreatment is an important alternative strategy for biorefining lignocellulose and has attracted increasing attention in recent years. However, current designs for this pretreatment mainly focus on using various white rot fungi, overlooking the bacteria. To the best of our knowledge, for the first time, we evaluated the potential contribution of bacteria to lignocellulose pretreatment, with and without a physicochemical process, based on the bacterial strain Pandoraea sp. B-6 (hereafter B-6) that was isolated from erosive bamboo slips. Moreover, the mechanism of the improvement of reducing sugar yield by bacteria was elucidated via analyses of the physicochemical changes of corn stover (CS) before and after pretreatment. The digestibility of CS pretreated with B-6 was equivalent to that of untreated CS. The recalcitrant CS surface provided fewer mediators for contact with the extracellular enzymes of B-6. A pre-erosion strategy using a tetrahydrofuran-water co-solvent system was shown to destroy the recalcitrant CS surface. The optimal condition for pre-erosion showed a 6.5-fold increase in enzymatic digestibility compared with untreated CS. The pre-erosion of CS can expose more phenolic compounds that were chelated to oxidized Mn 3+ and also provided mediators for combination with laccase, which was attributable to B-6 pretreatment. B-6 pretreatment following pre-erosion exhibited a sugar yield that was 91.2 mg/g greater than that of pre-erosion alone and 7.5-fold higher than that of untreated CS. This pre-erosion application was able to destroy the recalcitrant CS surface, thus leading to a rough and porous architecture that better facilitated the diffusion and transport of lignin derivatives. This enhanced the ability of laccase and manganese peroxidase secreted by B-6 to improve the efficiency of this biological pretreatment. Bacteria were not found useful alone as a biological pretreatment, but they significantly improved enzymatic digestion

Enhanced laccase production by Trametes versicolor using corn steep liquor as both nitrogen source and inducer.

PubMed

Wang, Feng; Hu, Jian-Hua; Guo, Chen; Liu, Chun-Zhao

2014-08-01

A highly efficient strategy for laccase production by Trametes versicolor was developed using corn steep liquor (CSL) as both a nitrogen source and a laccase inducer. At the optimal CSL concentration of 20 gL(-1), an extracellular laccase activity of 633.3 UL(-1) was produced after a culture period of only 5 days. This represented a 1.96-fold increase relative to control medium lacking CSL. The addition of crude phenolic extracts from CSL improved laccase production to 91.8% greater than the control. Sinapinic acid, present in CSL, caused a reduction in laccase production, vanillic acid and ferulic acid (also present in CSL) synergistically induced laccase production by more than 100% greater than the control medium. Vanillic acid and ferulic acid provided the main contribution to the enhancement of laccase production. This study provides a basis for understanding the induction mechanism of CSL for laccase production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of the potential of microbial combinations obtained from spent mushroom cultivation substrates for use in textile effluent decolorization.

PubMed

Singh, Rajender; Ahlawat, O P; Rajor, Anita

2012-12-01

The study presents variation in microbial population of Agaricus bisporus, Pleurotus sajor-caju and Volvariella volvacea spent substrates (SMS) along with ligninolytic enzymes activity and textile effluent decolorization potential of microorganisms isolated from these. The effect of temperature, pH, carbon sources and immobilizing agents on effluent decolorization using different combinations of these microorganisms has also been studied. SMS of P. sajor-caju harbored highest population and diversity of bacteria and fungi compared to other SMSs. Schizophyllum commune and Pezizomycotina sp. from P. sajor-caju SMS, exhibited highest activities of laccase (11.8 and 8.32U mL(-1)) and lignin peroxidase (339 and 318 UL(-1)), while Pseudomonas fluorescens of Manganese peroxidase. Highest decolorization was in presence of glucose and sucrose at 30°C, and microbial consortium comprised of the immobilized forms of S. commune and Pezizomycotina sp. on wheat straw and broth cultures of P. fluorescens, Bacillus licheniformis and Bacillus pumilus. Copyright © 2012 Elsevier Ltd. All rights reserved.

Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi.

PubMed

Dias, Albino A; Freitas, Gil S; Marques, Guilhermina S M; Sampaio, Ana; Fraga, Irene S; Rodrigues, Miguel A M; Evtuguin, Dmitry V; Bezerra, Rui M F

2010-08-01

Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities. (c) 2010 Elsevier Ltd. All rights reserved.

Biotreatment of textile effluent in static bioreactor by Curvularia lunata URM 6179 and Phanerochaete chrysosporium URM 6181.

PubMed

Miranda, Rita de Cássia M de; Gomes, Edelvio de Barros; Pereira, Nei; Marin-Morales, Maria Aparecida; Machado, Katia Maria Gomes; Gusmão, Norma Buarque de

2013-08-01

Investigations on biodegradation of textile effluent by filamentous fungi strains Curvularia lunata URM 6179 and Phanerochaete chrysosporium URM 6181 were performed in static bioreactors under aerated and non-aerated conditions. Spectrophotometric, HPLC/UV and LC-MS/MS analysis were performed as for to confirm, respectively, decolourisation, biodegradation and identity of compounds in the effluent. Enzymatic assays revealed higher production of enzymes laccase (Lac), lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) by P. chrysosporium URM 6181 in aerated bioreactor (2020; 39 and 392 U/l, respectively). Both strains decolourised completely the effluent after ten days and biodegradation of the most predominant indigo dye was superior in aerated bioreactor (96%). Effluent treated by P. chrysosporium URM 6181 accumulated a mutagenic metabolite derived from indigo. The C. lunata URM 6179 strain, showed to be more successful for assure the environmental quality of treated effluent. These systems were found very effective for efficient fungal treatment of textile effluent. Copyright © 2013 Elsevier Ltd. All rights reserved.

Magnetic mesoporous silica nanoparticles: fabrication and their laccase immobilization performance.

PubMed

Wang, Feng; Guo, Chen; Yang, Liang-rong; Liu, Chun-Zhao

2010-12-01

Newly large-pore magnetic mesoporous silica nanoparticles (MMSNPs) with wormhole framework structures were synthesized for the first time by using tetraethyl orthosilicate as the silica source and amine-terminated Jeffamine surfactants as template. Iminodiacerate was attached on these MMSNPs through a silane-coupling agent and chelated with Cu(2+). The Cu(2+)-chelated MMSNPs (MMSNPs-CPTS-IDA-Cu(2+)) showed higher adsorption capacity of 98.1 mg g(-1)-particles and activity recovery of 92.5% for laccase via metal affinity adsorption in comparison with MMSNPs via physical adsorption. The Michaelis constant (K(m)) and catalytic constant (k(cat)) of laccase immobilized on the MMSNPs-CPTS-IDA-Cu(2+) were 3.28 mM and 155.4 min(-1), respectively. Storage stability and temperature endurance of the immobilized laccase on MMSNPs-CPTS-IDA-Cu(2+) increased significantly, and the immobilized laccase retained 86.6% of its initial activity after 10 successive batch reactions operated with magnetic separation. 2010 Elsevier Ltd. All rights reserved.

Laccase-conjugated amino-functionalized nanosilica for efficient degradation of Reactive Violet 1 dye

NASA Astrophysics Data System (ADS)

Gahlout, Mayur; Rudakiya, Darshan M.; Gupte, Shilpa; Gupte, Akshaya

2017-08-01

Immobilization of enzyme with nanostructures enhances its ideal characteristics, which may allow the enzyme to become more stable and resistant. The present investigation deals with the formulation of laccase nanosilica conjugates to overcome the problems associated with its stability and reusability. Synthesized nanosilica and laccase nanoparticles were spherical shaped, with the mean size of 220 and 615 nm, respectively. Laccase nanoparticles had an optimum temperature of 55 °C and pH 4.0 for the oxidation of ABTS. Laccase nanoparticle retained 79% of residual activity till 20th cycle. It also showed 91% of its initial activity at lower temperatures even after 60 days. Laccase nanoparticles were applied for Reactive Violet 1 degradation wherein 96.76% of decolourization was obtained at pH 5.0 and 30 °C within 12 h. Toxicity studies on microbes and plants suggested that the degraded metabolites were less toxic than control dye. Thus, the method applied for immobilization increased storage stability and reusability of laccase, and therefore, it can be utilized for efficient degradation of azo dyes.

Immobilization of fungal laccase onto a nonionic surfactant-modified clay material: application to PAH degradation.

PubMed

Chang, Yi-Tang; Lee, Jiunn-Fwu; Liu, Keng-Hua; Liao, Yi-Fen; Yang, Vivian

2016-03-01

Nonionic surfactant-modified clay is a useful absorbent material that effectively removes hydrophobic organic compounds from soil/groundwater. We developed a novel material by applying an immobilized fungal laccase onto nonionic surfactant-modified clay. Low-water-solubility polycyclic aromatic hydrocarbons (PAHs) (naphthalene/phenanthrene) were degraded in the presence of this bioactive material. PAH degradation by free laccase was higher than degradation by immobilized laccase when the surfactant concentration was allowed to form micelles. PAH degradation by immobilized laccase on TX-100-modified clay was higher than on Brij35-modified clay. Strong laccase degradation of PAH can be maintained by adding surfactant monomers or micelles. The physical adsorption of nonionic surfactants onto clay plays an important role in PAH degradation by laccase, which can be explained by the structure and molecular interactions of the surfactant with the clay and enzyme. A system where laccase is immobilized onto TX-100-monomer-modified clay is a good candidate bioactive material for in situ PAHs bioremediation.

Effect of antibiotics on growth and laccase production from Cyathus bulleri and Pycnoporus cinnabarinus.

PubMed

Dhawan, Shikha; Lal, Rup; Hanspal, Manjit; Kuhad, Ramesh Chander

2005-08-01

The effect of nine different antibiotics (chloramphenicol, ampicillin trihydrate, kanamycin A monosulfate, neomycin sulfate, erythromycin, thiostrepton, tetracycline, apramycin sulfate and streptomycin sulfate) on growth and laccase production from Cyathus bulleri and Pycnoporus cinnabarinus has been investigated. All the antibiotics tested at a concentration of 200 mg/l affected the fungal growth, release of protein and laccase production to different extent. Inhibition in fungal growth was found to be positively correlated with increase in laccase production. Interestingly, apramycin sulfate inhibited biomass production (14.9-26.2%), nevertheless, it stimulated maximum laccase production (18.2 U/ml) in both the fungi. Increasing concentrations of apramycin sulfate enhanced laccase production from P. cinnabarinus but not from C. bulleri.

Transcriptional response of lignin-degrading enzymes to 17α-ethinyloestradiol in two white rots.

PubMed

Přenosilová, L; Křesinová, Z; Amemori, A Slavíková; Cajthaml, T; Svobodová, K

2013-05-01

Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g⁻¹ of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l⁻¹ EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Characterization and immobilization of Trametes versicolor laccase on magnetic chitosan-clay composite beads for phenol removal.

PubMed

Aydemir, Tülin; Güler, Semra

2015-01-01

Laccase from Trametes versicolor was immobilized on magnetic chitosan-clay composite beads by glutaraldehyde crosslinking. The physical, chemical, and biochemical properties of the immobilized laccase and its application in phenol removal were comprehensively investigated. The structure and morphology of the composite beads were characterized by SEM, TGA, and FTIR analyses. The immobilized laccase showed better storage stability and higher tolerance to the changes in pH and temperature compared with free laccase. Moreover, the immobilized laccase retained more than 75% of its original activity after 10 cycles. The efficiency of phenol removal by immobilized laccase was about 80% under the optimum conditions after 4 h.

Determination Hypoiodous Acid (HIO) By Peroxidase System Using Peroxidase Enzyme

NASA Astrophysics Data System (ADS)

Al-Baarri, A. N.; Legowo, A. M.; Widayat; Abduh, S. B. M.; Hadipernata, M.; Wisnubroto; Ardianti, D. K.; Susanto, M. N.; Yusuf, M.; Demasta, E. K.

2018-02-01

It has been understood that peroxidase enzyme including peroxidase serves as catalyzer to enzymatic reaction among hydrogen peroxide and halides, therefore this research was done for generating hypoiodous acid (HIO) from peroxidase system using peroxidase enzyme. Hydrogen peroxide, potassium iodide, and peroxidase enzyme were used to produce HIO. Determination the amount of formed HIO was done using 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) or ABTS as substrate through the colorimetric measurement of hydrogen peroxide residue during reaction process using at 412 nm. The result indicated that residual hydrogen peroxide showed the minimum concentration after 60 minutes reaction time. Because the reaction started at the beginning time of mixing, hydrogen peroxide was unable to be eliminated totally to produce HIO. The reaction of peroxidase system was able to determine the beginning of mixing process but the reaction process could not eliminate the initial concentration of hydrogen peroxide indicating the maximum amount of production of HIO could be determined. In conclusion, the less of H2O2, higher HIO obtained and peroxidase enzymes can accelerate the formation of HIO.

Structure and Biochemestry of Laccases from the Lignin-Degrading Basidiomycete, Ganoderma lucidum

DOE Office of Scientific and Technical Information (OSTI.GOV)

C.A.Reddy, PI

2005-06-30

G. lucidum is one of the most important and widely distributed ligninolytic white rot fungi from habitats such as forest soils, agricultural soils, and tropical mangrove ecosystems and produce laccases as an important family of lignin modifying enzymes. Biochemically, laccases are blue multi copper oxidases that couple four electron reduction of molecular oxygen to water. There is a growing interest in the use of laccases for a variety of industrial applications such as bio-pulping and biobleaching as well as in their ability to detoxify a wide variety of toxic environmental pollutants. These key oxidative enzymes are found in all themore » three domains of life: Eukaryota. Prokarya, and Archaea. Ganoderma lucidum (strain no.103561) produces laccase with some of the highest activity (17,000 micro katals per mg of protein) reported for any laccases to date. Our results showed that this organism produces at least 11 different isoforms of laccase based on variation in mol. weight and/or PI. Our Studies showed that the presence of copper in the medium yields 15- to 20-fold greater levels of enzyme by G. lucidum. Dialysation of extra cellular fluid of G. lucidum against 10mM sodium tartrate (pH5.5) gave an additional 15 to 17 fold stimulation of activity with an observed specific activity of 17,000 {micro}katals/mg protein. Dialysis against acetate buffer gave five fold increase in activity while dialysis against glycine showed inhibition of activity. Purification by FPLC and preparative gel electrophoresis gave purified fractions that resolved into eleven isoforms as separated by isoelectric focusing, and the PI,s were 4.7, 4.6, 4.5, 4.3, 4.2, 4.1, 3.8, 3.7, 3.5, 3.4 and 3.3. Genomic clones of laccase were isolated using G. lucidum DNA as a template and using inverse PCR and forward/reverse primers corresponding to the sequences of the conserved copper binding region in the N-terminal domain of one of the laccases of this organism. Inverse PCR amplication of Hind

Potential of extracellular enzymes from Trametes versicolor F21a in Microcystis spp. degradation.

PubMed

Du, Jingjing; Pu, Gaozhong; Shao, Chen; Cheng, Shujun; Cai, Ji; Zhou, Liang; Jia, Yong; Tian, Xingjun

2015-03-01

Studies have shown that microorganisms may be used to eliminate cyanobacteria in aquatic environments. The present study showed that the white-rot fungus Trametes versicolor F21a could degrade Microcystis aeruginosa. After T. versicolor F21a and Microcystis spp. were co-incubated for 60h, >96% of Microcystis spp. cells were degraded by T. versicolor F21a. The activities of extracellular enzymes showed that cellulase, β-glucosidase, protease, and laccase were vital to Microcystis spp. degradation in the early stage (0h to 24h), while β-glucosidase, protease, laccase, and manganese peroxidase in the late stage (24h to 60h). The positive and significant correlation of the degradation rate with these enzyme activities indicated that these enzymes were involved in the degradation rate of Microcystis spp. cells at different phases. It suggested that the extracellular enzymes released by T. versicolor F21a might be vital to Microcystis spp. degradation. The results of this study may be used to develop alternative microbial control agents for cyanobacterial control. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrochemical studies of a truncated laccase produced in Pichia pastoris

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gelo-Pujic, M.; Kim, H.H.; Butlin, N.G.

1999-12-01

The cDNA that encodes an isoform is laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms ofmore » their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon influences the rate of heterogeneous electron transfer between and electrode and the copper-containing active site. These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.« less

Peroxidase(s) in environment protection.

PubMed

Bansal, Neelam; Kanwar, Shamsher S

2013-01-01

Industrial discharges of untreated effluents into water bodies and emissions into air have deteriorated the quality of water and air, respectively. The huge amount of pollutants derived from industrial activities represents a threat for the environment and ecologic equilibrium. Phenols and halogenated phenols, polycyclic aromatic hydrocarbons (PAH), endocrine disruptive chemicals (EDC), pesticides, dioxins, polychlorinated biphenyls (PCB), industrial dyes, and other xenobiotics are among the most important pollutants. Peroxidases are enzymes that are able to transform a variety of compounds following a free radical mechanism, thereby yielding oxidized or polymerized products. The peroxidase transformation of these pollutants is accompanied by a reduction in their toxicity, due to loss of biological activity, reduction in the bioavailability, or the removal from aqueous phase, especially when the pollutant is found in water. The review describes the sources of peroxidases, the reactions catalyzed by them, and their applications in the management of pollutants in the environment.

[Decolorization of dyestuff and dying waste water by laccase solution with self-flocculent mycelial pellets of Coriolus versicolor].

PubMed

Wu, Mianbin; Xia, Liming

2002-06-01

Both laccase production by the white-rot fungus Coriolus versicolor and decolorization of dyestuff and dying waste water with crude solution of laccase were studied in this work. Laccase production meets the definition of secondary metabolism. For laccase production the optimum initial pH is 4.5. Addition of veratryl alcohol or elevated trace metals could both enhance the laccase activity, while Tween80 showed some inhibition. The immobilized mycelia of C. versicolor in polyurethane foam had less laccase production ability than mycelial pellets. A repeated batch cultivation process was found to be a very economical way for laccase harvest. The same pellets could be used for at least 14 times and average laccase activity of each batch could maintain 6.72 IU/mL. This method reduces the enzyme production course, medium consumption and the possibility of contamination, showing high efficient and great economic benefit. Good results were also obtained in decolorization experiments with the crude solution of laccase. With 3.3 IU/mL initial laccase activity, color removal of Acid Orange reached 98.5% after 24 h reaction. Also with 2.6 IU/mL initial laccase activity, color removal of dying waste water reached 93% after 24 h reaction.

Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

PubMed

Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

2015-01-01

A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation. Copyright © 2014 Elsevier B.V. All rights reserved.

Peroxidase(s) in Environment Protection

PubMed Central

Bansal, Neelam; Kanwar, Shamsher S.

2013-01-01

Industrial discharges of untreated effluents into water bodies and emissions into air have deteriorated the quality of water and air, respectively. The huge amount of pollutants derived from industrial activities represents a threat for the environment and ecologic equilibrium. Phenols and halogenated phenols, polycyclic aromatic hydrocarbons (PAH), endocrine disruptive chemicals (EDC), pesticides, dioxins, polychlorinated biphenyls (PCB), industrial dyes, and other xenobiotics are among the most important pollutants. Peroxidases are enzymes that are able to transform a variety of compounds following a free radical mechanism, thereby yielding oxidized or polymerized products. The peroxidase transformation of these pollutants is accompanied by a reduction in their toxicity, due to loss of biological activity, reduction in the bioavailability, or the removal from aqueous phase, especially when the pollutant is found in water. The review describes the sources of peroxidases, the reactions catalyzed by them, and their applications in the management of pollutants in the environment. PMID:24453894

Construction of a laccase chimerical gene: recombinant protein characterization and gene expression via yeast surface display.

PubMed

Bleve, G; Lezzi, C; Spagnolo, S; Rampino, P; Perrotta, C; Mita, G; Grieco, Francesco

2014-03-01

The ERY4 laccase gene from Pleurotus eryngii was expressed in Saccharomyces cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified to obtain chimerical enzymes derived from the substitution of N-, C- and both N- and C-terminal regions with the corresponding regions of Ery3 laccase, another laccase isoform of P. eryngii. The chimerical isoform named 4NC3, derived from the substitution of both N- and C-terminal regions, showed the best performances in terms of enzymatic activities, affinities for different substrates and stability at a broad range of temperatures and pHs. The chimerical 4NC3 laccase isoform was displayed on the cell surface of S. cerevisiae using the N-terminal fusion with either the Pir2 or the Flo1 S. cerevisiae proteins as anchor attachment sequence. Immunofluorescence microscopy and Western blot analyses confirmed the localization of 4NC3 on the yeast cell surface. The enzyme activity on specific laccase substrates revealed that 4NC3 laccase was immobilized in active form on the cell surface. To our knowledge, this is the first example of expression of a chimerical fungal laccase by yeast cell display.

One-pot synthesis of active copper-containing carbon dots with laccase-like activities.

PubMed

Ren, Xiangling; Liu, Jing; Ren, Jun; Tang, Fangqiong; Meng, Xianwei

2015-12-14

Herein, an effective strategy for designing a new type of nanozyme, blue fluorescent laccase mimics, is reported. Active copper-containing carbon dots (Cu-CDs) were synthesized through a simple, nontoxic and one-pot hydrothermal method, which showed favorable photoluminescence properties and good photostability under high-salt conditions or in a broad pH range (3.0-13.5). The Cu-CDs possessed intrinsic laccase-like activities and could catalyze the oxidation of the laccase substrate p-phenylenediamine (PPD) to produce a typical color change from colorless to brown. Poly(methacrylic acid sodium salt) (PMAA) not only was used as the carbon source and reducing agent, but also provided carboxyl groups to assist flocculation between Cu-CDs and polyacrylamide, which facilitated the removal of PPD. Importantly, the intrinsic fluorescence of the as-prepared Cu-CDs could indicate the presence of hydroquinone, one of the substrates of laccases, based on laccase mimics and fluorescence quenching.

Simultaneous production of laccase and degradation of bisphenol A with Trametes versicolor cultivated on agricultural wastes.

PubMed

Zeng, Shengquan; Zhao, Jie; Xia, Liming

2017-08-01

Solid state fermentation with Trametes versicolor was carried out on agricultural wastes containing bisphenol A (BPA). It was found that BPA degradation was along with the occurrence of laccase production, and wheat bran and corn straw were identified as suitable mixed substrates for laccase production. In the process of BPA degradation with T. versicolor, laccase activity increased rapidly at the 6th-10th day after inoculation. Moreover, BPA can enhance the production of laccase. After 10 days of fermentation, degradation rate of BPA exceeded 90% without the usage of mediators ABTS and acetosyringone at pH 4.0-8.0. In addition, metal ions did not affect the BPA degradation with T. versicolor. In vitro, the optimum pH range of BPA degradation with laccase was in the acidic region with the optimal performance of pH 5.0. Metal ions Cu 2+ , Zn 2+ , and Co 2+ showed little effect on BPA degradation. However, Fe 3+ and Fe 2+ substantially inhibited the BPA degradation. Natural mediator acetosyringone showed optimum enhancement on BPA degradation. Greater than 90% of the estrogenic activity of BPA was removed by T. versicolor and its laccase. Compared to in vitro degradation with laccase, this study shows that the process of simultaneous laccase production and BPA degradation with T. versicolor was more advantageous since BPA can enhance the laccase production, mediators were unnecessary, degradation rate was not affected by metal ions, and the applicable pH range was broader. This study concludes that T. versicolor and laccase have great potential to treat industrial wastewater containing BPA.

Electrochemical Studies of a Truncated Laccase Produced in Pichia pastoris

PubMed Central

Gelo-Pujic, Mirjana; Kim, Hyug-Han; Butlin, Nathan G.; Palmore, G. Tayhas R.

1999-01-01

The cDNA that encodes an isoform of laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI→LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (khet for LCCIa = 1.3 × 10−4 cm s−1). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis. PMID:10584012

Functional expression, production, and biochemical characterization of a laccase using yeast surface display technology.

PubMed

Bertrand, Brandt; Trejo-Hernández, María R; Morales-Guzmán, Daniel; Caspeta, Luis; Suárez Rodríguez, Ramón; Martínez-Morales, Fernando

2016-12-01

A Trametes versicolor laccase was functionally expressed on the membrane surface of Saccharomyces cerevisiae EBY100. Laccase expression was increased 6.57-fold by medium optimization and surpassed production by the native strain. Maximal laccase and biomass production reached 19 735 ± 1719 Ug -1 and 6.22 ± 0.53 gL -1 respectively, after 2 d of culture. Optimum oxidization of all substrates by laccase was observed at pH 3. Laccase showed high affinity towards substrates used with Km (mM) and Vmax (μmol min -1 ) values of 0.57 ± 0.0047 and 24.55 ± 0.64, 1.52 ± 0.52 and 9.25 ± 1.78, and 2.67 ± 0.12 and 11.26 ± 0.75, were reported for ABTS, 2, 6-DMP and GUA, respectively. EDTA and NaN 3 displayed none competitive inhibition towards laccase activity. The optimum temperature for activity was 50 °C; however, the enzyme was stable over a wide range of temperatures (25-70 °C). The biologically immobilized laccase showed high reusability towards phenolic substrates and low reusability with non-phenolic substrates. High affinity for a diversity phenolic compounds and great ethanol tolerance substantiates this laccase/yeast biocatalyst potential for application in the production of bioethanol. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Protection of Wood from Microorganisms by Laccase-Catalyzed Iodination

PubMed Central

Engel, J.; Thöny-Meyer, L.; Schwarze, F. W. M. R.; Ihssen, J.

2012-01-01

In the present work, Norway spruce wood (Picea abies L.) was reacted with a commercial Trametes versicolor laccase in the presence of potassium iodide salt or the phenolic compounds thymol and isoeugenol to impart an antimicrobial property to the wood surface. In order to assess the efficacy of the wood treatment, a leaching of the iodinated and polymerized wood and two biotests including bacteria, a yeast, blue stain fungi, and wood decay fungi were performed. After laccase-catalyzed oxidation of the phenols, the antimicrobial effect was significantly reduced. In contrast, the enzymatic oxidation of iodide (I−) to iodine (I2) in the presence of wood led to an enhanced resistance of the wood surface against all microorganisms, even after exposure to leaching. The efficiency of the enzymatic wood iodination was comparable to that of a chemical wood preservative, VP 7/260a. The modification of the lignocellulose by the laccase-catalyzed iodination was assessed by the Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) technique. The intensities of the selected lignin-associated bands and carbohydrate reference bands were analyzed, and the results indicated a structural change in the lignin matrix. The results suggest that the laccase-catalyzed iodination of the wood surface presents an efficient and ecofriendly method for wood protection. PMID:22865075

Characterization of lignocellulolytic enzymes from white-rot fungi.

PubMed

Manavalan, Tamilvendan; Manavalan, Arulmani; Heese, Klaus

2015-04-01

The development of alternative energy sources by applying lignocellulose-based biofuel technology is critically important because of the depletion of fossil fuel resources, rising fossil fuel prices, security issues regarding the fossil fuel supply, and environmental issues. White-rot fungi have received much attention in recent years for their valuable enzyme systems that effectively degrade lignocellulosic biomasses. These fungi have powerful extracellular oxidative and hydrolytic enzymes that degrade lignin and cellulose biopolymers, respectively. Lignocellulosic biomasses from either agricultural or forestry wastes are abundant, low-cost feedstock alternatives in nature but require hydrolysis into simple sugars for biofuel production. This review provides a complete overview of the different lignocellulose biomasses and their chemical compositions. In addition, a complete list of the white-rot fungi-derived lignocellulolytic enzymes that have been identified and their molecular structures, mechanism of action in lignocellulose hydrolysis, and biochemical properties is summarized in detail. These enzymes include ligninolytic enzymes (laccase, manganese peroxidase, lignin peroxidase, and versatile peroxidase) and cellulolytic enzymes (endo-glucanase, cellobiohydrolase, and beta-glucosidase). The use of these fungi for low-cost lignocellulolytic enzyme production might be attractive for biofuel production.

Effects and interactions of medium components on laccase from a marine-derived fungus using response surface methodology.

PubMed

D'Souza-Ticlo, Donna; Garg, Sandeep; Raghukumar, Chandralata

2009-11-25

The effects of various synthetic medium components and their interactions with each other ultimately impact laccase production in fungi. This was studied using a laccase-hyper-producing marine-derived basidiomycete, Cerrena unicolor MTCC 5159. Inducible laccases were produced in the idiophase only after addition of an inducer such as CuSO(4). Concentration of carbon and nitrogen acted antagonistically with respect to laccase production. A combination of low nitrogen and high carbon concentration favored both biomass and laccase production. The most favorable combination resulted in 917 U L(-1) of laccase. After sufficient growth had occurred, addition of a surfactant such as Tween 80 positively impacted biomass and increased the laccase activity to around 1,300 U L(-1). Increasing the surface to volume ratio of the culture vessel further increased its activity to almost 2,000 U L(-1).

Mechanism of salt-induced activity enhancement of a marine-derived laccase, Lac15.

PubMed

Li, Jie; Xie, Yanan; Wang, Rui; Fang, Zemin; Fang, Wei; Zhang, Xuecheng; Xiao, Yazhong

2018-04-01

Laccase (benzenediol: oxygen oxidoreductases, EC1.10.3.2) is a multi-copper oxidase capable of oxidizing a variety of phenolic and other aromatic organic compounds. The catalytic power of laccase makes it an attractive candidate for potential applications in many areas of industry including biodegradation of organic pollutants and synthesis of novel drugs. Most laccases are vulnerable to high salt and have limited applications. However, some laccases are not only tolerant to but also activated by certain concentrations of salt and thus have great application potential. The mechanisms of salt-induced activity enhancement of laccases are unclear as yet. In this study, we used dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, intrinsic fluorescence emission, circular dichroism, ultraviolet-visible light absorption, and an enzymatic assay to investigate the potential correlation between the structure and activity of the marine-derived laccase, Lac15, whose activity is promoted by low concentrations of NaCl. The results showed that low concentrations of NaCl exert little influence on the protein structure, which was partially folded in the absence of the salt; moreover, the partially folded rather than the fully folded state seemed to be favorable for enzyme activity, and this partially folded state was distinctive from the so-called 'molten globule' occasionally observed in active enzymes. More data indicated that salt might promote laccase activity through mechanisms involving perturbation of specific local sites rather than a change in global structure. Potential binding sites for chloride ions and their roles in enzyme activity promotion are proposed.

Genes associated with lignin degradation in the polyphagous white-rot pathogen Heterobasidion irregulare show substrate-specific regulation.

PubMed

Yakovlev, Igor A; Hietala, Ari M; Courty, Pierre-Emmanuel; Lundell, Taina; Solheim, Halvor; Fossdal, Carl Gunnar

2013-07-01

The pathogenic white-rot basidiomycete Heterobasidion irregulare is able to remove lignin and hemicellulose prior to cellulose during the colonization of root and stem xylem of conifer and broadleaf trees. We identified and followed the regulation of expression of genes belonging to families encoding ligninolytic enzymes. In comparison with typical white-rot fungi, the H. irregulare genome has exclusively the short-manganese peroxidase type encoding genes (6 short-MnPs) and thereby a slight contraction in the pool of class II heme-containing peroxidases, but an expansion of the MCO laccases with 17 gene models. Furthermore, the genome shows a versatile set of other oxidoreductase genes putatively involved in lignin oxidation and conversion, including 5 glyoxal oxidases, 19 quinone-oxidoreductases and 12 aryl-alcohol oxidases. Their genetic multiplicity and gene-specific regulation patterns on cultures based on defined lignin, cellulose or Norway spruce lignocellulose substrates suggest divergent specificities and physiological roles for these enzymes. While the short-MnP encoding genes showed similar transcript levels upon fungal growth on heartwood and reaction zone (RZ), a xylem defense tissue rich in phenolic compounds unique to trees, a subset of laccases showed higher gene expression in the RZ cultures. In contrast, other oxidoreductases depending on initial MnP activity showed generally lower transcript levels on RZ than on heartwood. These data suggest that the rate of fungal oxidative conversion of xylem lignin differs between spruce RZ and heartwood. It is conceivable that in RZ part of the oxidoreductase activities of laccases are related to the detoxification of phenolic compounds involved in host-defense. Expression of the several short-MnP enzymes indicated an important role for these enzymes in effective delignification of wood by H. irregulare. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification and evaluation of bioremediation potential of laccase isoforms produced by Cyathus bulleri on wheat bran.

PubMed

Vats, Arpita; Mishra, Saroj

2018-02-15

Multiplicity in laccases among lignin degrading fungal species is of interest as it confers the ability to degrade several types of lignocellulosics. The combination of laccases produced on such substrates could be beneficial for treatment of complex aromatics, including dyes. In this study, we report on production of high units (679.6Ug -1 substrate) of laccase on solid wheat bran (WB) by Cyathus bulleri. Laccase, purified from the culture filtrates of WB grown fungus, was effective for oxidation of veratryl alcohol, Reactive blue 21 and textile effluent without assistance of externally added mediators. De novo sequencing of the 'purified' laccase lead to identification of several peptides that originated from different laccase genes. Transcriptome analysis of the fungus, cultivated on WB, confirmed presence of 8 isozymes, that were re-amplified and sequenced from the cDNA prepared from WB grown fungus. The 8 isozymes were grouped into 3 classes, based on their sequence relationship with other basidiomycete laccases. The isoforms produced on WB decolorized (by ∼57%) and degraded textile effluent far more effectively, compared to laccase obtained from Basal salt cultivated fungus. The decolorization and degradation was also accompanied by more than 95% reduction in phytotoxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Middle-redox potential laccase from Ganoderma sp.: its application in improvement of feed for monogastric animals

PubMed Central

Sharma, Krishna Kant; Shrivastava, Bhuvnesh; Sastry, V. R. B.; Sehgal, Neeta; Kuhad, Ramesh Chander

2013-01-01

The variables influencing laccase production by white-rot fungus Ganoderma sp. rckk-02 were optimized employing response surface methodology. Malt extract (6.0% w/v), lignin (0.5% w/v) and pH (5.5) were found to be the most significant factors for enhanced laccase production by 7 fold (226.0 U/ml) as compared to unoptimized growth conditions (32.0 U/ml). The N-terminal sequence of laccase revealed its distinct amino acid profile (S- I- R- N- S- G), which suggested it as a novel enzyme. The Far-UV CD spectrum of the laccase showed single broad negative trough at around 213 nm, a typical signature of all β proteins. The laccase was found to fall in the range of middle redox potential laccases. Purified laccase at dosage of 2.5 Ug−1 body weight when supplemented with pelleted diet of rats, a significant improvement (p < 0.05) in nutrients digestibility without causing any elevation of blood stress enzymes was observed. PMID:23416696

Continuous adsorption and biotransformation of micropollutants by granular activated carbon-bound laccase in a packed-bed enzyme reactor.

PubMed

Nguyen, Luong N; Hai, Faisal I; Dosseto, Anthony; Richardson, Christopher; Price, William E; Nghiem, Long D

2016-06-01

Laccase was immobilized on granular activated carbon (GAC) and the resulting GAC-bound laccase was used to degrade four micropollutants in a packed-bed column. Compared to the free enzyme, the immobilized laccase showed high residual activities over a broad range of pH and temperature. The GAC-bound laccase efficiently removed four micropollutants, namely, sulfamethoxazole, carbamazepine, diclofenac and bisphenol A, commonly detected in raw wastewater and wastewater-impacted water sources. Mass balance analysis showed that these micropollutants were enzymatically degraded following adsorption onto GAC. Higher degradation efficiency of micropollutants by the immobilized compared to free laccase was possibly due to better electron transfer between laccase and substrate molecules once they have adsorbed onto the GAC surface. Results here highlight the complementary effects of adsorption and enzymatic degradation on micropollutant removal by GAC-bound laccase. Indeed laccase-immobilized GAC outperformed regular GAC during continuous operation of packed-bed columns over two months (a throughput of 12,000 bed volumes). Copyright © 2016 Elsevier Ltd. All rights reserved.

