Sample records for phenol oxidase laccase

  1. Enzymological characterization of EpoA, a laccase-like phenol oxidase produced by Streptomyces griseus.

    PubMed

    Endo, Kohki; Hayashi, Yutaka; Hibi, Takahiro; Hosono, Kuniaki; Beppu, Teruhiko; Ueda, Kenji

    2003-05-01

    Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N,N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42 mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40 degrees C, respectively. The enzyme retained 40% activity after preincubation at 70 degrees C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria. PMID:12801920

  2. Decolorization of phenolic effluents by soluble and immobilized phenol oxidases

    Microsoft Academic Search

    Susan Davis; Richard G. Burns

    1990-01-01

    Colour removal from phenplic industrial effluents by phenol oxidase enzymes and white-rot fungi was compared. Soluble laccase and horseradish peroxidase (HRP) removed colour from pulp mill (E), cotton mill hydroxide (OH) and cotton mill sulphide (S) effluents, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase in alginate beads improved decolorization by factors of 3.5 (OH) and 2

  3. 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

  4. Laccase versus laccase-like multi-copper oxidase: a comparative study of similar enzymes with diverse substrate spectra.

    PubMed

    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

  5. Oxidation of phenols by laccase and laccase-mediator systems.

    PubMed

    D'Acunzo, Francesca; Galli, Carlo; Masci, Bernardo

    2002-11-01

    To investigate how solubility and steric issues affect the laccase-catalysed oxidation of phenols, a series of oligomeric polyphenol compounds, having increasing size and decreasing solubility in water, was incubated with laccase. The extent of substrate conversion, and the nature of the products formed in buffered aqueous solutions, were compared to those obtained in the presence of an organic cosolvent, and also in the presence of two mediating species, i.e. N-hydroxyphthalimide (HPI) and 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO). This approach showed not only an obvious role of solubility, but also a significant role of the dimension of the substrate upon the enzymatic reactivity. In fact, reactivity decreases as substrate size increases even when solubility is enhanced by a cosolvent. This effect may be ascribed to limited accessibility of encumbered substrates to the enzyme active site, and can be compensated through the use of the appropriate mediator. While TEMPO was highly efficient at enhancing the reactivity of large, less soluble substrates, HPI proved less effective. In addition, whereas the laccase/HPI system afforded the same products as laccase alone, the use of TEMPO provided a different product with high specificity. These results offer the first evidence of the role of 'oxidation shuttles' that the mediators of laccase may have, but also suggest two promising routes towards an environmentally friendly process for kraft pulp bleaching: (a) the identification of mediators which, once oxidized by laccase, are able to target strategic functional groups present in lignin, and (b) the introduction of those strategic functional groups in an appropriate pretreatment. PMID:12392567

  6. Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

    PubMed Central

    2012-01-01

    Background Laccase-like multicopper oxidases have been reported in several Aspergillus species but they remain uncharacterized. The biocatalytic potential of the Aspergillus niger fungal pigment multicopper oxidases McoA and McoB and ascomycete laccase McoG was investigated. Results The laccase-like multicopper oxidases McoA, McoB and McoG from the commonly used cell factory Aspergillus niger were homologously expressed, purified and analyzed for their biocatalytic potential. All three recombinant enzymes were monomers with apparent molecular masses ranging from 80 to 110 kDa. McoA and McoG resulted to be blue, whereas McoB was yellow. The newly obtained oxidases displayed strongly different activities towards aromatic compounds and synthetic dyes. McoB exhibited high catalytic efficiency with N,N-dimethyl-p-phenylenediamine (DMPPDA) and 2,2-azino-di(3-ethylbenzthiazoline) sulfonic acid (ABTS), and appeared to be a promising biocatalyst. Besides oxidizing a variety of phenolic compounds, McoB catalyzed successfully the decolorization and detoxification of the widely used textile dye malachite green. Conclusions The A. niger McoA, McoB, and McoG enzymes showed clearly different catalytic properties. Yellow McoB showed broad substrate specificity, catalyzing the oxidation of several phenolic compounds commonly present in different industrial effluents. It also harbored high decolorization and detoxification activity with the synthetic dye malachite green, showing to have an interesting potential as a new industrial biocatalyst. PMID:23270588

  7. Laccase-mediated detoxification of phenolic compounds. [Rhizoctonia praticola

    SciTech Connect

    Bollag, J.M.; Shuttleworth, K.L.; Anderson, D.H. (Pennsylvania State Univ., University Park (USA))

    1988-12-01

    The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.

  8. Laccase immobilization on the electrode surface to design a biosensor for the detection of phenolic compound such as catechol.

    PubMed

    Nazari, Maryam; Kashanian, Soheila; Rafipour, Ronak

    2015-06-15

    Biosensors based on the coupling of a biological entity with a suitable transducer offer an effective route to detect phenolic compounds. Phenol and phenolic compounds are among the most toxic environmental pollutants. Laccases are multi-copper oxidases that can oxide phenol and phenolic compounds. A method is described for construction of an electrochemical biosensor to detect phenolic compounds based on covalent immobilization of laccase (Lac) onto polyaniline (PANI) electrodeposited onto a glassy carbon (GC) electrode via glutaraldehyde coupling. The modified electrode was characterized by voltammetry, Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM) techniques. The results indicated that laccase was immobilized onto modified GC electrode by the covalent interaction between laccase and terminal functional groups of the glutaraldehyde. The laccase immobilized modified electrode showed a direct electron transfer reaction between laccase and the electrode. Linear range, sensitivity, and detection limit for this biosensor were 3.2 × 10(-6) to 19.6 × 10(-6)M, 706.7 mAL mol(-1), 2.07 × 10(-6)M, respectively. PMID:25770936

  9. Laccase immobilization on the electrode surface to design a biosensor for the detection of phenolic compound such as catechol

    NASA Astrophysics Data System (ADS)

    Nazari, Maryam; Kashanian, Soheila; Rafipour, Ronak

    2015-06-01

    Biosensors based on the coupling of a biological entity with a suitable transducer offer an effective route to detect phenolic compounds. Phenol and phenolic compounds are among the most toxic environmental pollutants. Laccases are multi-copper oxidases that can oxide phenol and phenolic compounds. A method is described for construction of an electrochemical biosensor to detect phenolic compounds based on covalent immobilization of laccase (Lac) onto polyaniline (PANI) electrodeposited onto a glassy carbon (GC) electrode via glutaraldehyde coupling. The modified electrode was characterized by voltammetry, Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM) techniques. The results indicated that laccase was immobilized onto modified GC electrode by the covalent interaction between laccase and terminal functional groups of the glutaraldehyde. The laccase immobilized modified electrode showed a direct electron transfer reaction between laccase and the electrode. Linear range, sensitivity, and detection limit for this biosensor were 3.2 × 10-6 to 19.6 × 10-6 M, 706.7 mA L mol-1, 2.07 × 10-6 M, respectively.

  10. Laccase?catalysed oxidations of naturally occurring phenols: from in vivo biosynthetic pathways to green synthetic applications

    PubMed Central

    Jeon, Jong?Rok; Baldrian, Petr; Murugesan, Kumarasamy; Chang, Yoon?Seok

    2012-01-01

    Summary Laccases are oxidases that contain several copper atoms, and catalyse single?electron oxidations of phenolic compounds with concomitant reduction of oxygen to water. The enzymes are particularly widespread in ligninolytic basidiomycetes, but also occur in certain prokaryotes, insects and plants. Depending on the species, laccases are involved in various biosynthetic processes contributing to carbon recycling in land ecosystems and the morphogenesis of biomatrices, wherein low?molecular?weight naturally occurring phenols serve as key enzyme substrates. Studies of these in vivo synthetic pathways have afforded new insights into fungal laccase applicability in green synthetic chemistry. Thus, we here review fungal laccase?catalysed oxidations of naturally occurring phenols that are particularly relevant to the synthesis of fine organic chemicals, and we discuss how the discovered synthetic strategies mimic laccase?involved in vivo pathways, thus enhancing the green nature of such reactions. Laccase?catalysed in vivo processes yield several types of biopolymers, including those of cuticles, lignin, polyflavonoids, humus and the melanin pigments, using natural mono? or poly?phenols as building blocks. The in vivo synthetic pathways involve either phenoxyl radical?mediated coupling or cross?linking reactions, and can be adapted to the design of in vitro oxidative processes involving fungal laccases in organic synthesis; the laccase substrates and the synthetic mechanisms reflect in vivo processes. Notably, such in vitro synthetic pathways can also reproduce physicochemical properties (e.g. those of chromophores, and radical?scavenging, hydration and antimicrobial activities) found in natural biomaterials. Careful study of laccase?associated in vivo metabolic pathways has been rewarded by the discovery of novel green applications for fungal laccases. This review comprehensively summarizes the available data on laccase?catalysed biosynthetic pathways and associated applications in fine chemical syntheses. PMID:21791030

  11. Oxirane-immobilized Lentinula edodes laccase: stability and phenolics removal efficiency in olive mill wastewater

    Microsoft Academic Search

    Alessandro D’Annibale; Silvia Rita Stazi; Vittorio Vinciguerra; Giovanni Giovannozzi Sermanni

    2000-01-01

    Immobilization of Lentinula edodes laccase on Eupergit® C increased pH, thermal and proteolytic stability with slight modifications in laccase oxidation efficiency. Immobilized laccase proved to be efficiently stable in removing olive mill wastewater phenolics.

  12. Alginate\\/carbon composite beads for laccase and glucose oxidase encapsulation: application in biofuel cell technology

    Microsoft Academic Search

    Zoreh Khani; Claude Jolivalt; Marc Cretin; Sophie Tingry; Christophe Innocent

    2006-01-01

    Alginate–carbon beads were prepared in order to develop a biocompatible matrix for laccase and glucose oxidase immobilization for application in biofuel cell technology. The enzyme loading capacity was high (91%) in pure alginate beads for glucose oxidase. For laccase, the loading capacity was enhanced from 75% to 83% by introducing carbon. Desorption out of the matrix was controlled by the

  13. 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

  14. Laccase catalyzed synthesis of iodinated phenolic compounds with antifungal activity.

    PubMed

    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

  15. Phenol-oxidizing laccases from the termite gut.

    PubMed

    Coy, M R; Salem, T Z; Denton, J S; Kovaleva, E S; Liu, Z; Barber, D S; Campbell, J H; Davis, D C; Buchman, G W; Boucias, D G; Scharf, M E

    2010-10-01

    cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (?4 copper molecules per laccase protein molecule and ?3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion. PMID:20691784

  16. Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site-directed mutagenesis

    PubMed Central

    Sherif, Mohammed; Waung, Debbie; Korbeci, Bihter; Mavisakalyan, Valentina; Flick, Robert; Brown, Greg; Abou-Zaid, Mamdouh; Yakunin, Alexander F; Master, Emma R

    2013-01-01

    Summary Multicopper oxidases can act on a broad spectrum of phenolic and non-phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2?-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,6-dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where kcat and Km values at 60°C were 8.1 (±?0.8) s?1 and 0.9 (±?0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH?4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half-life of over 10?h at 80°C and over 7?h at 90°C. Site-directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10-fold increase in activity compared with the wild-type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta-position of aromatic substrates. Funding Information Funding for this research was provided by the Government of Ontario for the project ‘FFABnet: Functionalized Fibre and Biochemicals’ (ORF-RE-05-005), and the Natural Sciences and Engineering Research Council of Canada. PMID:23815400

  17. Gene structure and molecular analysis of the laccase-like multicopper oxidase (LMCO) gene family in Arabidopsis thaliana

    Microsoft Academic Search

    Bonnie C. McCaig; Richard B. Meagher; Jeffrey F. D. Dean

    2005-01-01

    Completed genome sequences have made it clear that multicopper oxidases related to laccase are widely distributed as multigene families in higher plants. Laccase-like multicopper oxidase (LMCO) sequences culled from GenBank and the Arabidopsis thaliana genome, as well as those from several newly cloned genes, were used to construct a gene phylogeny that clearly divided plant LMCOs into six distinct classes,

  18. Laccase: production by Trametes versicolor and application to denim washing

    Microsoft Academic Search

    Nurdan Ka??kara Pazarl?o?lu; Merih Sarii?ik; Azmi Telefoncu

    2005-01-01

    Laccase activity was monitored in synthetic media supplemented with various phenolics. Phenol was the most effective inducer. The white rot basidiomycete Trametes versicolor, grown in non-induced and phenol induced conditions, was tested for production of laccase (TvLac), lignin peroxidase (LiP), manganese peroxidase (MnP), arylalcohol oxidase (AAO) and polyphenol oxidase (PPO). Although no PPO, LiP and AAO were observed in the

  19. Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase-mediator systems.

    PubMed

    Baiocco, Paola; Barreca, Anna Maria; Fabbrini, Maura; Galli, Carlo; Gentili, Patrizia

    2003-01-01

    The oxidation of benzyl alcohols with the enzyme laccase, under mediation by appropriate mediator compounds, yields carbonylic products, whereas laccase can not oxidise these non-phenolic substrates directly. The oxidation step is performed by the oxidised form of the mediator (Med(ox)), generated on its interaction with laccase. The Med(ox) can follow either an electron transfer (ET) or a radical hydrogen atom transfer (HAT) route of oxidation of the substrates. Experimental evidence is reported that enables unambiguous assessment of the occurrence of either one the oxidation routes with each of the investigated mediators, namely, ABTS, HBT, HPI and VLA. Support to the conclusions is provided by (i) investigating the intermolecular selectivity of oxidation with appropriate substrates, (ii) attempting Hammett correlations for the oxidation of a series of 4-X-substituted benzyl alcohols, (iii) measuring the kinetic isotope effect, (iv) investigating the product pattern with suitable probe precursors. Based on these points, a HAT mechanism results to be followed by the laccase-HBT, laccase-HPI and laccase-VLA systems, whereas an ET route appears feasible in the case of the laccase-ABTS system. PMID:12929410

  20. Phenol-oxidizing laccases from the termite gut

    Microsoft Academic Search

    M. R. Coy; T. Z. Salem; J. S. Denton; E. S. Kovaleva; Z. Liu; D. S. Barber; J. H. Campbell; D. C. Davis; G. W. Buchman; D. G. Boucias; M. E. Scharf

    2010-01-01

    cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and

  1. Laccases and other polyphenol oxidases in ecto- and ericoid mycorrhizal fungi

    Microsoft Academic Search

    R. M. Burke; J. W. G. Cairney

    2002-01-01

    Polyphenol oxidases are known to be produced by a range of ectomycorrhizal (ECM) and ericoid mycorrhizal fungi. These enzymes include laccase (EC 1.10.3.2), catechol oxidase (EC 1.10.3.1) and tyrosinase (EC 1.14.18.1), between which there exists considerable overlap in substrate affinities. In this review we consider the nature and function of these enzymes, along with the difficulties associated with assigning precise

  2. Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar.

    PubMed

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

    2002-05-01

    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

  3. 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

  4. Phenol-oxidizing laccases from the termite gut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignmen...

  5. Inhibition of cellulose enzymatic hydrolysis by laccase-derived compounds from phenols.

    PubMed

    Oliva-Taravilla, Alfredo; Tomás-Pejó, Elia; Demuez, Marie; González-Fernández, Cristina; Ballesteros, Mercedes

    2015-05-01

    The presence of inhibitors compounds after pretreatment of lignocellulosic materials affects the saccharification and fermentation steps in bioethanol production processes. Even though, external addition of laccases selectively removes the phenolic compounds from lignocellulosic prehydrolysates, when it is coupled to saccharification step, lower hydrolysis yields are attained. Vanillin, syringaldehyde and ferulic acid are phenolic compounds commonly found in wheat-straw prehydrolysate after steam-explosion pretreatment. These three phenolic compounds were used in this study to elucidate the inhibitory mechanisms of laccase-derived compounds after laccase treatment. Reaction products derived from laccase oxidation of vanillin and syringaldehyde showed to be the strongest inhibitors. The presence of these products causes a decrement on enzymatic hydrolysis yield of a model cellulosic substrate (Sigmacell) of 46.6 and 32.6%, respectively at 24 h. Moreover, a decrease in more than 50% of cellulase and ?-glucosidase activities was observed in presence of laccase and vanillin. This effect was attributed to coupling reactions between phenoxyl radicals and enzymes. On the other hand, when the hydrolysis of Sigmacell was performed in presence of prehydrolysate from steam-exploded wheat straw a significant inhibition on enzymatic hydrolysis was observed independently of laccase treatment. This result pointed out that the other components of wheat-straw prehydrolysate are affecting the enzymatic hydrolysis to a higher extent than the possible laccase-derived products. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:700-706, 2015. PMID:25740593

  6. Removal of phenol and bisphenol-A catalyzed by laccase in aqueous solution

    PubMed Central

    2014-01-01

    Background Elimination of hazardous phenolic compounds using laccases has gained attention during recent decades. The present study was designed to evaluate the ability of the purified laccase from Paraconiothyrium variabile (PvL) for elimination of phenol and the endocrine disrupting chemical bisphenol A. Effect of laccase activity, pH, and temperature on the enzymatic removal of the mentioned pollutants were also investigated. Results After 30 min treatment of the applied phenolic pollutants in the presence of PvL (5 U/mL), 80% of phenol and 59.7% of bisphenol A was removed. Increasing of laccase activity enhanced the removal percentage of both pollutants. The acidic pH of 5 was found to be the best pH for elimination of both phenol and bisphenol A. Increasing of reaction temperature up to 50°C enhanced the removal percentage of phenol and bisphenol A to 96.3% and 88.3%, respectively. Conclusions To sum up, the present work introduced the purified laccase of P. variabile as an efficient biocatalyst for removal of one of the most hazardous endocrine disruptor bisphenol A. PMID:25031840

  7. Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum

    Microsoft Academic Search

    Renate Reiss; Julian Ihssen; Linda Thöny-Meyer

    2011-01-01

    BACKGROUND: Laccases are multi-copper oxidases that catalyze the one electron oxidation of a broad range of compounds. Laccase substrates include substituted phenols, arylamines and aromatic thiols. Such compounds are activated by the enzyme to the corresponding radicals. Owing to their broad substrate range laccases are considered to be versatile biocatalysts which are capable of oxidizing natural and non-natural industrial compounds,

  8. Optimization of laccase production by two strains of Ganoderma lucidum using phenolic and metallic inducers.

    PubMed

    Kuhar, Francisco; Papinutti, Leandro

    2014-01-01

    Ganoderma lucidum (Curtis) P. Karst is a white rot fungus that is able to degrade the lignin component in wood. The ability of two strains of this species to produce the ligninolytic enzyme laccase was assessed. After the evaluation of induction with heavy metals and phenolic compounds, it was found that among the tested substances, copper and ferulic acid are the best laccase inducers. It was also observed that the two types of inducers (phenolic and metallic) produce different electrophoretic patterns of laccase activity. Optimized concentrations of inducers were obtained through a factorial design and the thermal stability of optimized supernatants was studied at a wide range of acidic pH. We found that the enzyme is more thermostable at higher pH values. PMID:25011599

  9. Transformation of polycyclic aromatic hydrocarbons by laccase is strongly enhanced by phenolic compounds present in soil.

    PubMed

    Cañas, Ana I; Alcalde, Miguel; Plou, Francisco; Martínez, Maria Jesús; Martínez, Angel T; Camarero, Susana

    2007-04-15

    Efficient transformation of several polycyclic aromatic hydrocarbons (PAHs) was obtained using a fungal laccase in the presence of phenolic compounds related to those formed in nature during the turnover of lignin and humus. The effect of these natural mediators, namely vanillin, acetovanillone, acetosyringone, syringaldehyde, 2,4,6-trimethylphenol, p-coumaric acid, ferulic acid, and sinapic acid, was compared with that of synthetic mediators such as 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). Anthracene was significantly degraded by laccase in the absence of mediators, whereas benzo[a]pyrene and pyrene were weakly transformed (less than 15% after 24 h). Vanillin, acetovanillone, 2,4,6-trimethylphenol, and, above all, p-coumaric acid strongly promoted the removal of PAHs by laccase. 9,10-Anthraquinone was the main product detected from anthracene oxidation by all the laccase-mediator systems. The yield of anthraquinone formed was directly correlated with the amount of p-coumaric acid used. This compound resulted in a better laccase mediator than ABTS and close similarity to HBT, attaining 95% removal of anthracene and benzo[a]pyrene and around 50% of pyrene within 24 h. Benzo[a]pyrene 1,6-, 3,6-, and 6,12-quinones were produced during benzo[a]pyrene oxidation with laccase and p-coumaric acid, HBT, or ABTS as mediators, although use of the latter mediator gave further oxidation products that were not produced by the two other systems. PMID:17533865

  10. Phenol oxidase activity in secondary transformed peat-moorsh soils

    NASA Astrophysics Data System (ADS)

    Sty?a, K.; Szajdak, L.

    2009-04-01

    The chemical composition of peat depends on the geobotanical conditions of its formation and on the depth of sampling. The evolution of hydrogenic peat soils is closely related to the genesis of peat and to the changes in water conditions. Due to a number of factors including oscillation of ground water level, different redox potential, changes of aerobic conditions, different plant communities, and root exudes, and products of the degradation of plant remains, peat-moorsh soils may undergo a process of secondary transformation conditions (Sokolowska et al. 2005; Szajdak et al. 2007). Phenol oxidase is one of the few enzymes able to degrade recalcitrant phenolic materials as lignin (Freeman et al. 2004). Phenol oxidase enzymes catalyze polyphenol oxidation in the presence of oxygen (O2) by removing phenolic hydrogen or hydrogenes to from radicals or quinines. These products undergo nucleophilic addition reactions in the presence or absence of free - NH2 group with the eventual production of humic acid-like polymers. The presence of phenol oxidase in soil environments is important in the formation of humic substances a desirable process because the carbon is stored in a stable form (Matocha et al. 2004). The investigations were carried out on the transect of peatland 4.5 km long, located in the Agroecological Landscape Park host D. Chlapowski in Turew (40 km South-West of Pozna?, West Polish Lowland). The sites of investigation were located along Wysko? ditch. The following material was taken from four chosen sites marked as Zbechy, Bridge, Shelterbelt and Hirudo in two layers: cartel (0-50cm) and cattle (50-100cm). The object of this study was to characterize the biochemical properties by the determination of the phenol oxidize activity in two layers of the four different peat-moors soils used as meadow. The phenol oxidase activity was determined spectrophotometrically by measuring quinone formation at ?max=525 nm with catechol as substrate by method of Perucci et al. (2000). In peat the highest activities of phenol oxidase was observed in the combinations marked as Shelterbelt and whereas the lowest - in Zbechy, Bridge and Hirudo. Activities of this enzyme in peat ranged from 15.35 to 38.33 ?mol h-1g d.m soil. Increased activities of phenol oxidase have been recorded on the depth 50-100cm - catotelm (21.74-38.33 ?mol h-1g d.m soil) in comparison with the depth 0-50cm - acrotelm (15.35-28.32 ?mol h-1g d.m soil). References Freeman, C., Ostle N.J., Fener, N., Kang H. 2004. A regulatory role for phenol oxidase during decomposition in peatlands. Soil Biology and Biochemistry, 36, 1663-1667. Matocha Ch.J., Haszler G.R., Grove J.H. 2004. Nitrogen fertilization suppresses soil phenol oxidase enzyme activity in no-tillage systems. Soil Science, 169/10, 708-714. Perucci P., Casucci C., Dumontet S. 2000. An improved method to evaluate the o-diphenol oxidase activity of soil. Soil Biology and Biochemistry, 32, 1927-1933. Sokolowska Z., Szajdak L., Matyka-Sarzy?ska D. 2005. Impact of the degree of secondary transformation on amid-base properties of organic compounds in mucks. Geoderma, 127, 80-90. Szajdak L., Szczepa?ski M., Bogacz A. 2007. Impact of secondary transformation of peat-moorsh soils on the decrease of nitrogen and carbon compounds in ground water. Agronomy Research, 5/2, 189-200.

  11. Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli.

    PubMed

    Ihssen, Julian; Reiss, Renate; Luchsinger, Ronny; Thöny-Meyer, Linda; Richter, Michael

    2015-01-01

    Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development. PMID:26068013

  12. Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli

    PubMed Central

    Ihssen, Julian; Reiss, Renate; Luchsinger, Ronny; Thöny-Meyer, Linda; Richter, Michael

    2015-01-01

    Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development. PMID:26068013

  13. Concerted electron/proton transfer mechanism in the oxidation of phenols by laccase.

    PubMed

    Galli, Carlo; Madzak, Catherine; Vadalà, Raffaella; Jolivalt, Claude; Gentili, Patrizia

    2013-12-16

    This study aimed to assess structural requirements in the enzyme/substrate interactions that are responsible for tuning the enzymatic reactivity. To better assess the role of the aspartic residue in the substrate-binding pocket of basidiomycete-type laccases, we compared the catalytic efficiency of wild-type enzymes to that of a mutant in which carboxylic acid residue Asp206 was changed to alanine. Oxidation efficiency towards phenolic substrates by laccases of Trametes villosa, Trametes versicolor and a T. versicolor D206A mutant was studied at two pH values. By the Hammett approach and Marcus analysis, we obtained unambiguous evidence that the oxidation takes place by a concerted electron/proton transfer (EPT) mechanism, and that at pH 5 (optimum pH for enzyme activity) the phenolic proton is transferred to Asp206 during the concerted electron/proton transfer process. PMID:24151197

  14. Immobilization of defined laccase combinations for enhanced oxidation of phenolic contaminants.

    PubMed

    Ammann, Erik M; Gasser, Christoph A; Hommes, Gregor; Corvini, Philippe F-X

    2014-02-01

    Immobilization is an important method to increase enzyme stability and allow enzyme reuse. One interesting application in the field of environmental biotechnology is the immobilization of laccase to eliminate phenolic contaminants via oxidation. Fumed silica nanoparticles have interesting potential as support material for laccase immobilization via sorption-assisted immobilization in the perspective of applications such as the elimination of micropollutants in aqueous phases. Based on these facts, the present work aimed to formulate laccase-nanoparticle conjugates with defined laccase combinations in order to obtain nanobiocatalysts, which are active over a broad range of pH values and possess a large substrate spectrum to suitably address pollution by multiple contaminants. A multi-enzymatic approach was investigated by immobilizing five different types of laccases originating from a Thielavia genus, Coriolopsis polyzona, Cerrena unicolor, Pleurotus ostreatus, and Trametes versicolor onto fumed silica nanoparticles, separately and in combinations. The laccases differed concerning their pH optima and substrate affinity. Exploiting their differences allowed the formulation of tailor-made nanobiocatalysts. In particular, the production of a nanobiocatalyst could be achieved that retained a higher percentage of its relative activity over the tested pH range (3-7) compared to the dissolved or separately immobilized enzymes. Furthermore, a nanobiocatalyst could be formulated able to oxidize a broader substrate range than the dissolved or separately immobilized enzymes. Thereby, the potential of the nanobiocatalyst for application in biochemical oxidation applications such as the elimination of multiple target pollutants in biologically treated wastewater has been illustrated. PMID:23812279

  15. Enzymatic grafting of simple phenols on flax and sisal pulp fibres using laccases.

    PubMed

    Aracri, Elisabetta; Fillat, Amanda; Colom, José F; Gutiérrez, Ana; Del Río, José C; Martínez, Angel T; Vidal, Teresa

    2010-11-01

    Flax and sisal pulps were treated with two laccases (from Pycnoporus cinnabarinus, PcL and Trametes villosa, TvL, respectively), in the presence of different phenolic compounds (syringaldehyde, acetosyringone and p-coumaric acid in the case of flax pulp, and coniferaldehyde, sinapaldehyde, ferulic acid and sinapic acid in the case of sisal pulp). In most cases the enzymatic treatments resulted in increased kappa number of pulps suggesting the incorporation of the phenols into fibres. The covalent binding of these compounds to fibres was evidenced by the analysis of the treated pulps, after acetone extraction, by pyrolysis coupled with gas chromatography/mass spectrometry in the absence and/or in the presence of tetramethylammonium hydroxide (TMAH) as methylating agent. The highest extents of phenol incorporation were observed with the p-hydroxycinnamic acids, p-coumaric and ferulic acids. The present work shows for the first time the use of analytical pyrolysis as an effective approach to study fibre functionalization by laccase-induced grafting of phenols. PMID:20580550

  16. 199mHg-derivatives of ascorbate oxidase and laccase: selective depletion and blocking of Cu-sites

    NASA Astrophysics Data System (ADS)

    Butz, T.; Tröger, W.; Messerschmidt, A.; Thoenes, U.; Huber, R.

    1993-03-01

    We report on the199mHg nuclear quadrupole interaction (NQI) of Hg-derivatives of the blue oxidases ascorbate oxidase (AO) and laccase (LAC). For fully reconstituted enzymes, three different NQIs were observed. The assignment of these NQIs to the type-1, -2, and -3 Cusites is based on type-2 depleted AO, on blocking studies with inactive Hg prior to199mHg/carrier reconstitution, and on the population ratio observed for fully reconstituted LAC. The NQIs for both enzymes are similar, suggesting similar Cu-sites. The type-2 site is preferentially reconstituted, contrary to expectations. Neither the blocking nor the depletion is as selective as expected.

  17. Co-occurrence of the Multicopper Oxidases Tyrosinase and Laccase in Lichens in Sub-order Peltigerineae

    PubMed Central

    LAUFER, ZSANETT; BECKETT, RICHARD P.; MINIBAYEVA, FARIDA V.

    2006-01-01

    • Background and Aims Following previous findings of high extracellular redox activity in lichens and the presence of laccases in lichen cell walls, the work presented here additionally demonstrates the presence of tyrosinases. Tests were made for the presence of tyrosinases in 40 species of lichens, and from selected species their cellular location and molecular weights were determined. The effects of stress and inhibitors on enzyme activity were also studied. • Methods Tyrosinase and laccase activities were assayed spectrophotometrically using a variety of substrates. The molecular mass of the enzymes was estimated using polyacrylamide gel electrophoresis. • Key Results Extracellular tyrosinase and laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from the sub-order Peltigerineae, all displayed significant tyrosinase and laccase activity, while activity was low or absent in other species tested. Representatives from both groups of lichens displayed low peroxidase activities. Identification of the enzymes as tyrosinases was confirmed by the ability of lichen thalli or leachates derived by shaking lichens in distilled water to metabolize substrates such as l-dihydroxyphenylalanine (DOPA), tyrosine and epinephrine readily in the absence of hydrogen peroxide, the sensitivity of the enzymes to the inhibitors cyanide, azide and hexylresorcinol, activation by SDS and having typical tyrosinase molecular masses of approx. 60?kDa. Comparing different species within the Peltigerineae showed that the activities of tyrosinases and laccase were correlated to each other. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. • Conclusions Cell walls of lichens in sub-order Peltigerineae have much higher activities and a greater diversity of cell wall redox enzymes compared with other lichens. Possible roles of tyrosinases include melanization, removal of toxic phenols or quinones, and production of herbivore deterrents. PMID:16950829

  18. Exploring laccase-like multicopper oxidase genes from the ascomycete Trichoderma reesei: a functional, phylogenetic and evolutionary study

    PubMed Central

    2010-01-01

    Background The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, Trichoderma reesei is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. T. reesei has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) to validate the function of a candidate LMCO gene from T. reesei, and ii) to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection. Results After homologous overproduction of a candidate LMCO gene, extracellular laccase activity was detected when ABTS or SRG were used as substrates, and the recombinant protein was purified to homogeneity followed by biochemical characterization. The recombinant protein, called TrLAC1, has a molecular mass of 104 kDa. Optimal temperature and pH were respectively 40-45°C and 4, by using ABTS as substrate. TrLAC1 showed broad pH stability range of 3 to 7. Temperature stability revealed that TrLAC1 is not a thermostable enzyme, which was also confirmed by unfolding studies monitored by circular dichroism. Evolutionary studies were performed to shed light on the LMCO family, and the phylogenetic tree was reconstructed using maximum-likelihood method. LMCO and classical laccases were clearly divided into two distinct groups. Finally, Darwinian selection was tested, and the results showed that positive selection drove the evolution of sequences leading to well-known laccases involved in ligninolysis. Positively-selected sites were observed that could be used as targets for mutagenesis and functional studies between classical laccases and LMCO from T. reesei. Conclusions Homologous production and evolutionary studies of the first LMCO from the biomass-degrading fungus T. reesei gives new insights into the physicochemical parameters and biodiversity in this family. PMID:20735824

  19. Primary structure of a Japanese lacquer tree laccase as a prototype enzyme of multicopper oxidases

    Microsoft Academic Search

    Kazutomo Nitta; Kunishige Kataoka; Takeshi Sakurai

    2002-01-01

    The cDNA library of the Japanese lacquer tree (Rhus vernicifera) was constructed by the reverse transcription of mRNA. A cDNA encoding laccase was amplified by PCR using primers based on the N-terminal amino acid sequences of the purified laccase and its peptide fragments formed by digestions with chymotrypsin and trypsin, and subcloned. The laccase cDNA clone contained a single, large

  20. Laccase biosensors based on different enzyme immobilization strategies for phenolic compounds determination.

    PubMed

    Casero, E; Petit-Domínguez, M D; Vázquez, L; Ramírez-Asperilla, I; Parra-Alfambra, A M; Pariente, F; Lorenzo, E

    2013-10-15

    Different enzyme immobilization approaches of Trametes versicolor laccase (TvL) onto gold surfaces and their influence on the performance of the final bioanalytical platforms are described. The laccase immobilization methods include: (i) direct adsorption onto gold electrodes (TvL/Au), (ii) covalent attachment to a gold surface modified with a bifunctional reagent, 3,3'-Dithiodipropionic acid di (N-succinimidyl ester) (DTSP), and (iii) integration of the enzyme into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface (TvL/MPTS/Au). The characterization and applicability of these biosensors are described. Characterization is performed in aqueous acetate buffer solutions using atomic force microscopy (AFM), providing valuable information concerning morphological data at the nanoscale level. The response of the three biosensing platforms developed, TvL/Au, TvL/DTSP/Au and TvL/MPTS/Au, is evaluated in the presence of hydroquinone (HQ), used as a phenolic enzymatic substrate. All systems exhibit a clear electrocatalytic activity and HQ can be amperometrically determined at -0.10 V versus Ag/AgCl. However, the performance of biosensors - evaluated in terms of sensitivity, detection limit, linear response range, reproducibility and stability - depends clearly on the enzyme immobilization strategy, which allows establishing its influence on the enzyme catalytic activity. PMID:24054609

  1. Purification, characterization, and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum

    Microsoft Academic Search

    Didem Sutay Kocabas; Ufuk Bakir; Simon E. V. Phillips; Michael J. McPherson; Zumrut B. Ogel

    2008-01-01

    A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains\\u000a heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol\\u000a oxidase activities were most

  2. Biofuel cell for generating power from methanol substrate using alcohol oxidase bioanode and air-breathed laccase biocathode.

    PubMed

    Das, Madhuri; Barbora, Lepakshi; Das, Priyanki; Goswami, Pranab

    2014-09-15

    We report here an alcohol oxidase (AOx) based third generation bioanode for generating power from methanol substrate in a fuel cell setup using air breathed laccase biocathode. A composite three dimensional microporous matrix containing multiwalled carbon nanotubes, carbon paste and nafion was used as electroactive support for immobilization of the enzymes on toray carbon paper as supporting electrode in the fabrication of the bioelectrodes. Polyethylenimine was used to electrostatically stabilize the AOx (pI 4.3) on the anode operating on direct electrochemistry principle. Osmium tetroxide on poly (4-vinylpyridine) was used to wire the laccase for electron transfer in the biocathode. The enzymatic biofuel cell (EFC) generated an open circuit potential of 0.61 (±0.02) V with a maximum power density of 46 (±0.002) µW cm(-2) at an optimum of 1M methanol, 25 °C and an internal resistance of 0.024 µ?. The operation and storage half life (t1/2) of the EFC were 17.22 h and 52 days, respectively at a fixed load of 1.85 ?. The findings have demonstrated the feasibility of developing EFC using AOx based bioanode and laccase based biocathode without applying any toxic free mediator and metal electrode supports for generating electricity. PMID:24727604

  3. Potentialities of a membrane reactor with laccase grafted membranes for the enzymatic degradation of phenolic compounds in water.

    PubMed

    Chea, Vorleak; Paolucci-Jeanjean, Delphine; Sanchez, José; Belleville, Marie-Pierre

    2014-01-01

    This paper describes the degradation of phenolic compounds by laccases from Trametes versicolor in an enzymatic membrane reactor (EMR). The enzymatic membranes were prepared by grafting laccase on a gelatine layer previously deposited onto ?-alumina tubular membranes. The 2,6-dimethoxyphenol (DMP) was selected  from among the three different phenolic compounds tested (guaiacol, 4-chlorophenol and DMP) to study the performance of the EMR in dead end configuration. At the lowest feed substrate concentration tested (100 mg·L-1), consumption increased with flux (up to 7.9 × 103 mg·h-1·m-2 at 128 L·h-1·m-2), whereas at the highest substrate concentration (500 mg·L-1), it was shown that the reaction was limited by the oxygen content. PMID:25295628

  4. Treatment of halogenated phenolic compounds by sequential tri-metal reduction and laccase-catalytic oxidation.

    PubMed

    Dai, Yunrong; Song, Yonghui; Wang, Siyu; Yuan, Yu

    2015-03-15

    Halogenated phenolic compounds (HPCs) are exerting negative effects on human beings and ecological health. Zero-valence metal reduction can dehalogenate HPCs rapidly but cannot mineralize them. Enzymatic catalysis can oxidize phenolic compounds but fails to dehalogenate efficiently, and sometimes even produces more toxic products. In this study, [Fe|Ni|Cu] tri-metallic reduction (TMR) and laccase-catalytic oxidation (LCO) processes were combined to sequentially remove HPCs, including triclosan, tetrabromobisphenol A, and 2-bromo-4-fluorophenol in water. The kinetics, pH and temperature dependences of TMR and LCO were obtained. The detailed TMR, LCO, and TMR-LCO transformation pathways of three HPCs were well described based on the identification of intermediate products and frontier molecular orbitals (FMOs) theory. The results showed that the two-stage process worked synergically: TMR that reductively dehalogenated HPCs followed by LCO that completely removed dehalogenated products. TMR was proven to not only improve biodegradability of HPCs but also reduce the yield of potential carcinogenic by-products. Furthermore, a TMR-LCO flow reactor was assembled and launched for 256 h, during which >95% HPCs and >75% TOC were removed. Meanwhile, monitored by microorganism indicators, 83.2%-92.7% acute toxicity of HPCs was eliminated, and the genotoxicity, produced by LCO, was also avoided by using TMR as pretreatment process. PMID:25596562

  5. Engineering and Applications of fungal laccases for organic synthesis.

    PubMed

    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

  6. 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

  7. Novel phenolic biosensor based on a magnetic polydopamine-laccase-nickel nanoparticle loaded carbon nanofiber composite.

    PubMed

    Li, Dawei; Luo, Lei; Pang, Zengyuan; Ding, Lei; Wang, Qingqing; Ke, Huizhen; Huang, Fenglin; Wei, Qufu

    2014-04-01

    A novel phenolic biosensor was prepared on the basis of a composite of polydopamine (PDA)-laccase (Lac)-nickel nanoparticle loaded carbon nanofibers (NiCNFs). First, NiCNFs were fabricated by a combination of electrospinning and a high temperature carbonization technique. Subsequently, the magnetic composite was obtained through one-pot Lac-catalyzed oxidation of dopamine (DA) in an aqueous suspension containing Lac, NiCNFs, and DA. Finally, a magnetic glass carbon electrode (MGCE) was employed to separate and immobilize the composite; the modified electrode was then denoted as PDA-Lac-NiCNFs/MGCE. Fourier transform infrared (FT-IR) spectra and cyclic voltammetry (CV) analyses revealed the NiCNFs had good biocompatibility for Lac immobilization and greatly facilitated the direct electron transfer between Lac and electrode surface. The immobilized Lac showed a pair of stable and well-defined redox peaks, and the electrochemical behavior of Lac was a surface-controlled process in pH 5.5 acetate buffer solution. The PDA-Lac-NiCNFs/MGCE for biosensing of catechol exhibited a sensitivity of 25 ?A mM(-1) cm(-2), a detection limit of 0.69 ?M (S/N = 3), and a linear range from 1 ?M to 9.1 mM, as well as good selectivity and stability. Meanwhile, this novel biosensor demonstrated its promising application in detecting catechol in real water samples. PMID:24606719

  8. Hydrogen peroxide produced by glucose oxidase affects the performance of laccase cathodes in glucose/oxygen fuel cells: FAD-dependent glucose dehydrogenase as a replacement.

    PubMed

    Milton, Ross D; Giroud, Fabien; Thumser, Alfred E; Minteer, Shelley D; Slade, Robert C T

    2013-11-28

    Hydrogen peroxide production by glucose oxidase (GOx) and its negative effect on laccase performance have been studied. Simultaneously, FAD-dependent glucose dehydrogenase (FAD-GDH), an O2-insensitive enzyme, has been evaluated as a substitute. Experiments focused on determining the effect of the side reaction of GOx between its natural electron acceptor O2 (consumed) and hydrogen peroxide (produced) in the electrolyte. Firstly, oxygen consumption was investigated by both GOx and FAD-GDH in the presence of substrate. Relatively high electrocatalytic currents were obtained with both enzymes. O2 consumption was observed with immobilized GOx only, whilst O2 concentration remained stable for the FAD-GDH. Dissolved oxygen depletion effects on laccase electrode performances were investigated with both an oxidizing and a reducing electrode immersed in a single compartment. In the presence of glucose, dramatic decreases in cathodic currents were recorded when laccase electrodes were combined with a GOx-based electrode only. Furthermore, it appeared that the major loss of performance of the cathode was due to the increase of H2O2 concentration in the bulk solution induced laccase inhibition. 24 h stability experiments suggest that the use of O2-insensitive FAD-GDH as to obviate in situ peroxide production by GOx is effective. Open-circuit potentials of 0.66 ± 0.03 V and power densities of 122.2 ± 5.8 ?W cm(-2) were observed for FAD-GDH/laccase biofuel cells. PMID:24121716

  9. 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-cellularly to a high level of 7200 U L-1 in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation. PMID:23092193

  10. Banana skin: a novel material for a low-cost production of laccase

    E-print Network

    Cruz, Johann Faccelo Osma

    2008-01-01

    Laccases (benzenodiol: oxygen oxidoreductases; EC 1.10.3.2) are multicopper oxidases of wide substrate specificity mainly found in white-rot fungi, which are the only microorganisms able to degrade the whole wood components, but they are also expressed in bacteria and higher plants. Laccases are used currently in biotechnological processes because this enzyme oxidizes both phenolic and non-phenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. In this work banana skin has been selected as a supporting material for laccase produntion because of its high content in carbohydrates, which due to their organic nature are easily metabolized by the fungus. In addition, its content in ascorbic acid exerts an inhibitory effect against bacteria. The activity of the produced laccase is tested in decoloration studies.

  11. Laccase?catalysed polymeric dye synthesis from plant?derived phenols for potential application in hair dyeing: Enzymatic colourations driven by homo? or hetero?polymer synthesis

    PubMed Central

    Jeon, Jong?Rok; Kim, Eun?Ju; Murugesan, Kumarasamy; Park, Hyo?Keun; Kim, Young?Mo; Kwon, Jung?Hee; Kim, Wang?Gi; Lee, Ji?Yeon; Chang, Yoon?Seok

    2010-01-01

    Summary Laccase efficiently catalyses polymerization of phenolic compounds. However, knowledge on applications of polymers synthesized in this manner remains scarce. Here, the potential of laccase?catalysed polymerization of natural phenols to form products useful in hair dyeing was investigated. All 15 tested phenols yielded coloured products after laccase treatment and colour diversity was attained by using mixtures of two phenolic monomers. After exploring colour differentiation pattern of 120 different reactions with statistical regression analysis, three monomer combinations, namely gallic acid and syringic acid, catechin and catechol, and ferulic acid and syringic acid, giving rise to brown, black, and red materials, respectively, were further characterized because such colours are commercially important for grey hair dyeing. Selected polymers could strongly absorb visible light and their hydrodynamic sizes ranged from 100 to 400?nm. Analyses of enzyme kinetic constants, liquid chromatography and electrospray ionization?mass spectrometry (ESI?MS) coupled with collision?induced dissociation MS/MS indicate that both monomers in reactions involving catechin and catechol, and ferulic acid and syringic acid, are coloured by heteropolymer synthesis, but the gallic acid/syringic acid combination is based on homopolymer mixture formation. Comparison of colour parameters from these three reactions with those of corresponding artificial homopolymer mixtures also supported the idea that laccase may catalyse either hetero? or homo?polymer synthesis. We finally used selected materials to dye grey hair. Each material coloured hair appropriately and the dyeing showed excellent resistance to conventional shampooing. Our study indicates that laccase?catalysed polymerization of natural phenols is applicable to the development of new cosmetic pigments. PMID:21255331

  12. Phenol contents, oxidase activities, and the resistance of coffee to the leaf miner Leucoptera coffeella.

    PubMed

    Ramiro, Daniel Alves; Guerreiro-Filho, Oliveiro; Mazzafera, Paulo

    2006-09-01

    We examined the role of phenolic compounds, and the enzymes peroxidase and polyphenol oxidase, in the expression of resistance of coffee plants to Leucoptera coffeella (Lepidoptera: Lyonetiidae). The concentrations of total soluble phenols and chlorogenic acid (5-caffeoylquinic acid), and the activities of the oxidative enzymes peroxidase (POD) and polyphenol oxidase (PPO), were estimated in leaves of Coffea arabica, C. racemosa, and progenies of crosses between these species, which have different levels of resistance, before and after attack by this insect. The results indicate that phenols do not play a central role in resistance to the coffee leaf miner. Differences were detected between the parental species in terms of total soluble phenol concentrations and activities of the oxidative enzymes. However, resistant and susceptible hybrid plants did not differ in any of these characteristics. Significant induction of chlorogenic acid and PPO was only found in C. racemosa, the parental donator of the resistance genes against L. coffeella. High-performance liquid chromatography (HPLC) analysis also showed qualitative similarity between hybrids and the susceptible C. arabica. These results suggest that the phenolic content and activities of POD and PPO in response to the attack by the leaf miner may not be a strong evidence of their participation in direct defensive mechanisms. PMID:16906360

  13. Laccases from Aureobasidium pullulans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. 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.

  15. 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

  16. Structural and functional characterization of two-domain laccase from Streptomyces viridochromogenes.

    PubMed

    Trubitsina, L I; Tishchenko, S V; Gabdulkhakov, A G; Lisov, A V; Zakharova, M V; Leontievsky, A A

    2015-05-01

    Laccase (EC 1.10.3.2) is one of the most common copper-containing oxidases found in many organisms and catalyses oxidation of primarily phenolic compounds by oxygen. A recently found bacterial laccase whose molecule is formed by two domains - the so called two-domain laccase (2DLac) or small laccase - has unusual resistance to inhibitors and an alkaline optimum of activity. The causes of these properties, as well as the biological function of two-domain laccases, are poorly understood. We performed an enzymatic and structural characterization of 2DLac from Streptomyces viridochromogenes (SvSL). It was cloned and overproduced in Escherichia coli. Phenolic compounds were oxidized in the presence of the enzyme under alkaline but not acidic conditions. Conversely, nonphenolic compounds were oxidized at acidic but not alkaline pH. SvSL catalysed oxidation of nonphenolic compounds more efficiently than that of phenols. Moreover, this two-domain laccase displayed a cytochrome c oxidase activity and exhibited no ferroxidase activity. The enzyme was resistant to specific inhibitors of copper-containing oxidases, such as NaN3 and NaF. We succeeded in generating X-ray quality crystals and solved their structure to a resolution of 2.4 Å. SvSL is a homotrimer in its native state. Comparison of its structure with that of a three-domain laccase revealed differences in the second coordination sphere of the T2/T3 centre and solvent channels. The role of these differences in the resistance of the enzyme to inhibitors and the activity at alkaline pH is under discussion. PMID:25778839

  17. Transformation of the fungicide cyprodinil by a laccase of Trametes villosa in the presence of phenolic mediators and humic acid.

    PubMed

    Kang, Ki-Hoon; Dec, Jerzy; Park, Heekyung; Bollag, Jean-Marc

    2002-11-01

    Xenobiotic chemicals can be transformed or covalently bound to humic materials by oxidoreductive enzymes present in terrestrial systems. Chemicals that are not substrates for oxidoreductive enzymes may undergo transformation in the presence of certain reactive compounds, which are often referred to as mediators. In this study, cyprodinil, a broad-spectrum fungicide, did not show any transformation when incubated alone with a laccase from Trametes villosa. It was transformed to a significant extent, however, when a mediator was present. All of the 13 tested mediators belonged to the group of naturally occurring phenols. With some exceptions (2,6-dimethoxyphenol, syringic acid, and ferulic acid), phenols substituted with one or two methoxy groups were very effective mediators. In experiments with 14C-labeled cyprodinil, the radioactive label was largely associated with brown transformation products that precipitated out of the aqueous solution. As determined by mass spectrometry, the products were mixed oligomers resulting from cross-coupling between cyprodinil and a mediator. The addition of large amounts of humic acid (HA) (400 mg/L) to the reaction mixtures involving the most effective mediators reduced cyprodinil transformation (42.6-68.6%) by 12-48%, probably due to an inhibitory effect. The inhibition decreased with decreasing concentration of HA. The addition of HA (400 mg/L) to the reaction mixtures involving the least effective mediators or no mediators (control) enhanced cyprodinil transformation (0.3-17.6%) by 2.9-17.1%, probably as a result of binding to HA. PMID:12448535

  18. Laccases of prokaryotic origin: enzymes at the interface of protein science and protein technology.

    PubMed

    Martins, Lígia O; Durão, Paulo; Brissos, Vânia; Lindley, Peter F

    2015-03-01

    The ubiquitous members of the multicopper oxidase family of enzymes oxidize a range of aromatic substrates such as polyphenols, methoxy-substituted phenols, amines and inorganic compounds, concomitantly with the reduction of molecular dioxygen to water. This family of enzymes can be broadly divided into two functional classes: metalloxidases and laccases. Several prokaryotic metalloxidases have been described in the last decade showing a robust activity towards metals, such as Cu(I), Fe(II) or Mn(II) and have been implicated in the metal metabolism of the corresponding microorganisms. Many laccases, with a superior efficiency for oxidation of organic compounds when compared with metals, have also been identified and characterized from prokaryotes, playing roles that more closely conform to those of intermediary metabolism. This review aims to present an update of current knowledge on prokaryotic multicopper oxidases, with a special emphasis on laccases, anticipating their enormous potential for industrial and environmental applications. PMID:25572294

  19. Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock

    PubMed Central

    2014-01-01

    Background Biofuel production from lignocellulosic material is hampered by biomass recalcitrance towards enzymatic hydrolysis due to the compact architecture of the plant cell wall and the presence of lignin. The purpose of this work is to study the ability of an industrially available laccase-mediator system to modify and remove lignin during pretreatment of wood (Eucalyptus globulus) feedstock, thus improving saccharification, and to analyze the chemical modifications produced in the whole material and especially in the recalcitrant lignin moiety. Results Up to 50% lignin removal from ground eucalypt wood was attained by pretreatment with recombinant Myceliophthora thermophila laccase and methyl syringate as mediator, followed by alkaline peroxide extraction in a multistage sequence. The lignin removal directly correlated with increases (approximately 40%) in glucose and xylose yields after enzymatic hydrolysis. The pretreatment using laccase alone (without mediator) removed up to 20% of lignin from eucalypt wood. Pyrolysis-gas chromatography/mass spectrometry of the pretreated wood revealed modifications of the lignin polymer, as shown by lignin markers with shortened side chains and increased syringyl-to-guaiacyl ratio. Additional information on the chemical modifications produced was obtained by two-dimensional nuclear magnetic resonance of the whole wood swollen in dimethylsulfoxide-d6. The spectra obtained revealed the removal of guaiacyl and syringyl lignin units, although with a preferential removal of the former, and the lower number of aliphatic side-chains per phenylpropane unit (involved in main ?-O-4? and ?-?? inter-unit linkages), in agreement with the pyrolysis-gas chromatography/mass spectrometry results, without a substantial change in the wood polysaccharide signals. However, the most noticeable modification observed in the spectra was the formation of C?-oxidized syringyl lignin units during the enzymatic treatment. Further insight into the modifications of lignin structure, affecting other inter-unit linkages and oxidized structures, was attained by nuclear magnetic resonance of the lignins isolated from the eucalypt feedstock after the enzymatic pretreatments. Conclusions This work shows the potential of an oxidative enzymatic pretreatment to delignify and improve cellulase saccharification of a hardwood feedstock (eucalypt wood) when applied directly on the ground lignocellulosic material, and reveals the main chemical changes in the pretreated material, and its recalcitrant lignin moiety, behind the above results. PMID:24401177

  20. Crystal structure of a blue laccase from Lentinus tigrinus: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases

    PubMed Central

    Ferraroni, Marta; Myasoedova, Nina M; Schmatchenko, Vadim; Leontievsky, Alexey A; Golovleva, Ludmila A; Scozzafava, Andrea; Briganti, Fabrizio

    2007-01-01

    Background Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O2 reduction to H2O. Results The structure of a laccase from the white-rot fungus Lentinus (Panus) tigrinus, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 Å. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, ?-?1:?1 peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (?3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled. Conclusion This is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology. PMID:17897461

  1. WILD OAT (AVENA FATUA L.) SEED PHENOLICS AND POLYPHENOL OXIDASE: POTENTIAL ROLES IN SEED LONGEVITY AND RESISTANCE TO DECAY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wild oat seeds can survive in a dormant state for five to seven years in cultivated soils. Long-term survival requires both dormancy and resistance to decay. Phenolic compounds and polyphenol oxidase (PPO) have been implicated in plant defense, although their interactions remain obscure. We have cha...

  2. Free phenolics and polyphenol oxidase (PPO): the factors affecting post-cut browning in eggplant (Solanum melongena).

    PubMed

    Mishra, Bibhuti Bhusan; Gautam, Satyendra; Sharma, Arun

    2013-08-15

    Polyphenol oxidase (PPO) catalyses oxidation of phenolics, which results in instant but differential browning in many cut fruits and vegetables, including eggplant. Eight cultivars of eggplant were characterised by their PPO specific activity, phenolic content, browning index, and PPO polymorphism. In fresh eggplant, browning was found to be dependent on both the phenolic content and PPO specific activity, whereas, total phenolic content played a major role in browning of stored fruits. Interestingly, although browning index increased in stored eggplant fruits, PPO activity reduced in four out of eight cultivars studied. Phenolic level was found to increase in all these cultivars during storage. Although a significant level of homology was observed in PPO nucleotide and conceptually translated protein sequence, two cultivars, which displayed highest PPO specific activity, differed in the 38 amino acid stretch in the peptide region 301-338. PMID:23561085

  3. Laccase from Pycnoporus cinnabarinus and phenolic compounds: can the efficiency of an enzyme mediator for delignifying kenaf pulp be predicted?

    PubMed

    Andreu, Glòria; Vidal, Teresa

    2013-03-01

    In this work, kenaf pulp was delignified by using laccase in combination with various redox mediators and the efficiency of the different laccase–mediator systems assessed in terms of the changes in pulp properties after bleaching. The oxidative ability of the individual mediators used (acetosyringone, syringaldehyde, p-coumaric acid, vanillin and actovanillone) and the laccase–mediator systems was determined by monitoring the oxidation–reduction potential (ORP) during process. The results confirmed the production of phenoxy radicals of variable reactivity and stressed the significant role of lignin structure in the enzymatic process. Although changes in ORP were correlated with the oxidative ability of the mediators, pulp properties as determined after the bleaching stage were also influenced by condensation and grafting reactions. As shown here, ORP measurements provide a first estimation of the delignification efficiency of a laccase–mediator system. PMID:23403063

  4. New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

    PubMed Central

    2013-01-01

    Background Fungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Results Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with ?max of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (?max of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast. Conclusions The high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators. PMID:24159930

  5. Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum

    PubMed Central

    2011-01-01

    Background Laccases are multi-copper oxidases that catalyze the one electron oxidation of a broad range of compounds. Laccase substrates include substituted phenols, arylamines and aromatic thiols. Such compounds are activated by the enzyme to the corresponding radicals. Owing to their broad substrate range laccases are considered to be versatile biocatalysts which are capable of oxidizing natural and non-natural industrial compounds, with water as sole by-product. Results A novel CotA-type laccase from Bacillus pumilus was cloned, expressed and purified and its biochemical characteristics are presented here. The molecular weight of the purified laccase was estimated to be 58 kDa and the enzyme was found to be associated with four copper atoms. Its catalytic activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine (SGZ) was investigated. The kinetic parameters KM and kcat for ABTS were 80 ± 4 ?M and 291 ± 2.7 s-1, for 2,6-DMP 680 ± 27 ?M and 11 ± 0.1 s-1 and for SGZ only kcat could be estimated to be 66 ± 1.5 s-1. The pH optimum for ABTS was 4, for 2,6-DMP 7 and for SGZ 6.5 and temperature optima for ABTS and 2,6-DMP were found to be around 70°C. The screening of 37 natural and non-natural compounds as substrates for B. pumilus laccase revealed 18 suitable compounds. Three of them served as redox mediators in the laccase-catalyzed decolorization of the dye indigocarmine (IC), thus assessing the new enzyme's biotechnological potential. Conclusions The fully copper loaded, thermostable CotA laccase from Bacillus pumilus is a versatile laccase with potential applications as an industrial biocatalyst. PMID:21266052

  6. [Activity and expression of laccase, tyrosinase, glucanase, and chitinase genes during morphogenesis of Lentinus edodes].

    PubMed

    Vetchinkina, E P; Gorshkov, V Iu; Ageeva, M V; Gogolev, Iu V; Nikitina, V E

    2015-01-01

    Activation of expression of the lcc4 and tir genes encoding laccase and tyrosinase was observed during transition of a xylotrophic basidiomycete Lentinus edodes from the vegetative to the generative growth stages. This was especially pronounced in the brown mycelial mat (the stage preceding formation of the fruiting bodies). Development of this structure was shown to be associated with a sharp increase of laccase and tyrosinase activities, as well as with rearrangements in the phenol oxidase complex. Formation of the tissues with thickened cell walls was associated with enhanced expression of the chi and exg1 genes encoding chitinase and glucanase, respectively. Exogenous treatment of the vegetative mycelium with laccase preparation from the brown mycelial mat promoted formation of this morphological structure. Activation of the lcc4, tir, chi, and exg1 genes may be used as a marker of readiness to fruition in xylotrophic fungi. PMID:25916150

  7. Bilirubin oxidase-like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass.

    PubMed

    Xie, Ning; Ruprich-Robert, Gwenaël; Silar, Philippe; Chapeland-Leclerc, Florence

    2015-03-01

    Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(?) and bod2(?) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(?) bod2(?) double mutant was changed. The bod1(?) and bod2(?) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(?) and bod2(?) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases. PMID:24947769

  8. Summer drought decreases soil fungal diversity and associated phenol oxidase activity in upland Calluna heathland soil.

    PubMed

    Toberman, Hannah; Freeman, Chris; Evans, Chris; Fenner, Nathalie; Artz, Rebekka R E

    2008-11-01

    Natural moisture limitation during summer drought can constitute a stress for microbial communities in soil. Given globally predicted increases in drought frequency, there is an urgent need for a greater understanding of the effects of drought events on soil microbial processes. Using a long-term field-scale drought manipulation experiment at Clocaenog, Wales, UK, we analysed fungal community dynamics, using internal transcribed spacer-denaturing gradient gel electrophoresis (DGGE), over a 1-year period in the 6th year of drought manipulation. Ambient seasonality was found to be the dominant factor driving variation in fungal community dynamics. The summer drought manipulation resulted in a significant decline in the abundance of dominant fungal species, both independently of, and in interaction with, this seasonal variation. Furthermore, soil moisture was significantly correlated with the changes in fungal diversity over the drought manipulation period. While the relationship between species diversity and functional diversity remains equivocal, phenol oxidase activity was decreased by the summer drought conditions and there was a significant correlation with the decline of DGGE band richness among the most dominant fungal species during the drought season. Climatically driven events such as droughts may have significant implications for fungal community diversity and therefore, have the potential to interfere with crucial ecosystem processes, such as organic matter decomposition. PMID:18662311

  9. Oxidation of the erythro and threo forms of the phenolic lignin model compound 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol by laccases and model oxidants.

    PubMed

    Bohlin, Christina; Lundquist, Knut; Jönsson, Leif J

    2009-10-01

    Mixtures of equal amounts of the erythro and threo forms of the phenolic arylglycerol beta-aryl ether 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol were oxidized (i) with laccases from Trametes versicolor, Agaricus bisporus, Myceliophthora thermophila and Rhus vernicifera, (ii) with laccase-mediator systems consisting of T. versicolor laccase and ABTS or HBT, and (iii) with various model oxidants including cerium(IV) ammonium nitrate (CAN), lignin peroxidase, Fenton's reagent, and lead(IV) tetraacetate (LTA). All the laccases exhibited a similar preferential degradation of the threo form. The mediator ABTS counteracted the threo preference of laccase, but the mediator HBT did not affect it. The outer-sphere model oxidants CAN and lignin peroxidase showed a preferential degradation of the threo form. LTA and Fenton's reagent did not exhibit any stereo-preference. The results suggest that laccases of different origin, primary structure, and redox potential behave as typical outer-sphere oxidants in their interaction with the diastereomers of the arylglycerol beta-aryl ether. PMID:19646732

  10. Production of laccase from Trametes versicolor by solid-state fermentation using olive leaves as a phenolic substrate.

    PubMed

    Aydino?lu, Tu?ba; 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. PMID:22763778

  11. Laccases from Aureobasidium pullulans.

    PubMed

    Rich, Joseph O; Leathers, Timothy D; Anderson, Amber M; Bischoff, Kenneth M; Manitchotpisit, Pennapa

    2013-06-10

    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 for laccase production. Enzymes from A. pullulans were distinct from those from lignin-degrading fungi and associated with pigment production. Laccases from strains in phylogenetic clade 5, which produced a dark vinaceous pigment, exhibited a temperature optimum of 50-60°C and were stable for an hour at 50°C, unlike enzymes from the lignin-degrading fungi Trametes versicolor and Pycnoporus cinnabarinus. Laccase purified from A. pullulans strain NRRL 50381, a representative of clade 5, was glycosylated but had a molecular weight of 60-70kDa after Endo H treatment. Laccase purified from strain NRRL Y-2568, which produced a dark olivaceous pigment, was also glycosylated, but had a molecular weight of greater than 100kDa after Endo H treatment. PMID:23683702

  12. Isolation and cDNA cloning of novel hydrogen peroxide-dependent phenol oxidase from the basidiomycete Termitomyces albuminosus.

    PubMed

    Johjima, T; Ohkuma, M; Kudo, T

    2003-05-01

    A novel hydrogen peroxide-dependent phenol oxidase (TAP) was isolated from the basidiomycete Termitomyces albuminosus. TAP is an extracellular monomeric enzyme with an estimated molecular weight of 67 kDa. The purified enzyme can oxidize various phenolic compounds in the presence of hydrogen peroxide, but cannot oxidize 3,4-dimethoxybenzyl (veratryl) alcohol. Mn(II) was not required for catalysis by TAP. The optimum pH for TAP activity was 2.3, which is the lowest known optimum pH for a fungal phenol oxidase. The cDNA encoding TAP was cloned with reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers based on the N-terminal amino acid sequence of TAP and 5' rapid amplification of cDNA ends (RACE)-PCR. The cDNA encodes a mature protein of 449 amino acids with a 55-amino-acid signal peptide. The deduced amino acid sequence of TAP showed 56% identity with dye-decolorizing heme peroxidase (DYP) from the ascomycete Geotrichum candidum Dec 1, but no homology with other known peroxidases from fungi. PMID:12698279

  13. Green oxidations with laccase-mediator systems.

    PubMed

    Wells, A; Teria, M; Eve, T

    2006-04-01

    Laccases are oxidase enzymes produced by 'white rot' fungi as part of a complex armoury of redox enzymes used to break down lignin--part of the carbon cycle of nature. Laccases alone or in combination with redox co-catalysts have been shown to oxidize xenobiotic compounds under conditions that can be described as 'green'. This paper describes some novel oxidations using the laccase-mediator method and some current limitations to the use of this technology. PMID:16545100

  14. 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, a supervised learning system was developed from the clusters. The models showed high performance with an overall accuracy of 99.03%, error of 0.49%, MCC of 0.9367, precision of 94.20%, sensitivity of 94.20%, and specificity of 99.47% in a 5-fold cross-validation test. In an independent test, our models still provide a high accuracy of 97.98%, error rate of 1.02%, MCC of 0.8678, precision of 87.88%, sensitivity of 87.88% and specificity of 98.90%. Conclusion This study provides a useful classification system for better understanding of Laccases from their physicochemical properties perspective. We also developed a publically available web tool for the characterization of Laccase protein sequences (http://lacsubpred.bioinfo.ucr.edu/). Finally, the programs used in the study are made available for researchers interested in applying the system to other enzyme classes (https://github.com/tweirick/SubClPred). PMID:25350584

  15. An amperometric biosensor based on laccase immobilized onto Fe?O?NPs/cMWCNT/PANI/Au electrode for determination of phenolic content in tea leaves extract.

    PubMed

    Rawal, Rachna; Chawla, Sheetal; Devender; Pundir, C S

    2012-09-10

    A method is described for the construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase onto iron oxide nanoparticles (Fe?O?NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto a gold (Au) electrode. The modified electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3s at pH 6.0 (0.1 M sodium acetate buffer) and 35°C, when operated at 0.3 V vs. Ag/AgCl. Linear range, detection limit were 0.1-10 ?M (lower concentration range) and 10-500 ?M (higher concentration range), and 0.03 ?M respectively. The sensor measured total phenolic content in tea leaves extract. The enzyme electrode lost 25% of its initial activity after its 150 uses over a period of 4 months, when stored at 4°C. PMID:22883551

  16. 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

  17. 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. PMID:25652594

  18. Can laccases catalyze bond cleavage in lignin?

    PubMed

    Munk, Line; Sitarz, Anna K; Kalyani, Dayanand C; Mikkelsen, J Dalgaard; Meyer, Anne S

    2015-01-01

    Modification of lignin is recognized as an important aspect of the successful refining of lignocellulosic biomass, and enzyme-assisted processing and upcycling of lignin is receiving significant attention in the literature. Laccases (EC 1.10.3.2) are taking the centerstage of this attention, since these enzymes may help degrading lignin, using oxygen as the oxidant. Laccases can catalyze polymerization of lignin, but the question is whether and how laccases can directly catalyze modification of lignin via catalytic bond cleavage. Via a thorough review of the available literature and detailed illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin model compounds; ii) For laccases to catalyze inter-unit bond cleavage in lignin substrates, the presence of a mediator system is required. Clearly, the higher the redox potential of the laccase enzyme, the broader the range of substrates, including o- and p-diphenols, aminophenols, methoxy-substituted phenols, benzenethiols, polyphenols, and polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin is proposed. PMID:25560931

  19. Purification and Characterization of an Extracellular, Thermo-Alkali-Stable, Metal Tolerant Laccase from Bacillus tequilensis SN4

    PubMed Central

    Sondhi, Sonica; Sharma, Prince; Saini, Shilpa; Puri, Neena; Gupta, Naveen

    2014-01-01

    A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (kcat/Km) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and ?-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2?-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu2+, Co2+, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications. PMID:24871763

  20. Localization of the C4 and C 3 pathways of photosynthesis in the leaves of Pennisetum purpureum and other C4 species. Insignificance of phenol oxidase.

    PubMed

    Ku, S B; Gutierrez, M; Edwards, G E

    1974-12-01

    Mesophyll protoplasts and bundle-sheath cells of Pennisetum purpureum Schum., a C4 plant with low phenol-oxidase activity, were enzymatically separated according to methods recently developed with sugarcane (Saccharum officinarum L.), maize (Zea mays L.), and sorghum (Sorghum bicolor L.). The phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase of the C4 pathway were found to be localized in the mesophyll protoplasts while ribulose-1,5-diphosphate (RuDP) carboxylase, phosphoribulokinase and NADP-malic enzyme were localized in the bundle-sheath cells. The levels of these enzyme activities in the leaf extracts and in certain cellular preparations of P. purpureum are sufficient to account for the rate of photosynthesis in the leaf. These results on the activities and distribution of photosynthetic enzymes with P. purpureum preparations are consistent with our previous evidence for cellular separation of the C4 and the reductive pentose-phosphate pathways in C4 species.With chlorogenic acid as the substrate, P. purpureum, Setaria lutescens (Weigel) Hubb. and Panicum texanum Buckl. have relatively low phenol-oxidase activity, similar to that found in spinach (Spinacia oleracea L.); while sorghum, sugarcane, maize, Panicum capillare L. and P. miliaceum L. have relatively high phenoloxidase activity, similar to that in tobacco (Nicotiana tabacum L.). C4 species having high phenol-oxidase activity have substantial activity of the enzyme in both mesophyll and bundle-sheath extracts. Since phenol oxidase is found in both cell types it is not logical to expect preferential inhibition of RuDP carboxylase or other photosynthetic enzymes through phenol oxidation in mesophyll extracts, as has been previously suggested. When dithiothreitol and polyvinylpyrrolidone were included in the enzyme extraction medium, the activity of RuDP carboxylase increased 10% in P. purpureum and 59% in sugarcane leaf extracts. PMID:24442563

  1. Targeted mutations in a Trametes villosa laccase. Axial perturbations of the T1 copper.

    PubMed

    Xu, F; Palmer, A E; Yaver, D S; Berka, R M; Gambetta, G A; Brown, S H; Solomon, E I

    1999-04-30

    Trametes villosa laccase was mutated on a tetrapeptide segment near the type 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Trametes laccases were expressed in an Aspergillus oryzae host and characterized. The E460S mutation failed to produce a transformant with meaningful expression. The F463L and A461E mutations did not significantly alter the molecular and enzymological properties of the laccase. In contrast, the F463M mutation resulted in a type 1 copper site with an EPR signal intermediate between that of the wild type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in kcat and Km. These effects are attributed to a significant perturbation of the T1 copper center caused by the coordination of the axial methionine (M463) ligand. PMID:10212209

  2. Extracellular and Intracellular Polyphenol Oxidases Cause Opposite Effects on Sensitivity of Streptomyces to Phenolics: A Case of Double-Edged Sword

    PubMed Central

    Yang, Han-Yu; Chen, Carton W.

    2009-01-01

    Many but not all species of Streptomyces species harbour a bicistronic melC operon, in which melC2 encodes an extracellular tyrosinase (a polyphenol oxidase) and melC1 encodes a helper protein. On the other hand, a melC-homologous operon (melD) is present in all sequenced Streptomyces chromosomes and could be isolated by PCR from six other species tested. Bioinformatic analysis showed that melC and melD have divergently evolved toward different functions. MelD2, unlike tyrosinase (MelC2), is not secreted, and has a narrower substrate spectrum. Deletion of melD caused an increased sensitivity to several phenolics that are substrates of MelD2. Intracellularly, MelD2 presumably oxidizes the phenolics, thus bypassing spontaneous copper-dependent oxidation that generates DNA-damaging reactive oxygen species. Surprisingly, melC+ strains were more sensitive rather than less sensitive to phenolics than melC? strains. This appeared to be due to conversion of the phenolics by MelC2 to more hydrophobic and membrane-permeable quinones. We propose that the conserved melD operon is involved in defense against phenolics produced by plants, and the sporadically present melC operon probably plays an aggressive role in converting the phenolics to the more permeable quinones, thus fending off less tolerant competing microbes (lacking melD) in the phenolic-rich rhizosphere. PMID:19826489

  3. Laccase engineering by rational and evolutionary design.

    PubMed

    Pardo, Isabel; Camarero, Susana

    2015-03-01

    Laccases are considered as green catalysts of great biotechnological potential. This has attracted a great interest in designing laccases a la carte with enhanced stabilities or activities tailored to specific conditions for different fields of application. Over 20 years, numerous efforts have been taken to engineer these multicopper oxidases and to understand their reaction mechanisms by site-directed mutagenesis, and more recently, using computational calculations and directed evolution tools. In this work, we review the most relevant contributions made in the field of laccase engineering, from the comprehensive study of their structure-function relationships to the tailoring of outstanding biocatalysts. PMID:25586560

  4. Developmental regulation of laccase levels in Aspergillus nidulans.

    PubMed Central

    Law, D J; Timberlake, W E

    1980-01-01

    Asexual spores (conidia) of Aspergillus nidulans contain a dark green pigment which is not present in other cell types. Synthesis of this pigment is catalyzed, in part, by a developmentally controlled p-diphenol oxidase, or laccase, encoded at the gamma A genetic locus (A. J. Clutterbuck, J. Gen. Microbiol. 70:423-435, 1972). We have investigated the mechanisms regulating expression of the gamma A gene of A. nidulans. Vegetative hyphae grown in submerged culture lacked detectable laccase enzyme activity and neither contained nor synthesized immunoprecipitable laccase protein. When such cultures were induced to conidiate by harvesting the cells onto filter papers and aerating them, laccase levels began to increase after 10 to 16 h, reached a peak at 20 to 36 h, and then declined slowly. Immunological assays showed that increases in laccase enzyme activity were (i) proceded by a transient rise in the relative rate of laccase protein synthesis and (ii) closely paralleled by increases in the amount of laccase protein. Addition of cycloheximide to cultures at any time after inducing conidiation inhibited further accumulation of laccase enzyme activity. These data are most consistent with increases in laccase levels being due to regulated, de novo synthesis of laccase protein. Addition of inhibitors of ribonucleic acid synthesis to conidiating cultures also inhibited further accumulation of laccase, suggesting that laccase expression is regulated by alterations in the transcriptional activity of the gamma A locus. Images PMID:7000747

  5. Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

    PubMed Central

    Jiang, Haobo; Wang, Yang; Kanost, Michael R.