Laccase Down-Regulation Causes Alterations in Phenolic Metabolism and Cell Wall Structure in Poplar1

PubMed Central

Ranocha, Philippe; Chabannes, Matthieu; Chamayou, Simon; Danoun, Saïda; Jauneau, Alain; Boudet, Alain-M.; Goffner, Deborah

2002-01-01

Laccases are encoded by multigene families in plants. Previously, we reported the cloning and characterization of five divergent laccase genes from poplar (Populus trichocarpa) xylem. To investigate the role of individual laccase genes in plant development, and more particularly in lignification, three independent populations of antisense poplar plants, lac3AS, lac90AS, and lac110AS with significantly reduced levels of laccase expression were generated. A repression of laccase gene expression had no effect on overall growth and development. Moreover, neither lignin content nor composition was significantly altered as a result of laccase suppression. However, one of the transgenic populations, lac3AS, exhibited a 2- to 3-fold increase in total soluble phenolic content. As indicated by toluidine blue staining, these phenolics preferentially accumulate in xylem ray parenchyma cells. In addition, light and electron microscopic observations of lac3AS stems indicated that lac3 gene suppression led to a dramatic alteration of xylem fiber cell walls. Individual fiber cells were severely deformed, exhibiting modifications in fluorescence emission at the primary wall/middle lamella region and frequent sites of cell wall detachment. Although a direct correlation between laccase gene expression and lignification could not be assigned, we show that the gene product of lac3 is essential for normal cell wall structure and integrity in xylem fibers. lac3AS plants provide a unique opportunity to explore laccase function in plants. PMID:12011346

Extraction and Application of Laccases from Shimeji Mushrooms (Pleurotus ostreatus) Residues in Decolourisation of Reactive Dyes and a Comparative Study Using Commercial Laccase from Aspergillus oryzae

PubMed Central

Teixeira, Ricardo Sposina S.; Pereira, Patrícia Maia; Ferreira-Leitão, Viridiana S.

2010-01-01

Oxidases are able to degrade organic pollutants; however, high costs associated with biocatalysts production still hinder their use in environmental biocatalysis. Our study compared the action of a commercial laccase from Aspergillus oryzae and a rich extract from Pleurotus ostreatus cultivation residues in decolourisation of reactive dyes: Drimaren Blue X-3LR (DMBLR), Drimaren Blue X-BLN (DMBBLN), Drimaren Rubinol X-3LR (DMR), and Drimaren Blue C-R (RBBR). The colour removal was evaluated by considering dye concentration, reaction time, absence or presence of the mediator ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and the source of laccase. The presence of ABTS was essential for decolourisation of DMR (80–90%, 1 h) and RBBR (80–90%, 24 h) with both laccases. The use of ABTS was not necessary in reactions containing DMBLR (85–97%, 1 h) and DMBBLN (63–84%, 24 h). The decolourisation of DMBBLN by commercial laccase showed levels near 60% while the crude extract presented 80% in 24 h. PMID:21052547

Electron beam-induced immobilization of laccase on porous supports for waste water treatment applications.

PubMed

Jahangiri, Elham; Reichelt, Senta; Thomas, Isabell; Hausmann, Kristin; Schlosser, Dietmar; Schulze, Agnes

2014-08-08

The versatile oxidase enzyme laccase was immobilized on porous supports such as polymer membranes and cryogels with a view of using such biocatalysts in bioreactors aiming at the degradation of environmental pollutants in wastewater. Besides a large surface area for supporting the biocatalyst, the aforementioned porous systems also offer the possibility for simultaneous filtration applications in wastewater treatment. Herein a "green" water-based, initiator-free, and straightforward route to highly reactive membrane and cryogel-based bioreactors is presented, where laccase was immobilized onto the porous polymer supports using a water-based electron beam-initiated grafting reaction. In a second approach, the laccase redox mediators 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and syringaldehyde were cross-linked instead of the enzyme via electron irradiation in a frozen aqueous poly(acrylate) mixture in a one pot set-up, yielding a mechanical stable macroporous cryogel with interconnected pores ranging from 10 to 50 µm in size. The membranes as well as the cryogels were characterized regarding their morphology, chemical composition, and catalytic activity. The reactivity towards waste- water pollutants was demonstrated by the degradation of the model compound bisphenol A (BPA). Both membrane- and cryogel-immobilized laccase remained highly active after electron beam irradiation. Apparent specific BPA removal rates were higher for cryogel- than for membrane-immobilized and free laccase, whereas membrane-immobilized laccase was more stable with respect to maintenance of enzymatic activity and prevention of enzyme leakage from the carrier than cryogel-immobilized laccase. Cryogel-immobilized redox mediators remained functional in accelerating the laccase-catalyzed BPA degradation, and especially ABTS was found to act more efficiently in immobilized than in freely dissolved state.

Immobilized laccase mediated dye decolorization and transformation pathway of azo dye acid red 27.

PubMed

Chhabra, Meenu; Mishra, Saroj; Sreekrishnan, Trichur Ramaswamy

2015-01-01

Laccases have good potential as bioremediating agents and can be used continuously in the immobilized form like many other enzymes. In the present study, laccase from Cyathus bulleri was immobilized by entrapment in Poly Vinyl Alcohol (PVA) beads cross-linked with either nitrate or boric acid. Immobilized laccase was used for dye decolorization in both batch and continuous mode employing a packed bed column. The products of degradation of dye Acid Red 27 were identified by LC MS/MS analysis. The method led to very effective (90%) laccase immobilization and also imparted significant stability to the enzyme (more than 70% after 5 months of storage at 4°C). In batch decolorization, 90-95% decolorization was achieved of the simulated dye effluent for up to 10-20 cycles. Continuous decolorization in a packed bed bioreactor led to nearly 90% decolorization for up to 5 days. The immobilized laccase was also effective in decolorization and degradation of Acid Red 27 in the presence of a mediator. Four products of degradation were identified by LC-MS/MS analysis. The immobilized laccase in PVA-nitrate was concluded to be an effective agent in treatment of textile dye effluents.

Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica.

PubMed

Guo, Mei; Lu, Fuping; Du, Lianxiang; Pu, Jun; Bai, Dongqing

2006-08-01

A cDNA encoding for laccase (Lcc1) was isolated from the ligninolytic fungus Trametes versicolor by reverse transcriptase polymerase chain reaction. The Lcc1 gene was subcloned into the Pichia methanolica expression vector pMETalphaA and transformed into the P. methanolica strains PMAD11 and PMAD16. The extracellular laccase activity of the PMAD11 recombinants was found to be 1.3-fold higher than that of the PMAD16 recombinants. The identity of the recombinant protein was further confirmed by immunodetection using the Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form. The effects of copper concentration, cultivation temperature, pH and methanol concentration in the BMMY on laccase expression were investigated. The laccase activity in the PMAD11 recombinant was up to 12.6 U ml(-1) by optimization.

Fungal Pretreatment of Sweet Sorghum Bagasse with Combined CuSO4-Gallic Acid Supplement for Improvement in Lignin Degradation, Selectivity, and Enzymatic Saccharification.

PubMed

Mishra, Vartika; Jana, Asim K

2017-09-01

Sweet sorghum (Sorghum sp.) has high biomass yield. Hydrolysis of lignocellulosic sweet sorghum bagasse (SSB) to fermentable sugar could be useful for manufacture of biofuel or other fermentation products. Pretreatment of lignocellulosic biomass to degrade lignin before enzymatic hydrolysis is a key step. Fungal pretreatment of SSB with combined CuSO 4 -gallic acid supplements in solid-state fermentation (SSF) to achieve higher lignin degradation, selectivity value (SV), and enzymatic hydrolysis to sugar was studied. Coriolus versicolor was selected due to high activities of ligninolytic enzymes laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), polyphenol oxidase (PPO), and arylalcohol oxidase (AAO) and low activities of cellulolytic enzymes CMCase, FPase, and β-glucosidase with high lignin degradation and SV in 20 days. CuSO 4 /gallic acid increased the activities of ligninolytic enzymes resulting in enhanced lignin degradations and SVs. Cumulative/synergistic effect of combined supplements further increased the activities of laccase, LiP, MnP, PPO, and AAO by 7.6, 14.6, 2.67, 2.06, and 2.15-folds, respectively (than control), resulting in highest lignin degradation 31.1 ± 1.4% w/w (1.56-fold) and SV 2.33 (3.58-fold). Enzymatic hydrolysis of pretreated SSB yielded higher (~2.2 times) fermentable sugar. The study showed combined supplements can improve fungal pretreatment of lignocellulosic biomass. XRD, SEM, FTIR, and TGA/DTG of SSB confirmed the results.

High-level coproduction, purification and characterisation of laccase and exopolysaccharides by Coriolus versicolor.

PubMed

Que, Youxiong; Sun, Shujing; Xu, Liping; Zhang, Yuye; Zhu, Hu

2014-09-15

In this study, a two-stage pH-shift fermentation process was developed for the coproduction of laccase and exopolysaccharides (EPS) by Coriolus versicolor. At the same time, laccase and EPS were purified and characterised in detail. The results showed that the highest laccase and EPS production reached 7680 U l(-1) and 8.2 g l(-1). Furthermore, the flow behaviour of fermentation broth was Newtonian and the maximum μ(ap) was 2.7×10(-3) Pa s. The MW of laccase was 64 kDa and it showed a pI value of 4.2. The CD analysis showed that laccase had a high α-helical content (68%). The MW of the purified EPS was determined to be 1.8×10(6) Da, consisting of carbohydrates (87.6%) and proteins (12.4%). The EPS consisted of 17 amino acids, mainly serine (11.3%), glutamic acid (12.60%), leucine (13.3%) and phenylalanine (9.4%) in protein moiety, and three monosaccharides (galactose, mannose and xylose). Copyright © 2014 Elsevier Ltd. All rights reserved.

Gram-scale production of a basidiomycetous laccase in Aspergillus niger.

PubMed

Mekmouche, Yasmina; Zhou, Simeng; Cusano, Angela M; Record, Eric; Lomascolo, Anne; Robert, Viviane; Simaan, A Jalila; Rousselot-Pailley, Pierre; Ullah, Sana; Chaspoul, Florence; Tron, Thierry

2014-01-01

We report on the expression in Aspergillus niger of a laccase gene we used to produce variants in Saccharomyces cerevisiae. Grams of recombinant enzyme can be easily obtained. This highlights the potential of combining this generic laccase sequence to the yeast and fungal expression systems for large-scale productions of variants. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Evaluation of laccase-mediator system (LMS) in the oxidation of veratryl alcohol

USDA-ARS?s Scientific Manuscript database

Identifying suitable reaction conditions remains an important task in the development of enzyme catalysis. Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both commercially...

Cost analysis in laccase production.

PubMed

Osma, Johann F; Toca-Herrera, José L; Rodríguez-Couto, Susana

2011-11-01

In this paper the cost of producing the enzyme laccase by the white-rot fungus Trametes pubescens under both submerged (SmF) and solid-state fermentation (SSF) conditions was studied. The fungus was cultured using more than 45 culture medium compositions. The cost of production was estimated by analyzing the cost of the culture medium, the cost of equipment and the operating costs. The cost of the culture medium represented, in all cases, the highest contribution to the total cost, while, the cost of equipment was significantly low, representing less than 2% of the total costs. The cultivation under SSF conditions presented a final cost 50-fold lower than the one obtained when culturing under SmF conditions at flask scale. In addition, the laccase production under SSF conditions in tray bioreactors reduced the final cost 4-fold compared to the one obtained under SSF conditions at flask scale, obtaining a final price of 0.04 cent €/U. Copyright © 2011 Elsevier Ltd. All rights reserved.

Reactivities of various mediators and laccases with kraft pulp and lignin model compounds.

PubMed

Bourbonnais, R; Paice, M G; Freiermuth, B; Bodie, E; Borneman, S

1997-12-01

Laccase-catalyzed oxygen delignification of kraft pulp offers some potential as a replacement for conventional chemical bleaching and has the advantage of requiring much lower pressure and temperature. However, chemical mediators are required for effective delignification by laccase, and their price is currently too high at the dosages required. To date, most studies have employed laccase from Trametes versicolor. We have found significant differences in reactivity between laccases from different fungi when they are tested for pulp delignification in the presence of the mediators 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). A more detailed study of T. versicolor laccase with ABTS and HBT showed that HBT gave the most extensive delignification over 2 h but deactivated the enzyme, and therefore a higher enzyme dosage was required. Other mediators, including 1-nitroso-2-naphthol-3,6-disulfonic acid, 4-hydroxy-3-nitroso-1-naphthalenesulfonic acid, promazine, chlorpromazine, and Remazol brilliant blue, were also tested for their ability to delignify kraft pulp. Studies with dimeric model compounds indicated that the mechanisms of oxidation by ABTS and HBT are different. In addition, oxygen uptake by laccase is much slower with HBT than with ABTS. It is proposed that the dication of ABTS and the 1-oxide radical of HBT, with redox potentials in the 0.8- to 0.9-V range, are required for pulp delignification.

Laccase Production from a Temperature and pH Tolerant Fungal Strain of Trametes hirsuta (MTCC 11397).

PubMed

Dhakar, Kusum; Pandey, Anita

2013-01-01

Laccase production by a temperature and pH tolerant fungal strain (GBPI-CDF-03) isolated from a glacial site in Indian Himalayan Region (IHR) has been investigated. The fungus developed white cottony mass on potato dextrose agar and revealed thread-like mycelium under microscope. ITS region analysis of fungus showed its 100% similarity with Trametes hirsuta. The fungus tolerated temperature from 4 to 48°C ± 2 (25°C opt.) and pH 3-13 (5-7 opt.). Molecular weight of laccase was determined approximately 45 kDa by native PAGE. Amplification of laccase gene fragment (corresponding to the copper-binding conserved domain) contained 200 bp. The optimum pH for laccase production, at optimum growth temperature, was determined between 5.5 and 7.5. In optimization experiments, fructose and ammonium sulfate were found to be the best carbon and nitrogen sources, respectively, for enhancing the laccase production. Production of laccase was favored by high carbon/nitrogen ratio. Addition of CuSO4 (up to 1.0 mM) induced laccase production up to 2-fold, in case of 0.4 mM concentration. Addition of organic solvents also induced the production of laccase; acetone showed the highest (2-fold) induction. The study has implications in bioprospecting of ecologically resilient microbial strains.

A cell wall-bound anionic peroxidase, PtrPO21, is involved in lignin polymerization in Populus trichocarpa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Chien-Yuan; Li, Quanzi; Tunlaya-Anukit, Sermsawat

2016-03-11

Class III peroxidases are members of a large plant-specific sequence-heterogeneous protein family. Several sequence-conserved homologs have been associated with lignin polymerization in Arabidopsis thaliana, Oryza sativa, Nicotiana tabacum, Zinnia elegans, Picea abies, and Pinus sylvestris. In Populus trichocarpa, a model species for studies of wood formation, the peroxidases involved in lignin biosynthesis have not yet been identified. To do this, we retrieved sequences of all PtrPOs from Peroxibase and conducted RNA-seq to identify candidates. Transcripts from 42 PtrPOs were detected in stem differentiating xylem (SDX) and four of them are the most xylem-abundant (PtrPO12, PtrPO21, PtrPO42, and PtrPO64). PtrPO21 showsmore » xylem-specific expression similar to that of genes encoding the monolignol biosynthetic enzymes. Using protein cleavage-isotope dilution mass spectrometry, PtrPO21 is detected only in the cell wall fraction and not in the soluble fraction. Downregulated transgenics of PtrPO21 have a lignin reduction of ~20% with subunit composition (S/G ratio) similar to wild type. The transgenics show a growth reduction and reddish color of stem wood. The modulus of elasticity (MOE) of the stems of the downregulated PtrPO21-line 8 can be reduced to ~60% of wild type. Differentially expressed gene (DEG) analysis of PtrPO21 downregulated transgenics identified a significant overexpression of PtPrx35, suggesting a compensatory effect within the peroxidase family. No significant changes in the expression of the 49 P. trichocarpa laccases (PtrLACs) were observed.« less

Laccase produced by a thermotolerant strain of Trametes trogii LK13

PubMed Central

Yan, Jinping; Chen, Yuhui; Niu, Jiezhen; Chen, Daidi; Chagan, Irbis

2015-01-01

Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%–50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g −1 ) along with low cellulose (2 U g −1 ) and xylanase (140 U g −1 ) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes. PMID:26221089

Unraveling the effects of laccase treatment on enzymatic hydrolysis of steam-exploded wheat straw.

PubMed

Oliva-Taravilla, Alfredo; Moreno, Antonio D; Demuez, Marie; Ibarra, David; Tomás-Pejó, Elia; González-Fernández, Cristina; Ballesteros, Mercedes

2015-01-01

Laccase enzymes are promising detoxifying agents during lignocellulosic bioethanol production from wheat straw. However, they affect the enzymatic hydrolysis of this material by lowering the glucose recovery yields. This work aimed at explaining the negative effects of laccase on enzymatic hydrolysis. Relative glucose recovery in presence of laccase (10IU/g substrate) with model cellulosic substrate (Sigmacell) at 10% (w/v) was almost 10% points lower (P<0.01) than in the absence of laccase. This fact could be due to an increase in the competition of cellulose binding sites between the enzymes and a slight inhibition of β-glucosidase activity. However, enzymatic hydrolysis and infrared spectra of laccase-treated and untreated wheat straw filtered pretreated residue (WS-FPR), revealed that a grafting process of phenoxy radicals onto the lignin fiber could be the cause of diminished accessibility of cellulases to cellulose in pretreated wheat straw. Copyright © 2014 Elsevier Ltd. All rights reserved.

Influence of very low doses of mediators on fungal laccase activity - nonlinearity beyond imagination

PubMed Central

Malarczyk, Elzbieta; Kochmanska-Rdest, Janina; Jarosz-Wilkolazka, Anna

2009-01-01

Laccase, an enzyme responsible for aerobic transformations of natural phenolics, in industrial applications requires the presence of low-molecular substances known as mediators, which accelerate oxidation processes. However, the use of mediators is limited by their toxicity and the high costs of exploitation. The activation of extracellular laccase in growing fungal culture with highly diluted mediators, ABTS and HBT is described. Two high laccase-producing fungal strains, Trametes versicolor and Cerrena unicolor, were used in this study as a source of enzyme. Selected dilutions of the mediators significantly increased the activity of extracellular laccase during 14 days of cultivation what was distinctly visible in PAGE technique and in colorimetric tests. The same mediator dilutions increased demethylation properties of laccase, which was demonstrated during incubation of enzyme with veratric acid. It was established that the activation effect was assigned to specific dilutions of mediators. Our dose-response dilution process smoothly passes into the range of action of homeopathic dilutions and is of interest for homeopaths. PMID:19732425

Manganese

USGS Publications Warehouse

Cannon, William F.; Kimball, Bryn E.; Corathers, Lisa A.; Schulz, Klaus J.; DeYoung,, John H.; Seal, Robert R.; Bradley, Dwight C.

2017-12-19

Manganese is an essential element for modern industrial societies. Its principal use is in steelmaking, where it serves as a purifying agent in iron-ore refining and as an alloy that converts iron into steel. Although the amount of manganese consumed to make a ton of steel is small, ranging from 6 to 9 kilograms, it is an irreplaceable component in the production of this fundamental material. The United States has been totally reliant on imports of manganese for many decades and will continue to be so for at least the near future. There are no domestic reserves, and although some large low-grade resources are known, they are far inferior to manganese ores readily available on the international market. World reserves of manganese are about 630 million metric tons, and annual global consumption is about 16 million metric tons. Current reserves are adequate to meet global demand for several decades. Global resources in traditional land-based deposits, including both reserves and rocks sufficiently enriched in manganese to be ores in the future, are much larger, at about 17 billion metric tons. Manganese resources in seabed deposits of ferromanganese nodules and crusts are larger than those on land and have not been fully quantified. No production from seabed deposits has yet been done, but current research and development activities are substantial and may bring parts of these seabed resources into production in the future. The advent of economically successful seabed mining could substantially alter the current scenario of manganese supply by providing a large new source of manganese in addition to traditional land-based deposits.From a purely geologic perspective, there is no global shortage of proven ores and potential new ores that could be developed from the vast tonnage of identified resources. Reserves and resources are very unevenly distributed, however. The Kalahari manganese district in South Africa contains 70 percent of the world’s identified resources

Degradation of phenanthrene by Trametes versicolor and its laccase.

PubMed

Han, Mun-Jung; Choi, Hyoung-Tae; Song, Hong-Gyu

2004-06-01

Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46% and 65% of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30 degrees C. Although the removal percentage of phenanthrene was highest (76.7%) at 10 mg/l of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of phenanthrene by laccase about 40% and 30%, respectively.

Gel-Based Purification and Biochemical Study of Laccase Isozymes from Ganoderma sp. and Its Role in Enhanced Cotton Callogenesis

PubMed Central

Kumar, Amit; Singh, Deepti; Sharma, Krishna K.; Arora, Sakshi; Singh, Amarjeet K.; Gill, Sarvajeet S.; Singhal, Barkha

2017-01-01

Basidiomycetous fungi, Ganoderma lucidum MDU-7 and Ganoderma sp. kk-02 secreted multiple laccase isozymes under diverse growth condition. Aromatic compounds and metal salts were also found to regulate the differential expression of laccase isozymes from both the Ganoderma sp. Laccase isozymes induced in the presence of copper from G. lucidum MDU-7 were purified by gel-based (native-PAGE) purification method. The purity of laccase isozymes was checked by zymogram and SDS-PAGE. The SDS-PAGE of purified proteins confirmed the multimeric nature of laccase isozymes. The molecular mass of isozymes was found to be in the range of 40–66 kDa. Further, the purified laccase isozymes and their peptides were confirmed with the help of MALDI-TOF peptide fingerprinting. The biochemical characterization of laccase isozymes viz. Glac L2, Glac L3, Glac L4, and Glac L5 have shown the optimum temperature in the range of 30°–45°C and pH 3.0. The Km values of all the laccase isozymes determined for guaiacol were (96–281 μM), ABTS (15–83 μM) and O-tolidine (78–724 μM). Further, laccase isozymes from G. lucidum whole genome were studied using bioinformatics tools. The molecular modeling and docking of laccase isozymes with different substrates showed a significant binding affinity, which further validates our experimental results. Interestingly, copper induced laccase of 40 U/ml in culture medium was found to significantly induce cotton callogenesis. Interestingly, all the laccase isozymes were found to have an antioxidative role and therefore capable in free radicals scavenging during callogenesis. This is the first detailed study on the biochemical characterization of all the laccase isozymes purified by a gel-based novel method. PMID:28473815

Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation.

PubMed Central

Muñoz, C; Guillén, F; Martínez, A T; Martínez, M J

1997-01-01

Two laccase isoenzymes produced by Pleurotus eryngii were purified to electrophoretic homogeneity (42- and 43-fold) with an overall yield of 56.3%. Laccases I and II from this fungus are monomeric glycoproteins with 7 and 1% carbohydrate content, molecular masses (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 65 and 61 kDa, and pIs of 4.1 and 4.2, respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for laccase I was reached at 65 degrees C and pH 4, and that for laccase II was reached at 55 degrees C and pH 3.5. Both isoenzymes are stable at high pH, retaining 60 to 70% activity after 24 h from pH 8 to 12. Their amino acid compositions and N-terminal sequences were determined, the latter strongly differing from those of laccases of other basidiomycetes. Antibodies against laccase I reacted with laccase II, as well as with laccases from Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus floridanus. Different hydroxy- and methoxy-substituted phenols and aromatic amines were oxidized by the two laccase isoenzymes from P. eryngii, and the influence of the nature, number, and disposition of aromatic-ring substituents on kinetic constants is discussed. Although both isoenzymes presented similar substrate affinities, the maximum rates of reactions catalyzed by laccase I were higher than those of laccase II. In reactions with hydroquinones, semiquinones produced by laccase isoenzymes were in part converted into quinones via autoxidation. The superoxide anion radical produced in the latter reaction dismutated, producing hydrogen peroxide. In the presence of manganous ion, the superoxide union was reduced to hydrogen peroxide with the concomitant production of manganic ion. These results confirmed that laccase in the presence of hydroquinones can participate in the production of both reduced oxygen species and manganic ions. PMID:9172335

Rice (Oryza sativa) Laccases Involved in Modification and Detoxification of Herbicides Atrazine and Isoproturon Residues in Plants.

PubMed

Huang, Meng Tian; Lu, Yi Chen; Zhang, Shuang; Luo, Fang; Yang, Hong

2016-08-24

Atrazine (ATR) and isoproturon (IPU) as herbicides have become serious environmental contaminants due to their overuse in crop production. Although ATR and IPU in soils are easily absorbed by many crops, the mechanisms for their degradation or detoxification in plants are poorly understood. This study identified a group of novel genes encoding laccases (EC 1.10.3.2) that are possibly involved in catabolism or detoxification of ATR and IPU residues in rice. Transcriptome profiling shows at least 22 differentially expressed laccase genes in ATR/IPU-exposed rice. Some of the laccase genes were validated by RT-PCR analysis. The biochemical properties of the laccases were analyzed, and their activities in rice were induced under ATR/IPU exposure. To investigate the roles of laccases in degrading or detoxifying ATR/IPU in rice, transgenic yeast cells (Pichia pastoris X-33) expressing two rice laccase genes (LOC_Os01g63180 and LOC_Os12g15680) were generated. Both transformants were found to accumulate less ATR/IPU compared to the control. The ATR/IPU-degraded products in the transformed yeast cells using UPLC-TOF-MS/MS were further characterized. Two metabolites, hydroxy-dehydrogenated atrazine (HDHA) and 2-OH-isopropyl-IPU, catalyzed by laccases were detected in the eukaryotic cells. These results indicate that the laccase-coding genes identified here could confer degradation or detoxification of the herbicides and suggest that the laccases could be one of the important enzymatic pathways responsible for ATR/IPU degradation/detoxification in rice.

Production of laccase by Pynoporus sanguineus using 2,5 - Xylidine and ethanol

PubMed Central

Valeriano, Viviane S.; Silva, Anna Maria F.; Santiago, Mariângela F.; Bara, Maria T. F.; Garcia, Telma A.

2009-01-01

Enzyme application in biotechnological and environmental processes has had increasing interest due to its efficiency, selectivity and mainly for being environmentally healthful, but these applications require a great volume of enzymes. In this work the effect of different concentrations of ethanol and 2,5-xylidine on growth and production of laccase by Pycnoporus sanguineus was investigated. In a medium containing 200 mg.L-1 of 2,5-xylidine or 50 g.L-1 of ethanol, the maximum activity of laccase was 2019 U.L-1 and 1035 U.L-1, respectively. No direct correlation between biomass and activity of laccase was observed for any of the inducers used during the tests. Ethanol concentrations, larger than or equal to 20 g.L-1, inhibited the radial growth of P. sanguineus. This study showed that ethanol, which has less toxicity and cost than the majority of the studied inducers, presents promising perspectives for laccase production by P. sanguineus. PMID:24031426

Structural and Phylogenetic Analysis of Laccases from Trichoderma: A Bioinformatic Approach

PubMed Central

Cázares-García, Saila Viridiana; Vázquez-Garcidueñas, Ma. Soledad; Vázquez-Marrufo, Gerardo

2013-01-01

The genus Trichoderma includes species of great biotechnological value, both for their mycoparasitic activities and for their ability to produce extracellular hydrolytic enzymes. Although activity of extracellular laccase has previously been reported in Trichoderma spp., the possible number of isoenzymes is still unknown, as are the structural and functional characteristics of both the genes and the putative proteins. In this study, the system of laccases sensu stricto in the Trichoderma species, the genomes of which are publicly available, were analyzed using bioinformatic tools. The intron/exon structure of the genes and the identification of specific motifs in the sequence of amino acids of the proteins generated in silico allow for clear differentiation between extracellular and intracellular enzymes. Phylogenetic analysis suggests that the common ancestor of the genus possessed a functional gene for each one of these enzymes, which is a characteristic preserved in T. atroviride and T. virens. This analysis also reveals that T. harzianum and T. reesei only retained the intracellular activity, whereas T. asperellum added an extracellular isoenzyme acquired through horizontal gene transfer during the mycoparasitic process. The evolutionary analysis shows that in general, extracellular laccases are subjected to purifying selection, and intracellular laccases show neutral evolution. The data provided by the present study will enable the generation of experimental approximations to better understand the physiological role of laccases in the genus Trichoderma and to increase their biotechnological potential. PMID:23383142

Fungal Growth and Manganese Peroxidase Production in a Deep Tray Solid-State Bioreactor, and In Vitro Decolorization of Poly R-478 by MnP.

PubMed

Zhao, Xinshan; Huang, Xianjun; Yao, Juntao; Zhou, Yue; Jia, Rong

2015-06-01

The growth of Irpex lacteus F17 and manganese peroxidase (MnP) production in a selfdesigned tray bioreactor, operating in solid-state conditions at a laboratory scale, were studied. The bioreactor was divided into three layers by three perforated trays. Agroindustrial residues were used both as the carrier of bound mycelia and as a nutrient medium for the growth of I. lacteus F17. The maximum biomass production in the bioreactor was detected at 60 h of fermentation, which was consistent with the CO2 releasing rate by the fungus. During the stationary phase of fungal growth, the maximum MnP activity was observed, reaching 950 U/l at 84 h. Scanning electron microscopy images clearly showed the growth situation of mycelia on the support matrix. Furthermore, the MnP produced by I. lacteus F17 in the bioreactor was isolated and purified, and the internal peptide sequences were also identified with mass spectrometry. The optimal activity of the enzyme was detected at pH 7 and 25 °C, with a long half-life time of 9 days. In addition, the MnP exhibited significant stability within a broad pH range of 4-7 and at temperature up to 55 °C. Besides this, the MnP showed the ability to decolorize the polymeric model dye Poly R-478 in vitro.

Laccase Catalyzed Synthesis of Iodinated Phenolic Compounds with Antifungal Activity

PubMed Central

Ihssen, Julian; Schubert, Mark; Thöny-Meyer, Linda; Richter, Michael

2014-01-01

Iodine is a well known antimicrobial compound. Laccase, an oxidoreductase which couples the one electron oxidation of diverse phenolic and non-phenolic substrates to the reduction of oxygen to water, is capable of oxidizing unreactive iodide to reactive iodine. We have shown previously that laccase-iodide treatment of spruce wood results in a wash-out resistant antimicrobial surface. In this study, we investigated whether phenolic compounds such as vanillin, which resembles sub-structures of softwood lignin, can be directly iodinated by reacting with laccase and iodide, resulting in compounds with antifungal activity. HPLC-MS analysis showed that vanillin was converted to iodovanillin by laccase catalysis at an excess of potassium iodide. No conversion of vanillin occurred in the absence of enzyme. The addition of redox mediators in catalytic concentrations increased the rate of iodide oxidation ten-fold and the yield of iodovanillin by 50%. Iodinated phenolic products were also detected when o-vanillin, ethyl vanillin, acetovanillone and methyl vanillate were incubated with laccase and iodide. At an increased educt concentration of 0.1 M an almost one to one molar ratio of iodide to vanillin could be used without compromising conversion rate, and the insoluble iodovanillin product could be recovered by simple centrifugation. The novel enzymatic synthesis procedure fulfills key criteria of green chemistry. Biocatalytically produced iodovanillin and iodo-ethyl vanillin had significant growth inhibitory effects on several wood degrading fungal species. For Trametes versicolor, a species causing white rot of wood, almost complete growth inhibition and a partial biocidal effect was observed on agar plates. Enzymatic tests indicated that the iodinated compounds acted as enzyme responsive, antimicrobial materials. PMID:24594755

Heterologous Expression of Peroxidases

NASA Astrophysics Data System (ADS)

de Weert, Sandra; Lokman, B. Christien

The industrial importance of peroxidases has led to much research in the past two decades on the development of a cost effective and efficient production process for peroxidases. Unfortunately, even today, no clear answers can be given to questions such as (1) should the peroxidase be expressed in bacteria, yeast, or fungi? (2) which is the optimal production strain (e.g., protease deficient, heme overproducing)? (3) which expression vector should be chosen? and (4) what purification method should be used? Strategies that have proven successful for one peroxidase can fail for another one; for each individual peroxidase, a new strategy has to be developed. This chapter gives an overview of the heterologous production of heme containing peroxidases in various systems. It focuses on the heterologous production of fungal peroxidases as they have been subject of considerable research for their industrial and environmental applications. An earlier study has also been performed by Conesa et al. [1] and is extended with recent proceedings.

Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici

PubMed Central

Feng, Bao Zhen; Li, Peiqian

2014-01-01

Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2′-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 °C, respectively. The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes. PMID:24948955

Reactivities of Various Mediators and Laccases with Kraft Pulp and Lignin Model Compounds

PubMed Central

Bourbonnais, R.; Paice, M. G.; Freiermuth, B.; Bodie, E.; Borneman, S.

1997-01-01

Laccase-catalyzed oxygen delignification of kraft pulp offers some potential as a replacement for conventional chemical bleaching and has the advantage of requiring much lower pressure and temperature. However, chemical mediators are required for effective delignification by laccase, and their price is currently too high at the dosages required. To date, most studies have employed laccase from Trametes versicolor. We have found significant differences in reactivity between laccases from different fungi when they are tested for pulp delignification in the presence of the mediators 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). A more detailed study of T. versicolor laccase with ABTS and HBT showed that HBT gave the most extensive delignification over 2 h but deactivated the enzyme, and therefore a higher enzyme dosage was required. Other mediators, including 1-nitroso-2-naphthol-3,6-disulfonic acid, 4-hydroxy-3-nitroso-1-naphthalenesulfonic acid, promazine, chlorpromazine, and Remazol brilliant blue, were also tested for their ability to delignify kraft pulp. Studies with dimeric model compounds indicated that the mechanisms of oxidation by ABTS and HBT are different. In addition, oxygen uptake by laccase is much slower with HBT than with ABTS. It is proposed that the dication of ABTS and the 1-oxide radical of HBT, with redox potentials in the 0.8- to 0.9-V range, are required for pulp delignification. PMID:16535747

Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris.

PubMed

O'Callaghan, J; O'Brien, M M; McClean, K; Dobson, A D W

2002-08-01

A cDNA encoding a laccase enzyme was isolated from a Trametes versicolor cDNA library. The gene was subcloned into the Pichia pastoris expression vector pPIC3.5 and transformed into the P. pastoris strains KM71 and GS115. Laccase-secreting transformants were selected by their ability to oxidise the substrate ABTS. No difference in laccase activity was observed between culture supernatants from GS115 (proteolytic) and KM71 (nonproteolytic) strains. The presence of at least 200 microM copper was necessary for optimal laccase activity in the culture supernatants. During growth of P. pastoris on minimal medium the pH of the medium was reduced to <3.0. If alanine was added to the medium the pH reduction was not as pronounced and at alanine concentrations >0.6% w/v the pH was kept constant for >7 days. Cultures in which the pH was maintained by alanine metabolism produced higher levels of laccase activity than those grown in the absence of alanine. This study describes the development of a medium that allows convenient pH control of P. pastoris without the need for continuous neutralisation.

The reactivity of phenolic and non-phenolic residual kraft lignin model compounds with Mn(II)-peroxidase from Lentinula edodes.

PubMed

Crestini, C; D'Annibale, A; Sermanni, G G; Saladino, R

2000-02-01

Three phenolic model compounds representing bonding patterns of residual kraft lignin were incubated with manganese peroxidase from Lentinula edodes. Extensive degradation of all the phenolic models, mainly occurring via side-chain benzylic oxidation, was observed. Among the tested model compounds the diphenylmethane alpha-5 phenolic model was found to be the most reactive, yielding several products showing oxidation and fragmentation at the bridging position. The non-phenolic 5-5' biphenyl and 5-5' diphenylmethane models were found unreactive.

Characterization, Molecular Cloning, and Differential Expression Analysis of Laccase Genes from the Edible Mushroom Lentinula edodes

PubMed Central

Zhao, J.; Kwan, H. S.

1999-01-01

The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus. PMID:10543802

Laccase modification of the physical properties of bark and pulp of loblolly pine and spruce pulp

Treesearch

William Kenealy; John Klungness; Mandla Tshabalala; Eric Horn; Masood Akhtar; Roland Gleisner; Gisela Buschle-Diller

2004-01-01

Pine bark, pine pulp, and spruce pulp were reacted with laccase in the presence of phenolic laccase substrates to modify the fiber surface properties. The acid-base and dispersive characteristics of these modified steam-treated thermomechanical loblolly pine pulps were determined by inverse gas chromatography. Different combinations of substrates with laccase modified...

A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris.