    1998-01-01

    Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle. PMID:9770467

  6. Laccase induction in fungi and laccase/N-OH mediator systems applied in paper mill effluent.

    PubMed

    Minussi, Rosana C; Pastore, Gláucia M; Durán, Nelson

    2007-01-01

    There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent. PMID:16376074

  7. Novel function of QM protein of shrimp (Penaeus japonicus) in regulation of phenol oxidase activity by interaction with hemocyanin.

    PubMed

    Xu, Jianyang; Wu, Suijie; Zhang, Xiaobo

    2008-01-01

    Since it was proposed to be a tumor suppressor in 1991, the QM protein has attracted intensive studies in plants, animals and fungi. Up to date, however, the function of QM protein remains unknown. In this investigation, it was found that the shrimp QM gene (designated as PjQM) was significantly up-regulated in virus- resistant shrimp, suggesting that the PjQM was involved in shrimp immunity. The GST pull-down assays showed that the PjQM protein interacted with shrimp hemocyanin and myosin, indicating that the PjQM protein might participate in prophenoloxidation (proPO) activation system of shrimp immunity. As revealed by RNAi assays, it was demonstrated for the first time that the QM protein could regulate the activity of phenol oxidase, a key enzyme in the proPO activation system of invertebrate immunity. This discovery showed a novel aspect of QM protein in arthropod immune response, which contributed a better understanding to the still poorly understood molecular events involved in innate immunity of arthropods. PMID:18453755

  8. Thermal inactivation kinetics of Rabdosia serra (Maxim.) Hara leaf peroxidase and polyphenol oxidase and comparative evaluation of drying methods on leaf phenolic profile and bioactivities.

    PubMed

    Lin, Lianzhu; Lei, Fenfen; Sun, Da-Wen; Dong, Yi; Yang, Bao; Zhao, Mouming

    2012-10-15

    Inactivation kinetics of peroxidase and polyphenol oxidase in fresh Rabdosia serra leaf were determined by hot water and steam blanching. Activation energy (52.30 kJ mol(-1)) of polyphenol oxidase inactivation was higher than that (20.15 kJ mol(-1)) of peroxidase. Water blanching at 90 °C or steam blanching at 100 °C for 90 s was recommended as the preliminary treatment for the retention of phenolics. Moreover, comparative evaluation of drying methods on the phenolics profiles and bioactivities of R. serra leaf were conducted. The results indicated that only intact leaf after freeze drying retained the initial quality. The sun- and air-dried leaves possessed identical phenolic profiles. The homogenised leaf (after freeze-drying) possessed a lower level of phenolics due to enzymatic degradation. Good antioxidant activities were detected for the sun- and air-dried leaves. There was insignificant difference in anti-tyrosinase and anti-?-glucosidase activities among sun-, air-, and freeze-dried leaves. PMID:23442652

  9. Phenols

    NASA Astrophysics Data System (ADS)

    Weber, Manfred; Weber, Markus

    Up to the end of the nineteenth century, phenol was recovered primarily from coal tar. With the commercialization of the phenolic resins, the demand for phenol grew significantly. Currently, the cumene-to-phenol process is the predominant synthetic route for the production of phenol. It is accompanied by acetone as a co-product. Cumene is oxidized with oxygen to form cumene hydroperoxide. The peroxide is subsequently decomposed to phenol and acetone, using a strong mineral acid as catalyst. The products are purified in a series of distillation columns. The cumene-to-phenol process is described in more detail in this chapter. An overview is given about synthetic routes via direct oxidation of benzene. None of these alternative routes has been commercialized. The chapter also gives an overview of global supply and use of phenol in 2008. Finally, the main natural sources and synthetic routes for cresols, xylenols, resorcinol, and bisphenol-A are described. These components are used as comonomers for special phenolic resins.

  10. Norway spruce (Picea abies) laccases: characterization of a laccase in a lignin-forming tissue culture.

    PubMed

    Koutaniemi, Sanna; Malmberg, Heli A; Simola, Liisa K; Teeri, Teemu H; Kärkönen, Anna

    2015-04-01

    Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce (Picea abies (L.) Karst.) laccases utilizing available genomic data. As many as 292 laccase encoding sequences (genes, gene fragments, and pseudogenes) were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five full-length laccase cDNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture. This laccase has an acidic pH optimum (pH 3.8-4.2) for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5??M; however, the laccase has a lower catalytic efficiency (V(max)/K(m)) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants. PMID:25626739

  11. 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

  12. Laccase production by the white-rot fungus Termitomyces clypeatus.

    PubMed

    Bose, Shilpi; Mazumder, Sharmishtha; Mukherjee, Mina

    2007-04-01

    Laccase was detected in the culture filtrate of white-rot fungus Termitomyces clypeatus. The enzyme was found at the late phase of submerged growth in a medium containing glucose or cellulose as the carbon source. The present study indicates that laccase produced by T. clypeatus is an intracellular enzyme, released in the medium due to cell lysis at the end of the growing phase. Laccase produced by T. clypeatus is different from the extracellular polyphenol oxidase of T. albuminosus, also produced at the late phase of growth. This is the first report of laccase production by a Termitomyces sp. PMID:17440914

  13. Phenols

    NSDL National Science Digital Library

    Reich, Ieva

    These organic chemistry exam/quiz questions all focus on the topic of phenols. The questions are divided into sections based on the following concepts: pericyclic reaction, claisen rearangement, extraction, synthesis mechanisms of reaction, and quinones.

  14. Copper Induction of Laccase Isoenzymes in the Ligninolytic Fungus Pleurotus ostreatus

    PubMed Central

    Palmieri, Gianna; Giardina, Paola; Bianco, Carmen; Fontanella, Bianca; Sannia, Giovanni

    2000-01-01

    Pleurotus ostreatus is a white rot basidiomycete that produces several extracellular laccase isoenzymes, including phenol oxidase A1b (POXA1b), POXA2, and POXC. POXC was the most abundant isoenzyme produced under all of the growth conditions examined in this study. Copper was the most efficient inducer of laccase activity among the putative inducers tested. The amounts of all of the previously described laccase isoenzymes increased substantially in copper-supplemented cultures. Under these conditions expression of POX isoenzymes was regulated at the level of gene transcription. It is worth noting that poxa1b mRNA was the most abundant induced transcript at all of the growth times analyzed, and the amount of this mRNA increased until day 7. The discrepancy between the poxa1b transcript and protein amounts can be explained by the presence of a high level of the protein in P. ostreatus cellular extract, which indicated that the POXA1b isoenzyme could be inefficiently secreted and/or that its physiological activity could occur inside the cell or on the cell wall. Moreover, the POXA1b isoenzyme behaved uniquely, as its activity was maximal on the second day of growth and then decreased. An analysis performed with protease inhibitors revealed that the loss of extracellular POXA1b activity could have been due to the presence of specific proteases secreted into the copper-containing culture medium that affected the extracellular POXA1b isoenzyme. PMID:10698752

  15. 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. PMID:24225344

  16. Alkali and halide-resistant catalysis by the multipotent oxidase from Marinomonas mediterranea.

    PubMed

    Jimenez-Juarez, Nuria; Roman-Miranda, Rosa; Baeza, Alejandro; Sánchez-Amat, Antonio; Vazquez-Duhalt, Rafael; Valderrama, Brenda

    2005-04-20

    The incorporation of fungal laccases into novel applications has been delayed mainly due to their intrinsic sensitivity towards halides and alkaline conditions. In order to explore new sources of enzymes we evaluated the multipotent polyphenol oxidase PPO1 from the marine bacterium Marinomonas mediterranea. Here we report that, in contrast to its fungal counterparts, PPO1 remained functional above neutral pH presenting high specificity for phenolic compounds, in particular for methoxyl-substituted mono-phenols and catechols. These properties, in addition to its tolerance towards chloride (up to 1 M) and its elevated redox potential at neutral pH (0.9 V), suggest this enzyme may be an interesting candidate for specific applications such as the Amperometric determination of phenolic compounds and bio-fuel cells. PMID:15831249

  17. Assessment of Antioxidant and Phenolic Compound Concentrations as well as Xanthine Oxidase and Tyrosinase Inhibitory Properties of Different Extracts of Pleurotus citrinopileatus Fruiting Bodies

    PubMed Central

    Alam, Nuhu; Yoon, Ki Nam; Lee, Kyung Rim; Kim, Hye Young; Shin, Pyung Gyun; Cheong, Jong Chun; Yoo, Young Bok; Shim, Mi Ja; Lee, Min Woong

    2011-01-01

    Cellular damage caused by reactive oxygen species has been implicated in several diseases, thus establishing a significant role for antioxidants in maintaining human health. Acetone, methanol, and hot water extracts of Pleurotus citrinopileatus were evaluated for their antioxidant activities against ?-carotene-linoleic acid and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, reducing power, ferrous ion-chelating abilities, and xanthine oxidase inhibitory activities. In addition, the tyrosinase inhibitory effects and phenolic compound contents of the extracts were also analyzed. Methanol and acetone extracts of P. citrinopileatus showed stronger inhibition of ?-carotene-linoleic acid compared to the hot water extract. Methanol extract (8 mg/mL) showed a significantly high reducing power of 2.92 compared to the other extracts. The hot water extract was more effective than the acetone and methanole extracts for scavenging DPPH radicals. The strongest chelating effect (92.72%) was obtained with 1.0 mg/mL of acetone extract. High performance liquid chromatography analysis detected eight phenolic compounds, including gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid, naringenin, hesperetin, formononetin, and biochanin-A, in an acetonitrile and hydrochloric acid (5 : 1) solvent extract. Xanthine oxidase and tyrosinase inhibitory activities of the acetone, methanol, and hot water extracts increased with increasing concentration. This study suggests that fruiting bodies of P. citrinopileatus can potentially be used as a readily accessible source of natural antioxidants. PMID:22783067

  18. A 24.7-kDa copper-containing oxidase, secreted by Thermobifida fusca, significantly increasing the xylanase/cellulase-catalyzed hydrolysis of sugarcane bagasse.

    PubMed

    Chen, Cheng-Yu; Hsieh, Zhi-Shen; Cheepudom, Jatuporn; Yang, Chao-Hsun; Meng, Menghsiao

    2013-10-01

    Thermobifida fusca is a moderately thermophilic soil bacterium belonging to Actinobacteria. It has been known for its capability to degrade plant cell wall polymers except lignin and pectin. To know whether it can produce enzymes to facilitate lignin degradation, the extracellular proteins bound to sugarcane bagasse were harvested and identified by liquid chromatography tandem mass spectrometry. Among the identified proteins, a putative copper-containing polyphenol oxidase of 241 amino acids, encoded by the locus Tfu_1114, was thought to presumably play a role in lignin degradation. This protein (Tfu1114) was thus expressed in E. coli and characterized. Similarly to common laccases, Tfu1114 is able to catalyze the oxidation reaction of phenolic and nonphenolic lignin related compounds such as 2,6-dimethoxyphenol and veratryl alcohol. More interestingly, it can significantly enhance the enzymatic hydrolysis of bagasse by xylanase and cellulase. Tfu1114 is stable against heat, with a half-life of 4.7 h at 90 °C, and organic solvents. It is sensitive to ethylenediaminetetraacetic acid and reducing agents but resistant to sodium azide, a potent inhibitor of laccases. Atomic absorption spectroscopy indicated that the ratio of copper to the protein monomer is 1, instead of 4, a feature of classical laccases. All these data suggest that Tfu1114 is a novel oxidase with laccase-like activity, potentially useful in biotechnology application. PMID:23377789

  19. 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. PMID:24951276

  20. Bilirubin Oxidase Activity of Bacillus subtilis CotA

    PubMed Central

    Sakasegawa, Shin-ichi; Ishikawa, Hidehiko; Imamura, Shigeyuki; Sakuraba, Haruhiko; Goda, Shuichiro; Ohshima, Toshihisa

    2006-01-01

    The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein. PMID:16391148

  1. First description of a laccase-like enzyme in soil algae

    Microsoft Academic Search

    Benjamin Otto; Dietmar Schlosser; Werner Reisser

    2010-01-01

    Laccases (EC 1.10.3.2) are versatile multi-copper oxidases so far found in higher plants, fungi, insects, prokaryotes and\\u000a lichens. In the present study, the production of an extracellular laccase-like enzyme by the coccoid green soil alga Tetracystis aeria was investigated and the enzyme was partly characterized, thereby providing the first description of a laccase-like enzyme\\u000a in soil algae. Enzyme production in

  2. Characterization of radical intermediates in laccase-mediator systems. A multifrequency EPR, ENDOR and DFT/PCM investigation.

    PubMed

    Brogioni, Barbara; Biglino, Daniele; Sinicropi, Adalgisa; Reijerse, Edward J; Giardina, Paola; Sannia, Giovanni; Lubitz, Wolfgang; Basosi, Riccardo; Pogni, Rebecca

    2008-12-28

    Suitable low molecular-weight compounds, called mediators, can be used in combination with the phenol-oxidase enzyme laccase to indirectly oxidize large organic substrates, such as environmental pollutants, which are not laccase natural substrates. The oxidation of two different synthetic redox mediators, violuric acid (VIO) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) has been studied under catalysis of two laccases from white-rot fungi (Trametes versicolor and Pleurotus ostreatus). VIO was selected as a prototype of the -NOH type of mediators and compared to ABTS, a well-known two-step redox system. To characterize the radical intermediates formed from both mediators after the enzymatic oxidation, a multifrequency EPR approach has been adopted. The radical species have been investigated employing 9.4 GHz (X-band), 34 GHz (Q-band) and 244 GHz (high field) EPR and pulse electron nuclear double resonance (ENDOR) techniques. Theoretical calculations based on density functional theory (DFT/PCM) have been performed to support and further interpret the experimental EPR and ENDOR data. This integrated approach allowed us to obtain a complete characterization of both radicals and to elucidate the type of the radical state (neutral or cationic). PMID:19060974

  3. Prediction model based on decision tree analysis for laccase mediators.

    PubMed

    Medina, Fabiola; Aguila, Sergio; Baratto, Maria Camilla; Martorana, Andrea; Basosi, Riccardo; Alderete, Joel B; Vazquez-Duhalt, Rafael

    2013-01-10

    A Structure Activity Relationship (SAR) study for laccase mediator systems was performed in order to correctly classify different natural phenolic mediators. Decision tree (DT) classification models with a set of five quantum-chemical calculated molecular descriptors were used. These descriptors included redox potential (?°), ionization energy (E(i)), pK(a), enthalpy of formation of radical (?(f)H), and OH bond dissociation energy (D(O-H)). The rationale for selecting these descriptors is derived from the laccase-mediator mechanism. To validate the DT predictions, the kinetic constants of different compounds as laccase substrates, their ability for pesticide transformation as laccase-mediators, and radical stability were experimentally determined using Coriolopsis gallica laccase and the pesticide dichlorophen. The prediction capability of the DT model based on three proposed descriptors showed a complete agreement with the obtained experimental results. PMID:23199741

  4. Secretion of laccase and manganese peroxidase by Pleurotus strains cultivate in solid-state using Pinus spp. sawdust

    PubMed Central

    Camassola, Marli; da Rosa, Letícia O.; Calloni, Raquel; Gaio, Tamara A.; Dillon, Aldo J.P.

    2013-01-01

    Pleurotus species secrete phenol oxidase enzymes: laccase (Lcc) and manganese peroxidase (MnP). New genotypes of these species show potential to be used in processes aiming at the degradation of phenolic compounds, polycyclic aromatic hydrocarbons and dyes. Hence, a screening of some strains of Pleurotus towards Lcc and MnP production was performed in this work. Ten strains were grown through solid-state fermentation on a medium based on Pinus spp. sawdust, wheat bran and calcium carbonate. High Lcc and MnP activities were found with these strains. Highest Lcc activity, 741 ± 245 U gdm?1 of solid state-cultivation medium, was detected on strain IB11 after 32 days, while the highest MnP activity occurred with strains IB05, IB09, and IB11 (5,333 ± 357; 4,701 ± 652; 5,999 ± 1,078 U gdm?1, respectively). The results obtained here highlight the importance of further experiments with lignocellulolytic enzymes present in different strains of Pleurotus species. Such results also indicate the possibility of selecting more valuable strains for future biotechnological applications, in soil bioremediation and biological biomass pre-treatment in biofuels production, for instance, as well as obtaining value-added products from mushrooms, like phenol oxidase enzymes. PMID:24159307

  5. Measuring phenol oxidase and peroxidase activities with pyrogallol, L-DOPA, and ABTS: Effect of assay conditions and soil type

    E-print Network

    German, Donovan P.

    carbon export. Measuring oxidative enzyme activities in soils is more problematic than assaying assay methods Extracellular enzymes Lignin Decomposition Soil carbon a b s t r a c t Microbial phenol hydrolytic enzyme activities because of the non-specific, free radical nature of the re- actions and complex

  6. Optimum conditions for inducing laccase production in Lentinus crinitus.

    PubMed

    Valle, J S; Vandenberghe, L P S; Santana, T T; Almeida, P H; Pereira, A M; Linde, G A; Colauto, N B; Soccol, C R

    2014-01-01

    Laccases are environmentally friendly alternatives in many important applications such as in bioremediation, biopulping, textile, and the food industry. They have wide substrate specificity, can oxidize a broad range of compounds, and show potential for use in various industrial processes. Therefore, developing methods to increase laccase production is important. In the current study, we aimed to identify optimum conditions for inducing laccase production in the basidiomycete Lentinus crinitus cultivated under varying nitrogen concentrations and in the presence of potential inducers of laccase production, including copper and phenolic compounds. Peak enzymatic activity (11,977 U/L) occurred at higher nitrogen concentrations (2.8 g/L nitrogen). Regardless of the nitrogen concentration, addition of copper increased the laccase activity and decreased mycelial growth, with maximum laccase activity (14,320 U/L) observed at the highest nitrogen concentration combined with 150 mM CuSO4. In addition, ethanol (0.5 or 1.0 mM) and guaiacol (1.5 mM) increased laccase production to 15,000, 14,800, and 14,850 U/L, respectively. Our findings highlighted the optimum conditions for producing L. crinitusderived laccase as potential alternatives to the conventional production and application of the enzyme. PMID:25366749

  7. Molecular characterization of a blue-copper laccase, TILA, of Aspergillus nidulans

    Microsoft Academic Search

    Mario Scherer; Reinhard Fischer

    2001-01-01

    Laccases are blue-copper enzymes, which oxidize phenolic substrates and thereby reduce molecular oxygen. They are widespread within fungi and are involved in lignin degradation or secondary metabolism such as pigment biosynthesis. Many fungi contain several laccases, not all of whose functions are known. In Aspergillus nidulans one, yA, is expressed during asexual development and converts a yellow precursor to the

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

    NASA Astrophysics Data System (ADS)

    Zhukova, Yu. N.; Zhukhlistova, N. E.; Lyashenko, A. V.; Morgunova, E. Yu.; Zaitsev, V. N.; Mikha?lov, A. M.

    2007-09-01

    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. In 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.

  9. Quantitative analysis of phenolic metabolites from different parts of Angelica keiskei by HPLC-ESI MS/MS and their xanthine oxidase inhibition.

    PubMed

    Kim, Dae Wook; Curtis-Long, Marcus J; Yuk, Heung Joo; Wang, Yan; Song, Yeong Hun; Jeong, Seong Hun; Park, Ki Hun

    2014-06-15

    Angelica keiskei is used as popular functional food stuff. However, quantitative analysis of this plant's metabolites has not yet been disclosed. The principal phenolic compounds (1-16) within A. keiskei were isolated, enabling us to quantify the metabolites within different parts of the plant. The specific quantification of metabolites (1-16) was accomplished by multiple reaction monitoring (MRM) using a quadruple tandem mass spectrometer. The limit of detection and limit of quantitation were calculated as 0.4-44 ?g/kg and 1.5-148 ?g/kg, respectively. Abundance and composition of these metabolites varied significantly across different parts of plant. For example, the abundance of chalcones (12-16) decreased as follows: root bark (10.51 mg/g)>stems (8.52 mg/g)>leaves (2.63 mg/g)>root cores (1.44 mg/g). The chalcones were found to be responsible for the xanthine oxidase (XO) inhibition shown by this plant. The most potent inhibitor, xanthoangelol inhibited XO with an IC50 of 8.5 ?M. Chalcones (12-16) exhibited mixed-type inhibition characteristics. PMID:24491695

  10. 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

  11. A novel Lentinula edodes laccase and its comparative enzymology suggest guaiacol-based laccase engineering for bioremediation.

    PubMed

    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

  12. Bilirubin oxidase bioelectrocatalytic cathodes: the impact of hydrogen peroxide.

    PubMed

    Milton, Ross D; Giroud, Fabien; Thumser, Alfred E; Minteer, Shelley D; Slade, Robert C T

    2014-01-01

    Mediator-less, direct electro-catalytic reduction of oxygen to water by bilirubin oxidase (Myrothecium sp.) was obtained on anthracene-modified, multi-walled carbon nanotubes. H2O2 was found to significantly and irreversibly affect the electro-catalytic activity of bilirubin oxidase, whereas similar electrodes comprised of laccase (Trametes versicolor) were reversibly inhibited. PMID:24185735

  13. Laccase activity tests and laccase inhibitors.

    PubMed

    Johannes, C; Majcherczyk, A

    2000-03-10

    Sulfhydryl organic compounds described as laccase inhibitors: dithiothreitol, thioglycolic acid, cysteine, diethyldithiocarbamic acid, and sodium azide were tested for their activity toward laccase of Trametes versicolor in different test systems utilising 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2, 6-dimethoxyphenol as enzyme substrates. Only sodium azide acted as a true laccase inhibitor and showed no significant interference with the enzyme tests. All other substances did not significantly inhibit the laccase activity and the previously reported inhibitory effects result from the reductions of the reaction products such as ABTS radical cation and diquinone or subsequent non-enzymatic interactions during substrate oxidation. The latter apparently forms a complex with unreacted ABTS displaying varied spectral characteristics and resulting in an underestimation of enzyme activity. PMID:10725542

  14. Effects of laccase-natural mediator systems on kenaf pulp.

    PubMed

    Andreu, Glòria; Vidal, Teresa

    2011-05-01

    This paper reports the first application of laccase-mediator systems (LMS) to kenaf pulp. Five natural phenolic compounds (acetosyringone, syringaldehyde, p-coumaric acid, vanillin and acetovanillone) were used as mediators in combination with laccase in an L stage in order to elucidate their effect on delignification. After LMS treatment, pulp samples were subjected to two alkaline treatments (an E or P stage). The results obtained were compared with those provided by the laccase-1-hydroxybenzotriazole (HBT) system. All natural mediators increased kappa number, decreased brightness and changed optical properties of the pulp after the L stage, suggesting that natural mediators tend to couple to fibers during a laccase-mediator treatment. The greatest delignification and bleaching effects after the P stage were obtained with syringaldehyde and acetosyringone, providing an effective means for delignifying kenaf, whereas those based on the other three could be used to functionalize kenaf with a view to obtaining pulp with novel properties. PMID:21444198

  15. 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. PMID:25192855

  16. Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization

    Microsoft Academic Search

    Mahdi Mohammadian; Mehrnoosh Fathi-Roudsari; Nasrin Mollania; Arastoo Badoei-Dalfard; Khosro Khajeh

    2010-01-01

    Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation\\u000a ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification\\u000a and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase\\u000a (CotA) from Bacillus sp. HR03 and then expressed

  17. Molecular cloning and functional characterization of a unique multipotent polyphenol oxidase from Marinomonas mediterranea

    Microsoft Academic Search

    Antonio Sanchez-Amat; Patricia Lucas-El??o; Eva Fernández; Jose Carlos Garc??a-Borrón; Francisco Solano

    2001-01-01

    Marinomonas mediterranea is a recently isolated melanogenic marine bacterium containing laccase and tyrosinase activities. These activities are due to the expression of two polyphenol oxidases (PPOs), a blue multicopper laccase and an SDS-activated tyrosinase. The gene encoding the first one, herein denominated M. mediterranea PpoA, has been isolated by transposon mutagenesis, cloned and expressed in Escherichia coli. Its predicted amino

  18. Which Copper is Paramagnetic in the Type 2\\/Type 3 Cluster of Laccase?

    Microsoft Academic Search

    Terri L. Fraterrigo; Catherine Miller; Bengt Reinhammar; D. R. McMillin

    1999-01-01

    Understanding the structure and function of the three copper atoms in the dioxygen reduction site of the blue oxidases such\\u000a as laccase has been a long standing challenge. In the case of a widely studied derivative, known as type 2-depleted laccase,\\u000a the removal of one copper from the cluster abolishes the EPR signal of the so-called type?2 copper. However, the

  19. Potential roles of laccases on virulence of Heterobasidion annosum s.s.

    PubMed

    Kuo, Hsiao-Che; Détry, Nicolas; Choi, Jaeyoung; Lee, Yong-Hwan

    2015-04-01

    Laccases, multi-copper-containing proteins, can catalyze the oxidation of phenolic substrates and have diverse functions such as a virulence factor in fungi. However, limited information can be found on the role of laccases in the interaction of Heterobasidion annosum s.s. to its host plant. Due to genome availability of the close-related species Heterobasidion irregulare, which contains 18 predicted laccase-encoding genes, phylogenetic analysis and gene expression profiling were performed. Eighteen laccase genes could be classified into 4 groups based on protein domains and phylogenetic analysis. However, there is no clear indication between phylogeny and domain compositions in laccases, and lifestyles of fungal species. The results of qRT-PCR showed that the expression of 8 laccase genes was highly up-regulated in Scots pine seedlings at 1 wpi. These data suggested that they might be involved in early stage of host infection. In addition, up-regulation of gene expression under glucose condition as a sole carbon source suggests that those laccases are not under carbon catabolite repression. Higher activities of laccase were observed in culture media containing cellulose, sucrose, or glucose compared to that of cellobiose as a sole carbon source. The highest mortality of Scots pine seedlings was observed when infected by H. annosum s.s. on extra carbon source as glucose. This was supported by the facts that glucose plays significant roles on up-regulation of laccase genes in planta and higher activity of laccase in H. annosum s.s.. Taking all together, laccases in H. annosum s.s. have diverse functions and a group of laccases may play a role during interactions with Scots pine seedlings. PMID:25757691

  20. In situ laccase-assisted overdyeing of denim using flavonoids.

    PubMed

    Guimarães, Clara; Kim, Suyeon; Silva, Carla; Cavaco-Paulo, Artur

    2011-10-01

    A laccase-mediated system for denim overdyeing using phenolic compounds was developed. Laccase from ascomycete Myceliophthora thermophila was able to oxidize phenolic compounds such as catechol and catechin and mediate their attachment to denim surfaces. Laccase-generated polymers gave rise to new coloration states from dark brown to green-yellow and replaced dyes in the overdyeing process. Process parameters, such as enzyme dosage, incubation time and presence of mediator, were studied by considering a compromise between the highest overdyeing level and lower energy/products consumption (2 U/mL laccase; 4 h incubation in the absence of mediator). Enzyme-generated polymers were followed by UV/Vis spectrophotometry and their level of attachment to denim surfaces was evaluated by means of spectral values quantification [k/s, Kubelka-Munk relationship (k=absorption coefficient, s=scattering coefficient)]. Overdyeing of denim with phenolics, such as catechol or catechin, was successfully achieved with acceptable levels in terms of durability. PMID:21751397

  1. Degradation of azo dyes by Trametes villosa laccase over long periods of oxidative conditions.

    PubMed

    Zille, Andrea; Górnacka, Barbara; Rehorek, Astrid; Cavaco-Paulo, Artur

    2005-11-01

    Trametes villosa laccase was used for direct azo dye degradation, and the reaction products that accumulated after 72 h of incubation were analyzed. Liquid chromatography-mass spectrometry (LC-MS) analysis showed the formation of phenolic compounds during the dye oxidation process as well as a large amount of polymerized products that retain azo group integrity. The amino-phenol reactions were also investigated by 13C-nuclear magnetic resonance and LC-MS analysis, and the polymerization character of laccase was shown. This study highlights the fact that laccases polymerize the reaction products obtained during long-term batch decolorization processes with azo dyes. These polymerized products provide unacceptable color levels in effluents, limiting the application of laccases as bioremediation agents. PMID:16269701

  2. Degradation of Azo Dyes by Trametes villosa Laccase over Long Periods of Oxidative Conditions

    PubMed Central

    Zille, Andrea; Górnacka, Barbara; Rehorek, Astrid; Cavaco-Paulo, Artur

    2005-01-01

    Trametes villosa laccase was used for direct azo dye degradation, and the reaction products that accumulated after 72 h of incubation were analyzed. Liquid chromatography-mass spectrometry (LC-MS) analysis showed the formation of phenolic compounds during the dye oxidation process as well as a large amount of polymerized products that retain azo group integrity. The amino-phenol reactions were also investigated by 13C-nuclear magnetic resonance and LC-MS analysis, and the polymerization character of laccase was shown. This study highlights the fact that laccases polymerize the reaction products obtained during long-term batch decolorization processes with azo dyes. These polymerized products provide unacceptable color levels in effluents, limiting the application of laccases as bioremediation agents. PMID:16269701

  3. Effect of various pollutants and soil-like constituents on laccase from Cerrena unicolor

    SciTech Connect

    Filazzola, M.T.; Sannino, F.; Rao, M.A.; Gianfreda, L.

    1999-12-01

    Laccase from Cerrena unicolor catalyses the oxidation of a wide range of aromatic compounds, either xenobiotic or naturally occurring phenols, leading to the formation of polymeric products. These are characterized by their low solubility and often may form precipitates or aggregates. The oxidizing efficiency of the enzyme is strictly dependent on the number of hydroxyl groups and the position of substituents on the phenolic molecules. During the reaction with some substrates, the enzyme is inactivated, because of possible adsorption of laccase molecules on newly formed polyphenols. By contrast, the oxidation of humic precursors (i.e., resorcinol, gallic acid, and pyrogallol) does not influence greatly the residual laccase activity. The triazinic herbicides, triazine and prometryn (2,4-bis(isopropylamino)-6-methylthio-s-triazine), are not substrates of laccase. They, however, inhibit laccase activity assayed with 2,4-dichlorophenol (2,4-DCP) or catechol as substrates. The reduction of substrate oxidation rates is usually accompanied by the retention of higher levels of residual enzymatic activity. These results, together with the slight recovery in laccase activity following dialysis of the assay mixture, provide further evidence that the enzyme may be incorporated into or adsorbed onto polyphenolic products, with a consequent reduction in the concentration of active forms of laccase.

  4. Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound

    PubMed Central

    Li, Kaichang; Xu, Feng; Eriksson, Karl-Erik L.

    1999-01-01

    Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2,2?-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both kcat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect kcat and the oxidation rate of dimer I. PMID:10347057

  5. Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound.

    PubMed

    Li, K; Xu, F; Eriksson, K E

    1999-06-01

    Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3, 4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both kcat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect kcat and the oxidation rate of dimer I. PMID:10347057

  6. Characterization and kinetic properties of the purified Trematosphaeria mangrovei laccase enzyme

    PubMed Central

    Atalla, M. Mabrouk; Zeinab, H. Kheiralla; Eman, R. Hamed; Amani, A. Youssry; Abeer, A. Abd El Aty

    2013-01-01

    The properties of Trematosphaeria mangrovei laccase enzyme purified on Sephadex G-100 column were investigated. SDS–PAGE of the purified laccase enzyme showed a single band at 48 kDa. The pure laccase reached its maximal activity at temperature 65 °C, pH 4.0 with Km equal 1.4 mM and Vmax equal 184.84 U/mg protein. The substrate specificity of the purified laccase was greatly influenced by the nature and position of the substituted groups in the phenolic ring. The pure laccase was tested with some metal ions and inhibitors, FeSO4 completely inhibited laccase enzyme and also highly affected by (NaN3) at a concentration of 1 mM. Amino acid composition of the pure enzyme was also determined. Carbohydrate content of purified laccase enzyme was 23% of the enzyme sample. The UV absorption spectra of the purified laccase enzyme showed a single peak at 260–280 nm. PMID:24235874

  7. 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

  8. 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

  9. Asymmetric diphenol formation by a fungal laccase.

    PubMed Central

    Bollag, J M; Liu, S Y; Minard, R D

    1979-01-01

    A laccase isolated from the fungus Rhizoctonia praticola catalyzed the cross-coupling of two differently halogenated phenols. When 2,4-dichlorophenol and 4-bromo-2-chlorophenol were incubated together with the enzyme, three dimers were formed and isolated by thin-layer chromatography. The molecular weights of these compounds were determined by mass spectrometry as 322, 410, and 366, which correspond with the respective dimers of each of the phenols and with a hybrid formed from both, tentatively assigned the structure 3,3',5'-trichloro-5-bromo-2,2'-diphenol. Gas chromatography-mass spectrometry analysis of these products and of their methylated derivatives lent support to these structural assignments. PMID:114116

  10. Which copper is paramagnetic in the type 2/type 3 cluster of laccase?

    PubMed

    Fraterrigo, T L; Miller, C; Reinhammar, B; McMillin, D R

    1999-04-01

    Understanding the structure and function of the three copper atoms in the dioxygen reduction site of the blue oxidases such as laccase has been a long standing challenge. In the case of a widely studied derivative, known as type 2-depleted laccase, the removal of one copper from the cluster abolishes the EPR signal of the so-called type 2 copper. However, the present studies of isotopically enriched protein from Polyporus versicolor show that the readily replaceable copper is not active in the low-temperature EPR spectrum of fungal laccase or its difluoride adduct. The same is true for the difluoride adduct of the tree enzyme. Thus, in type 2-depleted laccase the pattern of antiferromagnetic coupling is quite different from that of the native protein or the difluoride adduct. PMID:10499090

  11. Profile of natural redox mediators production of laccase-producing fungus Pleurotus ostreatus.

    PubMed

    Li, Xuanzhen; La, Guixiao; Cheng, Qian; Wang, Fengji; Feng, Fajie; Zhang, Bao; Zhang, Zhongyi

    2014-10-01

    Polycyclic aromatic hydrocarbons (PAHs) are highly toxic organic pollutants which are abundant and environmentally widespread. Anthracene is a simple PAH that can be oxidized by laccases, copper-containing oxidase enzymes, produced by some plants, fungi, and bacteria. In this work, the extracellular culture fluid (CF) of laccase-producing fungus Pleurotus ostreatus was separated to crude laccase (CL) and aqueous ultrafiltrate (AU) fractions. The rate of anthracene oxidation by CF was 68.7 % while oxidation by CL was only 27.8 %. The addition of AU enhanced anthracene oxidation rate by CL to 60.4 %, indicating that the natural redox-mediators were present in the CF. The laccase-catalyzed anthracene oxidation rate increased with increased AU concentration, implying that oxidation rate is positively related to the concentration of natural mediators when laccase activity is constant. The AU from fungal culture containing bran or straw enhanced laccase-catalyzed anthracene oxidation; this enhancement increased further with prolonged fungus-cultivation, implying that both bran and straw induce the natural mediators. Our findings suggest increasing natural mediator levels may be an alternative strategy to improve the biodegradability of laccase-producing fungi. PMID:25108623

  12. Polymerization of bisphenol A by purified laccase from Trametes villosa.

    PubMed

    Uchida, H; Fukuda, T; Miyamoto, H; Kawabata, T; Suzuki, M; Uwajima, T

    2001-09-21

    The metabolism of bisphenol A (BPA), an endocrine-disrupting chemical, was studied with a highly purified laccase from the basidiomycete Trametes villosa. The enzyme reaction products ranged widely from water-insoluble to -soluble compounds, one of which was previously identified as 4-isopropenylphenol. (1)H NMR and electron-impact mass spectrum analyses showed that one of the insoluble products was a BPA dimer, 5,5'-bis-[1-(4-hydroxy-phenyl)-1-methyl-ethyl]-biphenyl-2,2'-diol. Field-desorption mass spectrum analysis revealed BPA oligomers, some of which contained phenol, within the insoluble fraction. These results indicate that the laccase reaction may contain successive BPA polymerization, followed by either the addition of phenol to the formed oligomers or their decomposition to release 4-isopropenylphenol. PMID:11554734

  13. Molecular analysis of fungal communities and laccase genes in decomposing litter reveals differences among forest types but no impact of nitrogen deposition

    USGS Publications Warehouse

    Blackwood, C.B.; Waldrop, M.P.; Zak, D.R.; Sinsabaugh, R. L.