PubMed

Kittl, Roman; Mueangtoom, Kitti; Gonaus, Christoph; Khazaneh, Shima Tahvilda; Sygmund, Christoph; Haltrich, Dietmar; Ludwig, Roland

2012-01-20

Fungal laccases from basidiomycetous fungi are thoroughly investigated in respect of catalytic mechanism and industrial applications, but the number of reported and well characterized ascomycetous laccases is much smaller although they exhibit interesting catalytic properties. We report on a highly chloride tolerant laccase produced by the plant pathogen ascomycete Botrytis aclada, which was recombinantly expressed in Pichia pastoris with an extremely high yield and purified to homogeneity. In a fed-batch fermentation, 495 mg L(-1) of laccase was measured in the medium, which is the highest concentration obtained for a laccase by a yeast expression system. The recombinant B. aclada laccase has a typical molecular mass of 61,565 Da for the amino acid chain. The pI is approximately 2.4, a very low value for a laccase. Glycosyl residues attached to the recombinant protein make up for approximately 27% of the total protein mass. B. aclada laccase exhibits very low K(M) values and high substrate turnover numbers for phenolic and non-phenolic substrates at acidic and near neutral pH. The enzyme's stability increases in the presence of chloride ions and, even more important, its substrate turnover is only weakly inhibited by chloride ions (I(50)=1.4M), which is in sharp contrast to most other described laccases. This high chloride tolerance is mandatory for some applications such as implantable biofuel cells and laccase catalyzed reactions, which suffer from the presence of chloride ions. The high expression yield permits fast and easy production for further basic and applied research. Copyright © 2011 Elsevier B.V. All rights reserved.

Immobilization of laccase on a novel ZnO/SiO2 nano-composited support for dye decolorization

NASA Astrophysics Data System (ADS)

Li, Wei-Xun; Sun, Huai-Yan; Zhang, Rui-Feng

2015-07-01

ZnO nanowires were introduced into macroporous SiO2 by means of in situ hydrothermal growth. The obtained nano-composite was then used to immobilize laccase (secured from Trametes versicolor) through the process of static adsorption. The average loading amount was as high as 193.4 μmol-g-1. The immobilized laccase was proven to be an effective biocatalyst in the decolorization of two dyes: Remazol Brilliant Blue B, and Acid Blue 25. The decolorization percentage of Remazol Brilliant Blue B and Acid Blue 25 reached 93% and 82% respectively. The immobilized laccase exhibited enhanced thermal stability and pH adaptability compared to free laccase. After ten recycles, the immobilized laccase retained 42% decolorization catalytic activity.

Effect of amino acids and vitamins on laccase production by the bird's nest fungus Cyathus bulleri.

PubMed

Dhawan, Shikha; Kuhad, Ramesh Chander

2002-08-01

Various amino acids, their analogues and vitamins have shown stimulatory as well as inhibitory effects on laccase production by Cyathus bulleri. DL-methionine, DL-tryptophan, glycine and DL-valine stimulated laccase production, while L-cysteine monohydrochloride completely inhibited the enzyme production. Among vitamins tested biotin, riboflavin and pyridoxine hydrochloride were found to induce laccase production.

Xenobiotic Compounds Degradation by Heterologous Expression of a Trametes sanguineus Laccase in Trichoderma atroviride

PubMed Central

Balcázar-López, Edgar; Méndez-Lorenzo, Luz Helena; Batista-García, Ramón Alberto; Esquivel-Naranjo, Ulises; Ayala, Marcela; Kumar, Vaidyanathan Vinoth; Savary, Olivier; Cabana, Hubert; Herrera-Estrella, Alfredo; Folch-Mallol, Jorge Luis

2016-01-01

Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation. PMID:26849129

Expression and characterization of novel laccase gene from Pandoraea sp. ISTKB and its application.

PubMed

Kumar, Madan; Mishra, Arti; Singh, Shashi Shekhar; Srivastava, Shaili; Thakur, Indu Shekhar

2018-04-14

In the present study, a non-blue laccase gene from previously reported lignin degrading bacterium, Pandoraea sp. ISTKB, was isolated, cloned and expressed in E. coli. Bioinformatics analysis of sequence discovered twin-arginine translocation signal sequence, copper binding motifs and presence of more random coil compare to helices and sheets in structure. The enzyme was found to be active on wide pH range and the pH optima was observed at pH 4 and 8 on substrate 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 2,6-Dimethoxyphenol respectively. This is a thermophilic enzyme with maximum activity around 50-70 °C. The enzyme was further characterized by spectroscopy, reaction kinetics and effect of metal ions and inhibitors were studied. Compared to laccase alone; the treatment of dyes with laccase plus mediator resulted in enhanced decolorization of crystal violet, methylene blue, azure B, carmine and Congo red but the effect of mediator was not observed on trypan blue. Laccase treatment triggered polymerization on vanillic acid (VA) and kraft lignin (KL). Laccase plus mediator treatment reversed the polymerization and resulted in transformation or degradation of VA and KL. This thermophilic and alkalophilic non-blue laccase from Pandoraea sp. ISTKB is promising with prospective biotechnological application. Copyright © 2018. Published by Elsevier B.V.

Changes in selected enzyme activities during growth of pure and mixed cultures of the white-rot decay fungus Trametes versicolor and the potential biocontrol fungus Trichoderma harzianum.

PubMed

Freitag, M; Morrell, J J

1992-04-01

Two filamentous fungi, the white-rot fungus Trametes versicolor and the soil fungus and potential biocontrol organism Trichoderma harzianum, have been grown in pure and mixed cultures on low-N (0.4 mM) and high-N (4 mM) defined synthetic media to determine the activities of selected wood-degrading enzymes such as cellobiase, cellulase, laccase, and peroxidases. Growth characteristics and enzyme activities were examined for potential correlations. Such correlations would allow the use of simple enzyme assays for measuring biomass development and would facilitate predictions about competitiveness of species in mixed fungal cultures. Our results show that while laccase and Poly Red-478 peroxidase activities indicate survival of the decay fungus, none of the monitored extracellular enzymes can serve as a quantitative indicator for biomass accumulation. As expected, the level of available nitrogen affected the production of the enzymes monitored: in low-N media, specific cellobiase, specific cellulase, and peroxidase activities were enhanced, while laccase activities were reduced. Most importantly, laccase activities of Trametes versicolor, and to a smaller extent, cellobiase activities of both fungi, were significantly induced in mixed cultures of Trametes versicolor and Trichoderma harzianum.

[Impact of exogenous paraquat on enzyme exudation and biochemical changes of lignin degradation fungi].

PubMed

Zhao, Yunchen; Li, Jianlong; Chen, Yuru; Huang, Haixia; Yu, Zui

2009-08-01

To study the effect of exogenous oxygen, we added water solution of paraquat to 7 d cultures of Coriolus versicolor for the next 148 h. Enzyme exudation and biochemical process were investigated on the addition of paraquat. We found that compared with the control (without paraquat), the addition of 30 micromol/L paraquat stimulated the activity of manganese dependent peroxidase (MnP), lignin peroxidase (LiP), and laccases (Lac) 7, 2.5 and 1.3 times, respectively. Also, addition of paraquat enhanced activity of superoxide dismutase (SOD) and catalase (CAT) in the first 48 h. Impact of paraquat on ligninolytic enzymes was significant than that on antioxidant enzyme. Addition of paraquat enhanced phenolic compounds and formaldehyde of cultures too. And concentration of malondialdehyde was increased in the first 24 h. The results showed that addition of paraquat promoted oxidative stress, but the antioxidant systems of the fungal strain are sufficient to prevent mycelia from oxidative stress. As exogenous oxygen, paraquat might be a useful substrate in degradation of lignocellulose.

Removal of pigments from molasses wastewater by combining micro-electrolysis with biological treatment method.

PubMed

Chen, Ben; Tian, Xiaofei; Yu, Lian; Wu, Zhenqiang

2016-12-01

Pigments in molasses wastewater (MWW) effluent, such as melanoidins, were considered as kinds of the most recalcitrant and hazardous colorant contaminants to the environment. In this study, de-coloring the MWW by a synergistic combination of micro-electrolysis with bio-treatment was performed. Aiming to a high de-colorization yield, levels of nutrition source supplies, MWW dilution ratio, and micro-electrolysis reaction time were optimized accordingly. For a diluted (50 %, v/v) MWW, an maximum overall de-colorization yield (97.1 ± 0.5 %, for absorbance at 475 nm) was achieved through the bio-electrolysis treatment. In electrolysis bio-treatment, the positive effect of micro-electrolysis was also revealed by a promoted growth of fungal biomass as well as activities of ligninolytic enzymes. Activities of lignin peroxidase, manganese peroxidase, and laccase were promoted by 111.2, 103.9, and 7.7 %, respectively. This study also implied that the bio-treatment and the micro-electrolysis had different efficiencies on removal of pigments with distinct polarities.

Production of ligninolytic enzymes by solid-state fermentation using Pleurotus eryngii.

PubMed

Akpinar, Merve; Urek, Raziye Ozturk

2012-01-01

Pleurotus eryngii (DC.) Gillet (MCC58) was investigated for its ability to produce various ligninolytic enzymes such as laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AAO), and lignin peroxidase (LiP) by solid-state fermentation (SSF), which was carried out using a support substrate from the fruit juice industry. The chemical content of grape waste from this industry was studied. Also, the production patterns of these extracellular enzymes were researched during the growth of the organism for a period of 20 days and the protein, reducing sugar, and nitrogen levels were monitored during the stationary cultivation. The highest Lac activity was obtained as 2247.62 ± 75 U/L on day 10 in the presence of 750 µM Mn²⁺, while the highest MnP activity was attained as 2198.44 ± 65 U/L on day 15 in the presence of 500 µM Mn²⁺. Decolorization of methyl orange and reactive red 2 azo dyes was also achieved with ligninolytic enzymes, produced in SSF of P. eryngii.

Understanding Lignin-Degrading Reactions of Ligninolytic Enzymes: Binding Affinity and Interactional Profile

PubMed Central

Chen, Ming; Zeng, Guangming; Tan, Zhongyang; Jiang, Min; Li, Hui; Liu, Lifeng; Zhu, Yi; Yu, Zhen; Wei, Zhen; Liu, Yuanyuan; Xie, Gengxin

2011-01-01

Previous works have demonstrated that ligninolytic enzymes mediated effective degradation of lignin wastes. The degrading ability greatly relied on the interactions of ligninolytic enzymes with lignin. Ligninolytic enzymes mainly contain laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP). In the present study, the binding modes of lignin to Lac, LiP and MnP were systematically determined, respectively. Robustness of these modes was further verified by molecular dynamics (MD) simulations. Residues GLU460, PRO346 and SER113 in Lac, residues ARG43, ALA180 and ASP183 in LiP and residues ARG42, HIS173 and ARG177 in MnP were most crucial in binding of lignin, respectively. Interactional analyses showed hydrophobic contacts were most abundant, playing an important role in the determination of substrate specificity. This information is an important contribution to the details of enzyme-catalyzed reactions in the process of lignin biodegradation, which can be used as references for designing enzyme mutants with a better lignin-degrading activity. PMID:21980516

Mathematical model for Trametes versicolor growth in submerged cultivation.

PubMed

Tisma, Marina; Sudar, Martina; Vasić-Racki, Durda; Zelić, Bruno

2010-08-01

Trametes versicolor is a white-rot fungus known as a producer of extracellular enzymes such as laccase, manganese-peroxidase, and lignin-peroxidase. The production of these enzymes requires detailed knowledge of the growth characteristics and physiology of the fungus. Submerged cultivations of T. versicolor on glucose, fructose, and sucrose as sole carbon sources were performed in shake flasks. Sucrose hydrolysis catalyzed by the whole cells of T. versicolor was considered as one-step enzymatic reaction described with Michaelis-Menten kinetics. Kinetic parameters of invertase-catalyzed sucrose hydrolysis were estimated (K (m) = 7.99 g dm(-3) and V (m) = 0.304 h(-1)). Monod model was used for description of kinetics of T. versicolor growth on glucose and fructose as sole carbon sources. Growth associated model parameters were estimated from the experimental results obtained by independent experiments (mu(G)(max) = 0.14 h(-1), K(G)(S) = 8.06 g dm(-3), mu(F)(max) = 0.37 h(-1) and K(F)(S) = 54.8 g dm(-3)). Developed mathematical model is in good agreement with the experimental results.

Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyashenko, Andrey V.; Zhukhlistova, Nadegda E.; Gabdoulkhakov, Azat G.

2006-10-01

The crystallization and preliminary X-ray structure at 1.9 Å resolution of the fungal laccase from C. maxima are presented. Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 Å resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccasemore » structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 Å (R factor = 18.953%; R{sub free} = 23.835; r.m.s.d. bond lengths, 0.06 Å; r.m.s.d. bond angles, 1.07°) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002 ▶) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 Å X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO{sub 2} substituents.« less

A comparative genomic analysis of the oxidative enzymes potentially involved in lignin degradation by Agaricus bisporus.

PubMed

Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Fu, Bolei; Cullen, Dan

2013-06-01

The oxidative enzymatic machinery for degradation of organic substrates in Agaricus bisporus (Ab) is at the core of the carbon recycling mechanisms in this fungus. To date, 156 genes have been tentatively identified as part of this oxidative enzymatic machinery, which includes 26 peroxidase encoding genes, nine copper radical oxidase [including three putative glyoxal oxidase-encoding genes (GLXs)], 12 laccases sensu stricto and 109 cytochrome P450 monooxygenases. Comparative analyses of these enzymes in Ab with those of the white-rot fungus, Phanerochaete chrysosporium, the brown-rot fungus, Postia placenta, the coprophilic litter fungus, Coprinopsis cinerea and the ectomychorizal fungus, Laccaria bicolor, revealed enzyme diversity consistent with adaptation to substrates rich in humic substances and partially degraded plant material. For instance, relative to wood decay fungi, Ab cytochrome P450 genes were less numerous (109 gene models), distributed among distinctive families, and lacked extensive duplication and clustering. Viewed together with P450 transcript accumulation patterns in three tested growth conditions, these observations were consistent with the unique Ab lifestyle. Based on tandem gene arrangements, a certain degree of gene duplication seems to have occurred in this fungus in the copper radical oxidase (CRO) and the laccase gene families. In Ab, high transcript levels and regulation of the heme-thiolate peroxidases, two manganese peroxidases and the three GLX-like genes are likely in response to complex natural substrates, including lignocellulose and its derivatives, thereby suggesting an important role in lignin degradation. On the other hand, the expression patterns of the related CROs suggest a developmental role in this fungus. Based on these observations, a brief comparative genomic overview of the Ab oxidative enzyme machinery is presented. Copyright © 2013 Elsevier Inc. All rights reserved.

Ligninolytic peroxidase genes in the oyster mushroom genome: heterologous expression, molecular structure, catalytic and stability properties, and lignin-degrading ability

PubMed Central

2014-01-01

Background The genome of Pleurotus ostreatus, an important edible mushroom and a model ligninolytic organism of interest in lignocellulose biorefineries due to its ability to delignify agricultural wastes, was sequenced with the purpose of identifying and characterizing the enzymes responsible for lignin degradation. Results Heterologous expression of the class II peroxidase genes, followed by kinetic studies, enabled their functional classification. The resulting inventory revealed the absence of lignin peroxidases (LiPs) and the presence of three versatile peroxidases (VPs) and six manganese peroxidases (MnPs), the crystal structures of two of them (VP1 and MnP4) were solved at 1.0 to 1.1 Å showing significant structural differences. Gene expansion supports the importance of both peroxidase types in the white-rot lifestyle of this fungus. Using a lignin model dimer and synthetic lignin, we showed that VP is able to degrade lignin. Moreover, the dual Mn-mediated and Mn-independent activity of P. ostreatus MnPs justifies their inclusion in a new peroxidase subfamily. The availability of the whole POD repertoire enabled investigation, at a biochemical level, of the existence of duplicated genes. Differences between isoenzymes are not limited to their kinetic constants. Surprising differences in their activity T50 and residual activity at both acidic and alkaline pH were observed. Directed mutagenesis and spectroscopic/structural information were combined to explain the catalytic and stability properties of the most interesting isoenzymes, and their evolutionary history was analyzed in the context of over 200 basidiomycete peroxidase sequences. Conclusions The analysis of the P. ostreatus genome shows a lignin-degrading system where the role generally played by LiP has been assumed by VP. Moreover, it enabled the first characterization of the complete set of peroxidase isoenzymes in a basidiomycete, revealing strong differences in stability properties and providing

Characterization and cloning of laccase gene from Hericium coralloides NBRC 7716 suitable for production of epitheaflagallin 3-O-gallate.

PubMed

Itoh, Nobuya; Takagi, Shinya; Miki, Asami; Kurokawa, Junji

2016-01-01

Epitheaflagallin 3-O-gallate (ETFGg) is a minor polyphenol found in black tea extract, which has good physiological functions. It is synthesized from epigallocatechin gallate (EGCg) with gallic acid via laccase oxidation. Various basidiomycetes and fungi were screened to find a suitable laccase for the production of ETFGg. A basidiomycete, Hericium coralloides NBRC 7716, produced an appropriate extracellular laccase. The purified laccase produced twice the level of ETFGg compared with commercially available laccase from Trametes sp. The enzyme, termed Lcc2, is a monomeric protein with an apparent molecular mass of 67.2 kDa. The N-terminal amino acid sequence of Lcc2 is quite different from laccase isolated from the fruiting bodies of Hericium. Lcc2 showed similar substrate specificity to known laccases and could oxidize various phenolic substrates, including pyrogallol, gallic acid, and 2,6-dimethoxyphenol. The full-length lcc2 gene was obtained by PCR using degenerate primers, which were designed based on the N-terminal amino acid sequence of Lcc2 and conserved copper-binding sites of laccases, and 5'-, and 3'-RACE PCR with mRNA. The Lcc2 gene showed homology with Lentinula edodes laccase (sharing 77% amino acid identity with Lcc6). We successfully produced extracellular Lcc2 using a heterologous expression system with Saccharomyces cerevisiae. Moreover, it was confirmed that the recombinant laccase generates similar levels of ETFGg as the native enzyme. Copyright © 2015 Elsevier Inc. All rights reserved.

Evolved α-factor prepro-leaders for directed laccase evolution in Saccharomyces cerevisiae.

PubMed

Mateljak, Ivan; Tron, Thierry; Alcalde, Miguel

2017-11-01

Although the functional expression of fungal laccases in Saccharomyces cerevisiae has proven to be complicated, the replacement of signal peptides appears to be a suitable approach to enhance secretion in directed evolution experiments. In this study, twelve constructs were prepared by fusing native and evolved α-factor prepro-leaders from S. cerevisiae to four different laccases with low-, medium- and high-redox potential (PM1L from basidiomycete PM1; PcL from Pycnoporus cinnabarinus; TspC30L from Trametes sp. strain C30; and MtL from Myceliophthora thermophila). Microcultures of the prepro-leader:laccase fusions were grown in selective expression medium that used galactose as both the sole carbon source and as the inducer of expression so that the secretion and activity were assessed with low- and high-redox potential mediators in a high-throughput screening context. With total activity improvements as high as sevenfold over those obtained with the native α-factor prepro-leader, the evolved prepro-leader from PcL (α PcL ) most strongly enhanced secretion of the high- and medium-redox potential laccases PcL, PM1L and TspC30L in the microtiter format with an expression pattern driven by prepro-leaders in the order α PcL  > α PM 1L  ~ α native . By contrast, the pattern of the low-redox potential MtL was α native  > α PcL  > α PM 1L . When produced in flask with rich medium, the evolved prepro-leaders outperformed the α native signal peptide irrespective of the laccase attached, enhancing secretion over 50-fold. Together, these results highlight the importance of using evolved α-factor prepro-leaders for functional expression of fungal laccases in directed evolution campaigns. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Immobilization of Trametes versicolor cultures for improving laccase production in bubble column reactor intensified by sonication.

PubMed

Wang, Feng; Guo, Chen; Liu, Chun-Zhao

2013-01-01

The mycelia of Trametes versicolor immobilized in alginate beads provided higher laccase production than that in pelleted form. An efficient ultrasonic treatment enhanced laccase production from the immobilized T. versicolor cultures. The optimized treatment process consisted of exposing 36-h-old bead cultures to 7-min ultrasonic treatments twice with a 12-h interval using a fixed ultrasonic power and frequency (120 W, 40 kHz). Using the intensification strategy with sonication, laccase production increased by more than 2.1-fold greater than the untreated control in both flasks and bubble column reactors. The enhancement of laccase production by ultrasonic treatment is related to the improved mass transfer of nutrients and product between the liquid medium and the gel matrix. These results provide a basis for the large-scale and highly-efficient production of laccase using sonobioreactors.

Oxidation of anthracene and benzo[a]pyrene by laccases from Trametes versicolor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, P.J.; Dobson, A.D.W.; Kotterman, M.J.J.

1996-12-01

Polycyclic aromatic hydrocarbons, particularly benzene homologs, are highly toxic organic pollutants. One of the three major groups of extracellular oxidative enzymes involved in the white rot fungal lignin degradative process are laccases. This study presents evidence indicating that laccase has a role in PAH oxidation by white rot fungi. 36 refs., 5 figs., 1 tab.

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

PubMed

Wang, Jizhong; Yang, Chengli; Chen, Xing; Bao, Bingxin; Zhang, Xuan; Li, Dali; Du, Xingfan; Shi, Ruofu; Yang, Junfang; Zhu, Ronghui

2016-08-01

To find an efficient and cheap system for NAD(+) regeneration A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

Modeling of growth and laccase production by Pycnoporus sanguineus.

PubMed

Saat, Muhammad Naziz; Annuar, Mohamad Suffian Mohamad; Alias, Zazali; Chuan, Ling Tau; Chisti, Yusuf

2014-05-01

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L(-1 )days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L(-1).

Constitutive expression of Botrytis aclada laccase in Pichia pastoris

PubMed Central

Kittl, Roman; Gonaus, Christoph; Pillei, Christian; Haltrich, Dietmar; Ludwig, Roland

2012-01-01

The heterologous expression of laccases is important for their large-scale production and genetic engineering—a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL-1) and the AOX1 system (495 mgL-1) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg-1 GAP, 14.2 Umg-1 AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h). PMID:22705842

Plasticity of laccase generated by homeologous recombination in yeast.

PubMed

Cusano, Angela M; Mekmouche, Yasmina; Meglecz, Emese; Tron, Thierry

2009-10-01

Laccase-encoding sequences sharing 65-71% identity were shuffledin vivo by homeologous recombination. Yeast efficiently repaired linearized plasmids containing clac1, clac2 or clac5 Trametes sp. C30 cDNAs using a clac3 PCR fragment. From transformants secreting active variants, three chimeric laccases (LAC131, LAC232 and LAC535), each resulting from double crossovers, were purified, and their apparent kinetic parameters were determined using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and syringaldazine (SGZ) as substrates. At acidic pH, the apparent kinetic parameters of the chimera were not distinguishable from each other or from those obtained for the LAC3 enzyme used as reference. On the other hand, the pH tolerance of the variants was visibly extended towards alkaline pH values. Compared to the parental LAC3, a 31-fold increase in apparent k(cat) was observed for LAC131 at pH 8. This factor is one of the highest ever observed for laccase in a single mutagenesis step.

Kraft Pulp Biobleaching and Mediated Oxidation of a Nonphenolic Substrate by Laccase from Streptomyces cyaneus CECT 3335

PubMed Central

Arias, M. Enriqueta; Arenas, María; Rodríguez, Juana; Soliveri, Juan; Ball, Andrew S.; Hernández, Manuel

2003-01-01

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70°C, respectively. The activity was strongly enhanced in the presence of Cu2+, Mn2+, and Mg2+ and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes. PMID:12676669

Purification and characterization of two novel peroxidases from the dye-decolorizing fungus Bjerkandera adusta strain CX-9.

PubMed

Bouacem, Khelifa; Rekik, Hatem; Jaouadi, Nadia Zaraî; Zenati, Bilal; Kourdali, Sidali; El Hattab, Mohamed; Badis, Abdelmalek; Annane, Rachid; Bejar, Samir; Hacene, Hocine; Bouanane-Darenfed, Amel; Jaouadi, Bassem

2018-01-01

Two extracellular peroxidases from Bjerkandera adusta strain CX-9, namely a lignin peroxidase (called LiP BA45) and manganese peroxidase (called MnP BA30), were purified simultaneously by applying successively, ammonium sulfate precipitation-dialysis, Mono-S Sepharose anion-exchange and Sephacryl S-200 gel filtration and biochemically characterized. The sequence of their NH 2 -terminal amino acid residues showed high homology with those of fungi peroxidases. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzymes MnP BA30 and LiP BA45 were a monomers with a molecular masses 30125.16 and 45221.10Da, respectively. While MnP BA30 was optimally active at pH 3 and 70°C, LiP BA45 showed optimum activity at pH 4 and 50°C. The two enzymes were inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in their tertiary structures. The K m and V max for LiP BA45 toward 2,4-Dichlorolphenol (2,4-DCP) were 0.099mM and 9.12U/mg, respectively and for MnP BA30 toward 2,6-Dimethylphenol (2,6-DMP), they were 0.151mM and 18.60U/mg, respectively. Interestingly, MnP BA30 and LiP BA45 demonstrated higher catalytic efficiency than that of other tested peroxidases (MnP, LiP, HaP4, and LiP-SN) and marked organic solvent-stability and dye-decolorization efficiency. Data suggest that these peroxidases may be considered as potential candidates for future applications in distaining synthetic-dyes. Copyright © 2017 Elsevier B.V. All rights reserved.

Copper and dyes enhance laccase production in gamma-proteobacterium JB.

PubMed

Malhotra, Kanam; Sharma, Prince; Capalash, Neena

2004-07-01

Laccase production in gamma-proteobacterium JB was enhanced 13-fold by adding 0.1 mM CuSO(4) 24 h after the onset of growth. Ethidium bromide (2.5 microM), Malachite Green, Phenol Red and Thymol Blue (10 microM each) enhanced laccase production 17-, 19-, 4- and 2-fold, respectively. Among the fourteen aromatic/organic compounds tried, p-aminobenzoic acid and an industrial effluent, from where the organism was isolated, showed 1.2- and 1.26-fold increases in production.

Optimization of media components for laccase production by litter dwelling fungal isolate Fusarium incarnatum LD-3.

PubMed

Chhaya, Urvish; Gupte, Akshaya

2010-02-01

Laccase production by solid state fermentation (SSF) using an indigenously isolated litter dwelling fungus Fusarium incarnatum LD-3 was optimized. Fourteen medium components were screened by the initial screening method of Plackett-Burman. Each of the components was screened on the basis of 'p' (probability value) which was above 95% confidence level. Ortho-dianisidine, thiamine HCl and CuSO(4) . 5 H(2)O were identified as significant components for laccase production. The Central Composite Design response surface methodology was then applied to further optimize the laccase production. The optimal concentration of these three medium components for higher laccase production were (g/l): CuSO(4) . 5 H(2)O, 0.01; thiamine HCl, 0.0136 and ortho-dianisidine, 0.388 mM served as an inducer. Wheat straw, 5.0 g was used as a solid substrate. Using this statistical optimization method the laccase production was found to increase from 40 U/g to 650 U/g of wheat straw, which was sixteen times higher than non optimized medium. This is the first report on statistical optimization of laccase production from Fusarium incarnatum LD-3.

Oxidative polymerization of lignins by laccase in water-acetone mixture.

PubMed

Fiţigău, Ionița Firuța; Peter, Francisc; Boeriu, Carmen Gabriela

2013-01-01

The enzymatic oxidative polymerization of five technical lignins with different molecular properties, i.e. Soda Grass/Wheat straw Lignin, Organosolv Hardwood Lignin, Soda Wheat straw Lignin, Alkali pretreated Wheat straw Lignin, and Kraft Softwood was studied. All lignins were previously fractionated by acetone/water 50:50 (v/v) and the laccase-catalyzed polymerization of the low molecular weight fractions (Mw < 4000 g/mol) was carried out in the same solvent system. Reactivity of lignin substrates in laccase-catalyzed reactions was determined by monitoring the oxygen consumption. The oxidation reactions in 50% acetone in water mixture proceed with high rate for all tested lignins. Polymerization products were analyzed by size exclusion chromatography, FT-IR, and (31)P-NMR and evidence of important lignin modifications after incubation with laccase. Lignin polymers with higher molecular weight (Mw up to 17500 g/mol) were obtained. The obtained polymers have potential for applications in bioplastics, adhesives and as polymeric dispersants.

Kraft lignin biodegradation by Novosphingobium sp. B-7 and analysis of the degradation process.

PubMed

Chen, Yuehui; Chai, Liyuan; Tang, Chongjian; Yang, Zhihui; Zheng, Yu; Shi, Yan; Zhang, Huan

2012-11-01

This study focused on the biodegradation of kraft lignin (KL) by Novosphingobium sp. B-7 using KL as sole carbon source. Results revealed that Novosphingobium sp. B-7 reduced the chemical oxygen demand (COD) by 34.7% in KL mineral salt medium after 7days of incubation. Additionally, the maximum activities of manganese peroxidase (MnP) of 3229.8Ul(-1) and laccase (Lac) of 1275Ul(-1) were observed at 4th and 5th day, respectively. GC-MS analysis indicated that after incubated with Novosphingobium sp. B-7, low molecular weight alcohols and lignin-related monomer compounds such as ethanediol, p-hydroxy benzoic acid and vanillic acid were formed in the system, which strongly confirmed the degradation of KL by Novosphingobium sp. B-7. Copyright © 2012 Elsevier Ltd. All rights reserved.

Spore cells from BPA degrading bacteria Bacillus sp. GZB displaying high laccase activity and stability for BPA degradation.

PubMed

Das, Ranjit; Li, Guiying; Mai, Bixian; An, Taicheng

2018-06-04

Laccase has been applied extensively as a biocatalyst to remove different organic pollutants. This study characterized a spore-laccase from the bisphenol A (BPA)-degrading strain Bacillus sp. GZB. The spore-laccase was encoded with 513 amino acids, containing spore coat protein A (CotA). It showed optimal activity at 70 °C and pH = 7.2 in presence of 2, 6-dimethoxyphenol. At 60 °C, optimal activity was also seen at pH = 3.0 and pH = 6.8 with 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine, respectively. The spore-laccase was stable at high temperature, at acidic to alkaline pH values, and in the presence of different organic solvents. Spore-laccase activity was increased by introducing Cu 2+ , Mg 2+ , and Na + , but was strongly inhibited by Fe 2+ , Ag + , l-cysteine, dithiothreitol, and NaN 3 . The cotA gene was cloned and expressed in E. coli BL21 (DE3); the purified protein was estimated as having a molecular weight of ~63 kDa. Different synthetic dyes and BPA were effectively decolorized or degraded both by the spore laccase and recombinant laccase. When BPA oxidation was catalyzed using laccase, there was an initial formation of phenoxy radicals and further oxidation or CC bond cleavage of the radicals produced different organic acids. Detailed reaction pathways were developed based on nine identified intermediates. The acute toxicity decreased gradually during BPA degradation by laccase. This study is the first report about a genus of Bacillus that can produce a highly active and stable laccase to degrade BPA. Copyright © 2018 Elsevier B.V. All rights reserved.

Metal release and sequestration from black slate mediated by a laccase of Schizophyllum commune.

PubMed

Kirtzel, Julia; Scherwietes, Eric Leon; Merten, Dirk; Krause, Katrin; Kothe, Erika

2018-06-25

Schizophyllum commune is a filamentous basidiomycete which can degrade complex organic macromolecules like lignin by the secretion of a large repertoire of enzymes. One of these white rot enzymes, laccase, exhibits a broad substrate specificity and is able to oxidize a variety of substances including carbonaceous rocks. To investigate the role of laccase in bioweathering, laccase gene lcc2 was overexpressed, and the influence on weathering of black slate, originating from a former alum mine in Schmiedefeld, Germany, was examined. The metal release from the rock material was enhanced, associated with a partial metal accumulation into the mycelium. A sequestration of metals could be shown with fluorescent staining methods, and an accumulation of Zn, Cd, and Pb was visualized in different cell organelles. Additionally, we could show an increased metal resistance of the laccase overexpressing strain.

Analysis of in situ manganese(II) oxidation in the Columbia River and offshore plume: Linking microbial community structure to active biogeochemical cycles

PubMed Central

Anderson, C. R.; Davis, R. E.; Bandolin, N. S.; Baptista, A. M.; Tebo, B. M.

2017-01-01

The Columbia River is a major source of dissolved nutrients and trace metals for the west coast of North America. A large proportion of these nutrients are sourced from the Columbia River Estuary where coastal and terrestrial waters mix and resuspend particulate matter within the water column. As estuarine water is discharged off the coast it transports the particulate matter, dissolved nutrients and microorganisms forming nutrient rich and metabolically dynamic plumes. In this study, bacterial manganese oxidation within the plume and estuary was investigated during spring and neap tides. The microbial community proteome was fractionated and assayed for Mn oxidation activity. Proteins from the outer membrane and the loosely bound outer membrane fractions were separated using size exclusion chromatography and Mn(II)-oxidizing eluates were analyzed with tandem mass spectrometry to identify potential Mn oxidase protein targets. Multi-copper oxidase (MCO) and heme-peroxidase enzymes were identified in active fractions. T-RFLP cluster analysis indicates that the organisms oxidizing the most Mn(II) were sourced from the Columbia River estuary and nearshore coastal ocean. These organisms are producing up to 10 fM MnO2 cell−1 day−1. Evidence for the presence of Mn(II)-oxidizing bacterial isolates from the genera Aurantimonas, Rhodobacter, Bacillus, and Shewanella was found in T-RFLP profiles. Q-PCR was used to quantify the gene copies of the heme-peroxidase, Aurantimonas SSU rRNA and total bacterial SSU rRNA gene copies. The probes used suggested that Aurantimonas could only account for 1.7% of heme-peroxidase genes quantified suggesting that peroxidase driven manganese oxidation capabilities are widespread throughout other organisms in this environment. PMID:21418498

Sonochemical and hydrodynamic cavitation reactors for laccase/hydrogen peroxide cotton bleaching.

PubMed

Gonçalves, Idalina; Martins, Madalena; Loureiro, Ana; Gomes, Andreia; Cavaco-Paulo, Artur; Silva, Carla

2014-03-01

The main goal of this work is to develop a novel and environmental-friendly technology for cotton bleaching with reduced processing costs. This work exploits a combined laccase-hydrogen peroxide process assisted by ultrasound. For this purpose, specific reactors were studied, namely ultrasonic power generator type K8 (850 kHz) and ultrasonic bath equipment Ultrasonic cleaner USC600TH (45 kHz). The optimal operating conditions for bleaching were chosen considering the highest levels of hydroxyl radical production and the lowest energy input. The capacity to produce hydroxyl radicals by hydrodynamic cavitation was also assessed in two homogenizers, EmulsiFlex®-C3 and APV-2000. Laccase nanoemulsions were produced by high pressure homogenization using BSA (bovine serum albumin) as emulsifier. The bleaching efficiency of these formulations was tested and the results showed higher whiteness values when compared to free laccase. The combination of laccase-hydrogen peroxide process with ultrasound energy produced higher whiteness levels than those obtained by conventional methods. The amount of hydrogen peroxide was reduced 50% as well as the energy consumption in terms of temperature (reduction of 40 °C) and operating time (reduction of 90 min). Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica.

PubMed

Guo, Mei; Lu, Fuping; Pu, Jun; Bai, Dongqing; Du, Lianxiang

2005-11-01

A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETalphaA and expressed in Pichia methanolica. The laccase activity obtained with the Saccharomyces cerevisiae alpha-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the alpha-factor signal peptide was 9.79 U ml(-1). The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form.

A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye.

PubMed

Afreen, Sumbul; Shamsi, Tooba Naz; Baig, Mohd Affan; Ahmad, Nadeem; Fatima, Sadaf; Qureshi, M Irfan; Hassan, Md Imtaiyaz; Fatma, Tasneem

2017-01-01

A novel extracellular laccase enzyme produced from Spirulina platensis CFTRI was purified by ultrafiltration, cold acetone precipitation, anion exchange and size exclusion chromatography with 51.5% recovery and 5.8 purification fold. The purified laccase was a monomeric protein with molecular mass of ~66 kDa that was confirmed by zymogram analysis and peptide mass fingerprinting. The optimum pH and temperature of the enzyme activity was found at 3.0 and 30°C using ABTS as substrate but the enzyme was quite stable at high temperature and alkaline pH. The laccase activity was enhanced by Cu+2, Zn+2 and Mn+2. In addition, the dye decolorization potential of purified laccase was much higher in terms of extent as well as time. The purified laccase decolorized (96%) of anthraquinonic dye Reactive blue- 4 within 4 h and its biodegradation studies was monitored by UV visible spectra, FTIR and HPLC which concluded that cyanobacterial laccase can be efficiently used to decolorize synthetic dye and help in waste water treatment.