    2007-01-01

    The fungal community of the forest floor was examined as the cause of previously reported increases in soil organic matter due to experimental N deposition in ecosystems producing predominantly high-lignin litter, and the opposite response in ecosystems producing low-lignin litter. The mechanism proposed to explain this phenomenon was that white-rot basidiomycetes are more important in the degradation of high-lignin litter than of low-lignin litter, and that their activity is suppressed by N deposition. We found that forest floor mass in the low-lignin sugar-maple dominated system decreased in October due to experimental N deposition, whereas forest floor mass of high-lignin oak-dominated ecosystems was unaffected by N deposition. Increased relative abundance of basidiomycetes in high-lignin forest floor was confirmed by denaturing gradient gel electrophoresis (DGGE) and sequencing. Abundance of basidiomycete laccase genes, encoding an enzyme used by white-rot basidiomycetes in the degradation of lignin, was 5-10 times greater in high-lignin forest floor than in low-lignin forest floor. While the differences between the fungal communities in different ecosystems were consistent with the proposed mechanism, no significant effects of N deposition were detected on DGGE profiles, laccase gene abundance, laccase length heterogeneity profiles, or phenol oxidase activity. Our observations indicate that the previously detected accumulation of soil organic matter in the high-lignin system may be driven by effects of N deposition on organisms in the mineral soil, rather than on organisms residing in the forest floor. However, studies of in situ gene expression and temporal and spatial variability within forest floor communities will be necessary to further relate the ecosystem dynamics of organic carbon to microbial communities and atmospheric N deposition. ?? 2007 The Authors; Journal compilation ?? 2007 Society for Applied Microbiology and Blackwell Publishing Ltd.

  14. Structural insight into the oxidation of sinapic acid by CotA laccase.

    PubMed

    Xie, Tian; Liu, Zhongchuan; Liu, Qian; Wang, Ganggang

    2015-05-01

    Laccases can oxidize plenty of substrates by use of molecular oxygen as the final electron acceptor. The broad substrate spectrum is further expanded by using redox mediators in so-called laccase-mediator systems, but the structural studies on interactions between laccases and natural mediators are still absent. In this study, the crystal structure of CotA/sinapic acid complex is solved, structural comparison has revealed a novel substrate binding mode. The residue of His419 instead of His497 is bonding to the sinapic acid (SA) as the primary electron acceptor. Moreover, the binding of SA leads to 10° rotation on Arg416, our mutagenesis data exhibits that the residue Arg416 is crucial in the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and syringaldazine (SGZ). Furthermore, oxidation of several phenolic acids and one non-phenolic acid by CotA was investigated. By analyzing interactions between CotA and SA, it is indicated that the presence of methoxy groups in the ortho-position of the phenolic structure is crucial for the substrate recognition by CotA laccase. This work establishes structure-function relationships for laccase-natural mediator system. PMID:25799944

  15. Optical properties of sol-gel immobilized Laccase: a first step for its use in optical biosensing

    NASA Astrophysics Data System (ADS)

    Delfino, I.; Portaccio, M.; Della Ventura, B.; Manzo, G.; Mita, D. G.; Lepore, M.

    2012-04-01

    Laccases are cuproproteins belonging to the group of oxidoreductases that are able to catalyze the oxidation of various aromatic compounds (particularly phenols) with the concomitant reduction of oxygen to water. They are characterized by low substrate specificity and have a catalytic competence which widely varies depending on the source. Additionally, laccases have also very peculiar optical properties due to their copper active sites which participate to the reduction processes. All these characteristics make laccases very flexible biotic element for biotechnological applications. During the years, a number of studies have been devoted at exploiting catalytic properties of laccases and very few at profiting of their optical properties. Some preliminary studies by absorption, fluorescence FT-IR and Raman spectroscopies of commercial laccases have evidenced their potential usefulness for optical biosensing of phenol compounds as cathecol. Moreover the sol-gel process offers a convenient and versatile method for preparing optically transparent matrices at room temperature that can represent a very convenient support for laccase immobilization. Also for immobilised enzymes the above-mentioned techniques have allowed a detailed characterization of their optical properties that confirmed the potentials of laccases in optical biosensors and represented a fundamental step in the designing of an optimised optical biosensing scheme.

  16. Elucidation of the crystal structure of Coriolopsis caperata laccase: restoration of the structure and activity of the native enzyme from the T2-depleted form by copper ions.

    PubMed

    Glazunova, Olga A; Polyakov, Konstantin M; Fedorova, Tatyana V; Dorovatovskii, Pavel V; Koroleva, Olga V

    2015-04-01

    Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. A new laccase was isolated from the basidiomycete Coriolopsis caperata strain 0677 and its amino-acid sequence was determined. According to its physicochemical properties and spectroscopic features, the laccase from C. caperata is a high redox-potential blue laccase. Attempts to crystallize the native enzyme were unsuccessful. The copper type 2-depleted (T2D) laccase was prepared and crystallized. The structure of T2D laccase from C. caperata was solved at 1.6?Å resolution, and attempts to reconstruct the T2 copper centre were performed using Cu(+) and Cu(2+) ions. The structure of T2D+Cu(+) laccase was solved at 1.89?Å resolution. It was shown that the T2D+Cu(+) laccase structure contained four copper ions in the active site. Reconstruction could not be achieved when the T2D laccase crystals were treated with CuSO4. PMID:25849396

  17. Hydrotalcite-like anionic clays substituted with iron \\/ laccase, composites for biosensors applications

    Microsoft Academic Search

    Gabriela Carja; Gabriela Ciobanu; Gabriela Apostolescu; Sofronia Dranca; Nicolae Apostolescu

    2009-01-01

    Laccase - based biosensors are important for the selective determination of the phenolic compounds in the environmental matrices. The features of the enzyme immobilisation process and the characteristics of the inorganic porous matrix adsorbed on the electrode surface are both important for establishing the biosensor performances. This work presents the synthesis and physical-chemical characteristics of new hybrid materials based on

  18. Laccase-induced derivatization of unprotected amino acid L-tryptophan by coupling with p -hydroquinone 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide

    Microsoft Academic Search

    K. Manda; E. Hammer; A. Mikolasch; D. Gördes; K. Thurow; F. Schauer

    2006-01-01

    Summary.  We have studied the enzymatic derivatization of amino acids by use of the polyphenol oxidase laccase. Derivatization of L-tryptophan\\u000a was achieved by enzymatic crosslinking with the laccase substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide. The main product\\u000a (yield up to 70%) was identified as the quinoid compound 2-[2-(2-hydroxy-ethylcarbamoyl)-3,6-dioxo-cyclohexa-1,4-dienylamino]-3-(1H-indol-3-yl)-\\u000a propionic acid and demonstrates that laccase-catalyzed C–N-coupling occurred on the amino group of the aliphatic side chain.

  19. Laccases and their natural mediators: biotechnological tools for sustainable eco-friendly processes.

    PubMed

    Cañas, Ana I; Camarero, Susana

    2010-01-01

    Laccases are oxidoreductases which oxidize a variety of aromatic compounds using oxygen as the electron acceptor and producing water as by-product. The interest for these old enzymes (first described in 19th century) has progressively increased due to their outstanding biotechnological applicability. The presence of redox mediators is required for a number of biotechnological applications, providing the oxidation of complex substrates not oxidized by the enzyme alone. The efficiency of laccase-mediator systems to degrade recalcitrant compounds has been demonstrated, but still the high cost and possible toxicity of artificial mediators hamper their application at the industrial scale. Here, we present a general outlook of how alternative mediators can change this tendency. We focus on phenolic compounds related to lignin polymer that promotes the in vitro transformation of recalcitrant non-phenolic structures by laccase and are seemingly the natural mediators of laccases. The use of eco-friendly mediators easily available from lignocellulose, could contribute to the industrial implementation of laccases and the development of the 21th century biorefineries. PMID:20471466

  20. Biosensor based on laccase immobilized on plasma polymerized allylamine/carbon electrode.

    PubMed

    Ardhaoui, Malika; Bhatt, Sudhir; Zheng, Meihui; Dowling, Denis; Jolivalt, Claude; Khonsari, Farzaneh Arefi

    2013-08-01

    In this work, a simple and rapid method was used to functionalize carbon electrode in order to efficiently immobilize laccase for biosensor application. A stable allylamine coating was deposited using a low pressure inductively excited RF tubular plasma reactor under mild plasma conditions (low plasma power (10 W), few minutes) to generate high density amine groups (N/C ratio up to 0.18) on rough carbon surface electrodes. The longer was the allylamine plasma deposition time; the better was the surface coverage. Laccase from Trametes versicolor was physisorbed and covalently bound to these allylamine modified carbon surfaces. The laccase activities and current outputs measured in the presence of 2,2'-azinobis-(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) showed that the best efficiency was obtained for electrode plasma coated during 30 min. They showed also that for all the tested electrodes, the activities and current outputs of the covalently immobilized laccases were twice higher than the physically adsorbed ones. The sensitivity of these biocompatible bioelectrodes was evaluated by measuring their catalytic efficiency for oxygen reduction in the presence of ABTS as non-phenolic redox substrate and 2,6-dimethoxyphenol (DMP) as phenolic one. Sensitivities of around 4.8 ?A mg(-1)L and 2.7 ?A mg(-1)L were attained for ABTS and DMP respectively. An excellent stability of this laccase biosensor was observed for over 6 months. PMID:23706201

  1. 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. PMID:18459071

  2. Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system.

    PubMed

    Srebotnik, E; Hammel, K E

    2000-08-25

    Phenolic and nonphenolic (permethylated) synthetic [14C]lignins were depolymerized by Trametes villosa laccase in the presence of a radical mediator, 1-hydroxybenzotriazole (HOBT). Gel permeation chromatography of the treated lignins showed that approximately 10% of their substructures were cleaved. The system also cleaved a beta-O-4-linked model compound, 1-(4-ethoxy-3-methoxy-ring-[14C]phenyl)-2-(2-methoxyphenoxy)-propane- 1,3-diol, and a beta-1-linked model, 1, 2-bis-(3-methoxy-4-[14C]methoxyphenyl)-propane-1,3-diol, that represent nonphenolic substructures in lignin. High performance liquid chromatography of products from the oxidized models showed that they were produced in sufficient yields to account for the ability of laccase/HOBT to depolymerize nonphenolic lignin. PMID:10989177

  3. 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 (?E*) below 1.1 were measured for most dyes. PMID:10919791

  4. Nuclear track-based biosensors with the enzyme laccase

    NASA Astrophysics Data System (ADS)

    García-Arellano, H.; Fink, D.; Muñoz Hernández, G.; Vacík, J.; Hnatowicz, V.; Alfonta, L.

    2014-08-01

    A new type of biosensors for detecting phenolic compounds is presented here. These sensors consist of thin polymer foils with laccase-clad etched nuclear tracks. The presence of suitable phenolic compounds in the sensors leads to the formation of enzymatic reaction products in the tracks, which differ in their electrical conductivities from their precursor materials. These differences correlate with the concentrations of the phenolic compounds. Corresponding calibration curves have been established for a number of compounds. The sensors thus produced are capable to cover between 5 and 9 orders of magnitude in concentration - in the best case down to some picomoles. The sensor's detection sensitivity strongly depends on the specific compound. It is highest for caffeic acid and acid blue 74, followed by ABTS and ferulic acid.

  5. Induction, Isolation, and Characterization of Two Laccases from the White Rot Basidiomycete Coriolopsis rigida

    PubMed Central

    Saparrat, Mario C. N.; Guillén, Francisco; Arambarri, Angélica M.; Martínez, Angel T.; Martínez, María Jesús

    2002-01-01

    Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2?-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH2). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH2 extended the reactions catalyzed by these enzymes to the production of H2O2, the oxidation of Mn2+, the reduction of Fe3+, and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes. PMID:11916665

  6. Purification and Characterization of Laccase from Chaetomium thermophilium and Its Role in Humification

    PubMed Central

    Chefetz, Benny; Chen, Yona; Hadar, Yitzhak

    1998-01-01

    Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process. C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45°C both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70°C and had half-lives of 24 and 12 h at 40 and 50°C, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting. PMID:9726856

  7. Three-dimensional organization of three-domain copper oxidases: A review

    NASA Astrophysics Data System (ADS)

    Zhukhlistova, N. E.; Zhukova, Yu. N.; Lyashenko, A. V.; Za?tsev, V. N.; Mikha?lov, A. M.

    2008-01-01

    “Blue” copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrena maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.

  8. Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems.

    PubMed

    Johannes, C; Majcherczyk, A

    2000-02-01

    The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from Trametes versicolor and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was mediated by various laccase substrates (phenols and aromatic amines) or compounds produced and secreted by white rot fungi. The best natural mediators, such as phenol, aniline, 4-hydroxybenzoic acid, and 4-hydroxybenzyl alcohol were as efficient as the previously described synthetic compounds ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 1-hydroxybenzotriazole. The oxidation efficiency increased proportionally with the redox potentials of the phenolic mediators up to a maximum value of 0.9 V and decreased thereafter with redox potentials exceeding this value. Natural compounds such as methionine, cysteine, and reduced glutathione, containing sulfhydryl groups, were also active as mediator compounds. PMID:10653713

  9. Natural Mediators in the Oxidation of Polycyclic Aromatic Hydrocarbons by Laccase Mediator Systems

    PubMed Central

    Johannes, Christian; Majcherczyk, Andrzej

    2000-01-01

    The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from Trametes versicolor and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was mediated by various laccase substrates (phenols and aromatic amines) or compounds produced and secreted by white rot fungi. The best natural mediators, such as phenol, aniline, 4-hydroxybenzoic acid, and 4-hydroxybenzyl alcohol were as efficient as the previously described synthetic compounds ABTS [2,2?-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 1-hydroxybenzotriazole. The oxidation efficiency increased proportionally with the redox potentials of the phenolic mediators up to a maximum value of 0.9 V and decreased thereafter with redox potentials exceeding this value. Natural compounds such as methionine, cysteine, and reduced glutathione, containing sulfhydryl groups, were also active as mediator compounds. PMID:10653713

  10. Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity.

    PubMed

    Machczynski, Michael C; Vijgenboom, Erik; Samyn, Bart; Canters, Gerard W

    2004-09-01

    Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft. In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain. This protein is representative of a new family of enzymes--the two-domain laccases. Disruption of the corresponding gene abrogates laccase activity in the growth media. We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it. The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact. We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family. The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment. The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH. SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules. PMID:15295117

  11. 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. PMID:25272733

  12. Kinetic studies on the reaction between Trametes villosa laccase and dioxygen.

    PubMed

    Bukh, Christian; Lund, Martin; Bjerrum, Morten J

    2006-09-01

    A laccase from the fungus Trametes villosa (TviL) was investigated in order to elucidate the reaction mechanism of the reduction of dioxygen to water performed by this blue multi-copper oxidase. The ability of TviL to activate dioxygen was studied by stopped-flow experiments and under steady-state conditions. In the stopped-flow experiments TviL was reduced with a small excess of 4-hydroxyphenylacetic acid and afterwards the re-oxidation process was monitored by stopped-flow techniques by mixing with excess dioxygen. The reaction between reduced TviL and dioxygen was studied in the temperature range 10-35 degrees Celsius and with the concentration of dioxygen between 30 and 240microM. The observed rate constant k(obs) is found to be linear dependent on the dioxygen concentration and the observed second-order rate constant for the re-oxidation of reduced TviL is, at 25 degrees Celsius, determined to be 1.14x10(6)M(-1)s(-1). The activation energy, E(a), is from the same data determined to be 22kJmol(-1). Oxidation of different phenols (4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, guaiacolsulfonic acid and hydroquinone) under steady state conditions was investigated at concentrations of dioxygen ranging from 60 to 250microM. This line of experiments showed that TviL follows a ping-pong mechanism, and an observed second-order rate constant for the reaction with dioxygen of 7.1x10(5)M(-1)s(-1) at 25 degrees Celsius was found with 4-hydroxyphenylacetic acid as reducing substrate. The two kinetic methods resulted in observed rate constants of equal magnitudes for the reaction with dioxygen, which suggests that the rate limiting step(s) is (are) included in both the reactions studied by the two different techniques. PMID:16828870

  13. An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates.

    PubMed

    Tadesse, Mahelet Aweke; D'Annibale, Alessandro; Galli, Carlo; Gentili, Patrizia; Sergi, Federica

    2008-03-01

    Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4-0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on. PMID:18292878

  14. Laccase-Catalyzed Surface Modification of Thermo-Mechanical Pulp (TMP) for the Production of Wood Fiber Insulation Boards Using Industrial Process Water.

    PubMed

    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

  15. 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

  16. Hydrotalcite-like anionic clays substituted with iron / laccase, composites for biosensors applications

    NASA Astrophysics Data System (ADS)

    Carja, Gabriela; Ciobanu, Gabriela; Apostolescu, Gabriela; Dranca, Sofronia; Apostolescu, Nicolae

    2009-01-01

    Laccase - based biosensors are important for the selective determination of the phenolic compounds in the environmental matrices. The features of the enzyme immobilisation process and the characteristics of the inorganic porous matrix adsorbed on the electrode surface are both important for establishing the biosensor performances. This work presents the synthesis and physical-chemical characteristics of new hybrid materials based on iron containing layered double hydroxides / laccase. XRD and TGA-DTA analyses give information about the structural characteristics and thermal behaviour of the tested hybrids. The SEM images show the presence of a well crystallized texture of organized ensembles of platelets-like particles stacking on top of one another. The presence of iron in the substituted clay matrix is able to give rise to the specific redox properties that can be further used to tailor not only the laccase immobilisation process but also the biological sensing response of the biohybrid-transducer device.

  17. Polyphenol Oxidase: Characteristics and Mechanisms of Browning Control

    Microsoft Academic Search

    Christiane Queiroz; Maria Lúcia Mendes Lopes; Eliane Fialho; Vera Lúcia Valente-Mesquita

    2008-01-01

    Polyphenol oxidase, a copper-containing metalloprotein, catalyzes the oxidation of phenolic compounds to quinones, which produce brown pigments in wounded tissues. This enzymatic mechanism causes post harvest losses and mainly affects tropical fruits. In this article, some characteristics of polyphenol oxidase from different plants are reviewed and information about conventional and alternative methods to inactivate this enzyme is presented. Characterization of

  18. Direct electrochemistry of laccase immobilized on au nanoparticles encapsulated-dendrimer bonded conducting polymer: application for a catechin sensor.

    PubMed

    Rahman, Md Aminur; Noh, Hui-Bog; Shim, Yoon-Bo

    2008-11-01

    The direct electrochemistry of laccase was promoted by Au nanoparticle (AuNP)-encapsulated dendrimers (Den), which was applied for the detection of catechin. To increase the electrical properties, AuNPs were captured in the interiors of the dendrimer (Den-AuNPs) as opposed to attachment at the periphery of dendrimer. To prepare Den-AuNPs, the Au(III) ions were first coordinated in the interior of dendrimer with nitrogen ligands and then reduced to form AuNPs. The size of AuNPs encapsulated within the interior of the dendrimer was determined to be 1.7 +/- 0.4 nm. AuNPs-encapsulated dendrimers were then used to covalently immobilize laccase (PDATT/ Den(AuNPs)/laccase) through the formation of amide bonds between carboxylic acid groups of the dendrimer and the amine groups of laccase. Each layer of the PDATT/Den(AuNPs)/laccase probe was characterized using CV, EIS, QCM, XPS, SEM, and TEM. The PDATT/Den(AuNPs)/laccase probe displayed a well-defined direct electron-transfer (DET) process of laccase. The quasi-reversible redox peak of the Cu redox center of the laccase molecule was observed at -0.03/+0.13 V vs Ag/AgCl, and the electron-transfer rate constant was determined to be 1.28 s (-1). A catechin biosensor based on the electrocatalytic process by direct electrochemistry of laccase was developed. The linear range and the detection limit in the catechin analysis were determined to be 0.1-10 and 0.05 +/- 0.003 microM, respectively. Interference effects from various phenolic and polyphenolic compounds were also studied, and the general applicability of the biosensor was evaluated by selective analysis of real samples of catechin. PMID:18841943

  19. Oxidase Test Protocol

    NSDL National Science Digital Library

    American Society For Microbiology

    2010-11-11

    The oxidase test is used to detect the presence of the enzyme cytochrome oxidase in microorganisms.  While used as a taxonomic tool for many microorganisms, the test was established initially to differentiate Neisseria spp. (oxidase positive) from Acinetobacter (oxidase negative) and Pseudomonas spp. (oxidase positive) from the Enterobacteriaceae (oxidase negative).

  20. Composition and Distribution of Cell Wall Phenolic Compounds in Flax ( Linum usitatissimum L.) Stem Tissues

    Microsoft Academic Search

    T. A. Gorshkova; V. V. Salnikov; N. M. Pogodina; S. B. Chemikosova; E. V. Yablokova; A. V. Ulanov; M. V. Ageeva; J. E. G. van Dam; V. V. Lozovaya

    2000-01-01

    Cell wall phenolic compounds were analysed in xylem and bast fibre-rich peels of flax stems by biochemical, histochemical and ultrastructural approaches. Localization of cell wall phenolics by the enzyme-gold method using laccase revealed several gold particle distribution patterns. One of the major types (an even distribution of single gold particles) was present mainly in xylem, while the other (compact branched

  1. 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

  2. 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

  3. Formation of Halogenated Polyaromatic Compounds by Laccase Catalyzed Transformation of Halophenols.

    PubMed

    Lu, Junhe; Shao, Juan; Liu, Hui; Wang, Zunyao; Huang, Qingguo

    2015-07-21

    Laccases are a type of extracellular enzyme produced by fungi, bacteria, and plants. Laccase can catalyze one-electron oxidation of a variety of phenolic compounds using molecular oxygen as the electron acceptor. In this study, transformation of halophenols (XPs) in laccase-catalyzed oxidation processes was explored. We first examined the intrinsic reaction kinetics and found that the transformation of XPs appeared first order to the concentrations of both XPs and laccase. A numerical model was developed to describe the role of humic acid (HA) in this process. It was demonstrated that HA could reverse the oxidation of XPs by acting as the inner filtrator of XP radical intermediates formed upon reactions between the substrates and laccase. The extent of such reversion was proportional to HA concentration. MS analysis in combination with quantum chemistry computation suggested that coupling products were generated. XPs coupled to each via C-C or C-O-C pathways, generating hydroxyl polyhalogenated biphenyl ethers (OH-PCDEs) and hydroxyl polyhalogenated biphenyls, respectively. Many of these polyhalogenated products are known to be hazardous to the ecosystem and human health, but they are not synthetic chemicals. This study shed light on their genesis in the environmental media. PMID:26147794

  4. Purification and characterization of a laccase from the white-rot fungus Trametes multicolor.

    PubMed

    Leitner, Christian; Hess, Johann; Galhaup, Christiane; Ludwig, Roland; Nidetzky, Bernd; Kulbe, Klaus D; Haltrich, Dietmar

    2002-01-01

    The wood-degrading fungus Trametes multicolor secretes several laccase isoforms when grown on a simple medium containing copper in the millimolar range for stimulating laccase synthesis. The main isoenzyme laccase II was purified to apparent homogeneity from the culture supernatant by using anion-exchange chromatography and gel filtration. Laccase II is a monomeric glycoprotein with a molecular mass of 63 kDa as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, contains 18% glycosylation, and has a pI of 3.0. It oxidizes a variety of phenolic substrates as well as ferrocyanide and iodide. The pH optimum depends on the substrate employed and shows a bell-shaped pH activity profile with an optimum of 4.0 to 5.0 for the phenolic substrates, while the nonphenolic substrates ferrocyanide and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) show a monotonic pH profile with a rate decreasing with increasing pH. PMID:12018276

  5. Directed Evolution of Fungal Laccases

    PubMed Central

    Maté, Diana; García-Ruiz, Eva; Camarero, Susana; Alcalde, Miguel

    2011-01-01

    Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution. PMID:21966249

  6. Polyphenol oxidase activity in annual forage clovers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO)-mediated phenol reactions in red clover (Trifolium pratense L.) bind forage protein and reduce proteolysis, producing beneficial effects on forage protein degradability, silage fermentation, and soil-N cycling. We evaluated PPO activity in seven previously untested annual c...

  7. Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae.

    PubMed Central

    Berka, R M; Schneider, P; Golightly, E J; Brown, S H; Madden, M; Brown, K M; Halkier, T; Mondorf, K; Xu, F

    1997-01-01

    A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase. PMID:9251203

  8. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

    PubMed

    Peng, Zeyu; Dittmer, Neal T; Lang, Minglin; Brummett, Lisa M; Braun, Caroline L; Davis, Lawrence C; Kanost, Michael R; Gorman, Maureen J

    2015-04-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism. PMID:25701385

  9. 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 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. PMID:24019936

  10. 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. PMID:24019936

  11. 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??M, 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

  12. Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases.

    PubMed

    Xu, F

    1997-01-10

    The electronic absorption spectrum, susceptibility to fluoride inhibition, redox potential, and substrate turnover of several fungal laccases have been explored as a function of pH. The laccases showed a single spectrally detectable acid-base transition at pH 6-9 and a fluoride inhibition that diminished by increased pH (indicating a competition with hydroxide inhibition). Relatively small changes in the redox potentials (< or = 0.1 V) of laccase were observed over the pH 2.7-11. Under the catalysis of laccase, the apparent oxidation rates (kcat and kcat/Km) of two nonphenolic substrates, potassium ferrocyanide and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), decreased monotonically as the pH increased. In contrast, the apparent oxidation rates (kcat and kcat/Km) of three 2,6-dimethoxyphenols (whose pKa values range from 7.0 to 8.7) exhibited bell-shaped pH profiles whose maxima were distinct for each laccase but independent of the substrate. By correlating these pH dependences, it is proposed that the balance of two opposing effects, one generated by the redox potential difference between a reducing substrate and the type 1 copper of laccase (which correlates to the electron transfer rate and is favored for a phenolic substrate by higher pH) and another generated by the binding of a hydroxide anion to the type 2/type 3 coppers of laccase (which inhibits the activity at higher pH), contributes to the pH activity profile of the fungal laccases. PMID:8995383

  13. Expression and molecular properties of a new laccase of the white rot fungus Phlebia radiata grown on wood.

    PubMed

    Mäkelä, Miia R; Hildén, Kristiina S; Hakala, Terhi K; Hatakka, Annele; Lundell, Taina K

    2006-11-01

    Laccases are phenol-oxidizing, multicopper enzymes produced by fungi, plants, insects and bacteria. Fungal laccases are involved in ecologically important processes such as decomposition of lignocellulose (wood and plant material). In this work, in order to find out the role of fungal laccases upon wood colonisation and lignin decay, we describe expression of laccase-encoding genes in the white rot basidiomycete Phlebia radiata 79, when the fungus grows on its natural substrates, that is on softwood (Alnus incana) and hardwood (Picea abies). Clones for two laccase-encoding genes, the previously described Pr-lac1 and a new gene Pr-lac2 were characterized. Pr-lac2 coding region is interrupted by 12 introns and the deduced Lac2 protein displays a higher pI value (5.8) than Lac1 (pI 3.2-3.5). Phylogenetic analysis indicates differential evolution for the two laccases, and Lac2 demonstrates the highest sequence identity with Trametes laccases (66%). Transcripts of Pr-lac1 were the most abundant both in solid-state softwood and semi-solid hardwood cultures, as analyzed by competitive RT-PCR and Northern hybridization. On spruce wood chips, Pr-lac1 and Pr-lac2 were expressed within 2-3 weeks of growth together with manganese and lignin peroxidase-encoding genes. Our results indicate wood-promoted but time-dependent regulation of expression for the two, at protein and gene level distinct P. radiata laccases. PMID:16927090

  14. 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

    2013-01-01

    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

  15. Decolorization potential of some reactive dyes with crude laccase and laccase-mediated system.

    PubMed

    Sa?maz, Samet; Gedikli, Serap; Aytar, P?nar; Güngörmedi, Gökhan; Cabuk, Ahmet; Hür, Evrim; Unal, Arzu; Kolankaya, Nazif

    2011-02-01

    In this study, decolorization of dyestuffs, such as Reactive Red 198, Rem Blue RR, Dylon Navy 17, Rem Red RR, and Rem Yellow RR was studied using laccase and laccase-mediated system. The laccases are known to have an important potential for remediation of pollutants. Among these dyestuffs, decolorization of Rem Blue RR and Dylon Navy 17 was performed with crude laccase under optimized conditions. Vanillin was selected as laccase mediator after screening six different compounds with Rem Yellow RR, Reactive Red 198, and Rem Red RR as substrates. However, Rem Yellow RR was not decolorized by either laccase or laccase-mediated system. It is observed that the culture supernatant contained high laccase activity after treatment with catalase that was responsible for the decolorization. Besides, culture supernatant with high laccase activity as enzyme source was treated with catalase; in this way, the hypothesis that laccase was the enzyme responsible for decolorization was supported. The Rem Blue RR was decolorized with 64.84% under the optimum conditions and Dylon Navy 17 with 75.43% with crude laccase. However, using the laccase and vanillin, the decolorization of Reactive Red 198 and Rem Red RR was found to be 62% and 68%, respectively. Our study demonstrated that the decolorization abilities of laccase and/or laccase mediator systems were based on the types of mediator, the dye structure, and the standard experimental conditions. Also, the electrochemical behaviors of some samples were studied. The redox potentials of these samples were determined using cyclic voltammetry on glassy carbon electrode in phosphate buffer (pH 6) solution. PMID:20669054

  16. 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 ?-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

  17. Role of Laccase and Low Molecular Weight Metabolites from Trametes versicolor in Dye Decolorization

    PubMed Central

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

    2012-01-01

    The studies regarding decolorization of dyes by laccase may not only inform about the possible application of this enzyme for environmental purposes, but also may provide important information about its reaction mechanism and the influence of several factors that could be involved. In this paper, decolorization of crystal violet and phenol red was carried out with different fractions of extracellular liquids from Trametes versicolor cultures, in order to describe the role of laccase in this reaction. Moreover, the possible role of the low molecular weight metabolites (LMWMs) also produced by the fungus was evaluated. The results confirm the existence of a nonenzymatic decolorization factor, since the nonprotein fraction of the extracellular liquids from cultures of T. versicolor has shown decolorization capability. Several experiments were performed in order to identify the main compounds related to this ability, which are probably low molecular weight peroxide compounds. PMID:22566767

  18. Decolorization Potential of Some Reactive Dyes with Crude Laccase and Laccase-Mediated System

    Microsoft Academic Search

    Samet ?a?maz; Serap Gedikli; P?nar Aytar; Gökhan Güngörmedi; Ahmet Çabuk; Evrim Hür; Arzu Ünal; Nazif Kolankaya

    2011-01-01

    In this study, decolorization of dyestuffs, such as Reactive Red 198, Rem Blue RR, Dylon Navy 17, Rem Red RR, and Rem Yellow\\u000a RR was studied using laccase and laccase-mediated system. The laccases are known to have an important potential for remediation\\u000a of pollutants. Among these dyestuffs, decolorization of Rem Blue RR and Dylon Navy 17 was performed with crude

  19. Lignin-Derived Compounds as Efficient Laccase Mediators for Decolorization of Different Types of Recalcitrant Dyes

    PubMed Central

    Camarero, Susana; Ibarra, David; Martínez, María Jesús; Martínez, Ángel T.

    2005-01-01

    Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (<50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated. PMID:15812000

  20. Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes.

    PubMed

    Camarero, Susana; Ibarra, David; Martínez, María Jesús; Martínez, Angel T

    2005-04-01

    Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (< 50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated. PMID:15812000

  1. 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. PMID:22398306

  2. Amine Oxidases of Microorganisms

    Microsoft Academic Search

    O. V. Yagodina; E. B. Nikol'skaya; A. E. Khovanskikh; B. N. Kormilitsyn

    2002-01-01

    The review of works on amine oxidases of microorganisms is presented. Preparation, physical-chemical and kinetic properties of amine oxidases from archaebacteria Methanosarcina barkery, group of methane-producing archaebacteria, eubacteria, Sarcina lutea, Micrococcus rubens, M. lutea, representatives of Enterobacteriaceae family, such as Klebsiella and Escherichia, are considered. Besides, the amine oxidases obtained from mycelium of fungus Aspergillus niger are described. The works

  3. 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.

  4. Comparison of lignin derivatives as substrates for laccase-catalyzed scavenging of oxygen in coatings and films

    PubMed Central

    2014-01-01

    Background Lignin derivatives are phenylpropanoid biopolymers derived from pulping and biorefinery processes. The possibility to utilize lignin derivatives from different types of processes in advanced enzyme-catalyzed oxygen-scavenging systems intended for active packaging was explored. Laccase-catalyzed oxidation of alkali lignin (LA), hydrolytic lignin (LH), organosolv lignin (LO), and lignosulfonates (LS) was compared using oxygen-scavenging coatings and films in liquid and gas phase systems. Results When coatings containing lignin derivatives and laccase were immersed in a buffered aqueous solution, the oxygen-scavenging capability increased in the order LO?laccase and LO, LH or LA incubated in oxygen-containing gas in air-tight chambers and at a relative humidity (RH) of 100% showed that paperboard coated with LO and laccase reduced the oxygen content from 1.0% to 0.4% during a four-day period, which was far better than the results obtained with LA or LH. LO-containing coatings incubated at 92% RH also displayed activity, with a decrease in oxygen from 1.0% to 0.7% during a four-day period. The oxygen scavenging was not related to the content of free phenolic hydroxyl groups, which increased in the order LO?laccase and LO or LS were characterized using gel permeation chromatograpy, dynamic mechanical analysis, and wet stability. Conclusions The investigation shows that different lignin derivatives exhibit widely different properties as a part of active coatings and films. Results indicate that LS and LO were most suitable for the application studied and differences between them were attributed to a higher degree of laccase-catalyzed cross-linking of LS than of LO. Inclusion in active-packaging systems offers a new way to utilize some types of lignin derivatives from biorefining processes. PMID:24382027

  5. Mesosilica-coated ultrafine fibers for highly efficient laccase encapsulation

    NASA Astrophysics Data System (ADS)

    Wang, Shiwen; Chen, Wei; He, Sha; Zhao, Qilong; Li, Xiaohong; Sun, Jiashu; Jiang, Xingyu

    2014-05-01

    In this paper, we present a simple but efficient biomimetic method to encapsulate laccase on mesoporous silica-modified electrospun (ES) ultrafine fibers. Because of the mild immobilization conditions (room temperature, aqueous condition), the encapsulated laccase retained a high activity of 94%. Because of the protection from the silica layer, the laccase worked efficiently at 60 °C and retained a long-term activity in the presence of proteinase K. After recycling for 10 times the laccase still preserved 96% of its original reactivity. More remarkably, the immobilized laccase on fibers could completely recover its activity after thermal denature, while the free laccase permanently lost the activity. We also demonstrated that the laccase on silica-coated fibers exhibited an enhanced decolorization capability of Brilliant Blue KN-R (BBKN-R) as compared to the free laccase, showing its great potential for industrial applications.In this paper, we present a simple but efficient biomimetic method to encapsulate laccase on mesoporous silica-modified electrospun (ES) ultrafine fibers. Because of the mild immobilization conditions (room temperature, aqueous condition), the encapsulated laccase retained a high activity of 94%. Because of the protection from the silica layer, the laccase worked efficiently at 60 °C and retained a long-term activity in the presence of proteinase K. After recycling for 10 times the laccase still preserved 96% of its original reactivity. More remarkably, the immobilized laccase on fibers could completely recover its activity after thermal denature, while the free laccase permanently lost the activity. We also demonstrated that the laccase on silica-coated fibers exhibited an enhanced decolorization capability of Brilliant Blue KN-R (BBKN-R) as compared to the free laccase, showing its great potential for industrial applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01166j

  6. Possible role of laccase from Fusarium incarnatum UC-14 in bioremediation of Bisphenol A using reverse micelles system.

    PubMed

    Chhaya, Urvish; Gupte, Akshaya

    2013-06-15

    Bisphenol A [2,2 bis (4 hydroxyphenyl) propane] is widely used in the variety of industrial and residential applications such as the synthesis of polymers including polycarbonates, epoxy resins, phenol resins, polyesters and polyacrylates. BPA has been recognized as an Endocrine Disrupting Chemicals (EDC), thus it is necessary to assess its biodegradability or fate in the natural environment. In general, environmental pollutant such as BPA does not dissolve in aqueous media, owing to their high hydrophobicity, and hence non-aqueous catalysis can be employed to enhance biodegradability of phenolic environmental pollutant. Purified laccase hosted in reverse micelles using ternary system of isooctane: AOT [Bis (2-ethylhexyl) sulphosuccinate sodium salt)]:water having hydration ratio (Wo) of 30 with protein concentration of 43.5 ?g/ml was found to eliminate 91.43% of 200 ppm of Bisphenol A at 50 °C, pH-6.0 when incubated with laccase/Reverse Micelles system for 75 min. GC-MS analysis of isooctane soluble fractions detected the presence of 4,4'-(2 hydroxy propane 1,2 diyl) diphenol, bis (4-hydroxylphenyl) butenal and 2-(1-(4-hydroxyphenyl) vinyl) pent-2-enal indicated degradation of BPA by two oxidation steps and one ring opening step (C-C bond cleavage). Laccase/RM system exhibited several advantages for the oxidative degradation of hydrophobic phenols mainly because of the solubility of either enzyme or substrate was improved in organic media and the stable activity of laccase in organic media was achieved. PMID:23611799

  7. [Laccase-mediator systems and their applications: a review].

    PubMed

    Morozova, O V; Shumakovich, G P; Shleev, S V; Iaropolov, A I

    2007-01-01

    The mechanism of operation of laccase-mediator systems (LMSs) in xenobiotic degradation mediated by "true" redox mediators and laccase enhancing agents is considered. Structural formulae of most common laccase mediators and compounds that can be used as agents enhancing the enzyme operation are presented. Examples of LMS application in biotechnology are described. PMID:18038679

  8. Unfolding pathway of CotA-laccase and the role of copper on the prevention of refolding through aggregation of the unfolded state

    SciTech Connect

    Fernandes, Andre T. [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras (Portugal)] [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras (Portugal); Lopes, Carlos [Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro (Portugal)] [Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro (Portugal); Martins, Ligia O. [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras (Portugal)] [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras (Portugal); Melo, Eduardo Pinho, E-mail: emelo@ualg.pt [Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro (Portugal)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer CotA-laccase unfolds with an intermediate state. Black-Right-Pointing-Pointer Copper stabilizes the native and the intermediate state. Black-Right-Pointing-Pointer Copper binding to the unfolded state prevents refolding through protein aggregation. Black-Right-Pointing-Pointer Copper incorporation in CotA-laccase occurs as a later step during folding. -- Abstract: Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase CotA laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-CotA was described as a three-state process with accumulation of an intermediate in between the native and unfolded state. Copper stabilizes the native holo-CotA but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-CotA in a non-native coordination promoting CotA aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.

  9. Application of laccase-natural mediator systems to sisal pulp: an effective approach to biobleaching or functionalizing pulp fibres?

    PubMed

    Aracri, Elisabetta; Colom, Josep F; Vidal, Teresa

    2009-12-01

    The effects of laccase-natural mediator systems (LMS) on sisal pulp and their potential for either biobleaching or functionalizing (via radical-coupling) its fibres were investigated. The enzyme treatment (L stage) was followed by extraction with hydrogen peroxide in order to determine whether observable effects could be enhanced by removing LMS-modified lignin. Four different plant phenols [viz. the p-hydroxycinnamic compounds sinapic acid (SNC), ferulic acid (FRC), coniferyl aldehyde (CLD) and sinapyl aldehyde (SLD)] were used as laccase redox mediators and their effects on pulp and effluents compared with those of the synthetic compound 1-hydroxybenzotriazole (HBT). During the L stage performed with HBT, laccase underwent a loss of 99% and 78% of the initial activity, in the absence and presence of pulp, respectively. With natural mediators inactivation was markedly reduced, being the residual activity between 65% and 100% of the initial one, in the presence of pulp. The pulp was found to protect the enzyme against inactivation: the activity was only reduced by 45% in its presence. Under the operating conditions used the natural mediators proved less efficient than HBT in facilitating pulp bleaching; rather, they tended to bind to pulp fibres. This effect could be used to functionalize fibres in order to improve intrinsic properties of pulp or introducing novel ones (e.g. antimicrobial, antioxidant, optical properties, etc.). This paper shows for the first time the application of laccase-mediator systems to sisal pulp. PMID:19574042

  10. A Highly Efficient Recombinant Laccase from the Yeast Yarrowia lipolytica and Its Application in the Hydrolysis of Biomass

    PubMed Central

    Kalyani, Dayanand; Tiwari, Manish Kumar; Li, Jinglin; Kim, Sun Chang; Kalia, Vipin C.; Kang, Yun Chan; Lee, Jung-Kul

    2015-01-01

    A modified thermal asymmetric interlaced polymerase chain reaction was performed to obtain the first yeast laccase gene (YlLac) from the isolated yeast Yarrowia lipolytica. The 1557-bp full-length cDNA of YlLac encoded a mature laccase protein containing 519 amino acids preceded by a signal peptide of 19 amino acids, and the YlLac gene was expressed in the yeast Pichia pastoris. YlLac is a monomeric glycoprotein with a molecular mass of ~55 kDa as determined by polyacrylamide-gel electrophoresis. It showed a higher catalytic efficiency towards 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (kcat/Km = 17.5 s-1 ?M-1) and 2,6-dimethoxyphenol (kcat/Km = 16.1 s-1 ?M-1) than other reported laccases. The standard redox potential of the T1 site of the enzyme was found to be 772 mV. The highest catalytic efficiency of the yeast recombinant laccase, YlLac, makes it a good candidate for industrial applications: it removes phenolic compounds in acid-pretreated woody biomass (Populus balsamifera) and enhanced saccharification. PMID:25781945

  11. Dioxygen reactivity of laccase: dependence on laccase source, pH, and anion inhibition.

    PubMed

    Xu, F

    2001-08-01

    A study was carried out on the dioxygen reactivity of the laccases from Trametes villosa, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum, and Coprinus cinereus. At pH 5.5, these laccases showed an apparently constant Km (about 20-50 microM) for O2 with either 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) or methyl syringate as the reducing substrate, in contrast to the kcat, which varied up to 100-fold. O2 reactivity of the Trametes and Myceliophthora laccases was also studied at various pH and NaF concentrations. The apparent Km of Trametes and Myceliophthora laccases varied only slightly when pH changed from 3.0 to 8.0 or when the laccases were inhibited by F- at pH 5.5, although the apparent kcat were more significantly affected by both factors. The dependence of the apparent Km on the source of laccase, pH, and F- inhibition suggested that the fungal laccases might have a conserved O2 pocket and that the F- or OH- inhibition might affect the O2 reduction step (kcat) more than the O2 binding step (Km) under steady-state conditions. PMID:11694062

  12. Heterologous expression of a tannic acid-inducible laccase3 of Cryphonectria parasitica in Saccharomyces cerevisiae

    PubMed Central

    2010-01-01

    Background A tannic acid-inducible and mycoviral-regulated laccase3 (lac3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae. Results Laccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the lac3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned lac3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura- media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of lac3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura- media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the lac3 gene resulted in a reduced cell number of the lac3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without lac3 induction did not affect the growth of transformants. Conclusions The results suggest that expression of the lac3 gene has an inhibitory effect on the growth of transformed S. cerevisiae and that the controlled expression of lac3 is appropriate for the possible application of recombinant yeast to the treatment of phenolic compounds. PMID:20178646

  13. Effects of Metal Oxides on a Fungal Laccase Activity and Catechol Transformation

    NASA Astrophysics Data System (ADS)

    Ahn, M.; Dec, J.; Bollag, J.

    2003-12-01

    The transformation of naturally occurring phenols to humic polymers is generally catalyzed by various phenoloxidases commonly present in soil. Some poorly crystalline metal oxides and hydroxides may also participate in these reactions. In this study, catechol (0.1 M) was incubated with a fungal laccase (950 unit/mL) in the presence of poorly crystalline minerals (ferrihydrite; 50 mg/mL: birnessite; 1 mg/mL: aluminum hydroxide; 50 mg/mL) to examine the interaction between these soil components under field conditions. Birnessite had an inhibitory effect on the laccase-mediated transformation of catechol (by up to 40%). Enzyme inhibition was possibly caused by the rapid production of humic-like polymers by birnessite. An additional inhibitory effect was caused by Manganese ion released from birnessite as it oxidized catechol (up to 70% loss in enzyme activity). In contrast to birnessite, aluminum hydroxide had an additive effect on the disappearance of catechol despite the rapid adsorption of the enzyme by this mineral (Xm=6.18? g/mg). Apparently, the adsorbed laccase retained some enzyme activity. Ferrihydrite also had an additive effect on catechol transformation. However, as compared to aluminum hydroxide, ferrihydrite adsorbed less laccase (Xm=0.89? g/mg) and more humic-like polymers. Unlike birnessite, aluminum hydroxide and ferrihydrite released negligible amounts of metal ions. In conclusion, under field conditions, phenoloxidase activity may be diminished by the presence of birnessite, but the presence of either ferrihydrite or aluminum hydroxide is less likely to inhibit enzyme activity, and may even enhance substrate transformation.

  14. Label-free fluorometric method for monitoring conformational flexibility of laccase based on a selective laccase sensor.

    PubMed

    Qiu, Suyan; Lin, Zhenyu; Zhou, Yaomin; Li, Ruili; Zhang, Jinyan; Zhang, Dawen; Luo, Linguang; Guo, Longhua; Qiu, Bin; Chen, Guonan

    2013-11-19

    A facile and selective fluorescence sensor for laccase determination has been proposed depending on the interaction between 3-azidocoumarin and trametes versicolor (Tv) laccase in this paper. The azido group of 3-azidocoumarin that is electron-rich ?-nitrogen can directly interact with histidines that coordinate to three copper sites through hydrogen bonds and forms a new complex, which decreases the electron-donating ability of the azido group, leading to enhance the fluorescence intensity of the sensing system. Also, other common proteins have no significant interference for the proposed laccase sensor. Additionally, the proposed fluorescence sensor is extended to demonstrate the conformational flexibility of Tv laccase by the urea denaturant. A good consistency of the results obtained with the presented laccase sensor and CD spectra is performed. Furthermore, the relationship between the catalytic activity and the unfolding percentage of the unfolded Tv laccase through the proposed laccase sensor is also elucidated well. PMID:24117223

  15. Location of laccase in ordered mesoporous materials

    SciTech Connect

    Mayoral, Álvaro [Laboratorio de Microscopias Avanzadas, Instituto de Nanociencia de Aragon, Universidad de Zaragoza, Edificio I - D, Mariano Esquillor, 50018 Zaragoza (Spain); Gascón, Victoria; Blanco, Rosa M.; Márquez-Álvarez, Carlos; Díaz, Isabel, E-mail: idiaz@icp.csic.es [Instituto de Catálisis y Petroleoquímica, CSIC, c/Marie Curie 2, 28049 Madrid (Spain)

    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.

  16. White rot fungi laccases for biotechnological applications.

    PubMed

    Villalba, Laura L; Fonseca, María I; Giorgio, Martín; Zapata, Pedro D

    2010-06-01

    White rot fungi have an enzymatic system producing oxidative and hydrolytic enzymes that act on the degradation of certain components of the cell wall. They can be applied in several technological processes, such as paper industry, bio-fuels and environmental pollution. Laccases are multi-copper enzymes of wide substrate specificity and high non-specific oxidation capacity that use molecular oxygen to oxidize various aromatic compounds, and are highly relevant biotechnological applications. In this review, we present some significant patents on laccase production and recombinant DNA technology for diverse biotechnology applications. PMID:20550513

  17. Production, purification and biochemical characterization of two laccase isoforms produced by Trametes versicolor grown on oak sawdust.

    PubMed

    Martínez-Morales, Fernando; Bertrand, Brandt; Pasión Nava, Angélica A; Tinoco, Raunel; Acosta-Urdapilleta, Lourdes; Trejo-Hernández, María R

    2015-02-01

    Two laccase isoforms (lcc1 and lcc2) produced by Trametes versicolor, grown on oak sawdust under solid-state fermentation conditions, were purified and characterized. The two isoforms showed significant biochemical differences. Lcc1 and lcc2 had MWs of 60 and 100 kDa, respectively. Both isoforms had maximal activity at pH 3 with ABTS and 2,6-dimethyloxyphenol (DMP). Lcc1 was the most attractive isoform due to its greater affinity towards all the laccase substrates used. Lcc1 had Km values of 12, 10, 15 and 17 mM towards ABTS, DMP, guaiacol and syringaldazine, respectively. Lcc2 had equivalent values of 45, 47, 15 and 39 mM. The biochemical properties of lcc1 substantiate the potential of this enzyme for application in the treatment of contaminated water with low pH values and high phenolic content. PMID:25257594

  18. Structural and functional roles of glycosylation in fungal laccase from Lentinus sp.

    PubMed

    Maestre-Reyna, Manuel; Liu, Wei-Chun; Jeng, Wen-Yih; Lee, Cheng-Chung; Hsu, Chih-An; Wen, Tuan-Nan; Wang, Andrew H-J; Shyur, Lie-Fen

    2015-01-01

    Laccases are multi-copper oxidases that catalyze the oxidation of various organic and inorganic compounds by reducing O2 to water. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key ?-barrel topology. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn75, Asn238, and Asn458 were elucidated. Initial rate kinetic analysis revealed that the kcat, Km, and kcat/Km of nLcc4 with substrate ABTS were 3,382 s-1, 65.0 ± 6.5 ?M, and 52 s-1?M-1, respectively; and the values with lignosulfonic acid determined using isothermal titration calorimetry were 0.234 s-1, 56.7 ± 3.2 ?M, and 0.004 s-1?M-1, respectively. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were observed to maintain only 4-50% of the activity of the wild-type laccase. Molecular dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase. PMID:25849464

  19. Effects of Basidiomycete Laccase on Cercosporin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cercosporin is a perylenequinone pigment produced by fungi in the genus Cercospora which under light generates reactive oxygen species causing membrane damage and mortality of living cells. Our objectives were to evaluate the effects of laccase, a lignolitic copper-containing enzyme on the temporal ...

  20. Electrochemical redox transformations of T1 and T2 copper sites in native Trametes hirsuta laccase at gold electrode

    PubMed Central

    2004-01-01

    Mediatorless, electrochemically driven, redox transformations of T1 (type 1) and T2 copper sites in Trametes hirsuta laccase were studied by cyclic voltammetry and spectroelectrochemical redox titrations using bare gold electrode. DET (direct electron transfer) between the electrode and the enzyme was observed under anaerobic conditions. From analysis of experimental data it is concluded that the T2 copper site is in DET contact with gold. It was found that electron transfer between the gold surface and the T1 copper site progresses through the T2 copper site. From EPR measurements and electrochemical data it is proposed that the redox potential of the T2 site for high-potential ‘blue’ laccase is equal to about 400 mV versus NHE (normal hydrogen electrode) at pH 6.5. The hypothesis that the redox potentials of the T2 copper sites in low- and high-potential laccases/oxidases from totally different sources might be very similar, i.e. approx. 400 mV, is discussed. PMID:15453829

  1. Improved Folin-Ciocalteu assay of “total phenolic content” by removal of ascorbate and dehydroascorbate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The venerable and operationally simple Folin-Ciocalteu (F-C) assay for total phenolics can have severe limitations due to interference by ascorbic acid (AsA). For common fruit juices AsA interference can easily exceed the magnitude of the total phenolic signal itself. Ascorbate oxidase (AO) has been...

  2. Improved removal of ascorbate interference in the Folin-Ciocalteu assay of “total phenolic content"

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The venerable Folin-Ciocalteu (F-C) assay for total phenolics can have severe limitations due to interference by ascorbic acid (AsA). For common fruit juices AsA interference can easily exceed the magnitude of the total phenolic signal itself. Ascorbate oxidase (AO) has been a promising approach to ...

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

    Microsoft Academic Search

    J. ZHAO; H. S. KWAN

    1999-01-01

    The effect of different substrates and various developmental stages (mycelium growth, primordium appear- ance, 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,

  4. Integrated hot-compressed water and laccase-mediator treatments of Eucalyptus grandis fibers: structural changes of fiber and lignin.

    PubMed

    Wu, Jian-Quan; Wen, Jia-Long; Yuan, Tong-Qi; Sun, Run-Cang

    2015-02-18

    Eucalyptus grandis fibers were treated with hot-compressed water (HCW) and laccase mediator to enhance the fiber characteristics and to produce an active lignin substrate for binderless fiberboard production. The composition, morphology, and crystallinity index (CrI) analysis of fibers showed that the HCW treatment increased the CrI and lignin content of the treated fibers through partial removal of hemicelluloses. Simultaneously, the HCW treatment produced some granules and holes on the surface of the fibers, which possibly facilitated the accessibility of the laccase mediator. Milled wood lignins and enzymatic hydrolysis lignins isolated from the control and treated fibers were comparatively characterized. A reduction of molecular weight was observed, which indicated that a preferential degradation of lignin occurred after exposure to the laccase mediator. Quantitative (13)C, 2D-HSQC and (31)P NMR characterization revealed that the integrated treatment resulted in the cleavage of ?-O-4' linkages, removal of G' (oxidized ?-ketone) substructures, and an increase in the S/G ratio and free phenolic hydroxyls. PMID:25639522

  5. Decolorization of reactive dyes by immobilized laccase

    Microsoft Academic Search

    Patricio Peralta-Zamora; Cláudia M. Pereira; Elaine R. L. Tiburtius; Sandra G. Moraes; Maria A. Rosa; Rosana C. Minussi; Nelson Durán

    2003-01-01

    Immobilization of laccase by Trametes versicolor on silica chemically modified with imidazol groups, amberlite IRA-400, glass–ceramic chemically modified with carbodiimide\\/glutaraldehyde and by aminoprolyltriethoxysilane\\/glutaraldehyde and montmorillonite modified by aminoprolyltriethoxysilane\\/glutaraldehyde were afforded. These supports were used in the decolorization of textile reactive dyes (Remazol Brilliant Blue R, Remazol Black B, Reactive Orange 122 and Reactive Red 251). One of the most efficient

  6. Kinetics of phenol biotransformation

    Microsoft Academic Search

    P. Kumaran; Y. L. Paruchuri

    1997-01-01

    Phenol and its homologues are aromatics containing hydroxyl, methyl, amide and sulphonic groups attached to the benzenoid molecules. These molecules are, both, anthropogenic and xenobiotics. Phenols are environmental pollutants discharged through wastewaters from fossil fuel refining processes, phenol manufacturing plants, pharmaceutical and a variety of other industries. Phenols are toxic to several biochemical reactions. However, biological transformation of phenols to

  7. Fungal Laccases Degradation of Endocrine Disrupting Compounds

    PubMed Central

    Macellaro, Gemma; Cicatiello, Paola; Sannia, Giovanni

    2014-01-01

    Over the past decades, water pollution by trace organic compounds (ng/L) has become one of the key environmental issues in developed countries. This is the case of the emerging contaminants called endocrine disrupting compounds (EDCs). EDCs are a new class of environmental pollutants able to mimic or antagonize the effects of endogenous hormones, and are recently drawing scientific and public attention. Their widespread presence in the environment solicits the need of their removal from the contaminated sites. One promising approach to face this challenge consists in the use of enzymatic systems able to react with these molecules. Among the possible enzymes, oxidative enzymes are attracting increasing attention because of their versatility, the possibility to produce them on large scale, and to modify their properties. In this study five different EDCs were treated with four different fungal laccases, also in the presence of both synthetic and natural mediators. Mediators significantly increased the efficiency of the enzymatic treatment, promoting the degradation of substrates recalcitrant to laccase oxidation. The laccase showing the best performances was chosen to further investigate its oxidative capabilities against micropollutant mixtures. Improvement of enzyme performances in nonylphenol degradation rate was achieved through immobilization on glass beads. PMID:24829908

  8. Improving the decolorization for textile dyes of a metagenome-derived alkaline laccase by directed evolution.

    PubMed

    Liu, Yu Huan; Ye, Mao; Lu, Yi; Zhang, Xia; Li, Gang

    2011-08-01

    To obtain better performing laccases for textile dyes decolorization, random mutagenesis of Lac591, a metagenome-derived alkaline laccase, was carried out. After three rounds of error-prone PCR and high-throughput screening by assaying enzymatic activity toward the phenolic substrate 2,6-dimethoxyphenol (2,6-DMP), a mutant (Lac3T93) with remarkably improved enzymatic activity was obtained. Sequence analysis revealed that four amino acid substitutions (N40S, V55A, F62L, and E316V) were accumulated in the Lac3T93. Compared to the wild-type enzyme, the specific activity of Lac3T93 toward 2,6-DMP was increased to 4.8-fold (61.22 U/mg), and its optimal temperature and pH were changed to 60°C and 8.0 from 55°C and 7.5 of the wild-type enzyme, respectively. Furthermore, the degradation ability of Lac3T93 for textile dyes was investigated, and the new variant represented improved decolorization percentage for four industrial dyes with complex phenyl structure (Basic Blue 3, Methylene Blue, Bromophenol Blue, and Crystal Violet) and higher decolorization efficiency for Indigo Carmine than that of the parent enzyme. Furthermore, the decolorization percentage of Lac3T93 for five dyes in the absence of hydroxybenzotrizole (HBT) is clearly higher than those of the wild-type enzyme with 1 mM HBT, and HBT can further improve its decolorization ability. PMID:21523474

  9. Reactivity of Trametes laccases with fatty and resin acids.

    PubMed

    Karlsson, S; Holmbom, B; Spetz, P; Mustranta, A; Buchert, J

    2001-04-01

    Lipophilic extractives commonly referred to as wood pitch or wood resin can have a negative impact on paper machine runnability and product quality. The lipophilic extractives are composed mainly of fatty acids, resin acids, sterols, steryl esters and triglycerides. In this work, the suitability of laccases for the modification of fatty and resin acids was studied, using two model fractions. In the treatments, resin and fatty acid dispersions were treated with two different laccases, i.e. laccases from Trametes hirsuta and T. villosa. Different chromatographic methods were used to elucidate the effects of laccase treatments on the chemistry of the fatty and resin acids. Both laccases were able to modify the fatty and resin acids to some extent. In the case of fatty acids, a decrease in the amount of linoleic, oleic and pinolenic acids was observed, whereas the modification of resin acids resulted in a reduced amount of conjugated resin acids. PMID:11341313

  10. Production of a recombinant laccase from Pichia pastoris and biodegradation of chlorpyrifos in a laccase/vanillin system.

    PubMed

    Xie, Huifang; Li, Qi; Wang, Minmin; Zhao, Linguo

    2013-06-28

    The recombinant strain P. pastoris GS115-lccC was used to produce laccase with high activity. Factors influencing laccase expression, such as pH, methanol concentration, copper concentration, peptone concentration, shaker rotate speed, and medium volume were investigated. Under the optimal conditions, laccase activity reached 12,344 U/L on day 15. The recombinant enzyme was purified by precipitating and dialyzing to electrophoretic homogeneity, and was estimated to have a molecular mass of about 58 kDa. When guaiacol was the substrate, the laccase showed the highest activity at pH 5.0 and was stable when the pH was 4.5~6.0. The optimal temperature for the laccase to oxidize guaiacol was 60°C, but it was not stable at high temperature. The enzyme could remain stable at 30°C for 5 days. The recombinant laccase was used to degrade chlorpyrifos in several laccase/mediator systems. Among three synthetic mediators (ABTS, HBT, VA) and three natural mediators (vanillin, 2,6-DMP, and guaiacol), vanillin showed the most enhancement on degradation of chlorpyrifos. Both laccase and vanillin were responsible for the degradation of chlorpyrifos. A higher dosage of vanillin may promote a higher level of degradation of chlorpyrifos, and the 2-step addition of vanillin led to 98% chlorpyrifos degradation. The degradation of chlorpyrifos was faster in the L/V system (kobs = 0.151) than that in the buffer solution (kobs = 0.028). PMID:23676909

  11. The comparative study of a laccase-natural clinoptilolite-based catalyst activity and free laccase activity on model compounds.

    PubMed

    Donati, Enrica; Polcaro, Chiara M; Ciccioli, Piero; Galli, Emanuela

    2015-05-30

    For the first time a laccase from Trametes versicolor was immobilized on a natural clinoptilolite with Si/Al=5 to obtain a biocatalyst for environmental applications. Immobilization procedures exploiting adsorption and covalent binding were both tested, and only the last provided enough activity for practical applications. The optimal conditions for the immobilization of the enzyme on the support and the kinetic parameters for the free and covalent bonded laccase were determined. The laccase bonded to the zeolitic support showed a lower activity than the free laccase, but the pH and thermal stability were greater. 20 mg of dry biocatalyst containing 1 U of laccase were able to remove in 50h 73-78% of 2-chlorophenol and 2,4-dichlorophenol in relatively concentrated aqueous solutions (100 ?mol L(-1)). PMID:25710818

  12. Reduction of phenol content and toxicity in olive oil mill waste waters with the ligninolytic fungus Pleurotus ostreatus

    Microsoft Academic Search

    Luca Martirani; Paola Giardina; Liberato Marzullo; Giovanni Sannia

    1996-01-01

    Olive oil mill waste waters (OMW) constitute a major environmental problem because of the large amount produced and the toxicity of the phenolic compounds present. Several of these aromatic compounds can be assimilated to many of the components of lignin. Only few microorganisms, mainly “white-rot” basidiomycete, are able to degrade lignin by means of oxidative reactions catalysed by phenol oxidases

  13. Tuning laccase catalytic activity with phosphate functionalized carbon dots by visible light.

    PubMed

    Li, Hao; Guo, Sijie; Li, Chuanxi; Huang, Hui; Liu, Yang; Kang, Zhenhui

    2015-05-13

    The phosphate functionalized carbon dots (PCDs) with high biocompatibility and low toxicity can be used as efficient additives for the construction of laccase/PCDs hybrids catalyst. A series of experiments indicated that the activity of laccase/PCDs was higher than that of free laccase (increased by 47.7%). When laccase/PCDs hybrids catalyst was irradiated with visible light (laccase/PCDs-Light), its activity was higher than that of laccase/PCDs hybrids without light irradiation (increased by 92.1%). In the present system, the T1 Cu in laccase was combined with the phosphate group on PCDs, which can increase binding capacity of laccase/PCDs hybrids and substrate. Further, the visible light irradiation increased the donating and accepting electronic capability of the laccase/PCDs hybrids, improving their catalytic activity. PMID:25886497

  14. PtCu substrates subjected to AC and DC electric fields in a solution of benzene sulfonic acid-phenol as novel batteries and their use in glucose biofuel cells

    NASA Astrophysics Data System (ADS)

    Ammam, Malika; Fransaer, Jan

    2013-11-01

    We describe how bi-metal PtCu connected wires, immersed in a solution of benzene sulfonic acid (BSA)-phenol (P) or 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)-phenol (P), then subjected to simultaneous alternating current (AC) and direct current (DC) electric fields generate power. We discovered that PtCu substrate covered by the deposit containing (BSA-PP-Pt-Cu), abbreviated as PtCu(BSA-PP-Pt-Cu) electrode, plays the role of a substantial anode and cathode. The latter was related to the formation of micro-batteries in the deposited film (BSA-PP-Pt-Cu) that are able to take or deliver electrons from the deposited Pt and Cu, respectively. PP-BSA plays probably the role of bridge for proton conduction in the formed micro-batteries. The power density of the fuel cell (FC)-based PtCu(BSA-PP-Pt-Cu) anode and PtCu(BSA-PP-Pt-Cu) cathode in phosphate buffer solution pH 7.4 at room temperature reaches ˜10.8 ?W mm-2. Addition of enzymes, glucose oxidase at the anode and laccase at the cathode and, replacement of BSA by ABTS at the cathode in the deposited films increases the power density to 13.3 ?W mm-2. This new procedure might be of great relevance for construction of a new generation of FCs operating at mild conditions or boost the power outputs of BFCs and make them suitable for diverse applications.

  15. 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 ?l) 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. PMID:23861040

  16. Laccase immobilized on magnetic carriers for biotechnology applications

    NASA Astrophysics Data System (ADS)

    Rotková, Jana; Šuláková, Romana; Korecká, Lucie; Zdražilová, Pavla; Jandová, Miroslava; Lenfeld, Ji?í; Horák, Daniel; Bílková, Zuzana

    2009-05-01

    Laccase catalyzing the oxidation of p-diphenols has been applied in many industrial and biotechnology areas. Immobilized form of laccase has overcome the problem with contamination of the final product. Nevertheless sensitive enzymes immobilized to the matrix can be inactivated by the environmental conditions. The aim of this research was to prepare carrier with improved activity and responsible stability even under extreme reaction conditions. Laccase immobilized through carbohydrate moieties on magnetic hydrazide bead cellulose with a final activity of 0.63 I.U./1 ml of settled carrier confirmed that carriers with oriented immobilized enzyme might be useful in routine biocatalytic applications.

  17. Redox-mediated decolorization of synthetic dyes by fungal laccases.

    PubMed

    Claus, H; Faber, G; König, H

    2002-09-01

    Laccases from the lignin-degrading basidiomycetes Trametes versicolor, Polyporus pinisitus and the ascomycete Myceliophthora thermophila were found to decolorize synthetic dyes to different extents. Differences were attributed to the specific catalytic properties of the individual enzymes and to the structure of the dyes. Due to their higher oxidative capacities, the laccases from the two basidiomycetes decolorized dyes more efficiently than that of the ascomycete. The azo dye Direct Red 28, the indigoid Acid Blue 74 and anthraquinonic dyes were directly enzymatically decolorized within 16 h. The addition of 2 mM of the redox-mediator 1-hydroxybenzotriazole further improved and facilitated the decolorization of all nine dyes investigated. Laccases decolorized dyes both individually and in complex mixtures in the presence of bentonite or immobilized in alginate beads. Our data suggest that laccase/mediator systems are effective biocatalysts for the treatment of effluents from textile, dye or printing industries. PMID:12226723

  18. Enzymatic treatments of pulp using laccase and hydrophobic compounds.

    PubMed

    Garcia-Ubasart, Jordi; Esteban, Alberto; Vila, Carlos; Roncero, M Blanca; Colom, Josep F; Vidal, Teresa

    2011-02-01

    The aim of this work was to develop an innovative method for the internal sizing of paper by use of laccase and hydrophobic compounds. Nine different products containing hydrophobic moieties were tested in combination with laccase derived from Trametes villosa on Eucalyptus globulus kraft pulp in order to assess their internal sizing capability. The strongest internal sizing effect was obtained with lauryl gallate (LG). Heat treatment of the handsheets was found to increase the resistance to water absorption of internally sized samples significantly. Tests were conducted under variable operating conditions, including enzyme and reactant doses and treatment time. In addition to altering the water absorption rate, internal sizing with the laccase-LG treatments was found to affect the mechanical and optical properties of the handsheets. As shown in this work, treatments based on laccase and a hydrophobic compound (particularly lauryl gallate), can provide a new, effective biotechnological method for the internal sizing of paper. PMID:21050744

  19. Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from Bacillus clausii

    PubMed Central

    Brander, Søren; Mikkelsen, Jørn D.; Kepp, Kasper P.

    2014-01-01

    The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ?0.5–2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (KM) but to pH dependence of catalytic turnover: The kcat of B. clausii cotA was 1 s?1 at pH 6 and 5 s?1 at pH 8 in contrast to 6 s?1 at pH 6 and 2 s?1 at pH 8 for of B. subtilis cotA. Overall, kcat/KM was 10-fold higher for B. subtilis cotA at pHopt. While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500–700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ?20 minutes half-life at 80°C, less than the ?50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH?8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization. PMID:24915287

  20. NADPH oxidases and cancer.

    PubMed

    Roy, Krishnendu; Wu, Yongzhong; Meitzler, Jennifer L; Juhasz, Agnes; Liu, Han; Jiang, Guojian; Lu, Jiamo; Antony, Smitha; Doroshow, James H

    2015-06-01

    The mechanism by which reactive oxygen species (ROS) are produced by tumour cells remained incompletely understood until the discovery over the last 15 years of the family of NADPH oxidases (NOXs 1-5 and dual oxidases DUOX1/2) which are structural homologues of gp91phox, the major membrane-bound component of the respiratory burst oxidase of leucocytes. Knowledge of the roles of the NOX isoforms in cancer is rapidly expanding. Recent evidence suggests that both NOX1 and DUOX2 species produce ROS in the gastrointestinal tract as a result of chronic inflammatory stress; cytokine induction (by interferon-?, tumour necrosis factor ?, and interleukins IL-4 and IL-13) of NOX1 and DUOX2 may contribute to the development of colorectal and pancreatic carcinomas in patients with inflammatory bowel disease and chronic pancreatitis, respectively. NOX4 expression is increased in pre-malignant fibrotic states which may lead to carcinomas of the lung and liver. NOX5 is highly expressed in malignant melanomas, prostate cancer and Barrett's oesophagus-associated adenocarcinomas, and in the last it is related to chronic gastro-oesophageal reflux and inflammation. Over-expression of functional NOX proteins in many tissues helps to explain tissue injury and DNA damage from ROS that accompany pre-malignant conditions, as well as elucidating the potential mechanisms of NOX-related damage that contribute to both the initiation and the progression of a wide range of solid and haematopoietic malignancies. PMID:25818486

  1. NADH oxidase of plasma membranes

    Microsoft Academic Search

    D. James Morré; Andrew O. Brightman

    1991-01-01

    NADH oxidase is a cyanide-resistant and hormone-responsive oxidase intrinsic to the plasma membrane of both plant and animal cells. The activity has many unique characteristics that distinguish it from other oxidases and oxidoreductases of both organelles and internal membranes and from other oxidoreductases of the plasma membrane. Among these are resistance to inhibition by cyanide, catalase, superoxide dismutase, and phenylchloromer-curibenzoate.

  2. Expression and Characterization of a Recombinant Laccase with Alkalistable and Thermostable Properties from Streptomyces griseorubens JSD-1.

    PubMed

    Feng, Haiwei; Zhang, Dan; Sun, Yujing; Zhi, Yuee; Mao, Liang; Luo, Yanqing; Xu, Lurong; Wang, Lumei; Zhou, Pei

    2015-05-01

    Streptomyces griseorubens JSD-1 is a novel actinomycete that could grow efficiently upon lignin, and the ligninolytic genes active in this biotransformation were expected to be crucial. To investigate the molecular mechanism of utilizing lignin, genome sequencing was carried out to obtain its draft genome, which was deposited at GenBank under the accession No. JJMG00000000. Multiple copper oxidase (MCO) was obtained, which proved to be an extracellular enzyme and have relative high expression with the stimulation of ligninolytic materials. Judging from its putative 3D structure, the N-terminal of MCO was bared, which was fit for the linkage of poly-HIS10 tag. As a result, heterogeneous expression conditions of recombinant laccase was achieved with TransB(DE3) grown in a modified terrific broth (TB) medium with an extra addition of 0.5 % glucose at 30 °C until optical density at 600 nm (OD600) reached 0.8 when expression was induced by 25 ?M isopropyl ?-D-1-thiogalactopyranoside (IPTG) and also 100 ?M copper sulphate as supplement. Finally, it exhibited special characters of thermal robustness, alkaline activity profiles, high resistance to metallic ions and chemical inhibitors as well as dye decolourization. In summary, our findings illustrated the genetic basic of utilizing lignin in this isolate. Additionally, a novel laccase expected to be potential in agricultural and industrial application was expressed and characterized as well. PMID:25820450

  3. TRANSPARENT TESTA10 Encodes a Laccase-Like Enzyme Involved in Oxidative Polymerization of Flavonoids in Arabidopsis Seed CoatW?