A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye

PubMed Central

Afreen, Sumbul; Shamsi, Tooba Naz; Baig, Mohd Affan; Ahmad, Nadeem; Fatima, Sadaf; Qureshi, M. Irfan; Hassan, Md. Imtaiyaz

2017-01-01

A novel extracellular laccase enzyme produced from Spirulina platensis CFTRI was purified by ultrafiltration, cold acetone precipitation, anion exchange and size exclusion chromatography with 51.5% recovery and 5.8 purification fold. The purified laccase was a monomeric protein with molecular mass of ~66 kDa that was confirmed by zymogram analysis and peptide mass fingerprinting. The optimum pH and temperature of the enzyme activity was found at 3.0 and 30°C using ABTS as substrate but the enzyme was quite stable at high temperature and alkaline pH. The laccase activity was enhanced by Cu+2, Zn+2 and Mn+2. In addition, the dye decolorization potential of purified laccase was much higher in terms of extent as well as time. The purified laccase decolorized (96%) of anthraquinonic dye Reactive blue- 4 within 4 h and its biodegradation studies was monitored by UV visible spectra, FTIR and HPLC which concluded that cyanobacterial laccase can be efficiently used to decolorize synthetic dye and help in waste water treatment. PMID:28384218

Incorporation of copper ions into crystals of T2 copper-depleted laccase from Botrytis aclada

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osipov, E. M., E-mail: e.m.osipov@gmail.com; Polyakov, K. M.; Engelhardt Institute of Molecular Biology, Vavilova str. 32, Moscow 119991

2015-11-18

The restoration of the native form of laccase from B. aclada from the type 2 copper-depleted form of the enzyme was investigated. Copper ions were found to be incorporated into the active site after soaking the depleted enzyme in a Cu{sup +}-containing solution. Laccases belong to the class of multicopper oxidases catalyzing the oxidation of phenols accompanied by the reduction of molecular oxygen to water without the formation of hydrogen peroxide. The activity of laccases depends on the number of Cu atoms per enzyme molecule. The structure of type 2 copper-depleted laccase from Botrytis aclada has been solved previously. Withmore » the aim of obtaining the structure of the native form of the enzyme, crystals of the depleted laccase were soaked in Cu{sup +}- and Cu{sup 2+}-containing solutions. Copper ions were found to be incorporated into the active site only when Cu{sup +} was used. A comparative analysis of the native and depleted forms of the enzymes was performed.« less

NMR Studies of Peroxidases.

NASA Astrophysics Data System (ADS)

Veitch, Nigel Charles

Available from UMI in association with The British Library. Requires signed TDF. Peroxidases are a haem-containing group of enzymes with a wide diversity of function within biological systems. While a common characteristic is the ability to catalyse the conversion of hydrogen peroxide to water, it is the accompanying processes of hormone synthesis and degradation which have generated such a high level of interest. However, information at the molecular level is limited to a single well-resolved crystal structure, that of yeast cytochrome c peroxidase. This thesis presents a strategy for the investigation of peroxidase structure and function based on proton nuclear magnetic resonance spectroscopy, a technique which has the ability to address aspects of both protein structure and protein dynamics in solution. The application of one- and two-dimensional NMR techniques has been developed in the context of plant peroxidases, notably the isoenzyme HRP-C derived from the horseradish root. Characterisation of the proton NMR spectra of HRP -C in resting and ligated states provided new information enabling the structure of the binding site for aromatic donor molecules, such as indole-3-propionic, ferulic and benzhydroxamic acids, to be resolved. In order to overcome difficulties encountered with a protein of the complexity of peroxidase, additional information was obtained from chemical shift parameters and the use of peroxidase variants produced by site-directed mutagenesis. A comparative study using NMR spectroscopy was undertaken for wild-type recombinant HRP-C expressed in Escherichia coli, and two protein variants with substitutions made to residues located on the distal side of the haem pocket, Phe41 to Val and Arg38 to Lys. NMR analyses of a plant peroxidase from barley grains and the fungal peroxidase from Coprinus cinereus were also successful using methods conceived with HRP-C. Examination of three specifically constructed recombinant protein variants of C. cinereus

Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity.

PubMed

Mayolo-Deloisa, Karla; González-González, Mirna; Simental-Martínez, Jesús; Rito-Palomares, Marco

2015-03-01

Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme. Copyright © 2015 John Wiley & Sons, Ltd.

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR.

PubMed Central

D'Souza, T M; Boominathan, K; Reddy, C A

1996-01-01

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases. PMID:8837429

Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation.

PubMed

Awasthi, Manika; Jaiswal, Nivedita; Singh, Swati; Pandey, Veda P; Dwivedi, Upendra N

2015-09-01

Laccase, widely distributed in bacteria, fungi, and plants, catalyzes the oxidation of wide range of compounds. With regards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesis while fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual function of laccases using in-silico molecular docking and dynamics simulation approaches. Modeling and superimposition analyses of one each representative of plant and fungal laccases, namely, Populus trichocarpa and Trametes versicolor, respectively, revealed low level of similarity in the folding of two laccases at 3D levels. Docking analyses revealed significantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as compared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites were found to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analyses for the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetrameric lignin model compound remains attached to the active site of fungal laccase throughout the simulation period, while it protrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basis of binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than that of plant. The overall data suggested a situation favorable for the degradation of lignin polymer by fungal laccase while its synthesis by plant laccase.

Enhanced production of laccase from Coriolus versicolor NCIM 996 by nutrient optimization using response surface methodology.

PubMed

Arockiasamy, Santhiagu; Krishnan, Indira Packialakshmi Gurusamy; Anandakrishnan, Nimalanandan; Seenivasan, Sabitha; Sambath, Agalya; Venkatasubramani, Janani Priya

2008-12-01

Plackett and Burman design criterion and central composite design were applied successfully for enhanced production of laccase by Coriolus versicolor NCIM 996 for the first time. Plackett and Burman design criterion was applied to screen the significance of ten nutrients on laccase production by C. versicolor NCIM 996. Out of the ten nutrients tested, starch, yeast extract, MnSO(4), MgSO(4) x 7H(2)O, and phenol were found to have significant effect on laccase production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Plackett-Burman design. The optimized medium composition for production of laccase was (g/l): starch, 30.0; yeast extract, 4.53; MnSO(4), 0.002; MgSO(4) x 7H(2)O, 0.755; and phenol, 0.026, and the optimum laccase production was 6,590.26 (U/l), which was 7.6 times greater than the control.

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp.

PubMed

Ausec, Luka; Črnigoj, Miha; Šnajder, Marko; Ulrih, Nataša Poklar; Mandic-Mulec, Ines

2015-12-01

Laccases are oxidoreductases mostly studied in fungi, while bacterial laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli. The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general.

Combinatorial evaluation of the laccase-mediator system (LMS) in the oxidation of veratryl alcohol

USDA-ARS?s Scientific Manuscript database

Identifying suitable reaction conditions remains an important task in the development of practical enzyme catalysts. Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both co...

Development of a Saccharomyces cerevisiae Strain with Enhanced Resistance to Phenolic Fermentation Inhibitors in Lignocellulose Hydrolysates by Heterologous Expression of Laccase

PubMed Central

Larsson, Simona; Cassland, Pierre; Jönsson, Leif J.

2001-01-01

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose. PMID:11229906

Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase.

PubMed

Larsson, S; Cassland, P; Jönsson, L J

2001-03-01

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.

A Laccase with HIV-1 Reverse Transcriptase Inhibitory Activity from the Broth of Mycelial Culture of the Mushroom Lentinus tigrinus

PubMed Central

Xu, LiJing; Wang, HeXiang; Ng, TziBun

2012-01-01

A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC50 = 2.4 μM) was isolated from the broth of mycelial culture of the mushroom Lentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and other Lentinus tigrinus strain laccase. Its characteristics were different from previously reported laccase of other Lentinus tigrinus strain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities of Lentinus tigrinus laccase. PMID:22536022

Optimization and modeling of laccase production by Trametes versicolor in a bioreactor using statistical experimental design.

PubMed

Tavares, A P M; Coelho, M A Z; Agapito, M S M; Coutinho, J A P; Xavier, A M R B

2006-09-01

Experimental design and response surface methodologies were applied to optimize laccase production by Trametes versicolor in a bioreactor. The effects of three factors, initial glucose concentration (0 and 9 g/L), agitation (100 and 180 rpm), and pH (3.0 and 5.0), were evaluated to identify the significant effects and its interactions in the laccase production. The pH of the medium was found to be the most important factor, followed by initial glucose concentration and the interaction of both factors. Agitation did not seem to play an important role in laccase production, nor did the interaction agitation x medium pH and agitation x initial glucose concentration. Response surface analysis showed that an initial glucose concentration of 11 g/L and pH controlled at 5.2 were the optimal conditions for laccase production by T. versicolor. Under these conditions, the predicted value for laccase activity was >10,000 U/L, which is in good agreement with the laccase activity obtained experimentally (11,403 U/L). In addition, a mathematical model for the bioprocess was developed. It is shown that it provides a good description of the experimental profile observed, and that it is capable of predicting biomass growth based on secondary process variables.

[Degradation of fluorene and fluoranthene by the basidiomycete Pleurotus ostreatus].

PubMed

Pozdnyakova, N N; Chernyshova, M P; Grinev, V S; Landesman, E O; Koroleva, O V; Turkovskaya, O V

2016-01-01

The dependence of the degree of fluorene and fluoranthene degradation by the fungus Pleurotus ostreatus D1 on the culture medium composition has been studied. Polycyclic aromatic hydrocarbons (PAHs) have been transformed in Kirk’s medium (under conditions of laccase production) with the formation of a quinone metabolite and 9-fluorenone upon the use of fluoranthene and fluorene as substrates, respectively. More complete degradation with the formation of an intermediate metabolite, phthalic acid that has undergone subsequent utilization, has occurred in basidiomycete-rich medium (under the production of both laccase and versatile peroxidase). The formation of phthalic acid as a metabolite of fluoranthene degradation by lignolytic fungi has been revealed for the first time. The data allow the supposition that both extracellular laccase and laccase on the mycelium surface can participate in the initial stages of PAH metabolism, while versatile peroxidase is necessary for the oxidation of the formed metabolites. A scheme of fluorene metabolism by Pleurotus ostreatus D1 is suggested.

Molecular Phylogeny of Heme Peroxidases

NASA Astrophysics Data System (ADS)

Zámocký, Marcel; Obinger, Christian

All currently available gene sequences of heme peroxidases can be phylogenetically divided in two superfamilies and three families. In this chapter, the phylogenetics and genomic distribution of each group are presented. Within the peroxidase-cyclooxygenase superfamily, the main evolutionary direction developed peroxidatic heme proteins involved in the innate immune defense system and in biosynthesis of (iodinated) hormones. The peroxidase-catalase superfamily is widely spread mainly among bacteria, fungi, and plants, and particularly in Class I led to the evolution of bifunctional catalase-peroxidases. Its numerous fungal representatives of Class II are involved in carbon recycling via lignin degradation, whereas Class III secretory peroxidases from algae and plants are included in various forms of secondary metabolism. The family of di-heme peroxidases are predominantly bacteria-inducible enzymes; however, a few corresponding genes were also detected in archaeal genomes. Four subfamilies of dyp-type peroxidases capable of degradation of various xenobiotics are abundant mainly among bacteria and fungi. Heme-haloperoxidase genes are widely spread among sac and club fungi, but corresponding genes were recently found also among oomycetes. All described families herein represent heme peroxidases of broad diversity in structure and function. Our accumulating knowledge about the evolution of various enzymatic functions and physiological roles can be exploited in future directed evolution approaches for engineering peroxidase genes de novo for various demands.

Properties of the glycoprotein laccase immobilized by two methods.

PubMed

Froehner, S C; Eriksson, K

1975-01-01

Laccase (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) from Neurospora crassa has been immobilized by two different procedures: (1) Covalent attachment to Sepharose 4B activated with cyanogen bromide, and (2) Adsorption to Concanavalin A-Sepharose via the carbohydrate moiety. Except for small changes in the Michaelis-Menten constants, no differences were noted in the enzymological properties of the immobilized enzymes when compared to free enzyme. The carbohydrate moiety of laccase involved in the interaction with Concanavalin A does not appear to be closely associated with the active center since binding to the lectin has no effect on the enzymological parameters investigated.

Hybrid microfluidic fuel cell based on Laccase/C and AuAg/C electrodes.

PubMed

López-González, B; Dector, A; Cuevas-Muñiz, F M; Arjona, N; Cruz-Madrid, C; Arana-Cuenca, A; Guerra-Balcázar, M; Arriaga, L G; Ledesma-García, J

2014-12-15

A hybrid glucose microfluidic fuel cell composed of an enzymatic cathode (Laccase/ABTS/C) and an inorganic anode (AuAg/C) was developed and tested. The enzymatic cathode was prepared by adsorption of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and Laccase on Vulcan XC-72, which act as a redox mediator, enzymatic catalyst and support, respectively. The Laccase/ABTS/C composite was characterised by Fourier Transform Infrared (FTIR) Spectroscopy, streaming current measurements (Zeta potential) and cyclic voltammetry. The AuAg/C anode catalyst was characterised by Transmission electron microscopy (TEM) and cyclic voltammetry. The hybrid microfluidic fuel cell exhibited excellent performance with a maximum power density value (i.e., 0.45 mW cm(-2)) that is the highest reported to date. The cell also exhibited acceptable stability over the course of several days. In addition, a Mexican endemic Laccase was used as the biocathode electrode and evaluated in the hybrid microfluidic fuel cell generating 0.5 mW cm(-2) of maximum power density. Copyright © 2014 Elsevier B.V. All rights reserved.

Bacterial exopolysaccharides as a modern biotechnological tool for modification of fungal laccase properties and metal ion binding.

PubMed

Osińska-Jaroszuk, Monika; Jaszek, Magdalena; Starosielec, Magdalena; Sulej, Justyna; Matuszewska, Anna; Janczarek, Monika; Bancerz, Renata; Wydrych, Jerzy; Wiater, Adrian; Jarosz-Wilkołazka, Anna

2018-03-26

Four bacterial EPSs extracted from Rhizobium leguminosarum bv. trifolii Rt24.2, Sinorhizobium meliloti Rm1021, Bradyrhizobium japonicum USDA110, and Bradyrhizobium elkanii USDA76 were determined towards their metal ion adsorption properties and possible modification of Cerrena unicolor laccase properties. The highest magnesium and iron ion-sorption capacity (~ 42 and ~ 14.5%, respectively) was observed for EPS isolated from B. japonicum USDA110. An evident influence of EPSs on the stability of laccase compared to the control values (without EPSs) was shown after 30-day incubation at 25 °C. The residual activity of laccases was obtained in the presence of Rh76EPS and Rh1021EPS, i.e., 49.5 and 41.5% of the initial catalytic activity, respectively. This result was confirmed by native PAGE electrophoresis. The EPS effect on laccase stability at different pH (from 3.8 to 7.0) was also estimated. The most significant changes at the optimum pH value (pH 5.8) was observed in samples of laccase stabilized by Rh76EPS and Rh1021EPS. Cyclic voltamperometry was used for analysis of electrochemical parameters of laccase stabilized by bacterial EPS and immobilized on single-walled carbon nanotubes (SWCNTs) with aryl residues. Laccases with Rh76EPS and Rh1021EPS had an evident shift of the value of the redox potential compared to the control without EPS addition. In conclusion, the results obtained in this work present a new potential use of bacterial EPSs as a metal-binding component and a modulator of laccase properties especially stability of enzyme activity, which can be a very effective tool in biotechnology and industrial applications.

Cross-linking proteins by laccase: Effects on the droplet size and rheology of emulsions stabilized by sodium caseinate.

PubMed

Sato, A C K; Perrechil, F A; Costa, A A S; Santana, R C; Cunha, R L

2015-09-01

The aim of this work was to evaluate the influence of laccase and ferulic acid on the characteristics of oil-in-water emulsions stabilized by sodium caseinate at different pH (3, 5 and 7). Emulsions were prepared by high pressure homogenization of soybean oil with sodium caseinate solution containing varied concentrations of laccase (0, 1 and 5mg/mL) and ferulic acid (5 and 10mM). Laccase treatment and pH exerted a strong influence on the properties with a consequent effect on stability, structure and rheology of emulsions stabilized by Na-caseinate. At pH7, O/W emulsions were kinetically stable due to the negative protein charge which enabled electrostatic repulsion between oil droplets resulting in an emulsion with small droplet size, low viscosity, pseudoplasticity and viscoelastic properties. The laccase treatment led to emulsions showing shear-thinning behavior as a result of a more structured system. O/W emulsions at pH5 and 3 showed phase separation due to the proximity to protein pI, but the laccase treatment improved their stability of emulsions especially at pH3. At pH3, the addition of ferulic acid and laccase produced emulsions with larger droplet size but with narrower droplet size distribution, increased viscosity, pseudoplasticity and viscoelastic properties (gel-like behavior). Comparing laccase treatments, the combined addition of laccase and ferulic acid generally produced emulsions with lower stability (pH5), larger droplet size (pH3, 5 and 7) and higher pseudoplasticity (pH5 and 7) than emulsion with only ferulic acid. The results suggested that the cross-linking of proteins by laccase and ferulic acid improved protein emulsifying properties by changing functional mechanisms of the protein on emulsion structure and rheology, showing that sodium caseinate can be successfully used in acid products when treated with laccase. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biochemical characterization and molecular evidence of a laccase from the bird's nest fungus Cyathus bulleri.

PubMed

Vasdev, Kavita; Dhawan, Shikha; Kapoor, Rajeev Kumar; Kuhad, Ramesh Chander

2005-08-01

Cyathus bulleri, a bird's nest fungus, known to decolorize polymeric dye Poly R-478, was found to produce 8 U ml(-1) of laccase in malt extract broth. Laccase activity appeared as a single band on non-denaturing gel. Laccase was purified to homogeneity by anion exchange chromatography and gel filtration. The enzyme was a monomer with an apparent molecular mass of 60 kD, pI of 3.7 and was stable in the pH range of 2-6 with an optimum pH of 5.2. The optimal reaction temperature was 45 degrees C and the enzyme lost its activity above 70 degrees C. Enzyme could oxidize a broad range of various phenolic substrates. K(m) values for ABTS, 2,6-dimethoxyphenol, guaiacol, and ferulic acid were found to be 48.6, 56, 22, and 14 mM while K(cat) values were 204, 180, 95.6, and 5.2, respectively. It was completely inhibited by KCN, NaN(3), beta-mercaptoethanol, HgCl(2), and SDS, while EDTA had no effect on enzyme activity. The N-terminal amino acid sequence of C. bulleri laccase showed close homology to N-terminal sequences of laccase from other white-rot fungi. A 150 bp gene sequence encoding copper-binding domains I and II was most similar to the sequence encoding a laccase from Pycnoporus cinnabarinus with 74.8% level of similarity.

Studies on Acetone Powder and Purified Rhus Laccase Immobilized on Zirconium Chloride for Oxidation of Phenols

PubMed Central

Lu, Rong; Miyakoshi, Tetsuo

2012-01-01

Rhus laccase was isolated and purified from acetone powder obtained from the exudates of Chinese lacquer trees (Rhus vernicifera) from the Jianshi region, Hubei province of China. There are two blue bands appearing on CM-sephadex C-50 chromatography column, and each band corresponding to Rhus laccase 1 and 2, the former being the major constituent, and each had an average molecular weight of approximately 110 kDa. The purified and crude Rhus laccases were immobilized on zirconium chloride in ammonium chloride solution, and the kinetic properties of free and immobilized Rhus laccase, such as activity, molecular weight, optimum pH, and thermostability, were examined. In addition, the behaviors on catalytic oxidation of phenols also were conducted. PMID:22545205

Flocculation and haze removal from crude beer using in-house produced laccase from Trametes versicolor cultured on brewer's spent grain.

PubMed

Dhillon, Gurpreet Singh; Kaur, Surinder; Brar, Satinder Kaur; Verma, Mausam

2012-08-15

The potential of brewer's spent grain (BSG), a common waste from the brewing industry, as a support-substrate for laccase production by the well-known laccase producer Trametes versicolor ATCC 20869 under solid-state fermentation conditions was assessed. An attempt was made to improve the laccase production by T. versicolor through supplementing the cultures with inducers, such as 2,2-azino bis(3-ethylbenzthiazoline-6-sulfonic acid), copper sulfate, ethanol, gallic acid, veratryl alcohol, and phenol. A higher laccase activity of 13506.2 ± 138.2 IU/gds (gram dry substrate) was obtained with a phenol concentration of 10 mg/kg substrate in a tray bioreactor after 12 days of incubation time. The flocculation properties of the laccase treated crude beer samples have been studied by using various parameters, such as viscosity, turbidity, ζ potential, total polyphenols, and total protein content. The present results indicated that laccase (25 IU/L) showed promising results as a good flocculating agent. The laccase treatment showed better flocculation capacity compared to the industrial flocculation process using stabifix as a flocculant. The laccase treatments (25 IU/L) at 4 ± 1 °C and room temperature have shown almost similar flocculation properties without much variability. The study demonstrated the potential of in-house produced laccase using brewer's spent grain for the clarification and flocculation of crude beer as a sustainable alternative to traditional flocculants, such as stabifix and bentonite.

Laccase production by free and immobilized mycelia of Peniophora cinerea and Trametes versicolor: a comparative study.

PubMed

Silvério, Sara C; Moreira, Sérgio; Milagres, Adriane M F; Macedo, Eugénia A; Teixeira, José A; Mussatto, Solange I

2013-03-01

The production of laccase by immobilized mycelia of Peniophora cinerea and Trametes versicolor was studied. In an initial stage, experimental assays were performed in Erlenmeyer flasks using free and immobilized mycelium, and the performance of the fungal strains to produce the enzyme was compared. Both fungi adhered into the support material (a synthetic fiber), growing not only on the surface but also in the interspaces of the fibers. Immobilization of P. cinerea provided a 35-fold increase in laccase production when compared to the production obtained by using free mycelium. On the other hand, immobilization of T. versicolor caused a decrease in laccase activity. A comparison between the strains revealed that immobilized P. cinerea (3,500 U/L) surpassed the enzyme production by free T. versicolor (800 U/L). When the conditions that gave the best laccase production to each fungus were employed in a stirred tank bioreactor, very low laccase production was observed for both the cases, suggesting that shear stress and mycelia damage caused by the agitation impellers negatively affected the enzyme production.

Isolation and Physicochemical Characterization of Laccase from Ganoderma lucidum-CDBT1 Isolated from Its Native Habitat in Nepal.

PubMed

Shrestha, Prabin; Joshi, Bishnu; Joshi, Jarina; Malla, Rajani; Sreerama, Lakshmaiah

2016-01-01

At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely, Ganoderma lucidum -CDBT1 , Ganoderma japonicum, and Lentinula edodes , isolated from their native habitat in Nepal were screened for laccase production, and G. lucidum -CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation from G. lucidum -CDBT1. Presence of lignin (5 g/L) and copper sulfate (30  μ M) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced by G. lucidum -CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants, K m , V max , and K cat , determined using 2,2'-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110  μ M, 36  μ mol/min/mg, and 246 min -1 , respectively. The isolated thermostable laccase will be used in future experiments for delignification process.

Production of Trametes pubescens laccase under submerged and semi-solid culture conditions on agro-industrial wastes.

PubMed

Gonzalez, Juan C; Medina, Sandra C; Rodriguez, Alexander; Osma, Johann F; Alméciga-Díaz, Carlos J; Sánchez, Oscar F

2013-01-01

Laccases are copper-containing enzymes involved in the degradation of lignocellulosic materials and used in the treatment of phenol-containing wastewater. In this study we investigated the effect of culture conditions, i.e. submerged or semi-solid, and copper supplementation on laccase production by Trametespubescens grown on coffee husk, soybean pod husk, or cedar sawdust. The highest specific laccase activity was achieved when the culture was conducted under submerged conditions supplemented with copper (5 mM), and using coffee husk as substrate. The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2). Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3. In addition, they showed similar stability and electro-chemical properties. At optimal culture conditions laccase activity was 7.69 ± 0.28 U mg(-1) of protein for the crude extract, and 0.08 ± 0.001 and 2.86 ± 0.05 U mg(-1) of protein for Lac1 and Lac2, respectively. In summary, these results show the potential of coffee husk as an important and economical growth medium to produce laccase, offering a new alternative use for this common agro-industrial byproduct.

Production of Trametes pubescens Laccase under Submerged and Semi-Solid Culture Conditions on Agro-Industrial Wastes

PubMed Central

Rodriguez, Alexander; Osma, Johann F.; Alméciga-Díaz, Carlos J.; Sánchez, Oscar F.

2013-01-01

Laccases are copper-containing enzymes involved in the degradation of lignocellulosic materials and used in the treatment of phenol-containing wastewater. In this study we investigated the effect of culture conditions, i.e. submerged or semi-solid, and copper supplementation on laccase production by Trametes pubescens grown on coffee husk, soybean pod husk, or cedar sawdust. The highest specific laccase activity was achieved when the culture was conducted under submerged conditions supplemented with copper (5 mM), and using coffee husk as substrate. The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2). Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3. In addition, they showed similar stability and electro-chemical properties. At optimal culture conditions laccase activity was 7.69±0.28 U mg-1 of protein for the crude extract, and 0.08±0.001 and 2.86±0.05 U mg-1 of protein for Lac1 and Lac2, respectively. In summary, these results show the potential of coffee husk as an important and economical growth medium to produce laccase, offering a new alternative use for this common agro-industrial byproduct. PMID:24019936

Radical Scavenging by Acetone: A New Perspective to Understand Laccase/ABTS Inactivation and to Recover Redox Mediator.

PubMed

Liu, Hao; Zhou, Pandeng; Wu, Xing; Sun, Jianliang; Chen, Shicheng

2015-11-04

The biosynthetic utilization of laccase/mediator system is problematic because the use of organic cosolvent causes significant inhibition of laccase activity. This work explored how the organic cosolvent impacts on the laccase catalytic capacity towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in aqueous solution. Effects of acetone on the kinetic constants of laccase were determined and the results showed Km and Vmax varied exponentially with increasing acetone content. Acetone as well as some other cosolvents could transform ABTS radicals into its reductive form. The content of acetone in media significantly affected the radical scavenging rates. Up to 95% of the oxidized ABTS was successfully recovered in 80% (v/v) acetone in 60 min. This allows ABTS recycles at least six times with 70%-75% of active radicals recovered after each cycle. This solvent-based recovery strategy may help improve the economic feasibility of laccase/ABTS system in biosynthesis.

Potentiality of a ceramic membrane reactor for the laccase-catalyzed removal of bisphenol A from secondary effluents.

PubMed

Arca-Ramos, A; Eibes, G; Feijoo, G; Lema, J M; Moreira, M T

2015-11-01

In this study, the removal of bisphenol A (BPA) by laccase in a continuous enzymatic membrane reactor (EMR) was investigated. The effects of key parameters, namely, type of laccase, pH, and enzyme activity, were initially evaluated. Once optimal conditions were determined, the continuous removal of the pollutant in an EMR was assessed in synthetic and real biologically treated wastewaters. The reactor configuration consisted of a stirred tank reactor coupled to a ceramic membrane, which prevented the sorption of the pollutant and allowed the recovery and recycling of laccase. Nearly complete removal of BPA was attained under both operation regimes with removal yields above 94.5 %. In experiments with real wastewater, the removal of BPA remained high while the presence of colloids and certain ions and the formation of precipitates on the membrane potentially affected enzyme stability and made necessary the periodic addition of laccase. Polymerization and degradation were observed as probable mechanisms of BPA transformation by laccase.

LacSubPred: predicting subtypes of Laccases, an important lignin metabolism-related enzyme class, using in silico approaches

PubMed Central

2014-01-01

Background Laccases (E.C. 1.10.3.2) are multi-copper oxidases that have gained importance in many industries such as biofuels, pulp production, textile dye bleaching, bioremediation, and food production. Their usefulness stems from the ability to act on a diverse range of phenolic compounds such as o-/p-quinols, aminophenols, polyphenols, polyamines, aryl diamines, and aromatic thiols. Despite acting on a wide range of compounds as a family, individual Laccases often exhibit distinctive and varied substrate ranges. This is likely due to Laccases involvement in many metabolic roles across diverse taxa. Classification systems for multi-copper oxidases have been developed using multiple sequence alignments, however, these systems seem to largely follow species taxonomy rather than substrate ranges, enzyme properties, or specific function. It has been suggested that the roles and substrates of various Laccases are related to their optimal pH. This is consistent with the observation that fungal Laccases usually prefer acidic conditions, whereas plant and bacterial Laccases prefer basic conditions. Based on these observations, we hypothesize that a descriptor-based unsupervised learning system could generate homology independent classification system for better describing the functional properties of Laccases. Results In this study, we first utilized unsupervised learning approach to develop a novel homology independent Laccase classification system. From the descriptors considered, physicochemical properties showed the best performance. Physicochemical properties divided the Laccases into twelve subtypes. Analysis of the clusters using a t-test revealed that the majority of the physicochemical descriptors had statistically significant differences between the classes. Feature selection identified the most important features as negatively charges residues, the peptide isoelectric point, and acidic or amidic residues. Secondly, to allow for classification of new Laccases

Crystal structures of E. coli laccase CueO at different copper concentrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Xu; Wei Zhiyi; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101

2007-03-02

CueO protein is a hypothetical bacterial laccase and a good laccase candidate for large scale industrial application. Four CueO crystal structures were determined at different copper concentrations. Low copper occupancy in apo-CueO and slow copper reconstitution process in CueO with exogenous copper were demonstrated. These observations well explain the copper dependence of CueO oxidase activity. Structural comparison between CueO and other three fungal laccase proteins indicates that Glu106 in CueO constitutes the primary counter-work for reconstitution of the trinuclear copper site. Mutation of Glu106 to a Phe enhanced CueO oxidation activity and supported this hypothesis. In addition, an extra {alpha}-helixmore » from Leu351 to Gly378 covers substrate biding pocket of CueO and might compromises the electron transfer from substrate to type I copper.« less

Effects of laccase on lignin depolymerization and enzymatic hydrolysis of ensiled corn stover.

PubMed

Chen, Qin; Marshall, Megan N; Geib, Scott M; Tien, Ming; Richard, Tom L

2012-08-01

The aim of this study was to explore the synergies of laccase, a ligninolytic enzyme, with cellulose and hemicellulase amendments on ensiled corn stover. Molecular signals of lignin decomposition were observed by tetramethylammonium hydroxide thermochemolysis and gas chromatography-mass spectroscopy (TMAH-GC-MS) analysis. The significant findings suggest that ensilage might provide a platform for biological pretreatment. By partially hydrolyzing cellulose and hemicellulose into soluble sugars, ensilage facilitates laccase penetration into the lignocellulose complex to enhance lignin degradation. Downstream cellulose hydrolysis was improved 7% with increasing laccase loading rate. These results demonstrate the potential of enzymes, either directly amended or expressed by microbes during ensilage, to maximize utilization of corn stover for cellulosic biofuels and other downstream fermentations. Copyright © 2012. Published by Elsevier Ltd.

Inactivation of a Pleurotus ostreatus versatile peroxidase-encoding gene (mnp2) results in reduced lignin degradation.

PubMed

Salame, Tomer M; Knop, Doriv; Levinson, Dana; Mabjeesh, Sameer J; Yarden, Oded; Hadar, Yitzhak

2014-01-01

Lignin biodegradation by white-rot fungi is pivotal to the earth's carbon cycle. Manganese peroxidases (MnPs), the most common extracellular ligninolytic peroxidases produced by white-rot fungi, are considered key in ligninolysis. Pleurotus ostreatus, the oyster mushroom, is a preferential lignin degrader occupying niches rich in lignocellulose such as decaying trees. Here, we provide direct, genetically based proof for the functional significance of MnP to P. ostreatus ligninolytic capacity under conditions mimicking its natural habitat. When grown on a natural lignocellulosic substrate of cotton stalks under solid-state culture conditions, gene and isoenzyme expression profiles of its short MnP and versatile peroxidase (VP)-encoding gene family revealed that mnp2 was predominately expressed. mnp2, encoding the versatile short MnP isoenzyme 2 was disrupted. Inactivation of mnp2 resulted in three interrelated phenotypes, relative to the wild-type strain: (i) reduction of 14% and 36% in lignin mineralization of stalks non-amended and amended with Mn(2+), respectively; (ii) marked reduction of the bioconverted lignocellulose sensitivity to subsequent bacterial hydrolyses; and (iii) decrease in fungal respiration rate. These results may serve as the basis to clarify the roles of the various types of fungal MnPs and VPs in their contribution to white-rot decay of wood and lignocellulose in various ecosystems. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Selective oxidation of lignin model compounds – a combinatorial application of the laccase-mediator system

USDA-ARS?s Scientific Manuscript database

Identifying suitable reaction conditions remains an important task in the development of practical enzyme catalysts. Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both comm...

Optimization of laccase production by Pleurotus ostreatus IMI 395545 using the Taguchi DOE methodology.

PubMed

Periasamy, Rathinasamy; Palvannan, Thayumanavan

2010-12-01

Production of laccase using a submerged culture of Pleurotus orstreatus IMI 395545 was optimized by the Taguchi orthogonal array (OA) design of experiments (DOE) methodology. This approach facilitates the study of the interactions of a large number of variables spanned by factors and their settings, with a small number of experiments, leading to considerable savings in time and cost for process optimization. This methodology optimizes the number of impact factors and enables to calculate their interaction in the production of industrial enzymes. Eight factors, viz. glucose, yeast extract, malt extract, inoculum, mineral solution, inducer (1 mM CuSO₄) and amino acid (l-asparagine) at three levels and pH at two levels, with an OA layout of L18 (2¹ × 3⁷) were selected for the proposed experimental design. The laccase yield obtained from the 18 sets of fermentation experiments performed with the selected factors and levels was further processed with Qualitek-4 software. The optimized conditions shared an enhanced laccase expression of 86.8% (from 485.0 to 906.3 U). The combination of factors was further validated for laccase production and reactive blue 221 decolorization. The results revealed an enhanced laccase yield of 32.6% and dye decolorization up to 84.6%. This methodology allows the complete evaluation of main and interaction factors. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Graphene oxide vs. reduced graphene oxide as carbon support in porphyrin peroxidase biomimetic nanomaterials.

PubMed

Socaci, C; Pogacean, F; Biris, A R; Coros, M; Rosu, M C; Magerusan, L; Katona, G; Pruneanu, S

2016-02-01

The paper describes the preparation of supramolecular assemblies of tetrapyridylporphyrin (TPyP) and its metallic complexes with graphene oxide (GO) and thermally reduced graphene oxide (TRGO). The two carbon supports are introducing different characteristics in the absorption spectra of the investigated nanocomposites. Raman spectroscopy shows that the absorption of iron-tetrapyridylporphyrin is more efficient on GO than TRGO, suggesting that oxygen functionalities are involved in the non-covalent interaction between the iron-porphyrin and graphene. The biomimetic peroxidase activity is investigated and the two iron-containing composites exhibit a better catalytic activity than each component of the assembly, and their cobalt and manganese homologues, respectively. The main advantages of this work include the demonstration of graphene oxide as a very good support for graphene-based nanomaterials with peroxidase-like activity (K(M)=0.292 mM), the catalytic activity being observed even with very small amounts of porphyrins (the TPyP:graphene ratio=1:50). Its potential application in the detection of lipophilic antioxidants (vitamin E can be measured in the 10(-5)-10(-4) M range) is also shown. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel ethanol-tolerant laccase, Tvlac, from Trametes versicolor.

PubMed

Chen, Lei; Yi, Xiaoming; Deng, Fajun; Fang, Wei; Zhang, Xuecheng; Wang, Xiaotang; Fang, Zemin; Xiao, Yazhong

2016-03-01

To produce and characterize novel laccases with ethanol tolerance from Trametes versicolor using agriculture by-products as energy source. Trametes versicolor 1017 produces two laccase isoenzymes with a total activity of 10 U ml(-1) within 8 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A novel isoenzyme, named Tvlac, was identified, purified and characterized. Its optimum pH and temperature were from 4.5 to 5 and 55 to 60 °C, respectively. Its activity was stimulated by ethanol at 10 % (v/v) which increased the V 0. The biochemical properties of Tvlac substantiate the potential of this enzyme for applications under an aqueous ethanol mixture environment.

Modification of lignocellulosic materials by laccase

Treesearch

William Kenealy; John Klungness; Mandla Tshabalala; Roland Gleisner; Eric Horn; Masood Akhtar; Hilda Zulaica-Villagomez; Gisela Buschle-Diller

2003-01-01

Altering the surface properties of pulp can enhance binding, increase paper strength, and decrease the cost of fiber. In this study, we modified lignocellulosic materials (bark and pulp) with laccase and selected substrates to change the nature of the pulp surface. Modified pulps were evaluated by the amount of methylene blue (a cationic dye) that would bind to the...