    PubMed Central

    Pourcel, Lucille; Routaboul, Jean-Marc; Kerhoas, Lucien; Caboche, Michel; Lepiniec, Loïc; Debeaujon, Isabelle

    2005-01-01

    The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV–visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase. PMID:16243908

  4. Lysyl oxidases: a novel multifunctional amine oxidase family.

    PubMed

    Csiszar, K

    2001-01-01

    Lysyl oxidase (LOX), a copper-containing amine oxidase, belongs to a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes. LOX has been traditionally known for one function, the extracellular catalysis of lysine-derived cross-links in fibrillar collagens and elastin. More recently, diverse roles have been attributed to lysyl oxidase and these novel activities cover a spectrum of diverse biological functions such as developmental regulation, tumor suppression, cell motility, and cellular senescence. Lysyl oxidase has also been shown to have both intracellular and intranuclear locations. The multifunctional properties of lysyl oxidase (LOX) and our recent discovery of three novel members of this amine oxidase family, LOX-like (LOXL), LOXL2, and LOXL3, indicate the possibility that these varied functions are performed in both intracellular and extracellular environments by individual novel members of the LOX amine-oxidase family. Structural similarities of the highly conserved copper-binding and lysyl-tyrosylquinone cofactor sites among the LOX and LOX-like proteins may result in similar amine oxidase activities. However, specific novel functions, such as a potential role in cell adhesion and cell growth control, will be determined by other, conserved domains such as the cytokine receptor-like domain that is shared by all LOXs and by multiple scavenger receptor cysteine-rich (SRCR) domains present in LOXL2 and LOXL3. Furthermore, these functions may be carried out in a temporally and spatially regulated fashion. PMID:11642359

  5. Monitoring endogenous enzymes during olive fruit ripening and storage: correlation with virgin olive oil phenolic profiles.

    PubMed

    Hachicha Hbaieb, Rim; Kotti, Faten; García-Rodríguez, Rosa; Gargouri, Mohamed; Sanz, Carlos; Pérez, Ana G

    2015-05-01

    The ability of olive endogenous enzymes ?-glucosidase, polyphenol oxidase (PPO) and peroxidase (POX), to determine the phenolic profile of virgin olive oil was investigated. Olives used for oil production were stored for one month at 20 °C and 4 °C and their phenolic content and enzymatic activities were compared to those of ripening olive fruits. Phenolic and volatile profiles of the corresponding oils were also analysed. Oils obtained from fruits stored at 4 °C show similar characteristics to that of freshly harvested fruits. However, the oils obtained from fruits stored at 20 °C presented the lowest phenolic content. Concerning the enzymatic activities, results show that the ?-glucosidase enzyme is the key enzyme responsible for the determination of virgin olive oil phenolic profile as the decrease in this enzyme activity after 3 weeks of storage at 20 °C was parallel to a dramatic decrease in the phenolic content of the oils. PMID:25529676

  6. Laccase-mediated degradation and reduction of toxicity of the postharvest fungicide imazalil

    Microsoft Academic Search

    Tatsuo Maruyama; Chiho Komatsu; Junji Michizoe; Shinji Sakai; Masahiro Goto

    2007-01-01

    The degradation of the postharvest fungicide imazalil by the oxidative enzyme laccase was investigated in the presence of a reaction mediator (a laccase\\/mediator system). Of various natural mediators tested, imazalil was degraded most effectively in the presence of 4-hydroxybenzoic acid while the absence of a mediator resulted in no degradation. In this system, the mediator compound was oxidized by laccase

  7. [Effect of Coriolus hirsutus laccase on atrazine adsorption and desorption by different types of soil ].

    PubMed

    Davidchik, V N; Kulikova, N A; Golubeva, L I; Stepanova, E V; Koroleva, O V

    2008-01-01

    Study of adsorption-desorption behavior of herbicide atrazine in soils of different geographical zones in the presence of Coriolus hirsutus laccase was performed. Laccase was shown to significantly increase adsorption coefficient and to facilitate irreversible adsorption of atrazine to soil. Supposably, laccase catalyzes oxidative binding of atrazine to soil. PMID:18924413

  8. Phenol removal pretreatment process

    DOEpatents

    Hames, Bonnie R. (Westminster, CO)

    2004-04-13

    A process for removing phenols from an aqueous solution is provided, which comprises the steps of contacting a mixture comprising the solution and a metal oxide, forming a phenol metal oxide complex, and removing the complex from the mixture.

  9. 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

  10. Bacterial versus fungal laccase: potential for micropollutant degradation.

    PubMed

    Margot, Jonas; Bennati-Granier, Chloé; Maillard, Julien; Blánquez, Paqui; Barry, David A; Holliger, Christof

    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

  11. Solidphase extraction of phenols

    Microsoft Academic Search

    I Rodr??guez; M. P Llompart; R Cela

    2000-01-01

    Sample preparation for phenol analysis using solid-phase extraction (SPE) is reviewed. The scope of the review has been restricted to the literature dealing with the analysis of phenols as the main objective. The use, advantages and disadvantages of silica sorbents, polymeric, functionalised, carbon-based and mixed available sorbents, when applied to the separation and preconcentration of phenols, as well as the

  12. Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties

    SciTech Connect

    Osipov, Evgeny [A. N. Bach Institute of Biochemistry, Leninsky Prospect 33/2, Moscow 119071 (Russian Federation); Polyakov, Konstantin [A. N. Bach Institute of Biochemistry, Leninsky Prospect 33/2, Moscow 119071 (Russian Federation); Engelhardt Institute of Molecular Biology, Vavilova Str. 32, Moscow 119991 (Russian Federation); Kittl, Roman [BOKU – University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Wien (Austria); Shleev, Sergey [RSC ‘Kurchatov Institute’, Acad. Kurchatov Sq. 1, Moscow 123182 (Russian Federation); Malmö University, 205 06 Malmö (Sweden); Dorovatovsky, Pavel [RSC ‘Kurchatov Institute’, Acad. Kurchatov Sq. 1, Moscow 123182 (Russian Federation); Tikhonova, Tamara, E-mail: ttikhonova@inbi.ras.ru [A. N. Bach Institute of Biochemistry, Leninsky Prospect 33/2, Moscow 119071 (Russian Federation); Hann, Stephan; Ludwig, Roland [BOKU – University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Wien (Austria); Popov, Vladimir [A. N. Bach Institute of Biochemistry, Leninsky Prospect 33/2, Moscow 119071 (Russian Federation); RSC ‘Kurchatov Institute’, Acad. Kurchatov Sq. 1, Moscow 123182 (Russian Federation)

    2014-11-01

    The structures of the ascomycetous B. aclada laccase and its L499M T1-site mutant have been solved at 1.7 Å resolution. The mutant enzyme shows a 140 mV lower redox potential of the type 1 copper and altered kinetic behaviour. The wild type and the mutant have very similar structures, which makes it possible to relate the changes in the redox potential to the L499M mutation Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. These enzymes contain four Cu atoms per molecule organized into three sites: T1, T2 and T3. In all laccases, the T1 copper ion is coordinated by two histidines and one cysteine in the equatorial plane and is covered by the side chains of hydrophobic residues in the axial positions. The redox potential of the T1 copper ion influences the enzymatic reaction and is determined by the nature of the axial ligands and the structure of the second coordination sphere. In this work, the laccase from the ascomycete Botrytis aclada was studied, which contains conserved Ile491 and nonconserved Leu499 residues in the axial positions. The three-dimensional structures of the wild-type enzyme and the L499M mutant were determined by X-ray crystallography at 1.7 Å resolution. Crystals suitable for X-ray analysis could only be grown after deglycosylation. Both structures did not contain the T2 copper ion. The catalytic properties of the enzyme were characterized and the redox potentials of both enzyme forms were determined: E{sub 0} = 720 and 580 mV for the wild-type enzyme and the mutant, respectively. Since the structures of the wild-type and mutant forms are very similar, the change in the redox potential can be related to the L499M mutation in the T1 site of the enzyme.

  13. Fed-batch SSCF using steam-exploded wheat straw at high dry matter consistencies and a xylose-fermenting Saccharomyces cerevisiae strain: effect of laccase supplementation

    PubMed Central

    2013-01-01

    Background Lignocellulosic bioethanol is expected to play an important role in fossil fuel replacement in the short term. Process integration, improvements in water economy, and increased ethanol titers are key considerations for cost-effective large-scale production. The use of whole steam-pretreated slurries under high dry matter (DM) conditions and conversion of all fermentable sugars offer promising alternatives to achieve these goals. Results Wheat straw slurry obtained from steam explosion showed high concentrations of degradation compounds, hindering the fermentation performance of the evolved xylose-recombinant Saccharomyces cerevisiae KE6-12 strain. Fermentability tests using the liquid fraction showed a higher number of colony-forming units (CFU) and higher xylose consumption rates when treating the medium with laccase. During batch simultaneous saccharification and co-fermentation (SSCF) processes, cell growth was totally inhibited at 12% DM (w/v) in untreated slurries. However, under these conditions laccase treatment prior to addition of yeast reduced the total phenolic content of the slurry and enabled the fermentation. During this process, an ethanol concentration of 19 g/L was obtained, corresponding to an ethanol yield of 39% of the theoretical yield. By changing the operation from batch mode to fed-batch mode, the concentration of inhibitors at the start of the process was reduced and 8 g/L of ethanol were obtained in untreated slurries with a final consistency of 16% DM (w/v). When fed-batch SSCF medium was supplemented with laccase 33 hours after yeast inoculation, no effect on ethanol yield or cell viability was found compared to untreated fermentations. However, if the laccase supplementation (21 hours after yeast inoculation) took place before the first addition of substrate (at 25 hours), improved cell viability and an increased ethanol titer of up to 32 g/L (51% of the theoretical) were found. Conclusions Laccase treatment in SSCF processes reduces the inhibitory effect that degradation compounds have on the fermenting microorganism. Furthermore, in combination with fed-batch operational mode, laccase supplementation allows the fermentation of wheat straw slurry at high DM consistencies, improving final ethanol concentrations and yields. PMID:24219973

  14. Immunogold Labelling to Localize Polyphenol Oxidase (PPO) During Wilting of Red Clover Leaf Tissue and the Effect of Removing Cellular Matrices on PPO Protection of Glycerol-Based Lipid in the Rumen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants. PPO catalyses the conversion of phenols to quinones which can react with nucleophilic cellular constituents (e.g. proteins), forming protein-phenol complexes that may reduce protei...

  15. 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

  16. Bioinformatic Analysis Reveals High Diversity of Bacterial Genes for Laccase-Like Enzymes

    PubMed Central

    Ausec, Luka; Zakrzewski, Martha; Goesmann, Alexander; Schlüter, Andreas; Mandic-Mulec, Ines

    2011-01-01

    Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three- domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications. PMID:22022440

  17. 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. PMID:25898738

  18. Effect of phenolic compounds on the allergenic properties of peanut extracts and peanut butter slurries.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenolic compounds (PCs) are phytochemicals and antioxidants with known health benefits. They are known to bind to proteins as soluble and insoluble complexes. As soluble complexes, with major peanut allergens formed in the presence of polyphenol oxidase (PPO), PCs have been shown to be able to redu...

  19. Degradation of bisphenol A by purified laccase from Trametes villosa.

    PubMed

    Fukuda, T; Uchida, H; Takashima, Y; Uwajima, T; Kawabata, T; Suzuki, M

    2001-06-15

    Degradation of bisphenol A (BPA), an endocrine-disrupting chemical, was studied with a purified laccase from the basidiomycete Trametes villosa. SDS--polyacrylamide gel electrophoresis of the purified laccase gave one single band with a mobility corresponding to MW 65 kDa. The absorption spectrum showed the characteristics of a blue copper protein with a maximum peak at 600 nm. HPLC analysis revealed that 2.2 micromol BPA were degraded by incubation with 1.5 units of the purified laccase in a total volume of 1.0 ml at pH 6.0 and 60 degrees C for 3 h. The enzyme reaction proceeded rapidly without requirement of mediators for the electron transfer. Isolation and identification of several reaction products are in progress, in which one product was identified as 4-isopropenylphenol by a gas chromatography--mass spectrophotometer. PMID:11396959

  20. Mesoporous silicas with tunable morphology for the immobilization of laccase.

    PubMed

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

    2014-01-01

    Siliceous ordered mesoporous materials (OMM) are gaining interest as supports for enzyme immobilization due to their uniform pore size, large surface area, tunable pore network and the introduction of organic components to mesoporous structure. We used SBA-15 type silica materials, which exhibit a regular 2D hexagonal packing of cylindrical mesopores of uniform size, for non-covalent immobilization of laccase. Synthesis conditions were adjusted in order to obtain supports with different particle shape, where those with shorter channels had higher loading capacity. Despite the similar isoelectric points of silica and laccase and the close match between the size of laccase and the pore dimensions of these SBA-15 materials, immobilization was achieved with very low leaching. Surface modification of macro-/mesoporous amorphous silica by grafting of amine moieties was proved to significantly increase the isoelectric point of this support and improve the immobilization yield. PMID:24886935

  1. 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 ?mol/U), followed by the laccase-only system (28.5 ?mol/U), while the TON of the laccase-HBT system was the lowest (14.9 ?mol/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. PMID:25772881

  2. Molecular Structure of Phenol

    NSDL National Science Digital Library

    2003-05-08

    Phenol is a crystalline solid that is colorless or white. It melts at about 41°C, boils at 182°C, and it is soluble in ethanol and ether and a little bit in water. In industry, phenol is essential for making certain artificial resin such as Bakelite. It is also a component of desinfectants, dyes, weed killers, insecticides, explosives, and many drugs such as ear and nose drops. However, breathing and dermal exposure to phenol is very harmful to the skin, eyes, and mucous membranes in humans. It is toxic when taken orally. An exposure to phenol may occur through breathing contaminated air, skin contact, and ingesting of phenol-containing pharmaceuticals. Tobacco smoke and certain foods contain phenol as well.

  3. Reaction mechanisms of the multicopper oxidase CueO from Escherichia coli support its functional role as a cuprous oxidase.

    PubMed

    Djoko, Karrera Y; Chong, Lee Xin; Wedd, Anthony G; Xiao, Zhiguang

    2010-02-17

    CueO from Escherichia coli is a multicopper oxidase (MCO) involved in copper tolerance under aerobic conditions. It features four copper atoms that act as electron transfer (T1) and dioxygen reduction (T2, T3; trinuclear) sites. In addition, it displays a methionine-rich insert which includes a helix that blocks physical access to the T1 site and which provides an extra labile site T4 adjacent to the T1 center. This T4 site is required for CueO function. Like many MCOs, CueO exhibits phenol oxidase activity with broad substrate specificity. Maximal activity with model substrate 2,6-dimethoxyphenol required stoichiometric occupation of T4 by Cu(II) (notation: Cu(II)-CueO). This was achieved in Mops buffer which has little affinity for Cu(2+). However, pH buffers that bind or precipitate Cu(2+) (Tris, BisTris, and KPi) generated enzyme with a vacant T4 site (notation: square-CueO) which has no phenol oxidase activity. Addition of excess Cu(2+) effectively generated a Cu(2+) buffer and recovered the activity partially or completely, depending upon the specific pH buffer. This phenomenon allowed reliable estimation of the affinity of T4 for Cu(II): K(D) = 5.5 x 10(-9) M. CueO is involved in copper tolerance and has been suggested to be a cuprous oxidase. The anion [Cu(I)(Bca)(2)](3-) (Bca = bicinchoninate) acted as a novel chromophoric substrate. It is a robust reagent, being air-stable and having a Cu(I) affinity comparable to those of periplasmic Cu(I) binding proteins. The influences of pH buffer composition and of excess Cu(2+) on cuprous oxidation were diametrically opposite to those seen for phenol oxidation, suggesting that square-CueO, not Cu(II)-CueO, is the resting form of the cuprous oxidase. Steady-state kinetics demonstrated that the intact anion [Cu(I)(Bca)(2)](3-), not "free" Cu(+), is the substrate that donates Cu(I) directly to T4. The data did not follow classical Michaelis-Menten kinetics but could be fitted satisfactorily by an extension that considered the effect of free ligand Bca. The K(m) term consists of two components, allowing estimation of the transient affinity of T4 for Cu(I): K(D) = 1.3 x 10(-13) M. It may be concluded that Cu(I) carried by [Cu(I)(Bca)(2)](3-) is oxidized only upon complete transfer of Cu(I) to T4. The transfer is required to induce a negative shift in the copper reduction potential to allow oxidation and electron transfer to the T1 site. The results provide compelling evidence that CueO is a cuprous oxidase. The new approach will have significant application to the study of metallo-oxidase enzymes. PMID:20088522

  4. Halogenated pesticide transformation by a laccase-mediator system.

    PubMed

    Torres-Duarte, Cristina; Roman, Rosa; Tinoco, Raunel; Vazquez-Duhalt, Rafael

    2009-10-01

    The transformation of organic halogenated pesticides by laccase-mediator system has been investigated. Twelve pesticides were assayed in the presence of nine different mediators. Acetosyringone and syringaldehyde showed to be the best mediators. The halogenated pesticides bromoxynil, niclosamide, bromofenoxim and dichlorophen were transformed by the laccase-syringaldehyde system showing catalytic activities of 48.8, 142.0, 166.2 and 1257.6nmolmin(-1)U(-1), respectively. The highest pesticide transformation rates were obtained with a mediator-substrate proportion of 5:1, one of the lowest reported so far for the laccase-mediator systems. The analysis of the main product from the dichlorophen transformation showed that an oxidative dehalogenation is involved in the catalytic mechanism. Adduct formation between the mediator syringaldehyde and the pesticides dichlorophen or bromoxynil was also found after enzymatic oxidation. The main goal of this work is to evaluate environmental-friendly mediators for the pesticide transformation, and the potential of laccase-mediator system to efficiently reduce the environmental impact of organic halogenated pesticides is discussed. PMID:19695672

  5. Synthetic dye decolorization by three sources of fungal laccase

    PubMed Central

    2012-01-01

    Decolorization of six synthetic dyes using three sources of fungal laccase with the origin of Aspergillus oryzae, Trametes versicolor, and Paraconiothyrium variabile was investigated. Among them, the enzyme from P. variabile was the most efficient which decolorized bromophenol blue (100%), commassie brilliant blue (91%), panseu-S (56%), Rimazol brilliant blue R (RBBR; 47%), Congo red (18.5%), and methylene blue (21.3%) after 3 h incubation in presence of hydroxybenzotriazole (HBT; 5 mM) as the laccase mediator. It was also observed that decolorization efficiency of all dyes was enhanced by increasing of HBT concentration from 0.1 mM to 5 mM. Laccase from A. oryzae was able to remove 53% of methylene blue and 26% of RBBR after 30 min incubation in absence of HBT, but the enzyme could not efficiently decolorize other dyes even in presence of 5 mM of HBT. In the case of laccase from T. versicolor, only RBBR was decolorized (93%) in absence of HBT after 3 h incubation. PMID:23369690

  6. Laccase production by diverse phylogenetic clades of Aureobasidium pullulans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Laccases (EC 1.10.3.2) have numerous potential industrial applications including the degradation of dyes and toxic materials. Novel sources of this enzyme would be desirable to improve activity yields and substrate specificities. In this study we tested 51 strains of A. pullulans representing 13 d...

  7. NADPH Oxidases in Vascular Pathology

    PubMed Central

    Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

  8. Bioavailability of Phenolic Compounds

    Microsoft Academic Search

    SIBEL KARAKAYA

    2004-01-01

    Phenolic compounds in foods originate from one of the main classes of secondary metabolites in plants. They are essential for the growth and reproduction of plants, and are produced as a response for defending injured plants against pathogens. In recent years, there is a growing interest in phenolic compounds and their presumed role in the prevention of various degenerative diseases,

  9. REVIEW OF CHLORINATED PHENOLS

    EPA Science Inventory

    The chlorinated phenols are a group of 19 isomers composed of phenol with substituted chlorines. These chemicals are readily soluble in organic solvents but only slightly soluble in water, except for the chlorophenate salts. Chlorophenols with less than 3 chlorines are not used e...

  10. Phenolic resin syntactic foams

    SciTech Connect

    McIlroy, H.M.

    1980-06-01

    Syntactic foams were prepared from blends of six phenolic resins and carbon microbubbles. The compressive strength of the phenolic resin foams is equivalent to the strength of foams made from a polyimide resin. Ammonia evolved during the cure diffuses rapidly and is not bound by the foam.

  11. [Alternative oxidase in industrial fungi].

    PubMed

    Gu, Shuai; Liu, Qiang; He, Hao; Li, Shuang

    2015-01-01

    Filamentous fungi have been used in industrial fermentation extensively. Based on non-phosphorylating electron transport process, alternative respiration pathway (ARP) acts as an energy overflow, which can balance carbon metabolism and electron transport, allow the continuance of tricarboxylic acid cycle without the formation of ATP, and permit the turnover of carbon skeletons. Alternative respiration pathway also plays an important role in the stress response of fungi and the physiological function of conditioned pathogen. Alternative oxidase (AOX) is the terminal oxidase responsible for the activity of alternative respiration pathway, which exists widely in higher plants, parts of fungi and algae. Owing to the property that alternative oxidase (AOX) is sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of cytochrome respiration, alternative respiration pathway by AOX is also named as cyanide-resistant respiration (CRR). In recent years, the study of the alternative respiration pathway and alternative oxidase has been a hot topic in the area involving cellular respiration metabolism. In this review we summarized the latest research advances about the functions of alternative respiration pathway and alternative oxidase in industrial fungi. PMID:26021078

  12. Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus

    Microsoft Academic Search

    Claudia Eggert; Ulrike Temp; Karl-Erik L. Eriksson

    1997-01-01

    The white-rot fungus, Pycnoporus cinnabarinus, provides an excellent model organism to elucidate the controversial role of laccase in lignin degradation. P. cinnabarinus produces laccase in one isoform as the predominant phenoloxidase in ligninolytic cultures, and neither LiP nor MnP are secreted. Yet, P. cinnabarinus degrades lignin very efficiently. In the present work, we show that laccase-less mutants of P. cinnabarinus

  13. Heterologous expression of a tannic acid-inducible laccase3 of Cryphonectria parasitica in Saccharomyces cerevisiae

    Microsoft Academic Search

    Jung-Mi Kim; Seung-Moon Park; Dae-Hyuk Kim

    2010-01-01

    BACKGROUND: A tannic acid-inducible and mycoviral-regulated laccase3 (lac3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae. RESULTS: Laccase activity in the culture broth of transformants

  14. Treatment of 2,4-dichlorophenol polluted soil with free and immobilized laccase.

    PubMed

    Ahn, Mi-Youn; Dec, Jerzy; Kim, Jang-Eok; Bollag, Jean-Marc

    2002-01-01

    Enzyme treatment is currently considered for remediation of terrestrial systems polluted with organic compounds. In this study, two soils from Pennsylvania with 2.8 or 7.4% organic matter contents (Soils 1 and 2, respectively) were amended with 14C-labeled 2,4-dichlorophenol (2,4-DCP) and incubated with a laccase from Trametes villosa (free or immobilized on montmorillonite). 2,4-DCP was either transformed to methanol-soluble polymeric products (11-32%) or covalently bound to soil organic matter (53-85%); unaltered 2,4-DCP could be recovered from soil by methanol extraction (0-38%) at the completion of a 14-d incubation period. In Soil 1, both free and immobilized laccase removed 100% of 2,4-DCP without regard for moisture conditions. In Soil 2, immobilized laccase removed more 2,4-DCP (about 95%, regardless of moisture conditions) than free enzyme (55, 75, and 90% at 30, 55, and 100% of maximum water-holding capacity, respectively). Binding of 2,4-DCP in the humin fraction was nearly the same for free and immobilized laccase. More 2,4-DCP, however, was bound to humic and fulvic acids in the presence of immobilized laccase than in the presence of free laccase. In general, immobilized laccase performed better than free laccase. However, for practical applications, the higher activity of immobilized laccase is offset by a 23% loss in enzyme activity during immobilization, which approximates the 30% increase in free laccase needed to achieve the same level of remediation. Furthermore, immobilized laccase is more costly than free T. villosa laccase. PMID:12371168

  15. Generation and characterization of transgenic poplar plants overexpressing a cotton laccase gene

    Microsoft Academic Search

    Ji Wang; Chenglong Wang; Mulan Zhu; Yang Yu; Yuebo Zhang; Zhiming Wei

    2008-01-01

    Laccases are copper-containing glycoproteins, which are widespread in higher plants as multigene families. To gain more insight\\u000a in the function of laccases in plants, especially potential role in lignification, we produced transgenic poplar plants overexpressing\\u000a a cotton laccase cDNA (GaLAC1) under the control of the cauliflower mosaic virus 35S promoter. As compared with untransformed control plants, transgenic\\u000a plants exhibited a

  16. Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii

    Microsoft Academic Search

    R. Campos; A. Kandelbauer; K. H. Robra; Artur Cavaco-Paulo; G. M. Gubitz

    2001-01-01

    The degradation of the textile dye indigo with purified laccases from the fungi Trametes hirsuta (THL1 and THL2) and Sclerotium rolfsii (SRL1) was studied. All laccases were able to oxidize indigo yielding isatin (indole-2,3-dione), which was further decomposed to anthranilic acid (2-aminobenzoic acid). Based on the oxygen consumption rate of the laccases during indigo degradation, a potential mechanism for the

  17. Studies on the mechanism of alcohol oxidase 

    E-print Network

    Menon, Vipin

    1994-01-01

    The flavoprotein alcohol oxidase from the yeast Candida boidinii catalyzes the oxidation of primary alcohols to aidehydes with transfer of the electrons to molecular oxygen to form hydrogen peroxide. The mechanism of alcohol oxidase with beta...

  18. Molecular Characterization of Laccase Genes from the Basidiomycete Coprinus cinereus and Heterologous Expression of the Laccase Lcc1

    PubMed Central

    Yaver, Debbie S.; Overjero, Maria Del Carmen; Xu, Feng; Nelson, Beth A.; Brown, Kim M.; Halkier, Torben; Bernauer, Sheryl; Brown, Stephen H.; Kauppinen, Sakari

    1999-01-01

    A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent Km of 21 ± 2 ?M and a catalytic constant of 200 ± 10 min?1 for O2 with 2,2?-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing. PMID:10543807

  19. Effect of Direct-Current Electric Field on Enzymatic Activity and the Concentration of Laccase.

    PubMed

    Wang, Chunxing; Zhang, Huiling; Ren, Dajun; Li, Qian; Zhang, Shuqin; Feng, Tao

    2015-09-01

    This work investigates the effect of direct-current electric field on the extracellular enzymatic activity, concentration and other experimental parameters of laccase from Trametes versicolor. The results showed that laccase could significantly contribute to the change of pH at the end of graphite electrode. In addition, it increased the electrical conductivity of the water. In the experiment, the optimum pH and catalytic pH range for laccase activity were 3.0 and pH 2.5-4.0. The application of 6 V direct current showed significant effects on the laccase enzyme activity. The activity of laccase was enhanced in the anodic region, but at the same time was strongly inhibited at the cathode. The electric charge characteristics of laccase were changed when exposed to electric field, and some laccases molecules moved to the anode, which produced a slight migration phenomenon. This study is the basis of combination of laccase and electrical technology, at the same time, providing a new direction of enhancing laccase activity. Compared to immobilization, using electric field is simple, no chemical additives, and great potential. PMID:26063937

  20. Electrochemical sensor for predicting transformer overload by phenol measurement.

    PubMed

    Bosworth, Timothy; Setford, Steven; Heywood, Richard; Saini, Selwayan

    2003-03-10

    Transformer overload is a significant problem to the power transmission industry, with severe safety and cost implications. Overload may be predicted by measuring phenol levels in the transformer-insulating oil, arising from the thermolytic degradation of phenol-formaldehyde resins. The development of two polyphenol oxidase (PPO) sensors, based on monitoring the enzymatic consumption of oxygen using an oxygen electrode, or reduction of enzymatically generated o-quinone at a screen-printed electrode (SPE), for the measurement of phenol in transformer oil is reported. Ex-service oils were prepared either by extraction into aqueous electrolyte-buffer, or by direct dilution in propan-2-ol, the latter method being more amenable to simple at-line operation. The oxygen electrode, with a sensitivity of 2.87 nA microg(-1) ml(-1), RSD of 7.0-19.9% and accuracy of +/-8.3% versus the industry standard International Electrotechnical Commission (IEC) method, proved superior to the SPE (sensitivity: 3.02 nA microg(-1) ml(-1); RSD: 8.9-18.3%; accuracy: +/-7.9%) and was considerably more accurate at low phenol concentrations. However, the SPE approach is more amenable to field-based usage for reasons of device simplicity. The method has potential as a rapid and simple screening tool for the at-site monitoring of phenol in transformer oils, thereby reducing incidences of transformer failure. PMID:18968967

  1. GLUCOSE OXIDASE REDUCES OXIDATION IN FROZEN SHRIMP

    E-print Network

    role oxygen can have during storage of foods (Scott, 1958). Glucose oxidase-catalase preparations are used to carry out the net reaction: 2 glucose + oxygen glucose oxidase > 2 gluconic acid. catalase of glucose oxidase -catalase would probably be more obvious in shrimp, which were packed in transparent bags

  2. Stereoselective Hydrogen Abstraction by Galactose Oxidase

    Microsoft Academic Search

    Stefan G. Minasian; Mei M. Whittaker; James W. Whittaker

    2004-01-01

    The fungal enzyme galactose oxidase is a radical copper oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on galactose oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state

  3. Degradation of endocrine disrupting chemicals by genetic transformants in Irpex lacteus with an inducible laccase gene of Phlebia tremellosa

    Microsoft Academic Search

    Hyunwoo Kum; Myung K. Kim; Hyoung T. Choi

    2009-01-01

    Irpex\\u000a lacteus was genetically transformed using an laccase expression vector to get increased laccase producing strains. Stable integration\\u000a of the vector was confirmed by PCR using the vector-specific primers, and the transformants showed increased laccase activities.\\u000a When the transformants were grown with several endocrine disrupting chemicals, laccase activity of each transformant was induced\\u000a up to six times higher than that

  4. Purification, Characterization, Molecular Cloning, and Expression of Two Laccase Genes from the White Rot BasidiomyceteTrametes villosa

    Microsoft Academic Search

    DEBBIE S. YAVER; FENG XU; ELIZABETH J. GOLIGHTLY; KIM M. BROWN; STEPHEN H. BROWN; MICHAEL W. REY; PALLE SCHNEIDER; TORBEN HALKIER; KRISTINE MONDORF

    1996-01-01

    Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a2,5-xylidine-inducedcultureofthewhiterotbasidiomyceteTrametesvillosa(PolyporuspinsitusorCorioluspin- situs). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondena- turing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have

  5. Overlap of laccases\\/cellobiose dehydrogenase activities during the decolourisation of anthraquinonic dyes with close chemical structures by Pycnoporus strains

    Microsoft Academic Search

    Sophie Vanhulle; Estelle Enaud; Marie Trovaslet; Nancy Nouaimeh; Christian-Marie Bols; Tajalli Keshavarz; Thierry Tron; Giovanni Sannia; Anne-Marie Corbisier

    2007-01-01

    Pycnoporus strains were used as model to understand the role of laccases in the in vivo decolourisation of three anthraquinonic dyes. The decolourisation capability of Pycnoporus sanguineus MUCL 41582 (PS7), which produces laccases as the main oxidative enzyme, was assayed and compared with the decolourisation capability of a control strain, Pycnoporus cinnabarinus MUCL 39533 (PC330) described as laccase-deficient strain. In

  6. Protoporphyrinogen Oxidase-Inhibiting Herbicides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protoporphyrinogen oxidase-inhibiting herbicides (also referred to as Protox- or PPO-inhibiting herbicides) were commercialized in the 1960s and their market share reached approximately 10% (total herbicide active ingredient output) in the late 1990’s. The wide-spread adoption of glyphosate-resista...

  7. Signal enhancement in polysaccharide based sensors for infections by incorporation of chemically modified laccase.

    PubMed

    Schneider, Konstantin P; Gewessler, Ulrike; Flock, Teresa; Heinzle, Andrea; Schenk, Verena; Kaufmann, Franz; Sigl, Eva; Guebitz, Georg M

    2012-05-15

    Bioresponsive polymers (BRPs) allow the detection of potentially pathogenic microorganisms. Here, peptidoglycan and cellulose based hydrogels were constructed with potential for diagnosis of wound infection or, for example, Aspergillosis, respectively. These systems respond to extracellular enzymes from microbes or enzymes secreted from the human immune system in case of infection. Laccases as 'enhanzymes' were incorporated into these devices for signal and stability enhancement when compared to simple dye release based systems. To retain the enhanzymes within the BRPs, they were either PEGylated laccase (Laccase_PEG) to increase size or methacrylated laccase (Laccase_MA) to allow covalent attachment to the polysaccharide matrices. PEGylation of Trametes hirsuta laccase led to a fivefold increase in size to 270kDa according to size exclusion chromatography (SEC). Likewise, successful methacrylation of the laccase was demonstrated by using reversed phase chromatography while SEC analysis proved covalent attachment of the enzyme to the methacrylated polysaccharide matrix. Upon incubation of peptidoglycan based BRPs with fluid from infected wounds, the difference to controls was four times higher for Laccase_PEG based signalling when compared to simple dye release. Similarly, the control signals (i.e. leaching) were considerably reduced in case of Laccase_MA incorporated in crosslinked peptidoglycan (PG) and carboxymethylcellulose (CMC) hydrogels for signalling. In addition, Laccase_MA catalysed colour formation enhanced the signal dramatically with factors between 100- and 600-fold. Laccase_MA was demonstrated to oxidise silica gel immobilised ferulic acid incorporated into the BRP with clearly visible colour changes of 4.5 ?E units according the CIELab concept upon incubation by trigger enzymes as well as infected wound fluids. PMID:22445491

  8. Preparation of magnetic chitosan nanoparticles and immobilization of laccase

    Microsoft Academic Search

    Hua Fang; Jun Huang; Liyun Ding; Mingtian Li; Zhao Chen

    2009-01-01

    The magnetic chitosan nanoparticles were prepared by reversed-phase suspension method using Span-80 as an emulsifier, glutaraldehyde\\u000a as cross-linking reagent. And the nanoparticles were characterized by TEM, FT-IR and hysteresis loop. The results show that\\u000a the nanoparticles are spherical and almost superparamagnetic. The laccase was immobilized on nanoparticles by adsorption and\\u000a subsequently by cross-linking with glutaraldehyde. The immobilization conditions and characterizations

  9. Some Properties of Laccase from the Latex of Lacquer Trees

    Microsoft Academic Search

    D. Keilin; T. Mann

    1940-01-01

    WE have previously shown1 that laccase, the oxidizing enzyme present in the latex of the Indo-Chinese lacquer tree (Rhus succedanea), is a copper-protein compound. Our purest preparation of this enzyme, which catalyses the oxidation of polyphenols and diamines, contained 0.154 per cent copper and 6.45 per cent nitrogen, corresponding to about 45 per cent protein. The remaining fraction of the

  10. Modulating oxidoreductase activity modifies the phenolic content of virgin olive oil.

    PubMed

    García-Rodríguez, Rosa; Romero-Segura, Carmen; Sanz, Carlos; Pérez, Ana G

    2015-03-15

    The effect of modifying polyphenol oxidase (PPO) and peroxidase (POX) activity during the extraction of virgin olive oil has been assessed in terms of its influence on the phenolic profile of the oil produced. These enzymes were modified by adding exogenous enzyme or specific inhibitors during the milling and subsequent kneading step, studying the effect on specific phenolic compounds in the oils. PPO is the main enzyme involved in phenolic oxidation at the milling step whereas POX activity seems to be the main influence during the kneading step. The data obtained suggest it is possible to increase the nutritional and organoleptic quality of virgin olive oil by inhibiting these enzymes during olive fruit processing. Treatment with the PPO inhibitor tropolone produced a twofold increase in the phenolic fraction, which would therefore seem to be an interesting strategy to improve the nutritional and organoleptic properties of virgin olive oil. PMID:25308681

  11. Basidiomycetes laccase and manganese peroxidase activity in submerged fermentation of food industry wastes

    Microsoft Academic Search

    Giorgi Songulashvili; Vladimir Elisashvili; Solomon P. Wasser; Eviatar Nevo; Yitzhak Hadar

    2007-01-01

    The evaluation of eighteen strains of basidiomycetes laccase and manganese peroxidase (MnP) activity in submerged fermentation of mandarin peelings and ethanol production waste showed that the expression of enzyme activity is species- and strain-dependent. While all species of the genus Trametes expressed comparatively high laccase activity, the activity of this enzyme among species of the genus Ganoderma varied from 192

  12. Identification of Single Nucleotide Polymorphism Markers in the Laccase Gene of Shiitake Mushrooms (Lentinula edodes)

    PubMed Central

    Kim, Ki-Hwan; Ka, Kang-Hyeon; Kang, Ji Hyoun; Kim, Sangil; Lee, Jung Won; Jeon, Bong-Kyun; Yun, Jung-Kuk

    2015-01-01

    We identified single nucleotide polymorphism (SNP) markers in the laccase gene to establish a line-diagnostic system for shiitake mushrooms. A total of 89 fungal isolates representing four lines, including Korean registered, Korean wild type, Chinese, and Japanese lines, were analyzed. The results suggest that SNP markers in the laccase gene can be useful for line typing in shiitake mushrooms. PMID:25892919

  13. The multigene family of fungal laccases and their expression in the white rot basidiomycete Flammulina velutipes.

    PubMed

    Wang, Wei; Liu, Fang; Jiang, Yuji; Wu, Guangmei; Guo, Lixian; Chen, Renliang; Chen, Bingzhi; Lu, Yuanping; Dai, Yucheng; Xie, Baogui

    2015-06-01

    Fungal laccases play important roles in matrix degradation. Eleven laccase genes, including three novel ones (designated lac1, lac2 and lac4) were identified after sequencing the entire genome of the edible, white-rot fungus Flammulina velutipes. Analysis using bioinformatics revealed that all of the laccases, except lac3, possess a signal peptide. These laccase proteins consist of 502-670 amino acids and have predicted molecular weights ranging from 55kDa to 74kDa. These proteins each contain four copper-binding sites, except for Lac10. Transcriptomes were sequenced at different developmental stages and in different fruiting body tissues to analyze if there was differential expression of laccase genes. The novel laccase gene lac4 exhibited the highest expression levels among all of the observed laccases at every developmental stage and in all fruiting body tissues examined. We conclude that laccases in F. velutipes play a role not only in lignin degradation, but also in fruiting body formation and development. PMID:25776201

  14. Influence of different magnetic composites carriers on the immobilization of laccase

    NASA Astrophysics Data System (ADS)

    Xiao, Haiyan; Huang, Jun; Li, Bin; Wang, Juntao; Jiang, Desheng

    2006-01-01

    Laccase (E.C.1.10.3.2) has been used in various fields and enzyme immobilization technology is an effective means to perform enzyme reuse and to improve its stability. Carrier materials play an important role in the application of an immobilized enzyme. Magnetic carriers have been widely used in the field of protein and enzyme immobilization. The most important parameters of magnetic carriers are size, structure, density of reactive surface groups and the superparamagnetic property. The copper tetraaminophthalocyanine (CuTAPc)- Fe 3O 4 nano particle composite and chitosan-Fe 3O 4 microspheres composite were successfully synthesized and characterized by FTIR spectra, XRD and SEM micrograph. Active amino groups of two magnetic carriers could be used to bind laccase via glutaraldehyde. The optimal pH of the two immobilized laccases were the same at pH 3.0. The optimal temperature of laccase immobilized on CuTAPc-Fe 3O 4 nano particle was 45°C and that of the chitosan-Fe 3O 4 microspheres was 55°C. The immobilization yields of the two immobilized laccases were 5mg/g and 16mg/g, respectively. The Km value of the laccase immobilized on CuTAPc-Fe 3O 4 nano particles was 23.8?M, lower than that of the laccase immobilized on chitosan-Fe 3O 4 microspheres, 171.1?M. The laccase immobilized on magnetic composites could be used as biological sensing materials for biosensor.