Use of sugarcane molasses by Pycnoporus sanguineus for the production of laccase for dye decolorization.

PubMed

Marim, R A; Oliveira, A C C; Marquezoni, R S; Servantes, J P R; Cardoso, B K; Linde, G A; Colauto, N B; Valle, J S

2016-10-17

Pycnoporus sanguineus is a white-rot basidiomycete that produces laccase as the only oxidoreductase; enzyme synthesis depends on cultivation variables, and fungal species and strain. Laccases have wide substrate specificity, oxidize a broad range of compounds, and show potential for use in dye decolorization. We evaluated laccase production in a recently isolated strain of P. sanguineus cultivated with sugarcane molasses as the only carbon source, and urea or yeast extract as the nitrogen source [at various nitrogen concentrations (0.4, 1.4, 2.4, 3.4, and 4.4 g/L)], supplemented with copper (0, 150, 200, 250, and 300 µM), with or without agitation. The enzymatic extract produced at laccase peak activity was tested for dye decolorization capability on Remazol Brilliant Blue R, Reactive Black 5, Reactive Red 195, and Reactive Yellow 145. The nitrogen source did not affect enzyme production and the higher nitrogen concentration (3.4 g/L nitrogen as urea) increased enzymatic activity. The addition of up to 300 µM of Cu did not affect laccase production, whereas cultivation with agitation increased the activity peak by 17%. The highest laccase activity was ~50,000 U/L on the ninth day of cultivation. After 24 h, decolorization was 80% for Remazol Brilliant Blue R, 9% for Reactive Yellow 145, 6% for Reactive Red 195, and 2% for Reactive Black 5. The enzymatic extract of P. sanguineus provides a potential alternative to wastewater treatment. A better understanding of the behavior of this fungus under various culture conditions would allow improvement of the enzyme production bioprocess.

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

D`Souza, T.M.; Boominathan, K.; Reddy, C.A.

1996-10-01

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequences of each of the PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum,more » Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases. 36 refs., 6 figs., 2 tabs.« less

Isolation and Physicochemical Characterization of Laccase from Ganoderma lucidum-CDBT1 Isolated from Its Native Habitat in Nepal

PubMed Central

Joshi, Jarina; Malla, Rajani

2016-01-01

At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely, Ganoderma lucidum-CDBT1, Ganoderma japonicum, and Lentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, and G. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation from G. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30 μM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced by G. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants, K m, V max, and K cat, determined using 2,2′-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110 μM, 36 μmol/min/mg, and 246 min−1, respectively. The isolated thermostable laccase will be used in future experiments for delignification process. PMID:27822471

Ecofriendly syntheses of phenothiazones and related structures facilitated by laccase – A comparative study

DOE PAGES

Cannatelli, Mark D.; Ragauskas, Arthur J.

2016-07-06

The biocatalytic synthesis of phenothiazones and related compounds has been achieved in an aqueous system under mild conditions facilitated by laccase oxidation. It was found that by coupling 2-aminothiophenol directly with 1,4-quinones, the product yields could be significantly increased compared to generating the 1,4-quinones in situ from the corresponding hydroquinones via laccase oxidation. However, laccase still proved to be pivotal for achieving highest product yields by catalyzing the final oxidation step. Furthermore, a difference in reactivity of aromatic and aliphatic amines toward 1,4-naphthoquinone is observed. Furthermore, this study provides a sustainable approach to the synthesis of a biologically important classmore » of compounds.« less

Enhanced phenol degradation in coking wastewater by immobilized laccase on magnetic mesoporous silica nanoparticles in a magnetically stabilized fluidized bed.

PubMed

Wang, Feng; Hu, Yiru; Guo, Chen; Huang, Wei; Liu, Chun-Zhao

2012-04-01

The immobilized laccase on magnetic mesoporous silica nanoparticles has been developed for efficient phenol degradation. The degradation rate of phenol by the immobilized laccase was 2-fold higher than that of the free laccase, and the immobilized laccase retained 71.3% of its initial degradation ability after 10 successive batch treatments of coking wastewater. The phenol degradation in the coking wastewater was enhanced in a continuous treatment process by the immobilized laccase in a magnetically stabilized fluidized bed (MSFB) because of good mixing and mass transfer. The degradation rate of phenol maintained more than 99% at a flow rate of less than 450mLh(-1) and decreased slowly to 91.5% after 40h of the continuous operation in the MSFB. The present work indicated that the immobilized laccase on magnetic mesoporous supports together with the MSFB provided a promising avenue for the continuous enzymatic degradation of phenolic compounds in industrial wastewater. Copyright © 2012 Elsevier Ltd. All rights reserved.

Genome-Wide Identification and Characterization of Novel Laccase Genes in the White-Rot Fungus Flammulina velutipes

PubMed Central

Kim, Hong-Il; Kwon, O-Chul; Kong, Won-Sik; Lee, Chang-Soo

2014-01-01

The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges, fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1 (Asn-454), fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac4 (Asn-402 and Asn-457). In addition, the first 19~20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes. PMID:25606003

Molecular dynamics simulation studies suggests unconventional roles of non-secretary laccases from enteropathogenic gut bacteria and Cryptococcus neoformans serotype D.

PubMed

Sharma, Krishna Kant; Singh, Deepti; Rawat, Surender

2018-04-01

Laccase in Cryptococcus neoformans is covalently linked to the carbohydrate moiety of the cell wall, which allows it to get access to the different substrates for catalyzing their oxidation and therefore plays a vital role in the virulence. The laccase gene (3.0 kb) from C. neoformans serotype D was amplified, cloned and sequenced for protein modeling, docking and simulation studies. The three dimensional homology models of laccase protein from C. neoformans and other pathogenic gut bacteria were docked with selected biomolecules like prostaglandins (PG), membrane phospholipids, neurotransmitters (serotonin) using GOLD software. The GOLDscore values of laccase from C. neoformans docked with prostaglandinH 2 (59.76), prostaglandinG 2 (59.45), prostaglandinE 2 (60.99), phosphatidylinositol (54.95), phosphatidylcholine (46.26), phosphatidylserine (55.26), arachidonic acid (53.08) and serotonin (46.22) were similar to the laccase from enteropathogenic bacteria but showed a better binding affinity as compared to that of the non-pathogenic bacteria (e.g. Bacillus safensis, Bacillus pumilus and Bacillus subtilis). The RMSD of MD simulation study done for 25 ns using laccase protein from C. neoformans complexed with phosphatidylcholine was found to be highly stable, followed by the laccase-PGE 2 and laccase-serotonin complexes. Furthermore, the binding free energy results were found to support the docking and MD simulation results. The present study implies that few candidate ligands can be intermediate substrate in the catalysis of microbial laccases, which can further play some crucial role in the cell signaling and pathogenesis of enteropathogenic gut micro flora and C. neoformans. Copyright © 2018 Elsevier Ltd. All rights reserved.

Statistical Optimization of Laccase Production and Delignification of Sugarcane Bagasse by Pleurotus ostreatus in Solid-State Fermentation

PubMed Central

Karp, Susan Grace; Faraco, Vincenza; Amore, Antonella; Letti, Luiz Alberto Junior; Thomaz Soccol, Vanete; Soccol, Carlos Ricardo

2015-01-01

Laccases are oxidative enzymes related to the degradation of phenolic compounds, including lignin units, with concomitant reduction of oxygen to water. Delignification is a necessary pretreatment step in the process of converting plant biomass into fermentable sugars. The objective of this work was to optimize the production of laccases and to evaluate the delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation. Among eight variables (pH, water activity, temperature, and concentrations of CuSO4, (NH4)2SO4, KH2PO4, asparagine, and yeast extract), copper sulfate and ammonium sulfate concentrations were demonstrated to significantly influence laccase production. The replacement of ammonium sulfate by yeast extract and the addition of ferulic acid as inducer provided increases of 5.7- and 2.0-fold, respectively, in laccase activity. Optimization of laccase production as a function of yeast extract, copper sulfate, and ferulic acid concentrations was performed by response surface methodology and optimal concentrations were 6.4 g/L, 172.6 μM, and 1.86 mM, respectively. Experimentally, the maximum laccase activity of 151.6 U/g was produced at the 5th day of solid-state fermentation. Lignin content in sugarcane bagasse was reduced from 31.89% to 26.36% after 5 days and to 20.79% after 15 days by the biological treatment of solid-state fermentation. PMID:26180784

[Induce of laccase from Trametes gallica and its degradation on neutral dyes and organophosphorus pesticides].

PubMed

Jing, De-Jun; Huang, Jian-Bo; Yang, Zhou-Ping; Hu, Rong; Cheng, Zi-Zhang; Huang, Qian-Ming

2011-12-01

The characteristics of the induction of laccase in Trametes gallica under different initial cultural pH, incubation time by different inducers were discussed, as well as the effects of temperature, pH and time on laccase degradation of six dyes and four organophosphors. The results showed that RB-bright blue, ABTS and o-toluidine affected the production of laccase at different levels, and ABTS was the best inductive agent in our test conditions, whose optimal initial pH and incubation time were 4.0 and 13 days, respectively. The appropriate reaction temperature of the laccase produced was 38 degrees C, and it got a good stability, for it could retain 78.6% of the enzyme activity after 20 min holding at 40 degrees C. Mediated by ABTS, the optimal temperature for laccase to degrade the six types of neutral dyes could be divided into two cases, that was 30 degrees C (neutral black, neutral bordeaux, neutral pink, methyl orange) and 60 degrees C (neutral dark yellow, cresol red), the optimal pH were 6.0 (neutral black), 2.0 (neutral bordeaux, neutral pink) and 4.0 (methyl orange, neutral dark yellow, cresol red), respectively, while the optimal times separately were 6 h (methyl orange, neutral dark yellow, cresol red), 12 h (neutral pink) and 24 h (neutral bordeaux). And using the same inductive agent, the best temperature for laccase to degrade dimethoate, chlorpyrifos, trichlorfon and parathion-pyridazine was 25 degrees C, the suitable time was 9 h, and the optimal pH was 10.0 for dimethoate, chlorpyrifos and parathion-pyridazine, and 8.0 for trichlorfon.

Enzymatic browning and biochemical alterations in black spots of pineapple [Ananas comosus (L.) Merr.].

PubMed

Avallone, Sylvie; Guiraud, Joseph-Pierre; Brillouet, Jean-Marc; Teisson, Claude

2003-08-01

Penicillium funiculosum Thom. was consistently isolated from pineapple-infected fruitlet (black spots). Polyphenol oxidase, peroxidase, and laccase activities were determined in extracts from contiguous and infected fruitlets. Healthy fruitlets showed a rather high level of polyphenol oxidase (optimum pH 7.0), and this activity was tremendously increased (X 10) in contiguous infected fruitlets. Furthermore, infected fruitlets also exhibited laccase activity (optimum pH 4.0), while peroxidase was rather constant in both fruitlets. Browning reactions were attributed to qualitative and quantitative modifications of the enzymatic equipment (polyphenol oxidase and laccase) (p < 0.0001). In infected fruiltets, sucrose and L-malic acid were present at significantly lower amounts than in healthy ones, likely owing to fungal metabolism (p < 0.0001), whereas cell wall material was three times higher, which could be viewed as a defense mechanism to limit expansion of the mycelium.

Hydrophobic modification of jute fiber used for composite reinforcement via laccase-mediated grafting

NASA Astrophysics Data System (ADS)

Dong, Aixue; Yu, Yuanyuan; Yuan, Jiugang; Wang, Qiang; Fan, Xuerong

2014-05-01

Jute fiber is a lignocellulosic material which could be utilized for reinforcement of composites. To improve the compatibility of hydrophilic jute fiber with hydrophobic resin, surface hydrophobization of the fiber is often needed. In this study, the feasibility of laccase-mediated grafting dodecyl gallate (DG) on the jute fiber was investigated. First, the grafting products were characterized by FT-IR, XPS, SEM and AFM. And then the grafting percentage (Gp) and the DG content of the modified jute were determined in terms of weighting and saponification, respectively. The parameters of the enzymatic grafting process were optimized to the target application. Lastly, the hydrophobicity of the jute fabrics was estimated by means of contact angle and wetting time. The mechanical properties and the fracture section of the jute fabric/polypropylene (PP) composites were studied. The results revealed covalently coupling of DG to the jute substrates mediated by laccase. The enzymatic process reached the maximum grafting rate of 4.16% when the jute fabric was incubated in the 80/20 (v/v, %) pH 3 0.2 M acetate buffer/ethanol medium with 1.0 U/mL laccase and 5 mM DG at 50 °C for 4 h. The jute fabric modified with laccase and DG showed increased contact angle of 111.49° and wetting time of at least 30 min, indicating that the surface hydrophobicity of the jute fabric was increased after the enzymatic graft modification with hydrophobic DG. The breaking strength of the modified jute fiber/PP composite was also increased and the fracture section became neat and regular due to the laccase-assisted grafting with DG.

Immobilized ligninolytic enzymes: An innovative and environmental responsive technology to tackle dye-based industrial pollutants - A review.

PubMed

Bilal, Muhammad; Asgher, Muhammad; Parra-Saldivar, Roberto; Hu, Hongbo; Wang, Wei; Zhang, Xuehong; Iqbal, Hafiz M N

2017-01-15

In the twenty-first century, chemical and associated industries quest a transition prototype from traditional chemical-based concepts to a greener, sustainable and environmentally-friendlier catalytic alternative, both at the laboratory and industrial scale. In this context, bio-based catalysis offers numerous benefits along with potential biotechnological and environmental applications. The bio-based catalytic processes are energy efficient than conventional methodologies under moderate processing, generating no and negligible secondary waste pollution. Thanks to key scientific advances, now, solid-phase biocatalysts can be economically tailored on a large scale. Nevertheless, it is mandatory to recover and reprocess the enzyme for their commercial feasibility, and immobilization engineering can efficiently accomplish this challenge. The first part of the present review work briefly outlines the immobilization of lignin-modifying enzymes (LMEs) including lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase of white-rot fungi (WRF). Whereas, in the second part, a particular emphasis has been given on the recent achievements of carrier-immobilized LMEs for the degradation, decolorization, or detoxification of industrial dyes and dye-based industrial wastewater effluents. Copyright © 2016 Elsevier B.V. All rights reserved.

A Ca-alginate particle co-immobilized with Phanerochaete chrysosporium cells and the combined cross-linked enzyme aggregates from Trametes versicolor.

PubMed

Li, Yanchun; Wang, Zhi; Xu, Xudong; Jin, Liqiang

2015-12-01

For improving stability of immobilized white-rot fungus to treat various effluents, Phanerochaete chrysosporium cells and the combined cross-link enzyme aggregates (combi-CLEAs) prepared from Trametes versicolor were co-immobilized into the Ca-alginate gel particles in this paper. The activity yields of obtained combi-CLEAs were 42.7% for lignin peroxidases (LiPs), 31.4% for manganese peroxidases (MnPs) and 40.4% for laccase (Lac), respectively. And their specific activities were 30.2U/g as combi-CLEAs-LiPs, 9.5 U/g as combi-CLEAs-MnPs and 28.4 U/g as combi-CLEAs-Lac. Further, the present of the combi-CLEAs in the particles extremely improved their ability to degrade the dyes. Compared to the immobilized Ph. chrysosporium without the combi-CLEAs, the co-immobilized particles enhanced the decolorized rate of Acid Violet 7 (from 45.2% to 93.4%) and Basic Fuchsin (from 12.1% to 67.9%). In addition, the addition of the combi-CLEAs improved the adaptability of the white-rot fungal particles to adverse environmental conditions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fabrication, characterization and application of laccase-nylon 6,6/Fe3+ composite nanofibrous membrane for 3,3'-dimethoxybenzidine detoxification.

PubMed

Jasni, M Jasmin Fathi; Sathishkumar, Palanivel; Sornambikai, Sundaram; Yusoff, Abdull Rahim Mohd; Ameen, Fuad; Buang, Nor Aziah; Kadir, Mohammed Rafiq Abdul; Yusop, Zulkifli

2017-02-01

In this study, laccase was immobilized on nylon 6,6/Fe 3+ composite (NFC) nanofibrous membrane and used for the detoxification of 3,3'-dimethoxybenzidine (DMOB). The average size and tensile strength of the NFC membrane were found to be 60-80 nm (diameter) and 2.70 MPa, respectively. The FTIR results confirm that the amine (N-H) group of laccase was attached with Fe 3+ particles and the carbonyl (C=O) group of NFC membrane via hydrogen bonding. The half-life of the laccase-NFC membrane storage stability was increased from 6 to 11 weeks and the reusability was significantly extended up to 43 cycles against ABTS oxidation. Enhanced electro-oxidation of DMOB by laccase was observed at 0.33 V and the catalytic current was found to be 30 µA. The DMOB-treated mouse fibroblast 3T3-L1 preadipocytes showed maximum (97 %) cell inhibition at 75 µM L -1 within 24 h. The cytotoxicity of DMOB was significantly decreased to 78 % after laccase treatment. This study suggests that laccase-NFC membrane might be a good candidate for emerging pollutant detoxification.

Optimization of laccase production from Marasmiellus palmivorus LA1 by Taguchi method of Design of experiments.

PubMed

Chenthamarakshan, Aiswarya; Parambayil, Nayana; Miziriya, Nafeesathul; Soumya, P S; Lakshmi, M S Kiran; Ramgopal, Anala; Dileep, Anuja; Nambisan, Padma

2017-02-13

Fungal laccase has profound applications in different fields of biotechnology due to its broad specificity and high redox potential. Any successful application of the enzyme requires large scale production. As laccase production is highly dependent on medium components and cultural conditions, optimization of the same is essential for efficient product production. Production of laccase by fungal strain Marasmiellus palmivorus LA1 under solid state fermentation was optimized by the Taguchi design of experiments (DOE) methodology. An orthogonal array (L8) was designed using Qualitek-4 software to study the interactions and relative influence of the seven selected factors by one factor at a time approach. The optimum condition formulated was temperature (28 °C), pH (5), galactose (0.8%w/v), cupric sulphate (3 mM), inoculum concentration (number of mycelial agar pieces) (6Nos.) and substrate length (0.05 m). Overall yield increase of 17.6 fold was obtained after optimization. Statistical optimization leads to the elimination of an insignificant medium component ammonium dihydrogen phosphate from the process and contributes to a 1.06 fold increase in enzyme production. A final production of 667.4 ± 13 IU/mL laccase activity paves way for the application of this strain for industrial applications. Study optimized lignin degrading laccases from Marasmiellus palmivorus LA1. This laccases can thus be used for further applications in different scales of production after analyzing the properties of the enzyme. Study also confirmed the use of taguchi method for optimizations of product production.

Recovery of manganese from manganese oxide ores in the EDTA solution

NASA Astrophysics Data System (ADS)

Zhang, Chao; Wang, Shuai; Cao, Zhan-fang; Zhong, Hong

2018-04-01

A new process has been experimentally and theoretically established for the recovery of manganese from manganese oxide ores, mainly including the reductive leaching of manganese by ethylenediaminetetraacetic acid (EDTA), EDTA recovery, and manganese electrolysis. The experimental conditions for this process were investigated. Moderate leaching environment by EDTA with the pH in the range of 5-6 is of benefit to leach manganese from some manganese oxide ores with high-content impurities, such as iron and aluminum. Most of EDTA can be recovered by acidification. A small amount of the residual EDTA in the electrolyte can prevent the generation of anode mud. In addition, trimanganese tetroxide (Mn3O4) can be obtained by the roasting of the EDTA-Mn crystallized product.

The study of ascorbate peroxidase, catalase and peroxidase during in vitro regeneration of Argyrolobium roseum.

PubMed

Habib, Darima; Chaudhary, Muhammad Fayyaz; Zia, Muhammad

2014-01-01

Here, we demonstrate the micropropagation protocol of Argyrolobium roseum (Camb.), an endangered herb exhibiting anti-diabetic and immune-suppressant properties, and antioxidant enzymes pattern is evaluated. Maximum callogenic response (60 %) was observed from leaf explant at 1.0 mg L(-1) 1-nephthalene acetic acid (NAA) and 0.5 mg L(-1) 6-benzyl aminopurine (BA) in Murashige and Skoog (MS) medium using hypocotyl and root explants (48 % each). Addition of AgNO3 and PVP in the culture medium led to an increase in callogenic response up to 86 % from leaf explant and 72 % from hypocotyl and root explants. The best shooting response was observed in the presence of NAA, while maximum shoot length and number of shoots were achieved based on BA-supplemented MS medium. The regenerated shoots were rooted and successfully acclimatized under greenhouse conditions. Catalase and peroxidase enzymes showed ascending pattern during in vitro plant development from seed while ascorbate peroxidase showed descending pattern. Totally reverse response of these enzymes was observed during callus induction from three different explants. During shoot induction, catalase and peroxidase increased at high rate while there was a mild reduction in ascorbate peroxidase activity. Catalase and peroxidase continuously increased; on the other hand, ascorbate peroxidase activity decreased during root development and acclimatization states. The protocol described here can be employed for the mass propagation and genetic transformation of this rare herb. This study also highlights the importance and role of ascorbate peroxidase, catalase, and peroxidase in the establishment of A. roseum in vitro culture through callogenesis and organogenesis.

Electrokinetic remediation of manganese and ammonia nitrogen from electrolytic manganese residue.

PubMed

Shu, Jiancheng; Liu, Renlong; Liu, Zuohua; Du, Jun; Tao, Changyuan

2015-10-01

Electrolytic manganese residue (EMR) is a solid waste found in filters after sulphuric acid leaching of manganese carbonate ore, which mainly contains manganese and ammonia nitrogen and seriously damages the ecological environment. This work demonstrated the use of electrokinetic (EK) remediation to remove ammonia nitrogen and manganese from EMR. The transport behavior of manganese and ammonia nitrogen from EMR during electrokinetics, Mn fractionation before and after EK treatment, the relationship between Mn fractionation and transport behavior, as well as the effects of electrolyte and pretreatment solutions on removal efficiency and energy consumption were investigated. The results indicated that the use of H2SO4 and Na2SO4 as electrolytes and pretreatment of EMR with citric acid and KCl can reduce energy consumption, and the removal efficiencies of manganese and ammonia nitrogen were 27.5 and 94.1 %, respectively. In these systems, electromigration and electroosmosis were the main mechanisms of manganese and ammonia nitrogen transport. Moreover, ammonia nitrogen in EMR reached the regulated level, and the concentration of manganese in EMR could be reduced from 455 to 37 mg/L. In general, the electrokinetic remediation of EMR is a promising technology in the future.

Comparison of the efficiency of bacterial and fungal laccases in delignification and detoxification of steam-pretreated lignocellulosic biomass for bioethanol production.

PubMed

De La Torre, María; Martín-Sampedro, Raquel; Fillat, Úrsula; Eugenio, María E; Blánquez, Alba; Hernández, Manuel; Arias, María E; Ibarra, David

2017-11-01

This study evaluates the potential of a bacterial laccase from Streptomyces ipomoeae (SilA) for delignification and detoxification of steam-exploded wheat straw, in comparison with a commercial fungal laccase from Trametes villosa. When alkali extraction followed by SilA laccase treatment was applied to the water insoluble solids fraction, a slight reduction in lignin content was detected, and after a saccharification step, an increase in both glucose and xylose production (16 and 6%, respectively) was observed. These effects were not produced with T. villosa laccase. Concerning to the fermentation process, the treatment of the steam-exploded whole slurry with both laccases produced a decrease in the phenol content by up to 35 and 71% with bacterial and fungal laccases, respectively. The phenols reduction resulted in an improved performance of Saccharomyces cerevisiae during a simultaneous saccharification and fermentation (SSF) process, improving ethanol production rate. This enhancement was more marked with a presaccharification step prior to the SSF process.

Laccase catalyzed elimination of morphine from aqueous systems.

PubMed

Huber, Daniela; Bleymaier, Klaus; Pellis, Alessandro; Vielnascher, Robert; Daxbacher, Andreas; Greimel, Katrin J; Guebitz, Georg M

2018-05-25

Pharmaceuticals contaminate the environment for several reasons, including metabolic excretion after intake, industrial waste and improper disposal. The narcotic drug morphine is commonly utilized for chronic pain management, and the distribution of morphine in aqueous systems and in waste waters is of high concern. Here, the removal of morphine by a laccase from Myceliophthora thermophila both in its free form as well as immobilized on Accurel MP1000 beads was investigated. Complete morphine elimination was achieved within 30 min for the free and the immobilized enzyme (70% bound protein) for concentrations between 1 and 1,000 mg L -1 according to LC-TOF mass spectrometry analysis. Higher morphine concentrations up to 60 g L -1 were also tested and total elimination was achieved within 6 h. Therefore, laccases are ideal candidates for removing morphine from aqueous systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Production of Laccase by Recombinant Yarrowia lipolytica from Molasses: Bioprocess Development Using Statistical Modeling and Increase Productivity in Shake-Flask and Bioreactor Cultures.

PubMed

Darvishi, Farshad; Moradi, Marzieh; Madzak, Catherine; Jolivalt, Claude

2017-03-01

Laccases are used in numerous applications, from green degradation of various xenobiotic compounds, waste detoxification, textile dye bleaching, and delignification of lignocellulose materials to biofuel production. In this study, the recombinant Yarrowia lipolytica YL4 strain carrying the white-rot fungus Trametes versicolor laccase IIIb gene was used for laccase production from beet molasses as an agro-industrial residue. Response surface methodology was used to statistical optimization of the production of laccase by Y. lipolytica using an industrial medium containing molasses which allows a six times increase in laccase activity compared to primary medium contains glucose after 144 h. In bioreactor cultivation after 48 h, laccase production reached to 3.7- and 22.5-fold more than optimized and primary media in shake-flask cultures, respectively. Laccase productivity in bioreactor (0.0937 U/h) was higher than shake-flask culture (0.0084 U/h). The present study provides valuable information about statistical optimization of bioprocess development for cost-effective production of laccase and other heterologous proteins in Y. lipolytica from beet molasses as sole carbon source, thus allowing the valorization and decreasing environmental pollution of this agro-industrial waste.

Manganese deficiency in Chlamydomonas results in loss of photosystem II and MnSOD function, sensitivity to peroxides, and secondary phosphorus and iron deficiency.

PubMed

Allen, Michael D; Kropat, Janette; Tottey, Stephen; Del Campo, José A; Merchant, Sabeeha S

2007-01-01

For photoheterotrophic growth, a Chlamydomonas reinhardtii cell requires at least 1.7 x 10(7) manganese ions in the medium. At lower manganese ion concentrations (typically <0.5 microm), cells divide more slowly, accumulate less chlorophyll, and the culture reaches stationary phase at lower cell density. Below 0.1 microm supplemental manganese ion in the medium, the cells are photosynthetically defective. This is accompanied by decreased abundance of D1, which binds the Mn(4)Ca cluster, and release of the OEE proteins from the membrane. Assay of Mn superoxide dismutase (MnSOD) indicates loss of activity of two isozymes in proportion to the Mn deficiency. The expression of MSD3 through MSD5, encoding various isoforms of the MnSODs, is up-regulated severalfold in Mn-deficient cells, but neither expression nor activity of the plastid Fe-containing superoxide dismutase is changed, which contrasts with the dramatically increased MSD3 expression and plastid MnSOD activity in Fe-deficient cells. Mn-deficient cells are selectively sensitive to peroxide but not methyl viologen or Rose Bengal, and GPXs, APX, and MSRA2 genes (encoding glutathione peroxidase, ascorbate peroxidase, and methionine sulfoxide reductase 2) are slightly up-regulated. Elemental analysis indicates that the Mn, Fe, and P contents of cells in the Mn-deficient cultures were reduced in proportion to the deficiency. A natural resistance-associated macrophage protein homolog and one of five metal tolerance proteins were induced in Mn-deficient cells but not in Fe-deficient cells, suggesting that the corresponding gene products may be components of a Mn(2+)-selective assimilation pathway.

Efficient, environmentally-friendly and specific valorization of lignin: promising role of non-radical lignolytic enzymes.

PubMed

Wang, Wenya; Zhang, Chao; Sun, Xinxiao; Su, Sisi; Li, Qiang; Linhardt, Robert J

2017-06-01

Lignin is the second most abundant bio-resource in nature. It is increasingly important to convert lignin into high value-added chemicals to accelerate the development of the lignocellulose biorefinery. Over the past several decades, physical and chemical methods have been widely explored to degrade lignin and convert it into valuable chemicals. Unfortunately, these developments have lagged because of several difficulties, of which high energy consumption and non-specific cleavage of chemical bonds in lignin remain the greatest challenges. A large number of enzymes have been discovered for lignin degradation and these are classified as radical lignolytic enzymes and non-radical lignolytic enzymes. Radical lignolytic enzymes, including laccases, lignin peroxidases, manganese peroxidases and versatile peroxidases, are radical-based bio-catalysts, which degrade lignins through non-specific cleavage of chemical bonds but can also catalyze the radical-based re-polymerization of lignin fragments. In contrast, non-radical lignolytic enzymes selectively cleave chemical bonds in lignin and lignin model compounds and, thus, show promise for use in the preparation of high value-added chemicals. In this mini-review, recent developments on non-radical lignolytic enzymes are discussed. These include recently discovered non-radical lignolytic enzymes, their metabolic pathways for lignin conversion, their recent application in the lignin biorefinery, and the combination of bio-catalysts with physical/chemical methods for industrial development of the lignin refinery.

Laccase induction by synthetic dyes in Pycnoporus sanguineus and their possible use for sugar cane bagasse delignification.

PubMed

Hernández, Christian; Farnet Da Silva, Anne-Marie; Ziarelli, Fabio; Perraud-Gaime, Isabelle; Gutiérrez-Rivera, Beatriz; García-Pérez, José Antonio; Alarcón, Enrique

2017-02-01

The use of synthetic dyes for laccase induction in vivo has been scarcely explored. We characterized the effect of adding different synthetic dyes to liquid cultures of Pycnoporus sanguineus on laccase production. We found that carminic acid (CA) can induce 722 % and alizarin yellow 317 % more laccase than control does, and they promoted better fungal biomass development in liquid cultures. Aniline blue and crystal violet did not show such positive effect. CA and alizarin yellow were degraded up to 95 % during P. sanguineus culturing (12 days). With this basis, CA was selected as the best inducer and used to evaluate the induction of laccase on solid-state fermentation (SSF), using sugarcane bagasse (SCB) as substrate, in an attempt to reach selective delignification. We found that laccase induction occurred in SSF, and a slight inhibition of cellulase production was observed when CA was added to the substrate; also, a transformation of SCB under SSF was followed by the 13 C cross polarization magic angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). Results showed that P. sanguineus can selectively delignify SCB, decreasing aromatic C compounds by 32.67 % in 16 days; O-alkyl C region (polysaccharides) was degraded less than 2 %; delignification values were not correlated with laccase activities. Cellulose-crystallinity index was increased by 27.24 % in absence of CA and 15.94 % when 0.01 mM of CA was added to SCB; this dye also inhibits the production of fungal biomass in SSF (measured as alkyl C gain). We conclude that CA is a good inducer of laccase in liquid media, and that P. sanguineus is a fungus with high potential for biomass delignification.

A Laccase with Antiproliferative and HIV-I Reverse Transcriptase Inhibitory Activities from the Mycorrhizal Fungus Agaricus placomyces

PubMed Central

Sun, Jian; Chen, Qing-Jun; Cao, Qing-Qin; Wu, Ying-Ying; Xu, Li-Jing; Zhu, Meng-Juan; Ng, Tzi-Bun; Wang, He-Xiang; Zhang, Guo-Qing

2012-01-01

A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7′-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, Km values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC50 of 1.8 μM, 1.7 μM, and 1.25 μM, respectively, signifying that it is an antipathogenic protein. PMID:23093860

Performance of an alkalophilic and halotolerant laccase from gamma-proteobacterium JB in the presence of industrial pollutants.

PubMed

Singh, Gursharan; Sharma, Prince; Capalash, Neena

2009-08-01

An alkalophilic and halotolerant laccase from gamma-proteobacterium JB catalyzed in high concentrations of organic solvents and various salts. The enzyme retained 80-100% activity in 10% concentration of dimethylsulfoxide (DMSO), ethanol, acetone or methanol; 100, 85 and 50% activity in 20 mM MgCl(2), 5.0 mM MnCl(2) and 0.1 mM CuCl(2); 140, 120 and 110% activity in 5.0 mM MnSO(4), 10 mM MgSO(4) and 1mM CaSO(4), respectively. Sodium halides inhibited the enzyme in the order: F(-)> Br(-)> I(-)> Cl(-). In 0.5 M NaCl, pH 6.0, laccase was approximately 60% active. Decolorization of indigo carmine by laccase at pH 9.0 was not inhibited even in the presence of 0.5 M NaCl. Release of chromophoric, reducing and hydrophobic compounds during biobleaching of straw rich-soda pulp by laccase was not inhibited when the enzyme was applied in the presence of 1 M NaCl at pH 8.0. Laccase retained 50% residual activity even when incubated with 5% calcium hypochlorite for 30 min.

Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts

PubMed Central

De Fine Licht, Henrik H.; Schiøtt, Morten; Rogowska-Wrzesinska, Adelina; Nygaard, Sanne; Roepstorff, Peter; Boomsma, Jacobus J.

2013-01-01

Leaf-cutting ants combine large-scale herbivory with fungus farming to sustain advanced societies. Their stratified colonies are major evolutionary achievements and serious agricultural pests, but the crucial adaptations that allowed this mutualism to become the prime herbivorous component of neotropical ecosystems has remained elusive. Here we show how coevolutionary adaptation of a specific enzyme in the fungal symbiont has helped leaf-cutting ants overcome plant defensive phenolic compounds. We identify nine putative laccase-coding genes in the fungal genome of Leucocoprinus gongylophorus cultivated by the leaf-cutting ant Acromyrmex echinatior. One of these laccases (LgLcc1) is highly expressed in the specialized hyphal tips (gongylidia) that the ants preferentially eat, and we confirm that these ingested laccase molecules pass through the ant guts and remain active when defecated on the leaf pulp that the ants add to their gardens. This accurate deposition ensures that laccase activity is highest where new leaf material enters the fungus garden, but where fungal mycelium is too sparse to produce extracellular enzymes in sufficient quantities to detoxify phenolic compounds. Phylogenetic analysis of LgLcc1 ortholog sequences from symbiotic and free-living fungi revealed significant positive selection in the ancestral lineage that gave rise to the gongylidia-producing symbionts of leaf-cutting ants and their non–leaf-cutting ant sister group. Our results are consistent with fungal preadaptation and subsequent modification of a particular laccase enzyme for the detoxification of secondary plant compounds during the transition to active herbivory in the ancestor of leaf-cutting ants between 8 and 12 Mya. PMID:23267060

Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts.

PubMed

De Fine Licht, Henrik H; Schiøtt, Morten; Rogowska-Wrzesinska, Adelina; Nygaard, Sanne; Roepstorff, Peter; Boomsma, Jacobus J

2013-01-08

Leaf-cutting ants combine large-scale herbivory with fungus farming to sustain advanced societies. Their stratified colonies are major evolutionary achievements and serious agricultural pests, but the crucial adaptations that allowed this mutualism to become the prime herbivorous component of neotropical ecosystems has remained elusive. Here we show how coevolutionary adaptation of a specific enzyme in the fungal symbiont has helped leaf-cutting ants overcome plant defensive phenolic compounds. We identify nine putative laccase-coding genes in the fungal genome of Leucocoprinus gongylophorus cultivated by the leaf-cutting ant Acromyrmex echinatior. One of these laccases (LgLcc1) is highly expressed in the specialized hyphal tips (gongylidia) that the ants preferentially eat, and we confirm that these ingested laccase molecules pass through the ant guts and remain active when defecated on the leaf pulp that the ants add to their gardens. This accurate deposition ensures that laccase activity is highest where new leaf material enters the fungus garden, but where fungal mycelium is too sparse to produce extracellular enzymes in sufficient quantities to detoxify phenolic compounds. Phylogenetic analysis of LgLcc1 ortholog sequences from symbiotic and free-living fungi revealed significant positive selection in the ancestral lineage that gave rise to the gongylidia-producing symbionts of leaf-cutting ants and their non-leaf-cutting ant sister group. Our results are consistent with fungal preadaptation and subsequent modification of a particular laccase enzyme for the detoxification of secondary plant compounds during the transition to active herbivory in the ancestor of leaf-cutting ants between 8 and 12 Mya.