  15. A novel non-blue laccase from Bacillus amyloliquefaciens: secretory expression and characterization.

    PubMed

    Chen, Biao; Xu, Wen-Qi; Pan, Xin-Ru; Lu, Lei

    2015-05-01

    Laccases are copper-containing enzymes which possess a promising potential in many industrial and environmental applications. Here we describe the cloning, extracellular expression and characterization of a novel non-blue laccase from Bacillus amyloliquefaciens in Pichia pastoris. The recombinant enzyme was secreted into the culture supernatant with high activity. It lacks the absorption band at 610 nm typical for blue laccases. However, electron paramagnetic resonance (EPR) spectrum proved the existence of type 1 copper center that was not detectable in the UV-visible spectrum. Metal content analysis revealed that the enzyme contains two copper ions, one iron ion and one zinc ion per protein molecular, suggesting that it is a novel non-blue laccase. The pH and temperature optima of the recombinant laccase were 6.6 and 60°C, respectively, and it was stable at pH 9.0 for 10 days. The enzyme activity was slightly activated by NaCl with concentration up to 200 mM. The purified laccase showed high efficiency in decolorizing reactive black 5 and indigo carmine, achieving more than 93% decolorization after 1h. The extreme robustness of the recombinant B. amyloliquefaciens laccase offers several advantages over most fungal laccases in various industrial applications. PMID:25709013

  16. Diversity of Ascomycete Laccase Gene Sequences in a Southeastern US Salt Marsh

    Microsoft Academic Search

    J. I. Lyons; S. Y. Newell; A. Buchan; M. A. Moran

    2003-01-01

    The diversity of ascomycete laccase sequences was surveyed in a southeastern US salt marsh using a degenerate primer set designed around copper binding sites conserved in fungal laccases. This gene was targeted for diversity analysis because of its potential function in lignin degradation in the salt marsh ecosystem and because few studies have assessed functional gene diversity in natural fungal

  17. Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT 20197.

    PubMed Central

    Mansur, M; Suárez, T; Fernández-Larrea, J B; Brizuela, M A; González, A E

    1997-01-01

    A new lignin-degrading basidiomycete, strain I-62 (CECT 20197), isolated from decayed wood exhibited both a high dephenolization activity and decolorization capacity when tested on effluents from the sugar cane by-product fermentation industry. It has been classified as a member of the Polyporaceae family. The major ligninolytic activity detected in culture supernatants of basidiomycete I-62 was a phenoloxidase (laccase), in conjunction with small amounts of manganese peroxidase. No lignin peroxidase was detected. Laccase activity was produced in either defined or complete media. Addition of veratryl alcohol as the inducer, in defined medium, enhanced laccase production 10-fold. The use of fructose instead of glucose as a carbon source resulted in a 100-fold increase in laccase specific activity. Native isoelectrofocusing gels stained with guaiacol revealed the presence of at least seven laccase isozymes, with the most intense band being detected at pI 3. Southern hybridization analysis indicated the presence of a laccase gene family in strain I-62. Three different genes coding for phenoloxidases, lcc1, lcc2, and lcc3, were cloned and characterized. The high degree of homology between laccases from strain I-62 and laccases from Trametes species suggests a phylogenetic proximity between this new isolated fungus and the genus Trametes. PMID:9212414

  18. Specificities of a chemically modified laccase from Trametes hirsuta on soluble and cellulose-bound substrates

    Microsoft Academic Search

    M. Schroeder; S. Heumann; C. J. S. M. Silva; A. Cavaco-Paulo; G. M. Guebitz

    2006-01-01

    Laccases could prevent fabrics and garments from re-deposition of dyes during washing and finishing processes by degrading the solubilized dye. However, laccase action must be restricted to solubilized dye molecules thereby avoiding decolorization of fabrics. Chemical modification of enzymes can provide a powerful tool to change the adsorption behaviour of enzymes on water insoluble polymers. Polyethylene glycol (PEG) was covalently

  19. Laccase: A Review of Its Past and Its Future in Bioremediation

    Microsoft Academic Search

    P. J. Strong; H. Claus

    2011-01-01

    Laccases are multicopper proteins that use molecular oxygen to oxidize a broad spectrum of organic compounds by a radical-catalyzed reaction mechanism. Many articles over the past 15 years have touted the diverse potential applications of laccase in various biotechnological processes. This review covers the natural roles of the enzyme, its structural properties, substrates, reaction mechanism, and inhibitors, as well as

  20. Enhancing catalytic performance of laccase via immobilization on chitosan/CeO2 microspheres.

    PubMed

    Lin, Jiahong; Fan, Ling; Miao, Runli; Le, Xueyi; Chen, Shi; Zhou, Xiaohua

    2015-07-01

    In this study, laccase from Trametes versicolor was immobilized onto chitosan/CeO2 microspheres (CCMS) by adsorption or covalent binding after activating the amine groups of chitosan with glutaraldehyde (GA). The results indicated that the laccase loading on chitosan/CeO2 microspheres was approximately 73mg/g under the optimum conditions (pH 5.4, 6h), and the activity recovery was 66.9%. In comparison with free laccase, the thermal and operational stabilities of the immobilized laccase were significantly improved. The catalytic activity of the immobilized laccase was also demonstrated by the decolorization of two reactive dyes (methyl red and orange II). The laccase immobilized on CCMS was very effective for the removal of textile dyes from an aqueous solution. The removal rates of methyl red and orange II by the immobilized laccase were 83.3% and 92.6%, respectively, which are much higher than that of free laccase (i.e., 49.0% and 67.1%, respectively). PMID:25841367

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase

    Microsoft Academic Search

    A. Leontievsky; N. Myasoedova; B. Baskunov; L. Golovleva; C. Bucke; C. Evans

    2001-01-01

    Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4°C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the

  3. 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. PMID:23867099

  4. 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. PMID:24458655

  5. Laccase detoxification of steam-exploded wheat straw for second generation bioethanol

    Microsoft Academic Search

    Miguel Jurado; Alicia Prieto; Ángeles Martínez-Alcalá; Ángel T. Martínez; María Jesús Martínez

    2009-01-01

    In this work we compared the efficiency of a laccase treatment performed on steam-exploded wheat straw pretreated under soft conditions (water impregnation) or harsh conditions (impregnation with diluted acid). The effect of several enzymatic treatment parameters (pH, time of incubation, laccase origin and loading) was analysed. The results obtained indicated that severity conditions applied during steam explosion have an influence

  6. Laccase production by Trametes hirsuta, characterization, and its capability of decoloring chlorophyll.

    PubMed

    Liu, Jiayang; Liu, Wenhua; Cai, Yujie; Liao, Xiangru; Huang, Qingguo; Liang, Xiaohui

    2014-01-01

    The present study focused on laccase production, characterization, and its involvement in chlorophyll decolorization. Extracellular laccase, with the highest activity of 11 U/ml on day 8, was efficiently produced from Trametes hirsuta in 5 l bioreactor with optimized media comprising dual carbon sources, glucose and water hyacinth. A laccase was then purified from the supernatant to homogeneity with purification fold of 9.51 and recovery of 39.8% and an estimated molecular mass of 62 kDa by SDS-PAGE. The laccase showed activity at pH 2-6 and temperature 30-80°C and was relatively thermally stable at below 70°C and neutral pH. The laccase was applied to decolorize chlorophyll under different factors: temperature, pH, mediator, metal ions, and enzyme dosage. Other fungal laccases were also found to be able to degrade chlorophyll with rating from 52% to 88% following 1 h treatment with two laccase dosages (5 or 10 U/ml) in the absence of any other mediators at room temperature. These findings may be an important step in developing new, important, and commercially viable industrial applications for laccase enzymes. PMID:25546943

  7. Isolation, culture optimization and physico-chemical characterization of laccase enzyme from Pleurotus fossulatus.

    PubMed

    Chowdhury, P; Hari, R; Chakraborty, B; Mandal, B; Naskar, S; Das, Nirmalendu

    2014-01-15

    Pleurotus fossulatus (Cooke) Sace is member of oyster mushroom can produced extracellular laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) in submerged fermentation. To analyze the optimum production for laccase P. fossulatus was cultured both in stationary and shaking condition in different media. Partial purification of laccase was done after 0-80% ammonium sulphate precipitation, followed by DEAE (Diethylaminoethyl) Sephadex (A-50) anion exchange chromatography. Potato-sucrose peptone (PSP) medium and Potato-dextrose (PD) medium showed highest laccase production in shaking and stationary conditions, respectively. Though the time required for optimum laccase production in stationary condition was much more than the shaking condition but the amount of laccase was about 2.75t greater in former condition. The laccase produced in stationary condition was more stable than the enzyme produced in shaking condition. The partially purified enzyme showed highest affinity towards o-dianisidine than guaiacol and ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) as evidenced by their K(m). The physico-chemical properties of the laccase suggested the significance of this enzyme in industrial applications. PMID:24783799

  8. Identification of Single Nucleotide Polymorphism Markers in the Laccase Gene of Shiitake Mushrooms (Lentinula edodes).

    PubMed

    Kim, Ki-Hwan; Ka, Kang-Hyeon; Kang, Ji Hyoun; Kim, Sangil; Lee, Jung Won; Jeon, Bong-Kyun; Yun, Jung-Kuk; Park, Sang Rul; Lee, Hyuk Je

    2015-03-01

    We identified single nucleotide polymorphism (SNP) markers in the laccase gene to establish a line-diagnostic system for shiitake mushrooms. A total of 89 fungal isolates representing four lines, including Korean registered, Korean wild type, Chinese, and Japanese lines, were analyzed. The results suggest that SNP markers in the laccase gene can be useful for line typing in shiitake mushrooms. PMID:25892919

  9. Characterization of immobilized laccase from Lentinula edodes and its use in olive-mill wastewater treatment

    Microsoft Academic Search

    Alessandro D’Annibale; Silvia Rita Stazi; Vittorio Vinciguerra; Elena Di Mattia; Giovanni Giovannozzi Sermanni

    1999-01-01

    This study is mainly concerned with the characterization of an immobilized laccase from the white-rot fungus Lentinula edodes for use in wastewater treatment. The enzyme was immobilized on chitosan by adsorption and subsequent cross-linking with glutaraldehyde. The immobilized laccase displayed a lower specific activity and a lower substrate affinity than the free enzyme. Nevertheless, the immobilized catalyst exhibited an appreciable

  10. Selective oxidation of lignin model compounds – a combinatorial application of the laccase-mediator system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Enzymological Characterization of Atm, the First Laccase from Agrobacterium sp. S5-1, with the Ability to Enhance In Vitro digestibility of Maize Straw

    PubMed Central

    Si, Wei; Wu, ZhaoWei; Wang, LiangLiang; Yang, MingMing; Zhao, Xin

    2015-01-01

    Laccase is an enzyme that catalyzes oxidation of phenolic compounds, diamines and aromatic amines. In this study, a novel laccase-like gene (atm) in a ligninolyitic isolate Agrobacterium sp. S5-1 from soil humus was identified and heterologously expressed in Escherichia coli. Atm exhibited its maximal activity at pH 4.5 and at 50°C. This enzyme was tolerant to high temperature, a broad range of pH, heavy metal ions (Co3+, Mn2+, Cu2+ and Ni2+, 20 mM) and all tested organic solvents. Furthermore, Atm significantly (p<0.05) increased dry matter digestibility of maize straw from 23.44% to 27.96% and from 29.53% to 37.10% after 8 or 24 h of digestion and improved acid detergent fiber digestibility from 5.81% to 10.33% and from 12.80% to 19.07% after 8 or 24 h of digestion, respectively. The combination of Atm and fibrolytic enzymes significantly (p<0.05) enhanced neutral detergent fiber digestibility from 19.02% to 24.55% after 24 h of digestion respectively. Results showed treatment with Atm effectively improved in vitro digestibility of maize straw, thus suggesting that Atm has an application potential for bioconversion of lignin rich agricultural byproducts into animal feed and cellulosic ethanol. PMID:26010258

  12. Total phenolic compounds and free phenol in softwood structural plywood

    SciTech Connect

    Tiedeman, G.T.; Isaacson, R.L. (Weyerhaeuser Co., Tacoma, WA (United States)); Sellers, T. Jr. (Mississippi Forest Products Lab., Mississippi State, MS (United States))

    1994-03-01

    Construction-grade plywood panels manufactured at five plywood mills were analyzed for total phenolic compounds and free phenol detection. Small samples of plywood were ground <1-mm-size powders. The samples were subjected to an ambient temperature, methylene chloride extraction, and tested for free phenol content by a gas chromatography-mass spectrometry method. The plywood samples were also analyzed for total phenolic compounds by a distillation-colorimetric method. The range of total phenolic compounds was 6.8 to 25.3 mg/kg and the range of free phenol was 0.090 to 0.73 mg/kg. The sources of phenolic compounds in plywood are wood components, the phenol-formaldehyde resin adhesive, and the ligno-cellulosic adhesive fillers. The source of free phenol in structural plywood is presumably the phenol-formaldehyde resin adhesive. The extraction procedures used in this study were extreme and are not typical for plywood in service. Yet the levels of phenolic compounds and free phenol detected were so low that they most often were beyond the quantitative accuracy of the test methods and instruments, requiring extrapolative techniques. The low levels are supportive of the fact that structural wood composites bonded with phenol-formaldehyde resins have been found to be very safe environmentally for multiple uses.

  13. Enhanced functionality and stabilization of a cold active laccase using nanotechnology based activation-immobilization.

    PubMed

    Mukhopadhyay, Arka; Dasgupta, Anjan Kr; Chakrabarti, Krishanu

    2015-03-01

    A simple nanotechnology based immobilization technique for imparting psychrostability and enhanced activity to a psychrophilic laccase has been described here. Laccase from a psychrophile was supplemented with Copper oxide nanoparticles (NP) corresponding to copper (NP-laccase), the cationic activator of this enzyme and entrapped in single walled nanotube (SWNT). The activity and stability of laccase was enhanced both at temperatures as low as 4°C and as high as 80°C in presence of NP and SWNT. The enzyme could be released and re-trapped (in SWNT) multiple times while retaining significant activity. Laccase, immobilized in SWNT, retained its activity after repeated freezing and thawing. This unique capability of SWNT to activate and stabilize cold active enzymes at temperatures much lower or higher than their optimal range may be utilized for processes that require bio-conversion at low temperatures while allowing for shifts to higher temperature if so required. PMID:25590281

  14. Potential applications of laccase-mediated coupling and grafting reactions: a review.

    PubMed

    Kudanga, Tukayi; Nyanhongo, Gibson S; Guebitz, Georg M; Burton, Stephanie

    2011-03-01

    Many industries are currently pursuing enzymatic approaches for developing green chemistry technologies mainly due to shortcomings of physico-chemical methods, growing environmental concerns, legal restrictions, and increasing scientific knowledge. Laccase-assisted reactions, in particular, are being intensively investigated as they are generally eco-friendly and have wide application potential. Laccases only require oxygen as co-substrate, they release water as the only by-product and have a wide substrate range which can be further extended by use of laccase-mediator systems. Consequently, research covering various applications of laccase has been rapidly increasing in recent years, particularly in the areas of coupling and grafting reactions. This review summarizes the advances that have been made in developing technologies based on laccase-mediated coupling and grafting reactions for potential application in areas such as environmental pollution control, modification of lignocellulose materials, food industry, biosensors, textile industry, pharmaceutical industry, and in organic synthesis. PMID:22112901

  15. Production of laccase from Pleurotus florida using agro-wastes and efficient decolorization of Reactive blue 198.

    PubMed

    Sathishkumar, P; Murugesan, K; Palvannan, T

    2010-08-01

    Pleurotus florida NCIM 1243 produced laccase as the dominant lignolytic enzyme during the dye decolorization. Banana peel was the best substrate for extracellular laccase production under solid state fermentation when compared to mandarin peel and cantaloupe peel. The maximum activity of laccase (5.4 U/g) was detected on the 10 day. The ratio of banana peel: mandarin peel: cantaloupe peel (5:2:3) showed increased production of laccase (6.8 U/g). P. florida produced two extracellular laccase isoenzymes (L1 and L2). The half life of laccase at 60 degrees C was 2 h and at 4 h it retained 25% residual activity. P. florida laccase showed high thermostability and an interesting difference was noticed in the behavior of laccase isoenzymes at different temperature. The L1 isoenzyme of laccase showed remarked thermostability at 60 degrees C in the native PAGE when compared to L2 isoenzyme. The optimum pH, temperature and enzyme concentration for maximum decolorization was found to be 4.5, 60 degrees C and 1.2 U/ml, respectively. Partially purified laccase enzyme showed excellent decolorization activity to Reactive blue 198. The maximum decolorization (96%) was observed at lower dye concentrations (50-100 ppm) which decreased markedly when the dye concentration was increased beyond 150 ppm. The thermostable laccase of P. florida could be effectively used to decolorize the synthetic dyes in the textile effluent and other biotechnological applications. PMID:20586068

  16. NADPH oxidases and cardiac remodelling

    Microsoft Academic Search

    Adam Nabeebaccus; Min Zhang; Ajay M. Shah

    2011-01-01

    A heart under chronic stress undergoes cardiac remodelling, a process that comprises structural and functional changes including\\u000a cardiomyocyte hypertrophy, interstitial fibrosis, contractile dysfunction, cell death and ventricular dilatation. Reactive\\u000a oxygen species (ROS)-dependent modulation of intracellular signalling is implicated in the development of cardiac remodelling.\\u000a Among the different ROS sources that are present in the heart, NADPH oxidases (NOXs) are particularly

  17. NADPH oxidase and cardiac failure.

    PubMed

    Kuroda, Junya; Sadoshima, Junichi

    2010-08-01

    Increases in oxidative stress in the heart play an important role in mediating hypertrophy, apoptosis, fibrosis, mitochondrial dysfunction, and the consequent development of heart failure. Although it has been widely believed that electron leakage from the mitochondrial electron transport chain is the primary source of oxidative stress in the failing heart, increasing lines of evidence suggest that enzymes which produce reactive oxygen species may also contribute to it. NADPH oxidases are transmembrane enzymes dedicated to producing superoxide (O(2)(-)) by transferring an electron from NAD(P)H to molecular oxygen. Nox4 is a major NADPH oxidase isoform expressed in the heart. Nox4 is localized primarily at mitochondria in cardiac myocytes, and upregulation of Nox4 hypertrophic stimuli enhances O(2)(-) production, apoptosis, and mitochondrial dysfunction, thereby playing an important role in mediating cardiac dysfunction. Since Nox4 could be a key molecule mediating oxidative stress and pathological hypertrophy, it may serve as an important target of heart failure treatment. In this review, the importance of NADPH oxidases as sources of increased oxidative stress in the failing heart and the role of Nox4 in mediating growth and death of cardiac myocytes are discussed. PMID:20559780

  18. NADPH Oxidase and Cardiac Failure

    PubMed Central

    Kuroda, Junya; Sadoshima, Junichi

    2011-01-01

    Increases in oxidative stress in the heart play an important role in mediating hypertrophy, apoptosis, fibrosis, mitochondrial dysfunction and the consequent development of heart failure. Although it has been widely believed that electron leakage from the mitochondrial electron transport chain is the primary source of oxidative stress in the failing heart, increasing lines of evidence suggest that enzymes which produce reactive oxygen species (ROS) may also contribute to it. NADPH oxidases are transmembrane enzymes dedicated to producing superoxide (O2-) by transferring an electron from NAD(P)H to molecular oxygen. Nox4 is a major NADPH oxidase isoform expressed in the heart. Nox4 is localized primarily at mitochondria in cardiac myocytes, and upregulation of Nox4 hypertrophic stimuli enhances O2- production, apoptosis, and mitochondrial dysfunction, thereby playing an important role in mediating cardiac dysfunction. Since Nox4 could be a key molecule mediating oxidative stress and pathological hypertrophy, it may serve as an important target of heart failure treatment. In this review, the importance of NADPH oxidases as sources of increased oxidative stress in the failing heart and the role of Nox4 in mediating growth and death of cardiac myocytes are discussed. PMID:20559780

  19. Decolorization of two synthetic dyes using the purified laccase of Paraconiothyrium variabile immobilized on porous silica beads

    PubMed Central

    2014-01-01

    Background Decolorization of hazardous synthetic dyes using laccases in both free and immobilized form has gained attention during the last decades. The present study was designed to prepare immobilized laccase (purified from Paraconiothyrium variabile) on porous silica beads followed by evaluation of both free and immobilized laccases for decolorization of two synthetic dyes of Acid Blue 25 and Acid Orange 7. Effects of laccase concentration, pH and temperature alteration, and presence of 1-hydroxybenzotriazole (HBT) as laccase mediator on decolorization pattern were also studied. In addition, the kinetic parameters (K m and V max ) of the free and immobilized laccases for each synthetic dye were calculated. Results Immobilized laccase represented higher temperature and pH stability compare to free one. 39% and 35% of Acid Blue 25 and Acid Orange 7 was decolorized, respectively after 65 min incubation in presence of the free laccase. In the case of immobilized laccase decolorization percent was found to be 76% and 64% for Acid Blue 25 and Acid Orange 7, respectively at the same time. Increasing of laccase activity enhanced decolorization percent using free and immobilized laccases. Relative decolorization of both applied dyes was increased after treatment by laccase-HBT system. After nine cycles of decolorization by immobilized laccase, 26% and 31% of relative activity were lost in the case of Acid Blue 25 and Acid Orange 7, respectively. Conclusions To sum up, the present investigation introduced the immobilized laccase of P. variabile on porous beads as an efficient biocatalyst for decolorization of synthetic dyes. PMID:24393474

  20. Inhibition of MAO A and B by some plant-derived alkaloids, phenols and anthraquinones

    Microsoft Academic Search

    L. D. Kong; Christopher H. K. Cheng; R. X. Tan

    2004-01-01

    A total of seventeen phytochemicals including seven alkaloids (piperine, strychnine, brucine, stachydrine, tetrandrine, frangchinoline and sinomenine), four phenols (paeonol, honokiol, magnolol and eugenol) and six anthraquinones (emodin, rhein, chrysorphanol, aloe-emodin, physcion and 1,8-dihydroxyanthraquinone) was examined for inhibitory activity of monoamine oxidase (MAO) A and B from rat brain mitochondrial. Among these compounds, piperine and paeonol were found to be inhibitory

  1. Spectral and catalytic properties of aryl-alcohol oxidase, a fungal flavoenzyme acting on polyunsaturated alcohols

    PubMed Central

    2005-01-01

    Spectral and catalytic properties of the flavoenzyme AAO (aryl-alcohol oxidase) from Pleurotus eryngii were investigated using recombinant enzyme. Unlike most flavoprotein oxidases, AAO does not thermodynamically stabilize a flavin semiquinone radical and forms no sulphite adduct. AAO catalyses the oxidative dehydrogenation of a wide range of unsaturated primary alcohols with hydrogen peroxide production. This differentiates the enzyme from VAO (vanillyl-alcohol oxidase), which is specific for phenolic compounds. Moreover, AAO is optimally active in the pH range of 5–6, whereas VAO has an optimum at pH 10. Kinetic studies showed that AAO is most active with p-anisyl alcohol and 2,4-hexadien-1-ol. AAO converts m- and p-chlorinated benzyl alcohols at a similar rate as it does benzyl alcohol, but introduction of a p-methoxy substituent in benzyl alcohol increases the reaction rate approx. 5-fold. AAO also exhibits low activity on aromatic aldehydes. 19F NMR analysis showed that fluorinated benzaldehydes are converted into the corresponding benzoic acids. Inhibition studies revealed that the AAO active site can bind a wide range of aromatic ligands, chavicol (4-allylphenol) and p-anisic (4-methoxybenzoic) acid being the best competitive inhibitors. Uncompetitive inhibition was observed with 4-methoxybenzylamine. The properties described above render AAO a unique oxidase. The possible mechanism of AAO binding and oxidation of substrates is discussed in the light of the results of the inhibition and kinetic studies. PMID:15813702

  2. Computational Analysis and Low-Scale Constitutive Expression of Laccases Synthetic Genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris

    PubMed Central

    Reyes-Guzmán, Edwin Alfredo; Poutou-Piñales, Raúl A.; Reyes-Montaño, Edgar Antonio; Pedroza-Rodríguez, Aura Marina; Rodríguez-Vázquez, Refugio; Cardozo-Bernal, Ángela M.

    2015-01-01

    Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2?-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZ?A-LaccGluc-Stop and pGAPZ?A-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and ?-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications. PMID:25611746

  3. A novel laccase from fresh fruiting bodies of the wild medicinal mushroom Tricholoma matsutake.

    PubMed

    Xu, Lijing; Zhu, Mengjuan; Chen, Xiao; Wang, Hexiang; Zhang, Guoqing

    2015-01-01

    The knowledge about biological activities of constituents from medicinal mushrooms belonging to the genus Tricholoma is limited. A 59-kDa laccase has now been purified from fresh fruiting bodies of the mushroom Tricholoma matsutake. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, affinity chromatography on ConA-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Of the various affinity and ion exchange chromatographic media employed, the laccase bound only on Con A-Sepharose. The activity of the laccase did not undergo major changes over the temperature range 20-80°C. However, all activity vanished following exposure to 100°C for 10 minutes. The enzyme activity varied only slightly over the pH range 3-5, with the optimal pH of 5, but exhibited a precipitous decline when the pH was increased to 6, and was undetectable at pH 8 and 9. The laccase showed activity in the decolorization of azo dyes without a mediator. Its N-terminal sequence demonstrated only slight resemblance to those of other mushroom laccases. The newly described laccase is distinctive from the previously isolated Tricholoma mushroom laccases in a number of aspects. PMID:25781157

  4. Strain-dependent response to Cu(2+) in the expression of laccase in Pycnoporus coccineus.

    PubMed

    Park, Ju-Wan; Kang, Hyeon-Woo; Ha, Byung-Suk; Kim, Sin-Il; Kim, Soonok; Ro, Hyeon-Su

    2015-05-01

    The effects of Cu(2+) on the activity and expression of laccase were investigated in seven different strains of Pycnoporus coccineus collected from different regions in Korea. Cu(2+) was toxic to mycelial growth at concentrations greater than 0.5 mM CuSO4 and showed complete growth inhibition at 1 mM in the liquid culture. However, Cu(2+) significantly upregulated the extracellular laccase activity at 0.2 mM in five strains of P. coccineus, IUM4209, IUM0032, IUM0450, IUM0470, and IUM4093, whereas two strains, IUM0253 and IUM0049, did not respond to Cu(2+), despite being closely related to the other five strains. Subsequent RT-PCR analysis also showed that the laccase mRNA was highly expressed only in the former five strains in the presence of Cu(2+). Taken together, these results indicate that Cu(2+) regulates expression of the laccase gene in a strain-dependent manner. The five strains commonly produced a single predominant laccase protein with a molecular weight of 68 kDa. Peptide sequencing revealed that the laccase was a homolog of Lcc1 of P. coccineus, which was isolated in China. The Cu(2+)-induced culture supernatants exhibited high degradation of polycyclic aromatic hydrocarbons, indicating that the 68-kDa laccase is the primary extracellular degradative enzyme in P. coccineus. PMID:25677944

  5. 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

  6. Laccase immobilization over multi-walled carbon nanotubes: Kinetic, thermodynamic and stability studies.

    PubMed

    Tavares, Ana P M; Silva, Cláudia G; Draži?, Goran; Silva, Adrián M T; Loureiro, José M; Faria, Joaquim L

    2015-09-15

    The biocatalytic performance of immobilized enzyme systems depends mostly on the intrinsic properties of both biomolecule and support, immobilization technique and immobilization conditions. Multi-walled carbon nanotubes (MWCNTs) possess unique features for enzyme immobilization by adsorption. Enhanced catalytic activity and stability can be achieved by optimization of the immobilization conditions and by investigating the effect of operational parameters. Laccase was immobilized over MWCNTs by adsorption. The hybrid material was characterized by Fourier transformed infrared (FTIR) spectroscopy, scanning and transmission electron microscopy (SEM and TEM, respectively). The effect of different operational conditions (contact time, enzyme concentration and pH) on laccase immobilization was investigated. Optimized conditions were used for thermal stability, kinetic, and storage and operational stability studies. The optimal immobilization conditions for a laccase concentration of 3.75?L/mL were a pH of 9.0 and a contact time of 30min (522 Ulac/gcarrier). A decrease in the thermal stability of laccase was observed after immobilization. Changes in ?S and ?H of deactivation were found for the immobilized enzyme. The Michaelis-Menten kinetic constant was higher for laccase/MWCNT system than for free laccase. Immobilized laccase maintained (or even increased) its catalytic performance up to nine cycles of utilization and revealed long-term storage stability. PMID:26002339

  7. [Immobilization of crude laccase onto anion exchange resin and its application in decoloration of malachite green].

    PubMed

    Qi, Xu-liang; Liu, Xiang; Liu, Bo; Wang, Lin; Wang, Xiao-chun; Fang, Chao

    2012-08-01

    Crude laccase from Trametes versicolor was immobilized onto anion exchange resin D201 by three methods, i.e., direct electrostatic adsorption (D201-Lac-I), crosslinking after electrostatic adsorption (D201-Lac-II) and electrostatic adsorption after treating D201 with glutaraldehyde (D201-Lac-III). Compared to direct electrostatic adsorption, the immobilized laccase amount of D201-Lac-II increased by 4.65 times but the laccase activity was decreased to 4.8%, while the laccase activity on D201-Lac-III increased by 2.99 times, with the immobilization amount decreased to 51%. Shadows of laccase aggregation on D201-Lac-III were found by transmission electron microscopy (TEM). Continuous batch decoloration of malachite green demonstrated that the decoloration efficiency of D201-Lac-III remained in the range of 40% to 55% for more than 210 hours, in addition, the enzyme activity on D201-Lac-III maintained unchanged while the activity of free laccase declined to less than 20% under the same condition. All of the results above indicated that D201-Lac-III had a significantly enhanced stability and good reusability. Considering the low price and simple production procedure of crude laccase, D201-Lac-III could be promising for water treatment purpose. PMID:23213900

  8. Modeling Based Structural Insights into Biodegradation of the Herbicide Diuron by Laccase-1 from Ceriporiopsis subvermispora

    PubMed Central

    Vieira, Ana Carolina; Marschalk, Cidnei; Biavatti, Débora Carina; Lorscheider, Carla Andréia; Peralta, Rosane Marina; Seixas, Flavio Augusto Vicente

    2015-01-01

    The herbicide diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is used in many agricultural crops and non-crop areas worldwide, leading to the pollution of the aquatic environment by soil leaching. White rot fungi and its lignin modifying enzymes, peroxidases and laccases, are responsible for its degradation. Therefore, it is of interest to explore the potential use of Ceriporiopsis subvermispora laccase (CersuLac1) in the biotransformation of this herbicide by using its enzyme laccase. However, the structure of laccase from Ceriporiopsis subvermispora is still unknown. Hence, a model of laccase was constructed using homology modeling. The model was further used to dock p-methylbenzoate in the presence of four copper ions to analyze molecular basis of its binding and interaction. The ligand-protein interaction is stereo-chemically favorable in nature. The presence of the single protonated Lys457 was necessary for catalysis, being coordinated by a cupper ion. The best pose of diuron on CersuLac1 has a theoretical Ki of 2.91 mM. This is comparable to the KM values for laccases from other organisms with similar compounds. Thus, we document the insights for the potential use of laccase from Ceriporiopsis subvermispora in the biotransfrormation of diuron.

  9. Phenol and phenolics from lignocellulosic biomass by catalytic microwave pyrolysis

    SciTech Connect

    Bu, Quan; Lei, Hanwu; Ren, Shoujie; Wang, Lu; Holladay, Johnathan E.; Zhang, Qin; Tang, Juming; Ruan, Roger

    2011-07-01

    Catalytic microwave pyrolysis of biomass using activated carbon was investigated to determine the effects of pyrolytic conditions on the yields of phenol and phenolics. The high concentrations of phenol (38.9%) and phenolics (66.9%) were obtained at the temperature of 589 K, catalyst-to-biomass ratio of 3:1 and retention time of 8 min. The increase of phenol and its derivatives compared to pyrolysis without catalysts has a close relationship with the decomposition of lignin under the performance of activated carbon. The concentration of esters was also increased using activated carbon as a catalyst. The high content of phenols obtained in this study can be used either directly as fuel after upgrading or as feedstock of biobased phenols for chemical industry.