Enhanced the enzymatic hydrolysis efficiency of wheat straw after combined steam explosion and laccase pretreatment.

PubMed

Qiu, Weihua; Chen, Hongzhang

2012-08-01

Laccase, capable of selectively degrading lignin while keeping cellulose intact, has been widely applied for the modification and bio-bleaching of pulp. In this study Sclerotium sp. laccase (MSLac) was employed in combination with steam explosion to evaluate the effect of this treatment on cellulose hydrolysis. Combined steam explosion with laccase pretreatment enhanced the cellulose conversion rate of wheat straw no matter in the case of successive (MSLac-Cel) and simultaneous (MSLac+Cel) MSLac and cellulase hydrolysis. The highest cellulose conversion rate of 84.23% was obtained when steam-exploded wheat straw (SEWS) (1.3 MPa, 5 min) was treated by MSLac+Cel at a laccase loading of 0.55 U g(-1) substrate. FT-IR and SEM analyses indicated that MSLac oxidized the phenol and changed electron configuration of the ring, which contributed to loosening the compact wrap of lignin-carbohydrate complex and consequently enhancing the enzymatic hydrolysis efficiency of cellulose. This article provided a promising method for lignocellulose bio-pretreatment. Copyright © 2012 Elsevier Ltd. All rights reserved.

Laccase-catalyzed α-arylation of benzoylacetonitrile with substituted hydroquinones

DOE PAGES

Cannatelli, Mark D.; Ragauskas, Arthur J.

2014-09-03

This article investigates a green method for α-arylation of a primary nitrile. Compounds possessing a benzylic nitrile have shown to exhibit biological activity. Laccases (benzenediol:oxygen oxidoreductase EC 1.10.3.2) were used as the catalysts to perform the cross-coupling reaction between benzoylacetonitrile and substituted hydroquinones. The corresponding 2-substituted 3-oxo-3-phenylpropanenitriles where synthesized in moderate to excellent yields in pH 7.0 buffered water under mild conditions. The substituent on the hydroquinone had a significant impact on product yields and regioselectivity of reaction. The use of laccases, which require O 2 as their only co-substrate and produce H 2O as their only by-product, to performmore » this transformation is an environmentally benign method for α-arylation of primary nitriles.« less

Melanoidin-containing wastewaters induce selective laccase gene expression in the white-rot fungus Trametes sp. I-62.

PubMed

González, Tania; Terrón, María Carmen; Yagüe, Susana; Junca, Howard; Carbajo, José María; Zapico, Ernesto Javier; Silva, Ricardo; Arana-Cuenca, Ainhoa; Téllez, Alejandro; González, Aldo Enrique

2008-03-01

Wastewaters generated from the production of ethanol from sugar cane molasses may have detrimental effects on the environment due to their high chemical oxygen demand and dark brown color. The color is mainly associated with the presence of melanoidins, which are highly recalcitrant to biodegradation. We report here the induction of laccases by molasses wastewaters and molasses melanoidins in the basidiomycetous fungus Trametes sp. I-62. The time course of effluent decolorization and laccase activity in the culture supernatant of the fungus were correlated. The expression of laccase genes lcc1 and lcc2 increased as a result of the addition of complete molasses wastewater and its high molecular weight fraction to fungal cultures. This is the first time differential laccase gene expression has been reported to occur upon exposure of fungal cultures to molasses wastewaters and their melanoidins.

Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bryan, Anthony C.; Jawdy, Sara; Gunter, Lee

Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand-full of laccases in plants have been functionally evaluated and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G06400, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl (S/G) ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent onmore » a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. Finally, we propose a model in which this particular laccase has a range of functions related to oxidation of phenolics that interact with lignin in the cell wall.« less

Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release

DOE PAGES

Bryan, Anthony C.; Jawdy, Sara; Gunter, Lee; ...

2016-04-15

Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand-full of laccases in plants have been functionally evaluated and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G06400, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl (S/G) ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent onmore » a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. Finally, we propose a model in which this particular laccase has a range of functions related to oxidation of phenolics that interact with lignin in the cell wall.« less

Manganese Health Research Program (MHRP)

DTIC Science & Technology

2008-01-01

NO3)2 Manganese sulphate or Manganese (II) sulphate – MnSO4 Manganese sulphide or Manganese (II) sulphide – MnS Manganese oxide – MnO Barium... sulphide or Manganese (II) sulphide – MnS 1344-43-0 Manganese oxide – MnO 7787-35-1 Barium manganate - BaMnO4 10294-64-1 Potassium manganate – K2MnO4...Characterization of welding fumes and their potential neurotoxic effects. International Workshop: Neurotoxic Metals- Lead, Mercury , and Manganese

Polypeptides having laccase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Tang, Lan; Duan, Junxin

The present invention relates to isolated polypeptides having laccase activity and polynucleotides encoding the polypeptides and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

On the diversity of the laccase gene: a phylogenetic perspective from Botryosphaeria rhodina (Ascomycota: Fungi) and other related taxa.

PubMed

Castilho, Flávio J D; Torres, Rodrigo A; Barbosa, Aneli M; Dekker, Robert F H; Garcia, José E

2009-02-01

The present study is the first describing the sequencing of a fragment of the copper-oxidase domain of a laccase gene in the family Botryosphaeriaceae. The aim of this work was to assess the degree of genetic and evolutionary relationships of a laccase gene from Botryosphaeria rhodina MAMB-05 with other ascomycete and basidiomycete laccase genes. The 193-amino acid sequences of the copper-oxidase domain from several different fungi, insects, a plant, and a bacterial species were retrieved from GenBank and aligned. Phylogenetic analyses were performed using neighbor-joining, maximum parsimony, and Bayesian inference methods. The organisms studied clustered into five gene clades: fungi (ascomycetes and basidiomycetes), insects, plants, and bacteria. Also, the topologies showed that fungal laccases of the ascomycetes and basidiomycetes are clearly separated into two distinct clusters. This evidence indicated that B. rhodina MAMB-05 and other closely related ascomycetes are a new biological resource given the biotechnological potential of their laccase genes.

Crystallization and preliminary X-ray diffraction analysis of the small laccase from Streptomyces coelicolor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Skálová, Tereza, E-mail: skalova@imc.cas.cz; Dohnálek, Jan; Institute of Physics, Academy of Sciences of the Czech Republic, Cukrovarnicka 10, 162 53 Praha 6

2007-12-01

The expression, purification and crystallization of the small laccase from S. coelicolor are reported. Diffraction data were collected to 3 Å resolution. The small bacterial laccase from the actinobacterium Streptomyces coelicolor which lacks the second of the three domains of the laccases structurally characterized to date was crystallized. This multi-copper phenol oxidase crystallizes in a primitive tetragonal lattice, with unit-cell parameters a = b = 179.8, c = 175.3 Å. The crystals belong to either space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2. The self-rotation function shows the presence of a noncrystallographic threefold axis in the structure. Phases willmore » be determined from the anomalous signal of the natively present copper ions.« less

Production of Laccase by a New Myrothecium verrucaria MD-R-16 Isolated from Pigeon Pea [Cajanus cajan (L.) Millsp.] and its Application on Dye Decolorization.

PubMed

Sun, Jiao; Guo, Na; Niu, Li-Li; Wang, Qing-Fang; Zang, Yu-Ping; Zu, Yuan-Gang; Fu, Yu-Jie

2017-04-23

The present study was conducted to screen a laccase-producing fungal endophyte, optimize fermentation conditions, and evaluate the decolorization ability of the laccase. A new fungal endophyte capable of laccase-producing was firstly isolated from pigeon pea and identified as Myrothecium verrucaria based on a ITS-rRNA sequences analysis. Meanwhile, various fermentation parameters on the laccase production were optimized via response surface methodology (RSM). The optimal fermentation conditions were a fermentation time of five days, temperature 30 °C and pH 6.22. Laccase activity reached 16.52 ± 0.18 U/mL under the above conditions. Furthermore, the laccase showed effective decolorization capability toward synthetic dyes (Congo red, Methyl orange, Methyl red, and Crystal violet) in the presence of the redox mediator ABTS, with more than 70% of dyes decolorizing after 24 h of incubation. Additionally, the activity of laccase was relatively stable with pH (4.5-6.5) and a temperature range of 35-55 °C. Therefore, the high laccase production of the strain and the new fungal laccase could provide a promising alterative approach for industrial and environmental applications.

Lignin degradation by selected fungal species.

PubMed

Knežević, Aleksandar; Milovanović, Ivan; Stajić, Mirjana; Lončar, Nikola; Brčeski, Ilija; Vukojević, Jelena; Cilerdžić, Jasmina

2013-06-01

As biological decomposition of plant biomass represents a popular alternative environmental-friendly and economically justified process, screening of ligninolytic enzyme systems of various fungal species is a topical study area. The goal of the study was to obtain clear insight into the dynamics of laccase, Mn-dependent peroxidase, and Mn-independent peroxidase activity and levels of wheat straw lignin degradation in seven wood-rotting fungi. The best laccase producers were Pleurotus ostreatus and Pleurotus eryngii. Lenzites betulinus and Fomitopsis pinicola were the best Mn-dependent peroxidase producers, and P. ostreatus the weakest one. The peak of Mn-independent peroxidase was noted in Dichomytus squalens, and the minimum value in P. ostreatus. The profiles of the three enzymes, obtained by isoelectric focusing, were variable depending on the species and cultivation period. D. squalens was the best lignin degrader (34.1% of total lignin amount), and P. ostreatus and P. eryngii the weakest ones (7.1% and 14.5%, respectively). Copyright © 2013 Elsevier Ltd. All rights reserved.

Nucleus-Selective Expression of Laccase Genes in the Dikaryotic Strain of Lentinula edodes.

PubMed

Ha, Byeongsuk; Lee, Sieun; Kim, Sinil; Kim, Minseek; Moon, Yoon Jung; Song, Yelin; Ro, Hyeon-Su

2017-12-01

In mating of Lentinula edodes , dikaryotic strains generated from certain monokaryotic strains such as the B2 used in this study tend to show better quality of fruiting bodies regardless of the mated monokaryotic strains. Unlike B2, dikaryotic strains generated from B16 generally show low yields, with deformed or underdeveloped fruiting bodies. This indicates that the two nuclei in the cytoplasm do not contribute equally to the physiology of dikaryotic L. edodes , suggesting an expression bias in the allelic genes of the two nuclei. To understand the role of each nucleus in dikaryotic strains, we investigated single nucleotide polymorphisms (SNPs) in laccase genes of monokaryotic strains to reveal nuclear origin of the expressed mRNAs in dikaryotic strain. We performed reverse transcription PCR (RT-PCR) analysis using total RNAs extracted from dikaryotic strains (A5B2, A18B2, and A2B16) as well as from compatible monokaryotic strains (A5, A18, and B2 for A5B2 and A18B2; A2 and B16 for A2B16). RT-PCR results revealed that Lcc1, Lcc2, Lcc4, Lcc7, and Lcc10 were the mainly expressed laccase genes in the L. edodes genome. To determine the nuclear origin of these laccase genes, the genomic DNA sequences in monokaryotic strains were analyzed, thereby revealing five SNPs in Lcc4 and two in Lcc7. Subsequent sequence analysis of laccase mRNAs expressed in dikaryotic strains revealed that these were almost exclusively expressed from B2-originated nuclei in A5B2 and A18B2 whereas B16 nucleus did not contribute to laccase expression in A2B16 strain. This suggests that B2 nucleus dominates the expression of allelic genes, thereby governing the physiology of dikaryons.

Nucleus-Selective Expression of Laccase Genes in the Dikaryotic Strain of Lentinula edodes

PubMed Central

Ha, Byeongsuk; Lee, Sieun; Kim, Sinil; Kim, Minseek; Moon, Yoon Jung; Song, Yelin

2017-01-01

In mating of Lentinula edodes, dikaryotic strains generated from certain monokaryotic strains such as the B2 used in this study tend to show better quality of fruiting bodies regardless of the mated monokaryotic strains. Unlike B2, dikaryotic strains generated from B16 generally show low yields, with deformed or underdeveloped fruiting bodies. This indicates that the two nuclei in the cytoplasm do not contribute equally to the physiology of dikaryotic L. edodes, suggesting an expression bias in the allelic genes of the two nuclei. To understand the role of each nucleus in dikaryotic strains, we investigated single nucleotide polymorphisms (SNPs) in laccase genes of monokaryotic strains to reveal nuclear origin of the expressed mRNAs in dikaryotic strain. We performed reverse transcription PCR (RT-PCR) analysis using total RNAs extracted from dikaryotic strains (A5B2, A18B2, and A2B16) as well as from compatible monokaryotic strains (A5, A18, and B2 for A5B2 and A18B2; A2 and B16 for A2B16). RT-PCR results revealed that Lcc1, Lcc2, Lcc4, Lcc7, and Lcc10 were the mainly expressed laccase genes in the L. edodes genome. To determine the nuclear origin of these laccase genes, the genomic DNA sequences in monokaryotic strains were analyzed, thereby revealing five SNPs in Lcc4 and two in Lcc7. Subsequent sequence analysis of laccase mRNAs expressed in dikaryotic strains revealed that these were almost exclusively expressed from B2-originated nuclei in A5B2 and A18B2 whereas B16 nucleus did not contribute to laccase expression in A2B16 strain. This suggests that B2 nucleus dominates the expression of allelic genes, thereby governing the physiology of dikaryons. PMID:29371806

Identification of a laccase from Ganoderma lucidum CBS 229.93 having potential for enhancing cellulase catalyzed lignocellulose degradation.

PubMed

Sitarz, Anna K; Mikkelsen, Jørn D; Højrup, Peter; Meyer, Anne S

2013-12-10

Based on a differential pre-screening of 44 white-rot fungi on a lignocellulose-supplemented minimal medium, four basidiomycetes were selected for further study: Ganoderma lucidum, Polyporus brumalis, Polyporus ciliatus and Trametes versicolor. Only G. lucidum was able to grow vividly on malt extract or minimal media supplemented with alkali lignin. When grown on malt extract or minimal medium supplemented with lignocellulose (sugar cane bagasse), the crude G. lucidum protein extract exhibited high laccase activity, ∼3U/mL toward syringaldazine. This activity was 13-17 fold higher than the corresponding activities of the crude protein extracts of P. brumalis, P. ciliatus and T. versicolor. Native PAGE electrophoresis of the crude G. lucidum extract confirmed the presence of an active laccase. The G. lucidum laccase had a molecular weight of ∼62.5kDa, and a Km value of 0.107mM (determined on ABTS). A partial amino acid sequence analysis of four short de novo sequenced peptides, defined after trypsin digest analysis using MALDI-TOF MS/MS analysis, revealed 64-100% homology to sequences in related laccases in the UniProt database, but also indicated that certain sequence stretches had low homology. Addition of the laccase-rich G. lucidum broth to lignocellulosic biomass (pretreated sugar cane bagasse) together with a state-of-the-art cellulase enzyme preparation (Cellic™CTec1) produced significantly increased cellulolytic yields, which were also better than those obtained with a T. versicolor laccase addition, indicating that the laccase from G. lucidum has unique properties that may be momentous in lignocellulosic biomass conversion. Copyright © 2013 Elsevier Inc. All rights reserved.

Laccase and lectin activities of intracellular proteins produced in a submerged culture of the xylotrophic basidiomycete Lentinus edodes.

PubMed

Vetchinkina, Elena P; Pozdnyakova, Natalia N; Nikitina, Valentina E

2008-10-01

The white-rot fungus Lentinus edodes produced D-melibiose-specific lectins and two laccase forms in a lignin-containing medium. The maxima of laccase and lectin activities coincided, falling within the period of active mycelial growth. The enzymes and lectins were isolated and purified by gel filtration followed by anion-exchange chromatography. The L. edodes lectins were found to be able to stabilize the activity of the fungus's own laccases. Lectin activity during the formation of lectin-enzyme complexes remained unchanged.

Genome-wide identification of multifunctional laccase gene family in cotton (Gossypium spp.); expression and biochemical analysis during fiber development

PubMed Central

Balasubramanian, Vimal Kumar; Rai, Krishan Mohan; Thu, Sandi Win; Hii, Mei Mei; Mendu, Venugopal

2016-01-01

The single-celled cotton fibers, produced from seed coat epidermal cells are the largest natural source of textile fibers. The economic value of cotton fiber lies in its length and quality. The multifunctional laccase enzymes play important roles in cell elongation, lignification and pigmentation in plants and could play crucial role in cotton fiber quality. Genome-wide analysis of cultivated allotetraploid (G. hirsutum) and its progenitor diploid (G. arboreum and G. raimondii) cotton species identified 84, 44 and 46 laccase genes, respectively. Analysis of chromosomal location, phylogeny, conserved domain and physical properties showed highly conserved nature of laccases across three cotton species. Gene expression, enzymatic activity and biochemical analysis of developing cotton fibers was performed using G. arboreum species. Of the total 44, 40 laccases showed expression during different stages of fiber development. The higher enzymatic activity of laccases correlated with higher lignin content at 25 DPA (Days Post Anthesis). Further, analysis of cotton fiber phenolic compounds showed an overall decrease at 25 DPA indicating possible incorporation of these substrates into lignin polymer during secondary cell wall biosynthesis. Overall data indicate significant roles of laccases in cotton fiber development, and presents an excellent opportunity for manipulation of fiber development and quality. PMID:27679939

Expression of a thermotolerant laccase from Pycnoporus sanguineus in Trichoderma reesei and its application in the degradation of bisphenol A.

PubMed

Zhao, Jie; Zeng, Shengquan; Xia, Ying; Xia, Liming

2018-04-01

The laccase gene from Pycnoporus sanguineus was cloned and inserted between the strong Pcbh1 promoter and the Tcbh1 terminator from Trichoderma reesei to form the recombinant plasmid pCH-lac. Using Agrobacterium-mediated technique, the pCH-lac was integrated into the chromosomes of T. reesei. Twenty positive transformants were obtained by employing hygromycin B as a selective agent. PCR was used to confirm that the laccase gene was integrated into the chromosomal DNA of T. reesei. Laccase production by recombinant transformants was performed in shaking flasks, and the activity of laccase reached 8.8 IU/mL after 96-h fermentation under a batch process, and 17.7 IU/mL after 144-h fermentation using a fed-batch process. SDS-PAGE analysis of the fermentation broth showed that the molecular mass of the protein was about 68 kDa, almost the same as that of the laccase produced by P. sanguineus, which indicated that laccase was successfully expressed in T. reesei and secreted out of the cells. The laccase produced by the recombinant T. reesei showed good thermal stability, and could degrade the toxic phenolic material bisphenol A efficiently, after 1-h reaction with 0.06 IU/mL laccase and 0.5 mmol/L ABTS as the mediator at 60 °C and pH 4.5, the degradation rate reached 95%, which demonstrated that it had great potential value in treating the household garbage and wastewater containing the bisphenol A. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Degradation of lindane and endosulfan by fungi, fungal and bacterial laccases.

PubMed

Ulčnik, A; Kralj Cigić, I; Pohleven, F

2013-12-01

The ability of two white-rot fungi (Trametes versicolor and Pleurotus ostreatus) and one brown-rot fungus (Gloeophyllum trabeum) to degrade two organochlorine insecticides, lindane and endosulfan, in liquid cultures was studied and dead fungal biomass was examined for adsorption of both insecticides from liquid medium. Lindane and endosulfan were also treated with fungal laccase and bacterial protein CotA, which has laccase activities. The amount of degraded lindane and endosulfan increased with their exposure period in the liquid cultures of both examined white-rot fungi. Endosulfan was transformed to endosulfan sulphate by T. versicolor and P. ostreatus. A small amount of endosulfan ether was also detected and its origin was examined. Degradation of lindane and endosulfan by a brown rot G. trabeum did not occur. Mycelial biomasses of all examined fungi have been found to adsorb lindane and endosulfan and adsorption onto fungal biomass should therefore be considered as a possible mechanism of pollutant removal when fungal degradation potentials are studied. Bacterial protein CotA performed more efficient degradation of lindane and endosulfan than fungal laccase and has shown potential for bioremediation of organic pollutants.

Laccase-catalyzed decolorization of malachite green: performance optimization and degradation mechanism.

PubMed

Yang, Jie; Yang, Xiaodan; Lin, Yonghui; Ng, Tzi Bun; Lin, Juan; Ye, Xiuyun

2015-01-01

Malachite green (MG) was decolorized by laccase (LacA) of white-rot fungus Cerrena sp. with strong decolorizing ability. Decolorization conditions were optimized with response surface methodology. A highly significant quadratic model was developed to investigate MG decolorization with LacA, and the maximum MG decolorization ratio of 91.6% was predicted under the conditions of 2.8 U mL(-1) LacA, 109.9 mg L(-1) MG and decolorization for 172.4 min. Kinetic studies revealed the Km and kcat values of LacA toward MG were 781.9 mM and 9.5 s(-1), respectively. UV-visible spectra confirmed degradation of MG, and the degradation mechanism was explored with liquid chromatography-mass spectrometry (LC-MS) analysis. Based on the LC-MS spectra of degradation products, LacA catalyzed MG degradation via two simultaneous pathways. In addition, the phytotoxicity of MG, in terms of inhibition on seed germination and seedling root elongation of Nicotiana tabacum and Lactuca sativa, was reduced after laccase treatment. These results suggest that laccase of Cerrena was effective in decolorizing MG and promising in bioremediation of wastewater in food and aquaculture industries.

Laccase-Catalyzed Decolorization of Malachite Green: Performance Optimization and Degradation Mechanism

PubMed Central

Yang, Jie; Yang, Xiaodan; Lin, Yonghui; Ng, Tzi Bun; Lin, Juan; Ye, Xiuyun

2015-01-01

Malachite green (MG) was decolorized by laccase (LacA) of white-rot fungus Cerrena sp. with strong decolorizing ability. Decolorization conditions were optimized with response surface methodology. A highly significant quadratic model was developed to investigate MG decolorization with LacA, and the maximum MG decolorization ratio of 91.6% was predicted under the conditions of 2.8 U mL-1 LacA, 109.9 mg L-1 MG and decolorization for 172.4 min. Kinetic studies revealed the Km and kcat values of LacA toward MG were 781.9 mM and 9.5 s-1, respectively. UV–visible spectra confirmed degradation of MG, and the degradation mechanism was explored with liquid chromatography–mass spectrometry (LC-MS) analysis. Based on the LC-MS spectra of degradation products, LacA catalyzed MG degradation via two simultaneous pathways. In addition, the phytotoxicity of MG, in terms of inhibition on seed germination and seedling root elongation of Nicotiana tabacum and Lactuca sativa, was reduced after laccase treatment. These results suggest that laccase of Cerrena was effective in decolorizing MG and promising in bioremediation of wastewater in food and aquaculture industries. PMID:26020270

Transcriptional and Enzymatic Profiling of Pleurotus ostreatus Laccase Genes in Submerged and Solid-State Fermentation Cultures

PubMed Central

Castanera, Raúl; Pérez, Gúmer; Omarini, Alejandra; Alfaro, Manuel; Pisabarro, Antonio G.; Faraco, Vincenza; Amore, Antonella

2012-01-01

The genome of the white rot basidiomycete Pleurotus ostreatus includes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription-quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) the Lacc2 and Lacc10 genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and the Lacc gene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors. PMID:22467498

Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor.

PubMed

Elegir, G; Bussini, D; Antonsson, S; Lindström, M E; Zoia, L

2007-12-01

In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.

Leaching of manganese from electrolytic manganese residue by electro-reduction.

PubMed

Shu, Jiancheng; Liu, Renlong; Liu, Zuohua; Chen, Hongliang; Tao, Changyuan

2017-08-01

In this study, an improved process for leaching manganese from electrolytic manganese residue (EMR) by electro-reduction was developed. The mechanisms of the electro-reduction leaching were investigated through X-ray diffraction, scanning electron microscopy, X-ray fluorescence, and Brunauer Emmett Teller. The results show that the electric field could change the surface charge distribution of EMR particles, and the high-valent manganese can be reduced by electric field. The leaching efficient of manganese reached 84.1% under the optimal leaching condition: 9.2 wt% H 2 SO 4 , current density of 25 mA/cm 2 , solid-to-liquid ratio of 1:5, and leaching time for 1 h. It is 37.9% higher than that attained without an electric field. Meanwhile, the manganese content in EMR decreased from 2.57% to 0.48%.

Loss of hfe function reverses impaired recognition memory caused by olfactory manganese exposure in mice.

PubMed

Ye, Qi; Kim, Jonghan

2015-03-01

Excessive manganese (Mn) in the brain promotes a variety of abnormal behaviors, including memory deficits, decreased motor skills and psychotic behavior resembling Parkinson's disease. Hereditary hemochromatosis (HH) is a prevalent genetic iron overload disorder worldwide. Dysfunction in HFE gene is the major cause of HH. Our previous study has demonstrated that olfactory Mn uptake is altered by HFE deficiency, suggesting that loss of HFE function could alter manganese-associated neurotoxicity. To test this hypothesis, Hfe-knockout (Hfe (-/-)) and wild-type (Hfe (+/+)) mice mice were intranasally-instilled with manganese chloride (MnCl2 5 mg/kg) or water daily for 3 weeks and examined for memory function. Olfactory Mn diminished both short-term recognition and spatial memory in Hfe (+/+) mice, as examined by novel object recognition task and Barnes maze test, respectively. Interestingly, Hfe (-/-) mice did not show impaired recognition memory caused by Mn exposure, suggesting a potential protective effect of Hfe deficiency against Mn-induced memory deficits. Since many of the neurotoxic effects of manganese are thought to result from increased oxidative stress, we quantified activities of anti-oxidant enzymes in the prefrontal cortex (PFC). Mn instillation decreased superoxide dismutase 1 (SOD1) activity in Hfe (+/+) mice, but not in Hfe (-/-) mice. In addition, Hfe deficiency up-regulated SOD1 and glutathione peroxidase activities. These results suggest a beneficial role of Hfe deficiency in attenuating Mn-induced oxidative stress in the PFC. Furthermore, Mn exposure reduced nicotinic acetylcholine receptor levels in the PFC, indicating that blunted acetylcholine signaling could contribute to impaired memory associated with intranasal manganese. Together, our model suggests that disrupted cholinergic system in the brain is involved in airborne Mn-induced memory deficits and loss of HFE function could in part prevent memory loss via a potential up-regulation of

Decolorization and detoxification of two textile industry effluents by the laccase/1-hydroxybenzotriazole system.

PubMed

Benzina, Ouafa; Daâssi, Dalel; Zouari-Mechichi, Héla; Frikha, Fakher; Woodward, Steve; Belbahri, Lassaad; Rodriguez-Couto, Susana; Mechichi, Tahar

2013-08-01

The aim of this work was to determine the optimal conditions for the decolorization and the detoxification of two effluents from a textile industry-effluent A (the reactive dye bath Bezactive) and effluent B (the direct dye bath Tubantin)-using a laccase mediator system. Response surface methodology (RSM) was applied to optimize textile effluents decolorization. A Box-Behnken design using RSM with the four variables pH, effluent concentration, 1-hydroxybenzotriazole (HBT) concentration, and enzyme (laccase) concentration was used to determine correlations between the effects of these variables on the decolorization of the two effluents. The optimum conditions for pH and concentrations of HBT, effluent and laccase were 5, 1 mM, 50 % and 0.6 U/ml, respectively, for maximum decolorization of effluent A (68 %). For effluent B, optima were 4, 1 mM, 75 %, and 0.6 U/ml, respectively, for maximum decolorization of approximately 88 %. Both effluents were treated at 30 °C for 20 h. A quadratic model was obtained for each decolorization through this design. The experimental and predicted values were in good agreement and both models were highly significant. In addition, the toxicity of the two effluents was determined before and after laccase treatment using Saccharomyces cerevisiae, Bacillus cereus, and germination of tomato seeds.

Laccase production in bioreactor scale under saline condition by the marine-derived basidiomycete Peniophora sp. CBMAI 1063.

PubMed

Mainardi, Pedro H; Feitosa, Valker A; Brenelli de Paiva, Livia B; Bonugli-Santos, Rafaella C; Squina, Fabio M; Pessoa, Adalberto; Sette, Lara D

2018-05-01

Laccase production in saline conditions is still poorly studied. The aim of the present study was to investigate the production of laccase in two different types of bioreactors by the marine-derived basidiomycete Peniophora sp. CBMAI 1063. The highest laccase activity and productivity were obtained in the Stirred Tank (ST) bioreactor, while the highest biomass concentration in Air-lift (AL) bioreactor. The main laccase produced was purified by ion exchange and size exclusion chromatography and appeared to be monomeric with molecular weight of approximately 55 kDa. The optimum oxidation activity was obtained at pH 5.0. The thermal stability of the enzyme ranged from 30 to 50 °C (120 min). The Far-UV Circular Dichroism revealed the presence of high β-sheet and low α-helical conformation in the protein structure. Additional experiments carried out in flask scale showed that the marine-derived fungus was able to produce laccase only in the presence of artificial seawater and copper sulfate. Results from the present study confirmed the fungal adaptation to marine conditions and its potential for being used in saline environments and/or processes. Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Comparative analyses of laccase-catalyzed amination reactions for production of novel β-lactam antibiotics.

PubMed

Mikolasch, Annett; Manda, Katrin; Schlüter, Rabea; Lalk, Michael; Witt, Sabine; Seefeldt, Simone; Hammer, Elke; Schauer, Frieder; Jülich, Wolf-Dieter; Lindequist, Ulrike

2012-01-01

Seven novel β-lactam antibiotics with activities against Gram-positive bacterial strains, among them methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, were synthesized by amination of 2,5-dihydroxyphenylacetic acid in usable yields (30-60%). These products protected mice against an infection with S. aureus lethal to the control animals. The results show the usefulness of laccase for the synthesis of potential new antibiotics, in addition to the interdependence of the laccase substrates, the amino coupling partners, and the product formation, yield, and activity. The syntheses of β-lactam antibiotics with 2,5-dihydroxyaromatic acid substructures (para-substituted) are then compared with those of 3,4-dihydroxyaromatic acid substructures (ortho-substituted). Para-substituted laccase substrates were better reaction partners in these syntheses than ortho-substituted compounds. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Extraction of manganese from electrolytic manganese residue by bioleaching.

PubMed

Xin, Baoping; Chen, Bing; Duan, Ning; Zhou, Changbo

2011-01-01

Extraction of manganese from electrolytic manganese residues using bioleaching was investigated in this paper. The maximum extraction efficiency of Mn was 93% by sulfur-oxidizing bacteria at 4.0 g/l sulfur after bioleaching of 9days, while the maximum extraction efficiency of Mn was 81% by pyrite-leaching bacteria at 4.0 g/l pyrite. The series bioleaching first by sulfur-oxidizing bacteria and followed by pyrite-leaching bacteria evidently promoted the extraction of manganese, witnessing the maximum extraction efficiency of 98.1%. In the case of sulfur-oxidizing bacteria, the strong dissolution of bio-generated sulfuric acid resulted in extraction of soluble Mn2+, while both the Fe2+ catalyzed reduction of Mn4+ and weak acidic dissolution of Mn2+ accounted for the extraction of manganese with pyrite-leaching bacteria. The chemical simulation of bioleaching process further confirmed that the acid dissolution of Mn2+ and Fe2+ catalyzed reduction of Mn4+ were the bioleaching mechanisms involved for Mn extraction from electrolytic manganese residues. Copyright © 2010 Elsevier Ltd. All rights reserved.

Laccase-Catalyzed Surface Modification of Thermo-Mechanical Pulp (TMP) for the Production of Wood Fiber Insulation Boards Using Industrial Process Water

PubMed Central

Schubert, Mark; Ruedin, Pascal; Civardi, Chiara; Richter, Michael; Hach, André; Christen, Herbert

2015-01-01

Low-density wood fiber insulation boards are traditionally manufactured in a wet process using a closed water circuit (process water). The water of these industrial processes contains natural phenolic extractives, aside from small amounts of admixtures (e.g., binders and paraffin). The suitability of two fungal laccases and one bacterial laccase was determined by biochemical characterization considering stability and substrate spectra. In a series of laboratory scale experiments, the selected commercial laccase from Myceliophtora thermophila was used to catalyze the surface modification of thermo-mechanical pulp (TMP) using process water. The laccase catalyzed the covalent binding of the phenolic compounds of the process water onto the wood fiber surface and led to change of the surface chemistry directly via crosslinking of lignin moieties. Although a complete substitution of the binder was not accomplished by laccase, the combined use of laccase and latex significantly improved the mechanical strength properties of wood fiber boards. The enzymatically-treated TMP showed better interactions with the synthetic binder, as shown by FTIR-analysis. Moreover, the enzyme is extensively stable in the process water and the approach requires no fresh water as well as no cost-intensive mediator. By applying a second-order polynomial model in combination with the genetic algorithm (GA), the required amount of laccase and synthetic latex could be optimized enabling the reduction of the binder by 40%. PMID:26046652

Wound-induced expression of horseradish peroxidase.

PubMed

Kawaoka, A; Kawamoto, T; Ohta, H; Sekine, M; Takano, M; Shinmyo, A

1994-01-01

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the β-glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.

Differential regulation of mnp2, a new manganese peroxidase-encoding gene from the ligninolytic fungus Trametes versicolor PRL 572

Treesearch

Tomas Johansson; Per Olof Nyman; Daniel Cullen

2002-01-01

A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO4 to cultures increased...

Laccase production by Coriolopsis caperata RCK2011: Optimization under solid state fermentation by Taguchi DOE methodology

PubMed Central

Nandal, Preeti; Ravella, Sreenivas Rao; Kuhad, Ramesh Chander

2013-01-01

Laccase production by Coriolopsis caperata RCK2011 under solid state fermentation was optimized following Taguchi design of experiment. An orthogonal array layout of L18 (21 × 37) was constructed using Qualitek-4 software with eight most influensive factors on laccase production. At individual level pH contributed higher influence, whereas, corn steep liquor (CSL) accounted for more than 50% of the severity index with biotin and KH2PO4 at the interactive level. The optimum conditions derived were; temperature 30°C, pH 5.0, wheat bran 5.0 g, inoculum size 0.5 ml (fungal cell mass = 0.015 g dry wt.), biotin 0.5% w/v, KH2PO4 0.013% w/v, CSL 0.1% v/v and 0.5 mM xylidine as an inducer. The validation experiments using optimized conditions confirmed an improvement in enzyme production by 58.01%. The laccase production to the level of 1623.55 Ugds−1 indicates that the fungus C. caperata RCK2011 has the commercial potential for laccase. PMID:23463372

Biobleaching of wheat straw-rich soda pulp with alkalophilic laccase from gamma-proteobacterium JB: optimization of process parameters using response surface methodology.

PubMed

Singh, Gursharan; Ahuja, Naveen; Batish, Mona; Capalash, Neena; Sharma, Prince

2008-11-01

An alkalophilic laccase from gamma-proteobacterium JB was applied to wheat straw-rich soda pulp to check its bleaching potential by using response surface methodology based on central composite design. The design was employed by selecting laccase units, ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) concentration and pH as model factors. The results of second order factorial design experiments showed that all three independent variables had significant effect on brightness and kappa number of laccase-treated pulp. Optimum conditions for biobleaching of pulp with laccase preparation (specific activity, 65 nkat mg(-1) protein) were 20 nkat g(-1) of pulp, 2mM ABTS and pH 8.0 which enhanced brightness by 5.89% and reduced kappa number by 21.1% within 4h of incubation at 55 degrees C, without further alkaline extraction of pulp. Tear index (8%) and burst index (18%) also improved for laccase-treated pulp as compared to control raw pulp. Treatment of chemically (CEH1H2) bleached pulp with laccase showed significant effect on release of chromophores, hydrophobic and reducing compounds. Laccase-prebleaching of raw pulp reduced the use of hypochlorite by 10% to achieve brightness of resultant hand sheets similar to the fully chemically bleached pulp.

Spectroscopic Analyses of the Biofuels-Critical Phytochemical Coniferyl Alcohol and Its Enzyme-Catalyzed Oxidation Products

DOE Office of Scientific and Technical Information (OSTI.GOV)

Achyuthan, Komandoor; Adams, Paul; Simmons, Blake

2011-07-13

Lignin composition (monolignol types of coniferyl, sinapyl or p-coumaryl alcohol) is causally related to biomass recalcitrance. We describe multiwavelength (220, 228, 240, 250, 260, 290, 295, 300, 310 or 320 nm) absorption spectroscopy of coniferyl alcohol and its laccase- or peroxidase-catalyzed products during real time kinetic, pseudo-kinetic and endpoint analyses, in optical turn on or turn off modes, under acidic or basic conditions. Reactions in microwell plates and 100 mu L volumes demonstrated assay miniaturization and high throughput screening capabilities. Bathochromic and hypsochromic shifts along with hyperchromicity or hypochromicity accompanied enzymatic oxidations by laccase or peroxidase. The limits of detectionmore » and quantitation of coniferyl alcohol averaged 2.4 and 7.1 mu M respectively, with linear trend lines over 3 to 4 orders of magnitude. Coniferyl alcohol oxidation was evident within 10 minutes or with 0.01 mu g/mL laccase and 2 minutes or 0.001 mu g/mL peroxidase. Detection limit improved to 1.0 mu M coniferyl alcohol with Km of 978.7 +/- 150.7 mu M when examined at 260 nm following 30 minutes oxidation with 1.0 mu g/mL laccase. Our assays utilized the intrinsic spectroscopic properties of coniferyl alcohol or its oxidation products for enabling detection, without requiring chemical synthesis or modification of the substrate or product(s). These studies facilitate lignin compositional analyses and augment pretreatment strategies for reducing biomass recalcitrance.« less

Manganese, Metallogenium, and Martian Microfossils

NASA Technical Reports Server (NTRS)

Stein, L. Y.; Nealson, K. H.

1999-01-01

Manganese could easily be considered an abundant element in the Martian regolith, assuming that the composition of martian meteorites reflects the composition of the planet. Mineralogical analyses of 5 SNC meteorites have revealed an average manganese oxide concentration of 0.48%, relative to the 0.1% concentration of manganese found in the Earth's crust. On the Earth, the accumulation of manganese oxides in oceans, soils, rocks, sedimentary ores, fresh water systems, and hydrothermal vents can be largely attributed to microbial activity. Manganese is also a required trace nutrient for most life forms and participates in many critical enzymatic reactions such as photosynthesis. The wide-spread process of bacterial manganese cycling on Earth suggests that manganese is an important element to both geology and biology. Furthermore, there is evidence that bacteria can be fossilized within manganese ores, implying that manganese beds may be good repositories for preserved biomarkers. A particular genus of bacteria, known historically as Metallogenium, can form star-shaped manganese oxide minerals (called metallogenium) through the action of manganese oxide precipitation along its surface. Fossilized structures that resemble metallogenium have been found in Precambrian sedimentary formations and in Cretaceous-Paleogene cherts. The Cretaceous-Paleogene formations are highly enriched in manganese and have concentrations of trace elements (Fe, Zn, Cu, and Co) similar to modern-day manganese oxide deposits in marine environments. The appearance of metallogenium-like fossils associated with manganese deposits suggests that bacteria may be preserved within the minerals that they form. Additional information is contained in the original extended abstract.

Kinetic evidence for the interactive inhibition of laccase from Trametes versicolor by pH and chloride.

PubMed

Raseda, Nasrin; Hong, Soonho; Kwon, O Yul; Ryu, Keungarp

2014-12-28

The interactive inhibitory effects of pH and chloride on the catalysis of laccase from Trametes versicolor were investigated by studying the alteration of inhibition characteristics of sodium chloride at different pHs for the oxidation of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid). At pH 3.0, the addition of sodium chloride (50 mM) brought about a 40-fold increase in Km(app) and a 4-fold decrease in Vmax(app). As the pH increased to 7.0, the inhibitory effects of sodium chloride became significantly weakened. The mixed-inhibition mechanism was successfully used to quantitatively estimate the competitive and uncompetitive inhibition strengths by chloride at two different pHs (pH 3.0 and 6.0). At pH 3.0, the competitive inhibition constant, Ki, was 0.35 mM, whereas the uncompetitive inhibition constant, Ki', was 18.1 mM, indicating that the major cause of the laccase inhibition by chloride is due to the competitive inhibition step. At a higher pH of 6.0, where the inhibition of the laccase by hydroxide ions takes effect, the inhibition of the laccase by chloride diminished to a great extent, showing increased values of both the competitive inhibition constant (Ki= 23.7 mM) and uncompetitive inhibition constant (Ki' = 324 mM). These kinetic results evidenced that the hydroxide anion and chloride share a common mechanism to inhibit the laccase activity.

New potential biocatalysts by laccase immobilization in PVA Cryogel type carrier.

PubMed

Stanescu, Michaela Dina; Fogorasi, Magdalena; Shaskolskiy, Boris L; Gavrilas, Simona; Lozinsky, Vladimir I

2010-04-01

Laccases are enzymes belonging to the Oxidoreductases class. These enzymes may be good biocatalysts for different processes, at laboratory and industrial levels. A successful use at industrial scale demands a higher stability of the enzyme. As an easy way to obtain longer life biocatalysts, the immobilization process is recommended. Thus, the paper presents different ways of obtaining new biocatalysts by a laccase covalent immobilization on a macroporous carrier based on poly(vinyl alcohol) cryogel. Different procedures of covalent immobilization are described, the newly obtained biocatalysts being characterized. According to the experimental data, the stability of the immobilized enzyme increased and the pH profile changed, compared with those of the free enzyme.

Comparison of two laccases from Trametes versicolor for application in the decolorization of dyes.

PubMed

Li, Qi; Ge, Lin; Cai, Junli; Pei, Jianjun; Xie, Jingcong; Zhao, Linguo

2014-04-01

It has been previously demonstrated that laccases exhibit great potential for use in several industrial and environmental applications. In this paper, two laccase isoenzyme genes, lccB and lccC, were cloned and expressed in Pichia pastoris GS115. The sequence analysis indicated that the lccB and lccC genes consisted of 1,563 and 1,584 bp, and their open reading frames encoded 520 and 527 amino acids, respectively. They had 72.7% degree of identity in nucleotides and 86.7% in amino acids. The expression levels of LccB and LccC were up to 32,479 and 34,231 U/l, respectively. The recombinant laccases were purified by ultrafiltration and (NH4)2SO4 precipitation, showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimal pH and temperature for LccB were 2.0 and 55°C with 2,2'-azino-bis-[3-ethylbenzthiazolinesulfonic acid (ABTS) as a substrate, whereas LccC exhibited optimal pH and temperature at 3.0 and 60°C. The apparent kinetic parameters of LccB were 0.43 mM for ABTS with a Vmax value of 51.28 U/mg, and the Km and Vmax values for LccC were 0.29 mM and 62.89 U/mg. The recombinant laccases were able to decolorize five types of dyes. Acid Violet 43 (100 g/ml) was completely decolorized by LccB or LccC (2 U/ml), and the decolorization of Reactive Blue KN-R (100 g/ml) was 91.6% by LccC (2 U/ml). Thus, the study characterizes useful laccase isoenzymes from T. versicolor that have the capability of being incorporated into the treatment of similar azo and anthraquinone dyes from dyeing industries.

Hybrid biobattery based on arylated carbon nanotubes and laccase.

PubMed

Stolarczyk, Krzysztof; Sepelowska, Małgorzata; Lyp, Dominika; Zelechowska, Kamila; Biernat, Jan F; Rogalski, Jerzy; Farmer, Kevin D; Roberts, Ken N; Bilewicz, Renata

2012-10-01

Single-walled carbon nanotubes (SWCNT) were covalently modified with anthracene and anthraquinone and used for the construction of cathodes for biocatalytic reduction of dioxygen. The nanotubes with aromatic groups casted onto the electrode increased the working surface of the electrode and enabled efficient direct electron transfer (DET) between the enzyme and the electrode. The aryl groups enter the hydrophobic pocket of the T1 center of laccase responsible for exchanging electrons with the substrate. Glassy carbon electrode covered with arylated SWCNT and coated with a layer of neutralized Nafion containing laccase was found to be a very efficient cathode in the hybrid battery. Zn wire covered with a Nafion film served as the anode. The cell parameters were determined: power density was 2 mW/cm(2) and the open circuit potential was 1.5 V. Copyright © 2011 Elsevier B.V. All rights reserved.

Production of laccase by Coriolus versicolor and its application in decolorization of dyestuffs: (II). Decolorization of dyes by laccase containing fermentation broth with or without self-immobilized mycelia.

PubMed

Lin, Jian-Ping; Lian, Wei; Xia, Li-Ming; Cen, Pei-Lin

2003-01-01

The capability of decolorization for commercial dyes by Coriolus versicolor fermentation broth containing laccase with or without immobilized mycelium was evaluated. With cell-free fermentation broth containing laccase, high decolorization ratio was achieved foracid orange 7, but not for the other dyes concerned. The immobilized mycelium was proved to be more efficient than the cell-free system. All the four dyestuffs studied were found being decolourized with certain extent by immobilized mycelium. The repeated-batch decolorization was carried out with satisfactory results. The experimental data showed that the continuous decolorization of wastewater from a printing and dyeing industry was possible by using the self-immobilized C. versicolor.

Peptide/laccase cocatalyzed asymmetric α-oxyamination of aldehydes.

PubMed

Akagawa, Kengo; Kudo, Kazuaki

2011-07-01

An asymmetric α-oxyamination could be successfully performed by a peptide catalyst and laccase. The combination of peptide catalysis and enzymatic air oxidation promoted the reaction smoothly in water without employing a metal reagent. The oxyaminated compounds could be obtained as both aldehyde and carboxylic acid products depending on the reaction conditions.

Laccase immobilization and insolubilization: from fundamentals to applications for the elimination of emerging contaminants in wastewater treatment.

PubMed

Ba, Sidy; Arsenault, Alexandre; Hassani, Thanina; Jones, J Peter; Cabana, Hubert

2013-12-01

Over the last few decades many attempts have been made to use biocatalysts for the biotransformation of emerging contaminants in environmental matrices. Laccase, a multicopper oxidoreductase enzyme, has shown great potential in oxidizing a large number of phenolic and non-phenolic emerging contaminants. However, laccases and more broadly enzymes in their free form are biocatalysts whose applications in solution have many drawbacks rendering them currently unsuitable for large scale use. To circumvent these limitations, the enzyme can be immobilized onto carriers or entrapped within capsules; these two immobilization techniques have the disadvantage of generating a large mass of non-catalytic product. Insolubilization of the free enzymes as cross-linked enzymes (CLEAs) is found to yield a greater volume ratio of biocatalyst while improving the characteristics of the biocatalyst. Ultimately, novel techniques of enzymes insolubilization and stabilization are feasible with the combination of cross-linked enzyme aggregates (combi-CLEAs) and enzyme polymer engineered structures (EPESs) for the elimination of emerging micropollutants in wastewater. In this review, fundamental features of laccases are provided in order to elucidate their catalytic mechanism, followed by different chemical aspects of the immobilization and insolubilization techniques applicable to laccases. Finally, kinetic and reactor design effects for enzymes in relation with the potential applications of laccases as combi-CLEAs and EPESs for the biotransformation of micropollutants in wastewater treatment are discussed.

Bacterial extracellular lignin peroxidase

DOEpatents

Crawford, Donald L.; Ramachandra, Muralidhara

1993-01-01

A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

A bacterial laccase from marine microbial metagenome exhibiting chloride tolerance and dye decolorization ability.

PubMed

Fang, Zemin; Li, Tongliang; Wang, Quan; Zhang, Xuecheng; Peng, Hui; Fang, Wei; Hong, Yuzhi; Ge, Honghua; Xiao, Yazhong

2011-02-01

Laccases are blue multicopper oxidases with potential applications in environmental and industrial biotechnology. In this study, a new bacterial laccase gene of 1.32 kb was obtained from a marine microbial metagenome of the South China Sea by using a sequence screening strategy. The protein (named as Lac15) of 439 amino acids encoded by the gene contains three conserved Cu(2+)-binding domains, but shares less than 40% of sequence identities with all of the bacterial multicopper oxidases characterized. Lac15, recombinantly expressed in Escherichia coli, showed high activity towards syringaldazine at pH 6.5-9.0 with an optimum pH of 7.5 and with the highest activity occurring at 45 °C. Lac15 was stable at pH ranging from 5.5 to 9.0 and at temperatures from 15 to 45 °C. Distinguished from fungal laccases, the activity of Lac15 was enhanced twofold by chloride at concentrations lower than 700 mM, and kept the original level even at 1,000 mM chloride. Furthermore, Lac15 showed an ability to decolorize several industrial dyes of reactive azo class under alkalescent conditions. The properties of alkalescence-dependent activity, high chloride tolerance, and dye decolorization ability make the new laccase Lac15 an alternative for specific industrial applications.

Mediator-assisted decolorization and detoxification of textile dyes/dye mixture by Cyathus bulleri laccase.

PubMed

Chhabra, Meenu; Mishra, Saroj; Sreekrishnan, T R

2008-12-01

Laccase from basidiomycete fungus Cyathus bulleri was evaluated for its ability to decolorize a number of reactive and acidic dyes in the presence of natural and synthetic mediators. The extent of decolorization was monitored at different mediator/dye concentrations and incubation time. Among the synthetic mediators, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was effective at low mediator/dye ratios and resulted in 80-95% decolorization at rates that varied from 226 +/- 4 nmol min(-1) mg(-1) for Reactive Orange 1 to 1,333 +/- 15 nmol min(-1) mg(-1) for Reactive Red 198. Other synthetic mediators like 1-hydroxybenzotriazole and violuric acid showed both concentration- and time-dependent increases in percent decolorization. Natural mediators like vanillin, on the other hand, were found to be less effective on all the dyes except Reactive Orange 1. Computed rates of decolorization were about twofold lower than that with ABTS. The laccase-ABTS system also led to nearly 80% decolorization for the simulated dye mixture. No clear correlation between laccase activity on the mediator and its ability to decolorize dyes was found, but pH had a significant effect: Optimum pH for decolorization coincided with the optimum pH for mediator oxidation. The treated samples were also evaluated for toxicity in model microbial systems. The laccase-mediator system appears promising for treatment of textile wastewaters.

Bioinspired production of magnetic laccase-biotitania particles for the removal of endocrine disrupting chemicals.

PubMed

Ardao, Inés; Magnin, Delphine; Agathos, Spiros N

2015-10-01

Microbial laccases are powerful enzymes capable of degrading lignin and other recalcitrant compounds including endocrine disrupting chemicals (EDCs). Efficient EDC removal on an industrial scale requires robust, stable, easy to handle and cost-effective immobilized biocatalysts. In this direction, magnetic biocatalysts are attractive due to their easy separation through an external magnetic field. Recently, a bioinspired immobilization technique that mimics the natural biomineralization reactions in diatoms has emerged as a fast and versatile tool for generating robust, cheap, and highly stable (nano) biocatalysts. In this work, bioinspired formation of a biotitania matrix is triggered on the surface of magnetic particles in the presence of laccase in order to produce laccase-biotitania (lac-bioTiO2 ) biocatalysts suitable for environmental applications using a novel, fast and versatile enzyme entrapment technique. Highly active lac-bioTiO2 particles have been produced and the effect of different parameters (enzyme loading, titania precursor concentration, pH, duration of the biotitania formation, and laccase adsorption steps) on the apparent activity yield of these biocatalysts were evaluated, the concentration of the titania precursor being the most influential. The lac-bioTiO2 particles were able to catalyze the removal of bisphenol A, 17α-ethinylestradiol and diclofenac in a mixture of six model EDCs and retained 90% of activity after five reaction cycles and 60% after 10 cycles. © 2015 Wiley Periodicals, Inc.

Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

PubMed

DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

2015-05-01

Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Overproduction of laccase and pectinase by microbial associations in solid substrate fermentation.

PubMed

Stoilova, Ivanka; Krastanov, Albert

2008-04-01

The growth and the enzymatic production of two microbial fungal associations were studied: Aspergillus niger and Fusarium moniliforme and Trametes versicolor and Aspergillus niger. The synergistic interrelations between the species of the first mixed culture increased the biosynthesis of alpha-amylase and pectinase. T. versicolor and A. niger proved to be compatible partners in the overproduction of the enzyme laccase, whose synthesis surpassed 8.4 times the enzymatic level in the monoculture, with both of the mixed microbial populations cocultivation facilitating the amplified synthesis of enzymes rather than their growth acceleration. A further proof of the presence of synergism established by the cultures was the enzyme volumetric productivities in both of the mixed microbial cultures, which increased parallel to the rise in the combined biomass synthesis. The competent selection of compatible partners can adjust the desired enzymatic levels and compositions in mixed fungal systems aimed at a number of specified designations. Thus, a very high level of laccase production (97,600 IU/g dry weight) was achieved. The chosen fungal strains produce a variety of different enzymes, but first microbial association produces mainly amylase and pectinase, necessary for their growth, and second association produces mainly laccase and pectinase.

Refolding of laccase in dilution additive mode with copper-based ionic liquid.

PubMed

Bae, Sang-Woo; Ahn, Kihun; Koo, Yoon-Mo; Ha, Sung Ho

2013-11-01

Ionic liquids (ILs) are molten salts which do not crystallize at room temperature. Tunable physicochemical properties of ILs including hydrophobicity and polarity facilitate their applications in many biological processes. In this study, a copper-based IL was employed in order to enhance the refolding efficiency of laccase from Trametes versicolor which requires copper as a cofactor. When 1-ethyl-3-methylimidazolium trichlorocuprate ([EMIM][CuCl₃]) was added to refolding buffer instead of urea, the laccase refolding yield was improved more than 2.7 times compared to the conventional refolding buffer which contains urea. When the refolding of laccase was carried out at different temperatures (4, 25, and 37 °C), the highest refolding yield was obtained at 25 °C. At low temperature, two conflicting effects, i.e., suppression of the aggregate formation and decrease of folding rate, influence the protein refolding. In contrast, a copper-based IL did not enhance the refolding of lysozyme, a non-copper-containing protein. From these results, we can conclude that this copper-based IL, [EMIM][CuCl₃], was exclusively effective on the refolding process of a copper-containing protein.

Prediction and optimization of the laccase-mediated synthesis of the antimicrobial compound iodine (I2).

PubMed

Schubert, M; Fey, A; Ihssen, J; Civardi, C; Schwarze, F W M R; Mourad, S

2015-01-10

An artificial neural network (ANN) and genetic algorithm (GA) were applied to improve the laccase-mediated oxidation of iodide (I(-)) to elemental iodine (I2). Biosynthesis of iodine (I2) was studied with a 5-level-4-factor central composite design (CCD). The generated ANN network was mathematically evaluated by several statistical indices and revealed better results than a classical quadratic response surface (RS) model. Determination of the relative significance of model input parameters, ranking the process parameters in order of importance (pH>laccase>mediator>iodide), was performed by sensitivity analysis. ANN-GA methodology was used to optimize the input space of the neural network model to find optimal settings for the laccase-mediated synthesis of iodine. ANN-GA optimized parameters resulted in a 9.9% increase in the conversion rate. Copyright © 2014 Elsevier B.V. All rights reserved.

Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae.

PubMed

Yano, Akira; Kikuchi, Sayaka; Nakagawa, Yuko; Sakamoto, Yuichi; Sato, Toshitsugu

2009-01-01

The shiitake mushroom, Lentinula edodes, has an extracelluar secretory-type laccase, Lcc1, and a fruiting-body-accumulation-type laccase, Lcc4. We previously reported the production of Lcc1 by plant cells, but had difficulty producing Lcc4. Here, we report the production of Lcc1 and Lcc4 by Aspergillus oryzae and the extracellular secretory production of Lcc4 using a modified secretion signal peptide (SP) from Lcc1. Sp-Lcc4 produced by A. oryzae had biochemical activities similar to Lcc4 produced by L. edodes. Lcc1 did not react with beta-(3,4-dihydroxyphenol) alanine (DOPA), but Lcc4 from L. edodes and A. oryzae could oxidize DOPA. K(M) values for the substrates 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate), 2,6-dimethoxyphenol, guaiacol, pyrogallol, and catechol were similar for Lcc4 and Sp-Lcc4. In conclusion, a non-secretory-type fungal laccase is secreted into the culture media with its original enzymatic properties by exploiting modified secretory signal peptide. 2008 Elsevier GmbH.

Catechol Removal from Aqueous Media Using Laccase Immobilized in Different Macro- and Microreactor Systems.

PubMed

Tušek, Ana Jurinjak; Šalić, Anita; Zelić, Bruno

2017-08-01

Laccase belongs to the group of enzymes that are capable to catalyze the oxidation of phenols. Since the water is only by-product in laccase-catalyzed phenol oxidations, it is ideally "green" enzyme with many possible applications in different industrial processes. To make the oxidation process more sustainable in terms of biocatalyst consumption, immobilization of the enzyme is implemented in to the processes. Additionally, when developing a process, choice of a reactor type plays a significant role in the total outcome.In this study, the use of immobilized laccase from Trametes versicolor for biocatalytic catechol oxidation was explored. Two different methods of immobilization were performed and compared using five different reactor types. In order to compare different systems used for catechol oxidation, biocatalyst turnover number and turnover frequency were calculated. With low consumption of the enzyme and good efficiency, obtained results go in favor of microreactors with enzyme covalently immobilized on the microchannel surface.

Degradation of synthetic pollutants in real wastewater using laccase encapsulated in core-shell magnetic copper alginate beads.

PubMed

Le, Thao Thanh; Murugesan, Kumarasamy; Lee, Chung-Seop; Vu, Chi Huong; Chang, Yoon-Seok; Jeon, Jong-Rok

2016-09-01

Immobilization of laccase has been highlighted to enhance their stability and reusability in bioremediation. In this study, we provide a novel immobilization technique that is very suitable to real wastewater treatment. A perfect core-shell system composing copper alginate for the immobilization of laccase (Lac-beads) was produced. Additionally, nFe2O3 was incorporated for the bead recycling through magnetic force. The beads were proven to immobilize 85.5% of total laccase treated and also to be structurally stable in water, acetate buffer, and real wastewater. To test the Lac-beads reactivity, triclosan (TCS) and Remazol Brilliant Blue R (RBBR) were employed. The Lac-beads showed a high percentage of TCS removal (89.6%) after 8h and RBBR decolonization at a range from 54.2% to 75.8% after 4h. Remarkably, the pollutants removal efficacy of the Lac-beads was significantly maintained in real wastewater with the bead recyclability, whereas that of the corresponding free laccase was severely deteriorated. Copyright © 2016 Elsevier Ltd. All rights reserved.

Functional magnetic mesoporous nanoparticles for efficient purification of laccase from fermentation broth in magnetically stabilized fluidized bed.

PubMed

Wang, Feng; Guo, Chen; Liu, Chun-Zhao

2013-12-01

A magnetically stabilized fluidized bed (MSFB) with the Cu(2+)-chelated magnetic mesoporous silica nanoparticles (MMSNPs-Cu(2+)) was established to purify laccase directly from the fermentation broth of Trametes versicolor. The MMSNPs-Cu(2+) particles in the MSFB maintained a stable bed expansion of two to threefold at a flow rate of 120-180 cm/h. At the optimal magnetic field intensity of 120 Gs, both the maximal Bodenstein number and the smallest axial dispersion coefficient were achieved, which resulted in a stable fluidization stage. The dynamic binding capacity of laccase in the MSFB decreased from 192.5 to144.3 mg/g when the flow velocity through the bed increased from 44.2 to 69.8 cm/h. The MSFB with MMSNPs-Cu(2+) achieved efficient laccase purification from the fermentation broth with 62.4-fold purification of laccase and 108.9 % activity yield. These results provided an excellent platform for the application of these magnetic mesoporous nanoparticles integrated with the MSFB in developing novel protein purification process.

Novel Applications of Peroxidase

NASA Astrophysics Data System (ADS)

Rob, Abdul; Ball, Andrew S.; Tuncer, Munir; Wilson, Michael T.

1997-02-01

The article entitled "Novel Biocatalysts Will Work Even Better for Industry" published recently in this Journal (1) was informative and interesting. However it touched only briefly on the application of peroxidase as catalyst. Here, we would like to mention in more detail the novel applications of peroxidase in agricultural, paper pulp, water treatment, pharmaceutical, and medical situations. Firstly, the peroxidase isolated from Phanerochaete chyrosporium has been shown to detoxify herbicides such as atrazine to less toxic compounds and would certainly find potential application in agriculture (2). Secondly, the peroxidase produced by Streptomyces thermoviolaceus may find application in the paper pulp industry as a delignifying agent (3). Thirdly, it has been shown that extracellular peroxidase produced by Streptomyces avermitilis can remove the intense color from paper-mill effluent obtained after semichemical alkaline pulping of wheat straw (4), and thus this enzyme might find application as a catalyst in water treatment plants. Fourthly, the heme-containing horseradish peroxidase enzyme has been exploited in several diagnostic applications in pharmaceutics and medicine, such as the detection of human immunodeficiency virus and cystic fibrosis (5-10). Finally, recent work from our laboratory has suggested that thermophilic nonheme peroxidase produced by Thermomonospora fusca BD25 may find medical use in the diagnosis of myocardial infarction (11, 12). Literature Cited 1. Wiseman, A. J. Chem. Educ. 1996, 73, 55-58. 2. Mougin, C. Appl. Environ. Microbiol. 1994, 60, 705-708. 3. McCarthy A. J.; Peace, W.; Broda, P. Appl. Microbiol. Technol. 1985, 23, 238-244. 4. Hernandez, M; Rodriguez J; Soliveri, J; Copa, J. L; Perez, M. I; Arias, M. E. Appl. Environ. Microbiol. 1994, 60, 3909-3913. 5. Hopfer, S. M.; Aslanzadeh, J. Ann. Clin. Lab. Sci. 1995, 25, 475-480. 6. Suzuki, K; Iman, M. J. Virol. Methods 1995, 55, 347-356. 7. Nielsen, K. J. Immunoassay 1995, 16, 183-197. 8

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

PubMed

Copete-Pertuz, Ledys S; Plácido, Jersson; Serna-Galvis, Efraím A; Torres-Palma, Ricardo A; Mora, Amanda

2018-07-15

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 process 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. Furthermore, the applicability 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. Copyright © 2018 Elsevier B.V. All rights reserved.

"Chitin-specific" peroxidases in plants.

PubMed

Maksimov, I V; Cherepanova, E A; Khairullin, R M

2003-01-01

The activity of various plant peroxidases and the ability of their individual isoforms to bind chitin was studied. Some increase in peroxidase activity was observed in crude extracts in the presence of chitin. Activated peroxidases of some species fell in the fraction not sorbed on chitin and those of other species can bind chitin. Only anionic isoperoxidases from oat (Avena sativa), rice (Oryza sativa), horseradish (Armoracia rusticana), garden radish (Raphanus sativus var. radicula), peanut (Arachis hypogaea), and tobacco (Nicotiana tabacum Link et Otto) were sorbed on chitin. Both anionic and cationic isoforms from pea (Pisum sativum), galega(Galega orientalis), cucumber (Cucumis sativus), and zucchini (Cucurbita pepo L.) were sorbed on chitin. Peroxidase activation under the influence of chitin was correlated to the processes that occur during hypersensitive reaction and lignification of sites, in which pathogenic fungus penetrates into a plant. The role of chitin-specific isoperoxidases in inhibition of fungal growth and connection of this phenomenon with structural characteristics of isoperoxidases are also discussed.

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics.

PubMed

Bertrand, Thomas; Jolivalt, Claude; Briozzo, Pierre; Caminade, Eliane; Joly, Nathalie; Madzak, Catherine; Mougin, Christian

2002-06-11

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.

Advanced Synthesis of Conductive Polyaniline Using Laccase as Biocatalyst.

PubMed

de Salas, Felipe; Pardo, Isabel; Salavagione, Horacio J; Aza, Pablo; Amougi, Eleni; Vind, Jesper; Martínez, Angel T; Camarero, Susana

2016-01-01

Polyaniline is a conductive polymer with distinctive optical and electrical properties. Its enzymatic synthesis is an environmentally friendly alternative to the use of harsh oxidants and extremely acidic conditions. 7D5L, a high-redox potential laccase developed in our lab, is the biocatalyst of choice for the synthesis of green polyaniline (emeraldine salt) due to its superior ability to oxidize aniline and kinetic stability at the required polymerization conditions (pH 3 and presence of anionic surfactants) as compared with other fungal laccases. Doses as low as 7.6 nM of 7D5L catalyze the polymerization of 15 mM aniline (in 24 h, room temperature, 7% yield) in the presence of different anionic surfactants used as doping templates to provide linear and water-soluble polymers. Aniline polymerization was monitored by the increase of the polaron absorption band at 800 nm (typical for emeraldine salt). Best polymerization results were obtained with 5 mM sodium dodecylbenzenesulfonate (SDBS) as template. At fixed conditions (15 mM aniline and 5mM SDBS), polymerization rates obtained with 7D5L were 2.5-fold the rates obtained with commercial Trametes villosa laccase. Moreover, polyaniline yield was notably boosted to 75% by rising 7D5L amount to 0.15 μM, obtaining 1g of green polyaniline in 1L-reaction volume. The green polymer obtained with the selected system (7D5L/SDBS) holds excellent electrochemical and electro-conductive properties displayed in water-dispersible nanofibers, which is advantageous for the nanomaterial to be readily cast into uniform films for different applications.

Advanced Synthesis of Conductive Polyaniline Using Laccase as Biocatalyst

PubMed Central

de Salas, Felipe; Pardo, Isabel; Salavagione, Horacio J.; Aza, Pablo; Amougi, Eleni; Vind, Jesper; Martínez, Angel T.; Camarero, Susana

2016-01-01

Polyaniline is a conductive polymer with distinctive optical and electrical properties. Its enzymatic synthesis is an environmentally friendly alternative to the use of harsh oxidants and extremely acidic conditions. 7D5L, a high-redox potential laccase developed in our lab, is the biocatalyst of choice for the synthesis of green polyaniline (emeraldine salt) due to its superior ability to oxidize aniline and kinetic stability at the required polymerization conditions (pH 3 and presence of anionic surfactants) as compared with other fungal laccases. Doses as low as 7.6 nM of 7D5L catalyze the polymerization of 15 mM aniline (in 24 h, room temperature, 7% yield) in the presence of different anionic surfactants used as doping templates to provide linear and water-soluble polymers. Aniline polymerization was monitored by the increase of the polaron absorption band at 800 nm (typical for emeraldine salt). Best polymerization results were obtained with 5 mM sodium dodecylbenzenesulfonate (SDBS) as template. At fixed conditions (15 mM aniline and 5mM SDBS), polymerization rates obtained with 7D5L were 2.5-fold the rates obtained with commercial Trametes villosa laccase. Moreover, polyaniline yield was notably boosted to 75% by rising 7D5L amount to 0.15 μM, obtaining 1g of green polyaniline in 1L-reaction volume. The green polymer obtained with the selected system (7D5L/SDBS) holds excellent electrochemical and electro-conductive properties displayed in water-dispersible nanofibers, which is advantageous for the nanomaterial to be readily cast into uniform films for different applications. PMID:27741301

Purification and characterization of the extracellular laccase produced by Trametes polyzona WR710-1 under solid-state fermentation.

PubMed

Chairin, Thanunchanok; Nitheranont, Thitinard; Watanabe, Akira; Asada, Yasuhiko; Khanongnuch, Chartchai; Lumyong, Saisamorn

2014-01-01

Laccase from Trametes polyzona WR710-1 was produced under solid-state fermentation using the peel from the Tangerine orange (Citrus reticulata Blanco) as substrate, and purified to homogeneity. This laccase was found to be a monomeric protein with a molecular mass of about 71 kDa estimated by SDS-PAGE. The optimum pH was 2.0 for ABTS, 4.0 for L-DOPA, guaiacol, and catechol, and 5.0 for 2,6-DMP. The K(m) value of the enzyme for the substrate ABTS was 0.15 mM, its corresponding V(max) value was 1.84 mM min(-1), and the k(cat)/K(m) value was about 3960 s(-1)  mM(-1). The enzyme activity was stable between pH 6.0 and 8.0, at temperatures of up to 40 °C. The laccase was inhibited by more than 50% in the presence of 20 mM NaCl, by 95% at 5 mM of Fe(2+), and it was completely inhibited by 0.1 mM NaN(3). The N-terminal amino acid sequence of this laccase is AVTPVADLQISNAGISPDTF, which is highly similar to those of laccases from other white-rot basidiomycetes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Refolding of laccase from Trametes versicolor using aqueous two phase systems: Effect of different additives.

PubMed

Sánchez-Trasviña, Calef; Mayolo-Deloisa, Karla; González-Valdez, José; Rito-Palomares, Marco

2017-07-21

Protein refolding is a strategy used to obtain active forms of proteins from inclusion bodies. On its part, laccase is an enzyme with potential for different biotechnological applications but there are few reports regarding its refolding which in many cases is considered inefficient due to the poor obtained refolding yields. Aqueous Two-Phase Systems (ATPS) have been used for the refolding of proteins getting acceptable recovery percentages since PEG presents capacity to avoid protein aggregation. In this work, 48 PEG-phosphate ATPS were analyzed to study the impact of different parameters (i.e. tie line length (TLL), volume ratio (V R ) and PEG molecular weight) upon the recovery and refolding of laccase. Additionally, since laccase is a metalloprotein, the use of additives (individually and in mixture) was studied with the aim of favoring refolding. Results showed that laccase presents a high affinity for the PEG-rich phase obtaining recovery values of up to 90%. Such affinity increases with increasing TLL and decreases when PEG molecular weight and V R increase. In denatured state, this PEG-rich phase affinity decreases drastically. However, the use of additives such as l-cysteine, glutathione oxidized, cysteamine and Cu +2 was critical in improving refolding yield values up to 100%. The best conditions for the refolding of laccase were obtained using the PEG 400gmol -1 , TLL 45% w/w, V R 3 ATPS and a mixture of 2.5mM cysteamine with 1mM Cu +2 . To our knowledge, this is the first time that the use of additives and the behavior of the mixture of such additives to enhance refolding performance in ATPS is reported. Copyright © 2017 Elsevier B.V. All rights reserved.

Electron transfer and reaction mechanism of laccases.

PubMed

Jones, Stephen M; Solomon, Edward I

2015-03-01

Laccases are part of the family of multicopper oxidases (MCOs), which couple the oxidation of substrates to the four electron reduction of O2 to H2O. MCOs contain a minimum of four Cu's divided into Type 1 (T1), Type 2 (T2), and binuclear Type 3 (T3) Cu sites that are distinguished based on unique spectroscopic features. Substrate oxidation occurs near the T1, and electrons are transferred approximately 13 Å through the protein via the Cys-His pathway to the T2/T3 trinuclear copper cluster (TNC), where dioxygen reduction occurs. This review outlines the electron transfer (ET) process in laccases, and the mechanism of O2 reduction as elucidated through spectroscopic, kinetic, and computational data. Marcus theory is used to describe the relevant factors which impact ET rates including the driving force, reorganization energy, and electronic coupling matrix element. Then, the mechanism of O2 reaction is detailed with particular focus on the intermediates formed during the two 2e(-) reduction steps. The first 2e(-) step forms the peroxide intermediate, followed by the second 2e(-) step to form the native intermediate, which has been shown to be the catalytically relevant fully oxidized form of the enzyme.

The novel role of fungal intracellular laccase: used to screen hybrids between Hypsizigus marmoreus and Clitocybe maxima by protoplasmic fusion.

PubMed

Xu, Jianzhong; Zhang, Junlan; Zhang, Weiguo; Hu, Kaihui

2012-08-01

Laccase has been proved important in decolorization of Remazol Brilliant Blue R (RBBR), oxidation of 2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, lignin degradation and fruiting-body formation. The decolorization of RBBR by laccase was firstly used to screen protoplast fusants. Fusants were obtained by protoplast fusion between the strains of Hypsizigus marmoreus and Clitocybe maxima, and two fusants (IM1 and IIIM5) were screened on PDA medium containing RBBR. These fusants were significant higher in laccase activity than H. marmoreus, nearly 413 and 395 times, respectively. Their hyphal growth rates were also remarkable higher than H. marmoreus, nearly 1.5 and 1.4 times, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed these fusants contained the laccase, and the molecular mass of the laccase was consistent with the laccase of C. maxima, nearly 62 kDa. The pileus color of the IM1 and IIIM5 also showed partial recombined characteristics comparing to the parental strains, while biological efficiency ratios were prominent higher than that of H. marmoreus, up to 14.58 and 10.87 %, respectively. Randomly amplified polymorphic DNA bands of fusants not only were similar to parental bands, but presented new non-parental bands. Using the Unweighted pair-group method together with mathematic averages method to gain a dendrogram, in which the fusants showed intra-cluster variations. Significantly, H. marmoreus was the dominant parent, while C. maxima were distant from the fusants. The differences among IM1, IIIM5 and H. marmoreus, and the similarities among IM1, IIIM5 and C. maxima indicated IM1 and IIIM5 were somatic hybrids of H. marmoreus and C. maxima. Accordingly, it is feasible to use laccase to screen fusants of H. marmoreus and C. maxima.

Insights into the development of fungal biomarkers for metal ecotoxicity assessment: case of Trametes versicolor exposed to copper.

PubMed

Lebrun, Jérémie D; Trinsoutrot-Gattin, Isabelle; Laval, Karine; Mougin, Christian

2010-04-01

The relationship between the physiological state of fungi and the response of their functional system to metals is not known, limiting the use of fungal enzymes as tools for assessing metal ecotoxicity in terrestrial ecosystems. The present study attempts to establish how the development phases modulate the secretion of enzymes in the filamentous fungus Trametes versicolor after exposure to Cu. For that purpose, extracellular hydrolases (acid and alkaline phosphatases, aryl-sulfatase, beta-glucosidase, beta-galactosidase, and N-acetyl-beta-glucosaminidase) and oxidoreductases (laccase, manganese and lignin peroxidases) were monitored in liquid cultures for 2 weeks. Copper was added during either the growth or the stationary phases at 20 or 200 ppm. Results of the present study showed that Cu at the highest concentration modifies the secretion of enzymes, regardless of the development phase to which the fungus was exposed. However, the sensitivity of enzyme responses to Cu depended on the phase development and the type of secreted enzyme. In a general way, the production of hydrolases was decreased by Cu, whereas that of oxidoreductases was highly increased. Furthermore, lignin peroxidase was not detected in control cultures and was specifically produced in the presence of Cu. In conclusion, fungal oxidoreductases may be enzymatic biomarkers of copper exposure for ecotoxicity assessment. (c) 2009 SETAC.

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

PubMed Central

da Luz, José Maria Rodrigues; Nunes, Mateus Dias; Paes, Sirlaine Albino; Torres, Denise Pereira; de Cássia Soares da Silva, Marliane; Kasuya, Maria Catarina Megumi

2012-01-01

The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse). The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase) and hydrolytic enzymes (cellulases, xylanases and tanases). Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6). These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes. PMID:24031982

Evaluating the potential of immobilized bacterial consortium for black liquor biodegradation.

PubMed

Paliwal, Rashmi; Uniyal, Shivani; Rai, J P N

2015-05-01

Two indigenous bacterial strains, Bacillus megaterium ETLB-1 (accession no. KC767548) and Pseudomonas plecoglossicida ETLB-3 (accession no. KC767547), isolated from soil contaminated with paper mill effluent, were co-immobilized on corncob cubes to investigate their biodegradation potential against black liquor (BL). Results exhibit conspicuous reduction in color and lignin of BL upto 913.46 Co-Pt and 531.45 mg l(-1), respectively. Reduction in chlorophenols up to 12 mg l(-1) was recorded with highest release of chloride ions, i.e., 1290 mg l(-1). Maximum enzyme activity for lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (LAC) was recorded as 5.06, 8.13, and 8.23 U ml(-1), respectively, during the treatment. Scanning electron microscopy (SEM) revealed successful immobilization of bacterial strains in porous structures of biomaterial. Gas chromatography/mass spectroscopy (GC/MS) showed formation of certain low molecular weight metabolites such as 4-hydroxy-benzoic acid, 3-hydroxy-4-methoxybenzaldehyde, ferulic acid, and t-cinnamic acid and removal of majority of the compounds (such as teratogenic phthalate derivatives) during the period of treatment. Results demonstrated that the indigenous bacterial consortium possesses excellent decolorization and lignin degradation capability which enables its commercial utilization in effluents treatment system.

Screening of Biodegradable Function of Indigenous Ligno-degrading Mushroom Using Dyes

PubMed Central

Cho, Soo-Muk; Seok, Soon-Ja; Kong, Won-Sik; Kim, Gyu-Hyun; Sung, Jae-Mo

2009-01-01

our findings using Poly-R 478 dye to evaluate the PAH-degrading activity of the identified strains, Coriolus versicolor, MKACC 52492 was selected as a favorable strain. Coriolus versicolor, which was collected from Mt. Yeogi in Suwon, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). PMID:23983508

Comparative Examination of the Olive Mill Wastewater Biodegradation Process by Various Wood-Rot Macrofungi

PubMed Central

Koutrotsios, Georgios; Zervakis, Georgios I.

2014-01-01

Olive mill wastewater (OMW) constitutes a major cause of environmental pollution in olive-oil producing regions. Sixty wood-rot macrofungi assigned in 43 species were evaluated for their efficacy to colonize solidified OMW media at initially established optimal growth temperatures. Subsequently eight strains of the following species were qualified: Abortiporus biennis, Ganoderma carnosum, Hapalopilus croceus, Hericium erinaceus, Irpex lacteus, Phanerochaete chrysosporium, Pleurotus djamor, and P. pulmonarius. Fungal growth in OMW (25%v/v in water) resulted in marked reduction of total phenolic content, which was significantly correlated with the effluent's decolorization. A. biennis was the best performing strain (it decreased phenolics by 92% and color by 64%) followed by P. djamor and I. lacteus. Increase of plant seeds germination was less pronounced evidencing that phenolics are only partly responsible for OMW's phytotoxicity. Laccase production was highly correlated with all three biodegradation parameters for H. croceus, Ph. chrysosporium, and Pleurotus spp., and so were manganese-independent and manganese dependent peroxidases for A. biennis and I. lacteus. Monitoring of enzymes with respect to biomass production indicated that Pleurotus spp., H. croceus, and Ph. chrysosporium shared common patterns for all three activities. Moreover, generation of enzymes at the early biodegradation stages enhanced the efficiency of OMW treatment. PMID:24987685

Production of laccase from Trametes versicolor by solid-state fermentation using olive leaves as a phenolic substrate.

PubMed

Aydinoğlu, Tuğba; Sargin, Sayit

2013-02-01

The aim of the present study was to investigate whether olive leaves were feasible as a substrate for laccase production by the white-rot fungus Trametes versicolor FPRL 28A INI under solid-state fermentation conditions. Different experiments were conducted to select the variables that allow obtaining high levels of laccase activity. In particular, the effects of the initial moisture content, substrate particle size, supplementation with inorganic and organic nitrogen sources were evaluated. Highest laccase activity (276.62 ± 25.67 U/g dry substrate) was achieved with 80 % initial moisture content and 1.4-1.6 mm particle size of the substrate supplemented with yeast extract (1 % (w/w) nitrogen). Such a high activity was obtained without any addition of inducers.

From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase.

PubMed Central

Jespersen, H M; Kjaersgård, I V; Ostergaard, L; Welinder, K G

1997-01-01

Ascorbate peroxidases are haem proteins that efficiently scavenge H2O2 in the cytosol and chloroplasts of plants. Database analyses retrieved 52 expressed sequence tags coding for Arabidopsis thaliana ascorbate peroxidases. Complete sequencing of non-redundant clones revealed three novel types in addition to the two cytosol types described previously in Arabidopsis. Analysis of sequence data available for all plant ascorbate peroxidases resulted in the following classification: two types of cytosol soluble ascorbate peroxidase designated cs1 and cs2; three types of cytosol membrane-bound ascorbate peroxidase, namely cm1, bound to microbodies via a C-terminal membrane-spanning segment, and cm2 and cm3, both of unknown location; two types of chloroplast ascorbate peroxidase with N-terminal transit sequences, the stromal ascorbate peroxidase (chs), and the thylakoid-bound ascorbate peroxidase showing a C-terminal transmembrane segment and designated cht. Further comparison of the patterns of conserved residues and the crystal structure of pea ascorbate peroxidase showed that active site residues are conserved, and three peptide segments implicated in interaction with reducing substrate are similar, excepting cm2 and cm3 types. A change of Phe-175 in cytosol types to Trp-175 in chloroplast types might explain the greater ascorbate specificity of chloroplast compared with cytosol ascorbate peroxidases. Residues involved in homodimeric subunit interaction are conserved only in cs1, cs2 and cm1 types. The proximal cation (K+)-binding site observed in pea ascorbate peroxidase seems to be conserved. In addition, cm1, cm2, cm3, chs and cht ascorbate peroxidases contain Asp-43, Asn-57 and Ser-59, indicative of a distal monovalent cation site. The data support the hypothesis that present-day peroxidases evolved by an early gene duplication event. PMID:9291097

Laccase SilA from Streptomyces ipomoeae CECT 3341, a key enzyme for the degradation of lignin from agricultural residues?

PubMed Central

Blánquez, Alba; Ball, Andrew S.; González-Pérez, José Antonio; Jiménez-Morillo, Nicasio T.; González-Vila, Francisco; Arias, M. Enriqueta

2017-01-01

The role of laccase SilA produced by Streptomyces ipomoeae CECT 3341 in lignocellulose degradation was investigated. A comparison of the properties and activities of a laccase-negative mutant strain (SilA−) with that of the wild-type was studied in terms of their ability to degrade lignin from grass lignocellulose. The yields of solubilized lignin (acid precipitable polymeric lignin, APPL) obtained from wheat straw by both strains in Solid State Fermentation (SSF) conditions demonstrated the importance of SilA laccase in lignin degradation with the wild-type showing 5-fold more APPL produced compared with the mutant strain (SilA−). Analytical pyrolysis and FT-IR (Fourier Transform Infrared Spectroscopy) confirmed that the APPL obtained from the substrate fermented by wild-type strain was dominated by lignin derived methoxyphenols whereas those from SilA− and control APPLs were composed mainly of polysaccharides. This is the first report highlighting the role of this laccase in lignin degradation. PMID:29112957

Laccase SilA from Streptomyces ipomoeae CECT 3341, a key enzyme for the degradation of lignin from agricultural residues?

PubMed

Blánquez, Alba; Ball, Andrew S; González-Pérez, José Antonio; Jiménez-Morillo, Nicasio T; González-Vila, Francisco; Arias, M Enriqueta; Hernández, Manuel

2017-01-01

The role of laccase SilA produced by Streptomyces ipomoeae CECT 3341 in lignocellulose degradation was investigated. A comparison of the properties and activities of a laccase-negative mutant strain (SilA-) with that of the wild-type was studied in terms of their ability to degrade lignin from grass lignocellulose. The yields of solubilized lignin (acid precipitable polymeric lignin, APPL) obtained from wheat straw by both strains in Solid State Fermentation (SSF) conditions demonstrated the importance of SilA laccase in lignin degradation with the wild-type showing 5-fold more APPL produced compared with the mutant strain (SilA-). Analytical pyrolysis and FT-IR (Fourier Transform Infrared Spectroscopy) confirmed that the APPL obtained from the substrate fermented by wild-type strain was dominated by lignin derived methoxyphenols whereas those from SilA- and control APPLs were composed mainly of polysaccharides. This is the first report highlighting the role of this laccase in lignin degradation.

Development of recombinant biocatalysts expressing laccase enzyme from Trametes versicolor

USDA-ARS?s Scientific Manuscript database

Increasing demands for sustainable energy necessitate the use of biorenewable sources such as agricultural and forestry wastes. A major challenge of using lignocellulosic biomass for biofuel production is the recalcitrant nature of the lignin structure. Laccase is a multi-copper oxidase that catal...

Laccase Gene Expression and Vinasse Biodegradation by Trametes hirsuta Strain Bm-2.

PubMed

Tapia-Tussell, Raúl; Pérez-Brito, Daisy; Torres-Calzada, Claudia; Cortés-Velázquez, Alberto; Alzate-Gaviria, Liliana; Chablé-Villacís, Rubí; Solís-Pereira, Sara

2015-08-19

Vinasse is the dark-colored wastewater that is generated by bioethanol distilleries from feedstock molasses. The vinasse that is generated from molasses contains high amounts of pollutants, including phenolic compounds and melanoindin. The goal of this work was to study the expression of laccase genes in the Trametes hirsuta strain Bm-2, isolated in Yucatan, Mexico, in the presence of phenolic compounds, as well as its effectiveness in removing colorants from vinasse. In the presence of all phenolic compounds tested (guaiacol, ferulic acid, and vanillic acid), increased levels of laccase-encoding mRNA were observed. Transcript levels in the presence of guaiacol were 40 times higher than those in the control. The lcc1 and lcc2 genes of T. hirsuta were differentially expressed; guaiacol and vanillin induced the expression of both genes, whereas ferulic acid only induced the expression of lcc2. The discoloration of vinasse was concomitant with the increase in laccase activity. The highest value of enzyme activity (2543.7 U/mL) was obtained in 10% (v/v) vinasse, which corresponded to a 69.2% increase in discoloration. This study demonstrates the potential of the Bm-2 strain of T. hirsuta for the biodegradation of vinasse.

Biochemical investigation of kraft lignin degradation by Pandoraea sp. B-6 isolated from bamboo slips.

PubMed

Shi, Yan; Chai, Liyuan; Tang, Chongjian; Yang, Zhihui; Zheng, Yu; Chen, Yuehui; Jing, Qingxiu

2013-12-01

Kraft lignin (KL) is the major pollutant in black liquor. The bacterial strain Pandoraea sp. B-6 was able to degrade KL without any co-substrate under high alkaline conditions. At least 38.2 % of chemical oxygen demand and 41.6 % of color were removed in 7 days at concentrations from 1 to 6 g L(-1). The optimum pH for KL degradation was 10 and the optimum temperature was 30 °C. The greatest activities of 2,249.2 U L(-1) for manganese peroxidase and 1,120.6 U L(-1) for laccase were detected on the third and fifth day at pH 10, respectively. Many small molecules, such as cinnamic acid, ferulic acid, 2-hydroxy benzyl alcohol, and vanillyl methyl ketone, were formed during the period of KL degradation based on GC-MS analysis. These results indicate that this strain has great potential for biotreatment of black liquor.

Enhanced biodegradation of asphalt in the presence of Tween surfactants, Mn(2+) and H2O2 by Pestalotiopsis sp. in liquid medium and soil.

PubMed

Yanto, Dede Heri Yuli; Tachibana, Sanro

2014-05-01

Asphalt and fractions thereof can contaminate water and soil environments. Forming as residues in distillation products in crude oil refineries, asphalts consist mostly of asphaltene instead of aliphatics, aromatics, and resins. The high asphaltene content might be responsible for the decrease in bioavailability to microorganisms and therefore reduce the biodegradability of asphalt in the environment. In this study, the effect on asphalt biodegradation by Pestalotiopsis sp. in liquid medium and soil of nonionic Tween surfactants in the presence of Mn2+ and H2O2 was examined. The degradation was enhanced by Tween 40 or Tween 80 (0.1%) in the presence of Mn2+ (1 mM) and H2O2 (0.05 mM). A Tween surfactant, Mn2+, and H2O2 can overcome bioavailability-mediated constraints and increase ligninolytic activities, particularly manganese peroxidase and laccase activities. The study is significant for the bioremediation of asphalt and/or viscous-crude oil-contaminated environments. Copyright © 2013 Elsevier Ltd. All rights reserved.

Long term repeated prescribed burning increases evenness in the basidiomycete laccase gene pool in forest soils.

PubMed

Artz, Rebekka R E; Reid, Eileen; Anderson, Ian C; Campbell, Colin D; Cairney, John W G

2009-03-01

Repeated prescribed burning alters the biologically labile fraction of nutrients and carbon of soil organic matter (SOM). Using a long-term (30 years) repeated burning experiment where burning has been carried out at a 2- or 4-year frequency, we analysed the effect of prescribed burning on gross potential C turnover rates and phenol oxidase activity in relation to shifts in SOM composition as observed using Fourier-transform infrared spectroscopy. In tandem, we assessed the genetic diversity of basidiomycete laccases. While the overall effect of burning was a decline in phenol oxidase activity, Shannon diversity and evenness of laccases was significantly higher in burned sites. Co-correspondence analysis of SOM composition and laccase operational taxonomic unit frequency data also suggested a strong correlation. While this correlation could indicate that the observed increase in laccase genetic diversity due to burning is due to increased resource diversity, a temporal replacement of the most abundant members of the assembly by an otherwise dormant pool of fungi cannot be excluded. As such, our results fit the intermediate disturbance hypothesis. Effects were stronger in plots burned in 2-year rotations, suggesting that the 4-year burn frequency may be a more sustainable practice to ensure the long-term stability of C cycling in such ecosystems.

Enhanced production of thermostable laccases from a native strain of Pycnoporus sanguineus using central composite design*

PubMed Central

Ramírez-Cavazos, Leticia I.; Junghanns, Charles; Nair, Rakesh; Cárdenas-Chávez, Diana L.; Hernández-Luna, Carlos; Agathos, Spiros N.; Parra, Roberto

2014-01-01

The production of thermostable laccases from a native strain of the white-rot fungus Pycnoporus sanguineus isolated in Mexico was enhanced by testing different media and a combination of inducers including copper sulfate (CuSO4). The best conditions obtained from screening experiments in shaken flasks using tomato juice, CuSO4, and soybean oil were integrated in an experimental design. Enhanced levels of tomato juice as the medium, CuSO4 and soybean oil as inducers (36.8% (v/v), 3 mmol/L, and 1% (v/v), respectively) were determined for 10 L stirred tank bioreactor runs. This combination resulted in laccase titer of 143 000 IU/L (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), pH 3.0), which represents the highest activity so far reported for P. sanguineus in a 10-L fermentor. Other interesting media resulting from the screening included glucose-bactopeptone which increased laccase activity up to 20 000 IU/L, whereas the inducers Acid Blue 62 and Reactive Blue 19 enhanced enzyme production in this medium 10 times. Based on a partial characterization, the laccases of this strain are especially promising in terms of thermostability (half-life of 6.1 h at 60 °C) and activity titers. PMID:24711355

Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria

PubMed Central

Vandieken, Verona; Pester, Michael; Finke, Niko; Hyun, Jung-Ho; Friedrich, Michael W; Loy, Alexander; Thamdrup, Bo

2012-01-01

Dissimilatory manganese reduction dominates anaerobic carbon oxidation in marine sediments with high manganese oxide concentrations, but the microorganisms responsible for this process are largely unknown. In this study, the acetate-utilizing manganese-reducing microbiota in geographically well-separated, manganese oxide-rich sediments from Gullmar Fjord (Sweden), Skagerrak (Norway) and Ulleung Basin (Korea) were analyzed by 16S rRNA-stable isotope probing (SIP). Manganese reduction was the prevailing terminal electron-accepting process in anoxic incubations of surface sediments, and even the addition of acetate stimulated neither iron nor sulfate reduction. The three geographically distinct sediments harbored surprisingly similar communities of acetate-utilizing manganese-reducing bacteria: 16S rRNA of members of the genera Colwellia and Arcobacter and of novel genera within the Oceanospirillaceae and Alteromonadales were detected in heavy RNA-SIP fractions from these three sediments. Most probable number (MPN) analysis yielded up to 106 acetate-utilizing manganese-reducing cells cm−3 in Gullmar Fjord sediment. A 16S rRNA gene clone library that was established from the highest MPN dilutions was dominated by sequences of Colwellia and Arcobacter species and members of the Oceanospirillaceae, supporting the obtained RNA-SIP results. In conclusion, these findings strongly suggest that (i) acetate-dependent manganese reduction in manganese oxide-rich sediments is catalyzed by members of taxa (Arcobacter, Colwellia and Oceanospirillaceae) previously not known to possess this physiological function, (ii) similar acetate-utilizing manganese reducers thrive in geographically distinct regions and (iii) the identified manganese reducers differ greatly from the extensively explored iron reducers in marine sediments. PMID:22572639

Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria.

PubMed

Vandieken, Verona; Pester, Michael; Finke, Niko; Hyun, Jung-Ho; Friedrich, Michael W; Loy, Alexander; Thamdrup, Bo

2012-11-01

Dissimilatory manganese reduction dominates anaerobic carbon oxidation in marine sediments with high manganese oxide concentrations, but the microorganisms responsible for this process are largely unknown. In this study, the acetate-utilizing manganese-reducing microbiota in geographically well-separated, manganese oxide-rich sediments from Gullmar Fjord (Sweden), Skagerrak (Norway) and Ulleung Basin (Korea) were analyzed by 16S rRNA-stable isotope probing (SIP). Manganese reduction was the prevailing terminal electron-accepting process in anoxic incubations of surface sediments, and even the addition of acetate stimulated neither iron nor sulfate reduction. The three geographically distinct sediments harbored surprisingly similar communities of acetate-utilizing manganese-reducing bacteria: 16S rRNA of members of the genera Colwellia and Arcobacter and of novel genera within the Oceanospirillaceae and Alteromonadales were detected in heavy RNA-SIP fractions from these three sediments. Most probable number (MPN) analysis yielded up to 10(6) acetate-utilizing manganese-reducing cells cm(-3) in Gullmar Fjord sediment. A 16S rRNA gene clone library that was established from the highest MPN dilutions was dominated by sequences of Colwellia and Arcobacter species and members of the Oceanospirillaceae, supporting the obtained RNA-SIP results. In conclusion, these findings strongly suggest that (i) acetate-dependent manganese reduction in manganese oxide-rich sediments is catalyzed by members of taxa (Arcobacter, Colwellia and Oceanospirillaceae) previously not known to possess this physiological function, (ii) similar acetate-utilizing manganese reducers thrive in geographically distinct regions and (iii) the identified manganese reducers differ greatly from the extensively explored iron reducers in marine sediments.

Tetra(p-tolyl)borate-functionalized solvent polymeric membrane: a facile and sensitive sensing platform for peroxidase and peroxidase mimetics.

PubMed

Wang, Xuewei; Qin, Wei

2013-07-22

The determination of peroxidase activities is the basis for enzyme-labeled bioaffinity assays, peroxidase-mimicking DNAzymes- and nanoparticles-based assays, and characterization of the catalytic functions of peroxidase mimetics. Here, a facile, sensitive, and cost-effective solvent polymeric membrane-based peroxidase detection platform is described that utilizes reaction intermediates with different pKa values from those of substrates and final products. Several key but long-debated intermediates in the peroxidative oxidation of o-phenylenediamine (o-PD) have been identified and their charge states have been estimated. By using a solvent polymeric membrane functionalized by an appropriate substituted tetraphenylborate as a receptor, those cationic intermediates could be transferred into the membrane from the aqueous phase to induce a large cationic potential response. Thus, the potentiometric indication of the o-PD oxidation catalyzed by peroxidase or its mimetics can be fulfilled. Horseradish peroxidase has been detected with a detection limit at least two orders of magnitude lower than those obtained by spectrophotometric techniques and traditional membrane-based methods. As an example of peroxidase mimetics, G-quadruplex DNAzymes were probed by the intermediate-sensitive membrane and a label-free thrombin detection protocol was developed based on the catalytic activity of the thrombin-binding G-quadruplex aptamer. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by laccase from Trametes versicolor covalently immobilized on amino-functionalized SBA-15.

PubMed

Bautista, Luis Fernando; Morales, Gabriel; Sanz, Raquel

2015-10-01

A covalent immobilization method based on glutaraldehyde and amino-functionalized SBA-15 supports has been successfully applied to covalently and stably immobilize laccase from Trametes versicolor. The resultant biocatalysts displayed high incorporation yields of enzyme and led to excellent biodegradation rates of selected HPAs models, i.e. naphthalene, phenanthrene and anthracene, in water. The nature of the hydrocarbon chain accompanying the amino group has been shown as determinant for the immobilization as well as for the activity and reusability of the materials. Thus, alkyl moieties displayed higher enzyme loadings than phenyl moieties, being more adequate the larger n-butyl tethering residue likely due to its higher mobility. Using the aminobutyl-based laccase-SBA-15, 82%, 73%, and 55% conversion of naphthalene, phenanthrene and anthracene, respectively, were achieved after 48 h, very close to the values obtained with free laccase under the same reaction conditions. On the other hand, aminopropyl-based laccase-SBA-15 biocatalysts displayed the best reusability properties, retaining higher activity after four repeated uses than the corresponding aminobutyl-based materials. Copyright © 2015 Elsevier Ltd. All rights reserved.

Influence of treatment conditions on the oxidation of micropollutants by Trametes versicolor laccase.

PubMed

Margot, Jonas; Maillard, Julien; Rossi, Luca; Barry, D A; Holliger, Christof

2013-09-25

Many organic compounds present at low concentrations in municipal wastewater, such as various pharmaceuticals and biocides, are recalcitrant in conventional wastewater treatment plants (WWTPs). To improve their biodegradation, oxidoreductase enzymes such as laccases were tested. The goal was to find optimal conditions for the transformation of two anti-inflammatory pharmaceuticals (diclofenac (DFC) and mefenamic acid (MFA)), one biocide (triclosan (TCN)) and one plastic additive (bisphenol A (BPA)) by Trametes versicolor laccase. Experiments were conducted in spiked solutions at different pH values (from 3 to 9), enzyme concentrations (70-1400 Ul(-1)), reaction times (0-26 hours) and temperatures (10, 25 and 40°C) following a Doehlert experimental design. A semi-empirical model was developed to understand better the combined effects of the four factors and to determine optimal values. This model was able to fit well the experimental data (R(2)>0.97) and showed good predictive ability. All four factors had a significant effect on the micropollutant oxidation with the greatest influence shown by pH. Results for single compounds were different from those obtained for mixtures of micropollutants. For instance, DFC transformation occurred at much higher rates in mixtures under alkaline conditions. Optimal conditions were compound-dependent, but were found to be between pH 4.5 to 6.5 and between 25°C to more than 40°C. A laccase concentration of 730 Ul(-1) was sufficient to obtain a high removal rate (>90%) of the four individual compounds (range of times: 40 min to 5 hours), showing the potential of laccases to improve biodegradation of environmentally persistent compounds. Copyright © 2013 Elsevier B.V. All rights reserved.

Manganese Deficiency in Chlamydomonas Results in Loss of Photosystem II and MnSOD Function, Sensitivity to Peroxides, and Secondary Phosphorus and Iron Deficiency1[W][OA

PubMed Central

Allen, Michael D.; Kropat, Janette; Tottey, Stephen; Del Campo, José A.; Merchant, Sabeeha S.

2007-01-01

For photoheterotrophic growth, a Chlamydomonas reinhardtii cell requires at least 1.7 × 107 manganese ions in the medium. At lower manganese ion concentrations (typically <0.5 μm), cells divide more slowly, accumulate less chlorophyll, and the culture reaches stationary phase at lower cell density. Below 0.1 μm supplemental manganese ion in the medium, the cells are photosynthetically defective. This is accompanied by decreased abundance of D1, which binds the Mn4Ca cluster, and release of the OEE proteins from the membrane. Assay of Mn superoxide dismutase (MnSOD) indicates loss of activity of two isozymes in proportion to the Mn deficiency. The expression of MSD3 through MSD5, encoding various isoforms of the MnSODs, is up-regulated severalfold in Mn-deficient cells, but neither expression nor activity of the plastid Fe-containing superoxide dismutase is changed, which contrasts with the dramatically increased MSD3 expression and plastid MnSOD activity in Fe-deficient cells. Mn-deficient cells are selectively sensitive to peroxide but not methyl viologen or Rose Bengal, and GPXs, APX, and MSRA2 genes (encoding glutathione peroxidase, ascorbate peroxidase, and methionine sulfoxide reductase 2) are slightly up-regulated. Elemental analysis indicates that the Mn, Fe, and P contents of cells in the Mn-deficient cultures were reduced in proportion to the deficiency. A natural resistance-associated macrophage protein homolog and one of five metal tolerance proteins were induced in Mn-deficient cells but not in Fe-deficient cells, suggesting that the corresponding gene products may be components of a Mn2+-selective assimilation pathway. PMID:17085511

In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

PubMed

Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

2012-05-01

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

Studying the effects of laccase treatment in a softwood dissolving pulp: cellulose reactivity and crystallinity.

PubMed

Quintana, Elisabet; Valls, Cristina; Barneto, Agustín G; Vidal, Teresa; Ariza, José; Roncero, M Blanca

2015-03-30

An enzymatic biobleaching sequence (LVAQPO) using a laccase from Trametes villosa in combination with violuric acid (VA) and then followed by a pressurized hydrogen peroxide treatment (PO) was developed and found to give high bleaching properties and meet dissolving pulp requirements: high brightness, low content of hemicellulose, satisfactory pulp reactivity, no significant cellulose degradation manifested by α-cellulose and HPLC, and brightness stability against moist heat ageing. The incorporation of a laccase-mediator system (LMS) to bleach sulphite pulps can be a good alternative to traditional bleaching processes since thermogravimetric analysis (TGA) showed that the laccase treatment prevented the adverse effect of hydrogen peroxide on fibre surface as observed during a conventional hydrogen peroxide bleaching treatment (PO). Although VA exhibited the best results in terms of bleaching properties, the performance of natural mediators, such as p-coumaric acid and syringaldehyde, was discussed in relation to changes in cellulose surface detected by TGA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enhancement of catalytic, reusability, and long-term stability features of Trametes versicolor IBL-04 laccase immobilized on different polymers.

PubMed

Asgher, Muhammad; Noreen, Sadia; Bilal, Muhammad

2017-02-01

In the current study, different bio-polymers such as agar-agar, polyacrylamide and gelatin were utilized as bolster materials for the immobilization of a fungal laccase through entrapment approach. Among the polymers, agar-agar matrix most firmly encapsulated the enzyme yielding significant laccase immobilization (79.65±2.55%). Immobilization prolonged the reaction time of laccase and agar-agar, polyacrylamide and gelatin entrapped laccases displayed maximum catalytic activities after 10.0, 15.0 and 10.0min of reaction, respectively, as compared to free counterpart (5.0min). It also increased the optimal temperature by 5.0-10°C and provided an alkaline shift of the pH optima to agar-agar and gelatin entrapped laccase, while, in case of polyacrylamide, optimum pH was displaced to acidic region. Kinetic data revealed that K m(app) values were slightly increased while V max values were decreased as compared to free counterpart. Polymers encapsulation led to significant improvement in activity against thermal denaturation. After 180min at 60°C, the enzymes preserved 28.1±0.9, 48.6±1.3 and 32.5±1.8% residual activities, respectively, whereas, the free enzyme was completely inactive. Immobilization enabled the enzymes to resist a number of different effectors including metal ions, inhibitors/denaturants and chelating agents. Moreover, the resulted modified laccases displayed good recycling capability for substrate-oxidation reactions in several successive batches. In summary, the tremendously improved attributes of polymers-encapsulated enzymes display a high potential for various applications in different industrial sectors. Copyright © 2016 Elsevier B.V. All rights reserved.

Reduced toxicity of malachite green decolorized by laccase produced from Ganoderma sp. rckk-02 under solid-state fermentation.

PubMed

Sharma, Abha; Shrivastava, Bhuvnesh; Kuhad, Ramesh Chander

2015-10-01

Statistical designs were applied for optimizing laccase production from a white-rot fungus, Ganoderma sp. rckk-02 under solid-state fermentation (SSF). Compared to unoptimized conditions [2,154 U/gds (Unit per gram of dry substrate)], the optimization process resulted in a 17.3-fold increase in laccase production (37,423 U/gds). The laccase produced was evaluated for its potential to decolorize a recalcitrant synthetic dye, malachite green. Laccase at dosage of 30 U/ml in presence of 1 mM of 1-hydroxybenzotriazole (HBT) almost completely decolorized 100 and 200 mg/l of malachite green in 16 and 20 h, respectively, at 30 °C, pH 5.5 and 150 rpm. While, higher dyes concentrations of 300, 400 and 500 mg/l were decolorized to 72, 62 and 55 % in 24, 28 and 32 h, respectively, under similar conditions. Furthermore, it was observed that the decolorized malachite green was less toxic towards the growth of five white-rot fungi tested viz. Crinipellis sp. RCK-1, Ganoderma sp. rckk-02, Coriolopsis Caperata RCK 2011, Phanerochaete chrysosporium K3 and Pycnoporous cinnabarinus PB. The present study demonstrates the potential of Ganoderma sp. rckk-02 to produce high titres of laccase under SSF, which can be exploited in conjunction with redox mediator for the decolorization of high concentrations of malachite green from water bodies.

Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Deepti; Rawat, Surender; Waseem, Mohd

The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, K{sub m} values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative propertymore » against BSA and Cu{sup 2+}/H{sub 2}O{sub 2} model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies. - Highlights: • Laccase from Yersinia enterocolitica strain 7 was expressed in Escherichia coli BL21 (DE3). • Recombinant laccase was found to be thermostable and alkali tolerant. • The in silico and experimental studied proves the biotransformation of NSAIDs. • Laccase binds to ligands differentially under different protonation state. • Laccase also possesses free radical scavenging property.« less

Effect of the inducers veratryl alcohol, Xylidine, and ligninosulphonates on activity and thermal stability and inactivation kinetics of laccase from Trametes versicolor.

PubMed

Saraiva, Jorge A; Tavares, Ana P M; Xavier, Ana M R B

2012-06-01

Laccase production from Trametes versicolor was improved in the presence of the inducers ligninosulphonates, veratryl alcohol, and xylidine respectively two-, four-, and eightfold. The thermal inactivation of the produced laccase, after partial purification with ammonium sulfate was kinetically investigated at various temperatures (60-70 °C) and pH values (3.5, 4.5, and 5.5). The inactivation process followed first-order kinetics for all conditions tested, except for veratryl alcohol, for which a constant activity level was observed at the end of the inactivation, also after first-order decay. Enzyme thermostability was affected by the type of inducer used in the culture medium for the production of laccase and also by the pH of incubation mixture. Generally, laccase stability increased with pH increment, being more stable at pH 5.5, except with xylidine. At pHs 4.5 and 5.5, the three inducers significantly increased laccase thermal stability, with the higher effect being observed for pH 5.5 and ligninosulphonates, where increment of half-life times ranged from 3- to 20-fold, depending on the temperature.

Functional Characterization of Epitheaflagallin 3-O-Gallate Generated in Laccase-Treated Green Tea Extracts in the Presence of Gallic Acid.

PubMed

Itoh, Nobuya; Kurokawa, Junji; Isogai, Yasuhiro; Ogasawara, Masaru; Matsunaga, Takayuki; Okubo, Tsutomu; Katsube, Yuji

2017-12-06

Epitheaflagallin (ETFG) and epitheaflagallin 3-O-gallate (ETFGg) are minor polyphenols in black tea extract that are enzymatically synthesized from epigallocatechin (EGC) and epigallocatechin gallate (EGCg), respectively, in green tea extract via laccase oxidation in the presence of gallic acid. The constituents of laccase-treated green tea extract in the presence of gallic acid are thus quite different from those of nonlaccase-treated green tea extract: EGC and EGCg are present in lower concentrations, and ETFG and ETFGg are present in higher concentrations. Additionally, laccase-treated green tea extract contains further polymerized catechin derivatives, comparable with naturally fermented teas such as oolong tea and black tea. We found that ETFGg and laccase-treated green tea extracts exhibit versatile physiological functions in vivo and in vitro, including antioxidative activity, pancreatic lipase inhibition, Streptococcus sorbinus glycosyltransferase inhibition, and an inhibiting effect on the activity of matrix metalloprotease-1 and -3 and their synthesis by human gingival fibroblasts. We confirmed that these inhibitory effects of ETFGg in vitro match well with the results obtained by docking simulations of the compounds with their target enzymes or noncatalytic protein. Thus, ETFGg and laccase-treated green tea extracts containing ETFGg are promising functional food materials with potential antiobesity and antiperiodontal disease activities.

Long term storage of Pleurotus ostreatus and Trametes versicolor isolates using different cryopreservation techniques and its impact on laccase activity.

PubMed

Eichlerová, Ivana; Homolka, Ladislav; Tomšovský, Michal; Lisá, Ludmila

2015-12-01

The strain Pleurotus ostreatus Florida f6, its 45 basidiospore-derived isolates (both monokaryons and dikaryons prepared in our laboratory), Trametes versicolor strain CCBAS 614 and 22 other T. versicolor isolates obtained from the sporocarps collected in distant localities were successfully preserved for 12 y using perlite and straw cryopreservation protocols. All tested isolates survived a 12-year storage in liquid nitrogen (LN) and their laccase production and Poly B411 decolorization capacity was preserved. Also mycelium extension rate and the types of colony appearance of individual isolates remained unchanged. Different cryopreservation techniques were also tested for the short time (24 h) and the long time (6 m) storage of the culture liquid with extracellular laccase produced by T. versicolor strain CCBAS 614. The results showed that 10 % glycerol was the most suitable cryopreservant. The absence of the cryopreservant did not cause high loss of laccase activity in the samples; the presence of DMSO (5 or 10 %) in LN-stored samples caused mostly a decrease of laccase activity. For the preservation of laccase activity in the liquid culture the storage in the freezer at -80 °C is more convenient than the storage in liquid nitrogen. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.