  10. 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. PMID:25301391

  11. 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. PMID:24043124

  12. Some new functions of amine oxidases

    Microsoft Academic Search

    B. Mondovì; P. Pietrangeli; L. Morpurgo; E. Masini; R. Federico; M. A. Mateescu; O. Befani; E. Agostinelli

    2003-01-01

    Two contrasting topics are examined in this account: the protective actions of amine oxidases (AOs) resulting from the elimination and\\/or modulation of the levels of polyamines and some biogenic amines, such as histamine, in anaphylactic shock and the cell damaging effect of AOs catabolic products. Other functions of the plasma copper-containing amine oxidase are considered; namely the modification of some

  13. Essential role of the N- and C-terminals of laccase from Pleurotus florida on the laccase activity and stability.

    PubMed

    Hu, Meirong; Zhou, Xue; Shi, Yiping; Lin, Jianhui; Irfan, Muhammad; Tao, Yong

    2014-11-01

    POXA1b is the most thermostable laccase isoenzyme from Pleurotus ostreatus. POXA1b is remarkably stable at alkaline pH (the t1/2 at pH 10 was 30 days), and its C-terminal affects its catalytic and stability properties. We cloned POXA1c from P. florida, which showed 99 % identity with POXA1b. POXA1c was functionally expressed in Pichia pastoris. The functions of the N and C termini of POXA1c were investigated using site-directed mutagenesis. Compared with POXA1c, the N-terminal R5V site effectively increased the specific activities for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and guaiacol by 2- and 3.5-fold, respectively. A C-terminal truncated mutant, POXA1c?13, also increased the specific activities for ABTS and guaiacol by 2.3- and 3.4-fold, respectively. A double mutant, POXA1c?13-R5V, combined the R5V and ?13 effects. The specific activity of this double mutant for ABTS was 1,321 U/mg, which indicated a 4-fold increase compared with the wild type. The role of residue V5 on laccase catalytic properties was also observed for laccases from Trametes versicolor and Rigidoporus lignosus. The specific activities of the V5R of the laccases from T. versicolor and R. lignosus were half of that of the wild type. The pH and thermal stability analysis of POXA1c and its mutants showed that the enzymes were remarkably stable because they showed 63 % residual activity after incubation for 108 h at 30 °C over a pH range of 4.5 to 9.0. Similar results were observed for POXA1c?13-R5V. POXA1c?13-R5V can be widely used in industrial biotechnology because of its excellent catalytic properties. PMID:25161036

  14. Forage polyphenol oxidase and ruminant livestock nutrition

    PubMed Central

    Lee, Michael R. F.

    2014-01-01

    Polyphenol oxidase (PPO) is predominately associated with the detrimental effect of browning fruit and vegetables, however, interest within PPO containing forage crops (crops to be fed to animals) has grown since the browning reaction was associated with reduced nitrogen (N) losses in silo and the rumen. The reduction in protein breakdown in silo of red clover (high PPO forage) increased the quality of protein, improving N-use efficiency [feed N into product N (e.g., Milk): NUE] when fed to ruminants. A further benefit of red clover silage feeding is a significant reduction in lipolysis (cleaving of glycerol-based lipid) in silo and an increase in the deposition of beneficial C18 polyunsaturated fatty acid (PUFA) in animal products, which has also been linked to PPO activity. PPOs protection of plant protein and glycerol based-PUFA in silo is related to the deactivation of plant proteases and lipases. This deactivation occurs through PPO catalyzing the conversion of diphenols to quinones which bind with cellular nucleophiles such as protein reforming a protein-bound phenol (PBP). If the protein is an enzyme (e.g., protease or lipase) the complexing denatures the enzyme. However, PPO is inactive in the anaerobic rumen and therefore any subsequent protection of plant protein and glycerol based-PUFA in the rumen must be as a result of events that occurred to the forage pre-ingestion. Reduced activity of plant proteases and lipases would have little effect on NUE and glycerol based-PUFA in the rumen due to the greater concentration of rumen microbial proteases and lipases. The mechanism for PPOs protection of plant protein in the rumen is a consequence of complexing plant protein, rather than protease deactivation per se. These complexed proteins reduce protein digestibility in the rumen and subsequently increase undegraded dietary protein flow to the small intestine. The mechanism for protecting glycerol-based PUFA has yet to be fully elucidated but may be associated with entrapment within PBP reducing access to microbial lipases or differences in rumen digestion kinetics of the forage and therefore not related to PPO activity. PMID:25538724

  15. Immunological comparison of sulfite oxidase

    SciTech Connect

    Pollock, V.; Barber, M.J. (Univ. South Florida College, Tampa (United States))

    1991-03-11

    Polyclonal antibodies (rabbit), elicited against FPLC-purified chicken and rat liver sulfite oxidase (SO), have been examined for inhibition and binding to purified chicken (C), rat (R), bovine (B), alligator (A) and shark (S) liver enzymes. Anti-CSO IgG cross-reacted with all five enzymes, with varying affinities, in the order CSO=ASO{gt}RSO{gt}BSO{gt}SSO. Anti-ROS IgG also cross-reacted with all five enzymes in the order RSO{gt}CSO=ASO{gt}BSO{gt}SSO. Anti-CSO IgG inhibited sulfite:cyt. c reductase (S:CR), sulfite:ferricyanide reductase (S:FR) and sulfite:dichlorophenolindophenol reductase (S:DR) activities of CSO to different extents (S:CR{gt}S:FR=S:DR). Similar differential inhibition was found for anti-ROS IgG and RSO S:CR, S:FR and S:DR activities. Anti-CSO IgG inhibited S:CR activities in the order CSO=ASO{much gt}SSO{gt}BSO. RSO was uninhibited. For anti-RSO IgG the inhibition order was RSO{gt}SSO{gt}BSO{gt}ASO. CSO was uninhibited. Anti-CSO and RSO IgGs partially inhibited Chlorella nitrate reductase (NR). Minor cross-reactivity was found for xanthine oxidase. Common antigenic determinants for all five SO's and NR are indicated.

  16. An alkali-stable enzyme with laccase activity from entophytic fungus and the enzymatic modification of alkali lignin.

    PubMed

    Weihua, Qiu; Hongzhang, Chen

    2008-09-01

    Mycelia Sterilia YY-5, an entophytic fungus, was isolated from Rhus chinensis Mill and its extracellular enzyme had a higher laccase activity (MS-Lac). After been purified by anion exchange and gel filtration chromatography, MS-Lac, which had a molecular mass of 45 kDa, was found to be an alkali-stable enzyme with an optimum pH of 10.0 and capable of retaining 80% activity after incubation for 72 h with syringaldazine as substrate. It was also found that syringaldazine had a higher affinity than 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as substrate for MS-Lac, which was determined in sodium phosphate buffer (pH 6.0, 0.1M) at 30 degrees C. Meanwhile, the lignin modification, catalyzed by MS-Lac, indicated that it could oxidize the phenolic hydroxyl, side chain substituent or carbonyl group of spruce alkali lignin in cetyltrimethylammonium bromide (CTAB) reversed micelles (20 mM, pH 6.0, W/O=40) and steam-exploded wheat straw alkali lignin in NaOH solution (20 mM, pH 10.0). PMID:18096384

  17. Effect of solvent phase transitions on enzymatic activity and structure of laccase from Coriolus hirsutus.

    PubMed

    Stepanova, E V; Fedorova, T V; Sorokina, O N; Volkov, V V; Koroleva, O V; Dembo, A T

    2009-04-01

    The effect of solvent phase transitions on catalytic activity and structure of the active site of laccase produced by the Basidiomycetes Coriolus hirsutus 072 was studied. As shown by small-angle X-ray scattering, laccase exists in solution as a mixture of monomeric and aggregated particles in the percent ratio 85:15. This ratio did not change on phase transitions. A complex nature of laccase activity dynamics during thawing and further heating to 20 degrees C was shown. Spontaneous oxidation of T1 copper center in the temperature range 12-20 degrees C was not observed. According to spectral data, the structure of laccase active sites including all copper centers of types T1, T2, and T3 changes during the phase transition. PMID:19463091

  18. Influence of process variables on the properties of laccase biobleached pulps.

    PubMed

    Martin-Sampedro, Raquel; Miranda, Jesús; García-Fuentevilla, Luisa L; Hernández, Manuel; Arias, Maria E; Diaz, Manuel J; Eugenio, Maria E

    2015-01-01

    A laccase stage can be used as a pre-treatment of a standard chemical bleaching sequence to reduce environmental concerns associated to this process. The importance of each independent variable and its influence on the properties of the bleached pulp have been studied in depth in this work, using an adaptive network-based fuzzy inference system (ANFIS) with four independent variables (laccase, buffer, mediator and oxygen) as input. Eucalyptus globulus kraft pulp was biobleached using a laccase from Pycnoporus sanguineus and a natural mediator (acetosyringone). Later, an alkaline extraction and a hydrogen peroxide treatment were applied. Most biobleaching processes showed a decrease in kappa number and an increase in brightness with no significant impact on the viscosity values, compared with the control. Oxygen was the variable with the smallest influence on the final pulp properties while the laccase and buffer solution showed a significant influence. PMID:25085529

  19. Degradation of phenol and phenolic compounds by Pseudomonas putida EKII

    Microsoft Academic Search

    Christel Hinteregger; Raimund Leitner; Michael Loidl; Andreas Ferschl; Franz Streichsbier

    1992-01-01

    The phenol-degrading strain Pseudomonas putida EKII was isolated from a soil enrichment culture and utilized phenol up to 10.6 mM (1.0 g·1 -1) as the sole source of carbon and energy. Furthermore, cresols, chlorophenols, 3,4-dimethylphenol, and 4-chloro-m-cresol were metabolized as sole substrates by phenol-grown resting cells of strain EKII. Under conditions of cell growth, degradation of these xenobiotics was achieved

  20. Effects of some alkyl phenols on methanogenic degradation of phenol

    SciTech Connect

    Wang, Y.T.; Suidan, M.T.; Pfeffer, J.T.; Najm, I.

    1988-05-01

    The effects of six phenolic compounds (o-, m-, and p-cresol and 2-,3-, and 4-ethylphenol) on the anaerobic biodegradation of phenol was examined in batch methanogenic cultures. Results showed that ethylphenols were more inhibitory of phenol degradation than were cresols. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.

  1. A psychrotolerant strain of Serratia marcescens (MTCC 4822) produces laccase at wide temperature and pH range.

    PubMed

    Kaira, Gaurav Singh; Dhakar, Kusum; Pandey, Anita

    2015-12-01

    A psychrotolerant bacterial strain of Serratia marcescens, originally isolated from a glacial site in Indian Himalayan Region (IHR), has been investigated for laccase production under different culture conditions. The bacterial strain was found to grow between 4 to 45°C (opt. 25°C) and 3 to 14 pH (opt. 5 pH) on prescribed growth medium, coinciding with production of laccase in laccase producing medium. However, the production of laccase was more consistent toward alkaline pH. Laccase enzyme was partially purified using gel filtration chromatography. The molecular mass of laccase was determined ~53 kDa on native PAGE. The Km and Vmax values were determined to be 0.10 mM and 50.00 ?M min(-1), respectively, with ABTS. Inoculum size (4.0% v/v at 1.5 O.D.) resulted in significantly higher production of laccase. Carbon and nitrogen sources also affected the laccase production significantly. All the carbon sources enhanced laccase production, xylose being the best enhancer (P?laccase production. Low molecular weight organic solvents significantly (P?laccase production up to 24 h of incubation with a decline in later incubation period. Production of laccase by the psychrotolerant bacterium in wide range of temperature and pH is likely to have inference in biotechnological processes. PMID:26054732

  2. Laccase-mediator biobleaching applied to a direct yellow dyed paper.

    PubMed

    Knutson, Kristina; Ragauskas, Arthur

    2004-01-01

    A laccase-mediator system (LMS) for biobleaching was applied to a bleached chemical pulp dyed with stilbene dye Direct Yellow 11. Of mediators tested, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) was found to be more effective than either violuric acid (VA) or N-hydroxybenzotriazole (HBT), which had been shown to be superior to ABTS when bleaching virgin chemical pulp. The laccase-ABTS system removed more than 60% of the color. PMID:15575730

  3. Improvement membrane filterability in nanofiltration of prehydrolysis liquor of kraft dissolving pulp by laccase treatment.

    PubMed

    Wang, Qiang; Liu, Shanshan; Yang, Guihua; Chen, Jiachuan

    2015-04-01

    In this work, laccase treatment was employed to enhance nanofiltration process by lignin removal. Results showed that the membrane filterability was increased in terms of deionized water flux and PHL filtration process. On the other hand, the hemicellulosic sugars were negligible affected and can be concentrated to 172 g/L, which was increased about 300% from the original one. The combined laccase-nanofiltration process provides an alternative approach to utilize hemicellulosic sugars of PHL in an environmentally friendly way. PMID:25643958

  4. Oxygen Activation during Oxidation of Methoxyhydroquinones by Laccase from Pleurotus eryngii

    PubMed Central

    Guillén, Francisco; Muñoz, Carmen; Gómez-Toribio, Víctor; Martínez, Angel T.; Jesús Martínez, María

    2000-01-01

    Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH2) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH2) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH2 more efficiently than it oxidized MBQH2; both the affinity and maximal velocity of oxidation were higher for DBQH2 than for MBQH2. Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q·? + O2 ? Q + O2·?). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O2·? on quinone formation rates. Then, the production of H2O2 in laccase reactions, as a consequence of O2·? dismutation, confirmed that semiquinones autoxidized. The highest H2O2 levels were obtained with DBQH2, indicating that DBQ·? autoxidized to a greater extent than did MBQ·?. Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O2·? consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O2·?, besides undergoing dismutation, reacts with Mn2+, Fe3+, and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 ?M concentrations of MBQ·? and DBQ·? from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ·? autoxidation rose to 13.8% and that of DBQ·? increased to 39.9%. PMID:10618219

  5. Laccase activity in soils: considerations for the measurement of enzyme activity.

    PubMed

    Eichlerová, Ivana; Šnajdr, Jaroslav; Baldrian, Petr

    2012-08-01

    Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing enzymes that catalyze the oxidative conversion of a variety of chemicals, such as mono-, oligo-, and polyphenols and aromatic amines. Laccases have been proposed to participate in the transformation of organic matter and xenobiotics as well as microbial interactions. Several laccase assays have been proposed and used in soils. Here, we show that the optimal pH conditions for the laccase substrates 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, pH 3-5), 2,6-dimethoxyphenol (4-5.5), L-3,4-dihydroxyphenylalanine (DOPA; 4-6), guaiacol (3.5-5), 4-methylcatechol (3.5-5), and syringaldazine (5.5-7.0) are similar between purified laccases from Trametes versicolor and Pyricularia sp. and soil extracts; the substrate affinities of purified enzymes (K(M)) and soil extracts were also similar. The laccase assays showed specificity overlap with tyrosinase and ligninolytic peroxidases when hydrogen peroxide is present. The ABTS oxidation assay is able to reliably detect the presence of 13.5 pg mL(-1) or 0.199×10(-12) mol mL(-1) of T. versicolor laccase, which is three times more sensitive than the 2,6-dimethoxyphenol-based assay and more than 40 times more sensitive than any of the other assays. The low molecular mass soil-derived compounds and the isolated fulvic and humic acids influence the laccase assays and should be removed from the soil extracts before measurements of the enzyme activity are performed. PMID:22475148

  6. Laccase from the medicinal mushroom Agaricus blazei : production, purification and characterization

    Microsoft Academic Search

    René Ullrich; Le Mai Huong; Nguyen Lan Dung; Martin Hofrichter

    2005-01-01

    The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l-1) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (-1) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein

  7. Enhanced Adsorption of Atrazine in Different Soils in the Presence of Fungal Laccase

    Microsoft Academic Search

    Natalia A. Kulikova; Valentina N. Davidchik; Elena V. Stepanova; Olga V. Koroleva

    Adsorption–desorption behavior of atrazine was studied in three soils belonging to different soil geographical zones. Experimental\\u000a investigations focused on the effect of laccase addition on adsorption and desorption of target chemical when present in solution.\\u000a Addition of laccase resulted in a dramatic increase in adsorption of the atrazine. Desorption was little or negligible. Hysteresis,\\u000a represented by hysteresis indices, was significantly

  8. Extracellular laccase production during hyphal interactions between Trichoderma sp. and Shiitake, Lentinula edodes

    Microsoft Academic Search

    J.-M. Savoie; G. Mata; C. Billette

    1998-01-01

    Lentinula edodes (Berk.) Pegler was cultivated in liquid media containing malt and yeast extract. Extracellular laccase activity, measured\\u000a in the culture fluids, was 5–18 times higher in cultures incubated for 29 days than in cultures incubated for 24 days. The\\u000a addition of water-soluble lignin derivatives or Trichoderma sp. in cultures of L. edodes incubated for 11 days increased laccase activity

  9. Potato and mushroom polyphenol oxidase activities are differently modulated by natural plant extracts.

    PubMed

    Kuijpers, Tomas F M; van Herk, Teunie; Vincken, Jean-Paul; Janssen, Renske H; Narh, Deborah L; van Berkel, Willem J H; Gruppen, Harry

    2014-01-01

    Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the outcome of such screening, this study compared the effect of 60 plant extracts on the activity of PPO from mushroom ( Agaricus bisporus , AbPPO) and PPO from potato ( Solanum tuberosum , StPPO). Some plant extracts had different effects on the two PPOs: an extract that inhibited one PPO could be an activator for the other. As an example of this, the mate ( Ilex paraguariensis ) extract was investigated in more detail. In the presence of mate extract, oxygen consumption by AbPPO was found to be reduced >5-fold compared to a control reaction, whereas that of StPPO was increased >9-fold. RP-UHPLC-MS analysis showed that the mate extract contained a mixture of phenolic compounds and saponins. Upon incubation of mate extract with StPPO, phenolic compounds disappeared completely and saponins remained. Flash chromatography was used to separate saponins and phenolic compounds. It was found that the phenolic fraction was mainly responsible for inhibition of AbPPO and activation of StPPO. Activation of StPPO was probably caused by activation of latent StPPO by chlorogenic acid quinones. PMID:24344979

  10. Advanced enzymatic elimination of phenolic contaminants in wastewater: a nano approach at field scale.

    PubMed

    Gasser, Christoph A; Yu, Liang; Svojitka, Jan; Wintgens, Thomas; Ammann, Erik M; Shahgaldian, Patrick; Corvini, Philippe F-X; Hommes, Gregor

    2014-04-01

    The removal of recalcitrant chemicals in wastewater treatment systems is an increasingly relevant issue in industrialized countries. The elimination of persistent xenobiotics such as endocrine-disrupting chemicals (EDCs) emitted by municipal and industrial sewage treatment plants remains an unsolved challenge. The existing efficacious physico-chemical methods, such as advanced oxidation processes, are resource-intensive technologies. In this work, we investigated the possibility to remove phenolic EDCs [i.e., bisphenol A (BPA)] by means of a less energy and chemical consuming technology. To that end, cheap and resistant oxidative enzymes, i.e., laccases, were immobilized onto silica nanoparticles. The resulting nanobiocatalyst produced at kilogram scale was demonstrated to possess a broad substrate spectrum regarding the degradation of recalcitrant pollutants. This nanobiocatalyst was applied in a membrane reactor at technical scale for tertiary wastewater treatment. The system efficiently removed BPA and the results of long-term field tests illustrated the potential of fumed silica nanoparticles/laccase composites for advanced biological wastewater treatment. PMID:24305739

  11. 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. PMID:19539671

  12. Immobilized laccase on activated poly(vinyl alcohol) microspheres for enzyme thermistor application.

    PubMed

    Bai, Xue; Gu, Haixin; Chen, Wei; Shi, Hanchang; Yang, Bei; Huang, Xin; Zhang, Qi

    2014-07-01

    Poly(vinyl alcohol) (PVA) microspheres were prepared by inverse suspension crosslinked method, with glutaraldehyde as a crosslinking agent. PVA microspheres activated with aldehyde groups were employed for Trametes versicolor laccase immobilization. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to characterize the activated PVA microspheres and PVA microspheres with immobilized laccase (Lac/PVA microspheres), which show that laccase was successfully immobilized on the PVA microspheres. The optimum pH and temperature coupling conditions for the immobilized laccase were determined to be 3.3 and 30 °C, respectively. Residual activity was also investigated by soaking the immobilized laccase in organic solvents at different concentrations, proving it chemically stable. Immobilized laccase exhibited good storage stability at 4 °C. The enzyme biosensor showed good performance in 2,2-azinobis(3-ethylthiazoline-6-sulfonate) and bisphenol A, with concentration ranges of 2 to 8 mM and 0.05 to 0.25 mM, respectively. Therefore, PVA microspheres may have high potential as support for enzyme thermistor applications. PMID:24760609

  13. 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 ?M, 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

  14. Laccase-Based CLEAs: Chitosan as a Novel Cross-Linking Agent

    PubMed Central

    Arsenault, Alexandre; Cabana, Hubert; Jones, J. Peter

    2011-01-01

    Laccase from Coriolopsis Polyzona was insolubilized as cross-linked enzyme aggregates (CLEAs) for the first time with chitosan as the cross-linking agent. Concentrations between 0.01 and 1.867?g/L of chitosan were used and between 0.05 and 600?mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The laccase was precipitated using ammonium sulphate and cross-linked simultaneously. Specific activity and thermal stability of these biocatalysts were measured. Activities of up to 737?U/g were obtained when 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was used as a substrate. Moreover, the stability of these biocatalysts was improved with regards to thermal degradation compared to free laccase when exposed to denaturing conditions of high temperature and low pH. The CLEAs stability against chemical denaturants was also tested but no significant improvement was detected. The total amount of ABTS to be oxidized during thermal degradation by CLEAs and free laccase was calculated and the insolubilized enzymes were reported to oxidize more substrate than free laccase. The formation conditions were analyzed by response surface methodology in order to determine an optimal environment for the production of efficient laccase-based CLEAs using chitosan as the cross-linking agent. After 24 hours of formation at pH 3 and at 4°C without agitation, the CLEAs exhibit the best specific activity. PMID:21811672

  15. Molecular Cloning and Expression in Saccharomyces cerevisiae of a Laccase Gene from the Ascomycete Melanocarpus albomyces

    PubMed Central

    Kiiskinen, Laura-Leena; Saloheimo, Markku

    2004-01-01

    The lac1 gene encoding an extracellular laccase was isolated from the thermophilic fungus Melanocarpus albomyces. This gene has five introns, and it encodes a protein consisting of 623 amino acids. The deduced amino acid sequence of the laccase was shown to have high homology with laccases from other ascomycetes. In addition to removal of a putative 22-amino-acid signal sequence and a 28-residue propeptide, maturation of the translation product of lac1 was shown to involve cleavage of a C-terminal 14-amino-acid extension. M. albomyces lac1 cDNA was expressed in Saccharomyces cerevisiae under the inducible GAL1 promoter. Extremely low production was obtained with the expression construct containing laccase cDNA with its own signal and propeptide sequences. The activity levels were significantly improved by replacing these sequences with the prepro sequence of the S. cerevisiae ?-factor gene. The role of the C-terminal extension in laccase production in S. cerevisiae was also studied. Laccase production was increased sixfold with the modified cDNA that had a stop codon after the native processing site at the C terminus. PMID:14711635

  16. 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

  17. 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. PMID:18075999

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

    SciTech Connect

    D`Souza, T.M.; Boominathan, K.; Reddy, C.A. [Michigan State Univ., East Lansing, MI (United States)

    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, 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.

  19. A comparative study on electrochemistry of laccase at two kinds of carbon nanotubes and its application for biofuel cell

    NASA Astrophysics Data System (ADS)

    Zheng, W.; Zhou, H. M.; Zheng, Y. F.; Wang, N.

    2008-05-01

    Direct electron transfer between laccase and a glassy carbon electrode modified with carbon nanotubes having a uniform inner tube diameter was observed by cyclic voltammetry in 0.10 M phosphate buffer. The formal potential of +530 mV ( vs. SCE) was very close to redox potential of T1 copper in laccase. No direct electron transfer between laccase and a glassy carbon electrode modified with carbon nanotubes having a tapered inner tube diameter was determined under the same condition. The possible application of the laccase-catalyzed O 2 reduction at these electrodes was successfully illustrated by constructing an ascorbate/O 2 biofuel cell.

  20. Overproduction of laccase from a newly isolated Ganoderma lucidum using the municipal food waste as main carbon and nitrogen supplement.

    PubMed

    Hailei, Wang; Ping, Li; Yuhua, Yang; Yufeng, Liu

    2015-05-01

    A strain of Ganoderma lucidum was separated and identified according to its morphological characteristics and phylogenetic data. The fungus is a laccase producer and it can secrete laccase using the municipal food waste (FW) as carbon and nitrogen supplement. After the statistic optimization, a laccase activity of 42,000 ± 600 U/l was obtained at 500 ml flask level and the activity is 12,000 U/l higher than that obtained by fermenting glucose and peptone, indicating that the use of FW to produce laccase not only reduces production cost, but also improves laccase activity. In 15 l bioreactor, FW is also suitable for laccase production and the maximum laccase activity reached 54,000 U/l. Moreover, some details of laccase overproduction using FW were investigated. The G. lucidum consumes FW by secreting a series of hydrolases and proteases and the improvement of laccase activity is because FW induces over-expression of three isoenzymes by polyacrylamide gel electrophoresis analysis. PMID:25533042

  1. Structural Insights into Sulfite Oxidase Deficiency

    SciTech Connect

    Karakas,E.; Wilson, H.; Graf, T.; Xiang, S.; Jaramillo-Busquets, S.; Rajagopalan, K.; Kisker, C.

    2005-01-01

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

  2. Phenoloxidase-mediated interactions of phenols and anilines with humic materials

    SciTech Connect

    Dec, J.; Bollag, J.M.

    2000-06-01

    Phenoloxidases present in terrestrial systems may contribute to the formation of humus through random coupling of a variety of aromatic compounds, including xenobiotic chemicals. Because of their structural similarity to natural substrates originating mainly from lignin decomposition, xenobiotic phenols and anilines can be readily incorporated into the soil organic matter, a phenomenon referred to as binding. The underlying mechanism of binding involves oxidation of the xenobiotic substrates to free radicals or quinone products that subsequently couple directly to humus or to naturally occurring phenols that also are subject to oxidation. The oxidation can be mediated by soil phenoloxidases as well as by abiotic catalysts. The ability of the enzymes to mediate the oxidation was demonstrated in a number of model studies, in which selected pollutants were incubated with humic monomers or natural humic acids in the presence of different phenoloxidases (laccase, peroxidase, tyrosinase). Analysis of the formed complexes by mass spectrometry and {sup 13}C nuclear magnetic resonance (NMR) spectroscopy left no doubt about the formation of covalent bonds between the pollutants and humic materials. Some bonds were formed at the chlorinated sites, leading to partial dehalogenation of the aromatic contaminants. Experimental data indicated that bound phenols and anilines were unlikely to adversely affect the environment; their release from humic complexes by soil microorganisms was very limited and once released, they were subjected to mineralization. For those reasons, phenoloxidases, which proved capable of mediating the underlying reaction, are currently considered as a tool for enhancing immobilization phenomena in soil.

  3. NADPH Oxidases and Angiotensin II Receptor Signaling

    PubMed Central

    Garrido, Abel Martin; Griendling, Kathy K.

    2010-01-01

    Over the last decade many studies have demonstrated the importance of reactive oxygen species (ROS) production by NADPH oxidases in angiotensin II (Ang II) signaling, as well as a role for ROS in the development of different diseases in which Ang II is a central component. In this review, we summarize the mechanism of activation of NADPH oxidases by Ang II and describe the molecular targets of ROS in Ang II signaling in the vasculature, kidney and brain. We also discuss the effects of genetic manipulation of NADPH oxidase function on the physiology and pathophysiology of the renin angiotensin system. PMID:19059306

  4. Effect of additives on enzyme-catalyzed polymerization of phenols and aromatic amines.

    PubMed

    D'Annibale, Alessandro; Stazi, Silvia Rita; Petruccioli, Maurizio

    2012-01-01

    Among biological approaches to the removal of aromatic amines and phenols from wastewater, the so-called enzyme-catalyzed polymerization and precipitation (ECPP) process relies on the use of oxidoreductases acting via radical mechanisms and characterized by a rather relaxed substrate specificity, such as laccase, tyrosinase and peroxidases. The main technical constraints of ECPP processes are due to a variety of enzyme deactivation phenomena occurring during catalysis and to the incomplete removal of oxidation products from solution. In order to put ECPP into practice, these drawbacks have to be either counteracted or minimized. Although several approaches, such as enzyme immobilization and reaction engineering, have been proposed to limit these constraints, this review is intended to provide a wide survey on some chemical additives with either protective or coagulating effects that have been so far employed for these purposes. PMID:22652869

  5. Transcriptional regulation of laccase and cellulase in relation to fruit body formation in the mycelium of Lentinula edodes on a sawdust-based substrate

    Microsoft Academic Search

    Shoji Ohga; Nam-Seok Cho; Christopher F. Thurston; David A. Wood

    2000-01-01

    Extracellular enzyme activities of laccase and cellulase and their transcriptional regulation were investigated at various\\u000a growth stages in a sawdust-based substrate forLentinula edodes. Changes of laccase and cellulase activities revealed a clear relationship with fruit body development stages. Laccase and\\u000a cellulase activities were regulated at the level of gene transcription. The level of laccase mRNA was maximal at the fully

  6. Laccase/AuAg Hybrid Glucose Microfludic Fuel Cell

    NASA Astrophysics Data System (ADS)

    López-González, B.; Cuevas-Muñiz, F. M.; Guerra-Balcázar, M.; Déctor, A.; Arjona, N.; Ledesma-García, J.; Arriaga, L. G.

    2013-12-01

    In this work a hybrid microfluidic fuel cell was fabricated and evaluated with a AuAg/C bimetallic material for the anode and an enzymatic cathode. The cathodic catalyst was prepared adsorbing laccase and ABTS on Vulcan carbon (Lac-ABTS/C). This material was characterized by FTIR-ATR, the results shows the presence of absorption bands corresponding to the amide bounds. The electrochemical evaluation for the materials consisted in cyclic voltammetry (CV). The glucose electrooxidation reaction in AuAg/C occurs around - 0.3 V vs. NHE. Both electrocatalytic materials were placed in a microfluidic fuel cell. The fuel cell was fed with PBS pH 5 oxygen saturated solution in the cathodic compartment and 5 mM glucose + 0.3 M KOH in the anodic side. Several polarization curves were performed and the maximum power density obtained was 0.3 mWcm-2 .

  7. Activation of polyphenol oxidase of chloroplasts.

    PubMed

    Tolbert, N E

    1973-02-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30 minutes. No single peptide from the digested trypsin was found to be the sole activating factor. About 0.25 mug of trypsin activated 50% the polyphenol oxidase activity in a standard chloroplast assay containing 2.1 mug of chlorophyll. Treatment of spinach chloroplasts with tris buffer or ethylenediamine tetraacetate extracted the ATPase activity, but the polyphenol oxidase activity remained with the broken plastids. However these treatments increased the latent polyphenol oxidase activity 50- to 100-fold.Chloroplasts from a second group of plants, including alfalfa, wheat, oats, peas, and sugarcane leaves, oxidized dihydroxyphenylalanine at a rate of 11 to 120 mumoles x mg(-1) chlorophyll x hr(-1). Polyphenol oxidase in these chloroplasts required a low intensity of red light for activity. Fifty or 75% activation of the oxidase in wheat chloroplasts required 4 to 6 foot candles of light and more light was required for alfalfa chloroplasts. Blue or far red light were ineffective. Trypsin was inhibitory. Upon aging chloroplasts from wheat leaves, but not alfalfa or peas, for 5 to 7 days at 4 C the total polyphenol oxidase activity did not increase, but the activation characteristics changed to those of chloroplasts from the spinach group. Chloroplasts from a third group of plants, including bean, tomato, and corn leaves, slowly oxidized dihydroxyphenylalanine in the dark and exhibited no latency. PMID:16658308

  8. DIVERSITY OF ASCOMYCETE LACCASE SEQUENCES AND CONTRIBUTIONS OF BACTERIA AND ASCOMYCETOUS FUNGI TO LIGNOCELLULOSE DEGRADATION IN A SOUTHEASTERN U.S. SALT MARSH

    Microsoft Academic Search

    JUSTINE ISABELLE; LYONS MORETA

    In the first part of this study, we used molecular tools to create a database of protein sequences for laccase genes of salt marsh ascomycetes. Laccase is one of the enzymes shown to be involved in fungally-mediated lignin degradation. We then used this database to identify laccase sequences in the natural decomposer community on blades of Spartina alterniflora in two

  9. Phenolic glucosides from Hasseltia floribunda

    Microsoft Academic Search

    Enkhmaa Dagvadorj; Kamel H. Shaker; Donald Windsor; Bernd Schneider; Wilhelm Boland

    2010-01-01

    The leaves of Hasseltia floribunda were examined for their chemical constituents. Twelve phenolic glucosides, namely three hydroxycyclohexenyl acyl glucosides, four acylated salicortin derivatives, and five coumaroyl salicin derivatives, were isolated along with eight known phenolic glycosides, six known flavones, and two known sesquiterpenoid cyclohexenone derivatives. The structures of the isolated compounds were elucidated by NMR spectroscopic and HRMS spectrometric methods

  10. MICROWAVE PHOTOCHEMISTRY OF SUBSTITUTED PHENOLS

    E-print Network

    Cirkva, Vladimir

    MICROWAVE PHOTOCHEMISTRY OF SUBSTITUTED PHENOLS V. Církva, J. Kurfürstová, M. Hájek Institute application for organic photochemistry has been shown only recently [5-11]. We disclosed the studies of microwave photochemistry of substituted phenols in an original photoche- mical reactor consisting of EDL

  11. 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 ?g/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 range of laccase to PPCPs not directly recognized by the enzyme, even in a primary effluent matrix. PMID:21237478

  12. Purification and characteristics of a low-molecular-weight peptide possessing oxidative capacity for phenol from Phanerochaete chrysosporium

    Microsoft Academic Search

    Ming Hu; Weican Zhang; Xuemei Lu; Peiji Gao

    2006-01-01

    A new low-molecular-weight peptide with phenol oxidase activity, named Pc factor, was isolated and purified from liquid culture\\u000a of a white-rot basidiomycete Phanerochaete chrysosporium. Its molecular weight was about 600 Da estimated by gel-filtration. Three amino acids Glu, Gly and Val were detected in hydrolysate.\\u000a Absorption peaks corresponding to amino acids and peptide were observed by UV and IR spectra

  13. Adsorption of phenols from wastewater.

    PubMed

    Ahmaruzzaman, M; Sharma, D K

    2005-07-01

    The present work involves an investigation of the possible use of coal, residual coal, and residual coal treated with H3PO4 as a means of removal of phenol from wastewater. The study was realized using batch experiments, with synthetic wastewater having phenol concentration of 1000 ppm. Other low-cost adsorbents such as petroleum coke, coke breeze, rice husk, and rice husk char have also been used. The effect of system variables such as pH, contact time, and temperature has been investigated. The suitability of the Freundlich, Langmuir, and Redlich-Peterson adsorption models to the equilibrium data was investigated for each phenol-adsorbent system. The results showed that the equilibrium data for all the phenol-sorbent systems fitted the Redlich-Peterson model best. Kinetic modeling of removal of phenols was done using the Lagergren first-order rate expression. A series of column experiments were performed to determine the breakthrough curves. PMID:15914144

  14. Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity

    Microsoft Academic Search

    Andrea Mattevi; Marco W Fraaije; Andrea Mozzarelli; Luca Olivi; Alessandro Coda; Willem JH van Berkel

    1997-01-01

    Background: Lignin degradation leads to the formation of a broad spectrum of aromatic molecules that can be used by various fungal micro-organisms as their sole source of carbon. When grown on phenolic compounds, Penicillium simplicissimum induces the strong expression of a flavin-containing vanillyl-alcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines

  15. Targeting NADPH oxidases in vascular pharmacology

    PubMed Central

    Schramm, Agata; Matusik, Pawe?; Osmenda, Grzegorz; Guzik, Tomasz J

    2012-01-01

    Oxidative stress is a molecular dysregulation in reactive oxygen species (ROS) metabolism, which plays a key role in the pathogenesis of atherosclerosis, vascular inflammation and endothelial dysfunction. It is characterized by a loss of nitric oxide (NO) bioavailability. Large clinical trials such as HOPE and HPS have not shown a clinical benefit of antioxidant vitamin C or vitamin E treatment, putting into question the role of oxidative stress in cardiovascular disease. A change in the understanding of the molecular nature of oxidative stress has been driven by the results of these trials. Oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Indeed, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin-angiotensin-aldosterone system inhibit NADPH oxidase activation and expression. Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans) and aliskiren, as well as spironolactone or eplerenone, have been discussed. Molecular aspects of NADPH oxidase regulation must be considered, while thinking about novel pharmacological targeting of this family of enzymes consisting of several homologs Nox1, Nox2, Nox3, Nox4 and Nox5 in humans. In order to properly design trials of antioxidant therapies, we must develop reliable techniques for the assessment of local and systemic oxidative stress. Classical antioxidants could be combined with novel oxidase inhibitors. In this review, we discuss NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin. Therefore, our efforts must focus on generating small molecular weight inhibitors of NADPH oxidases, allowing the selective inhibition of dysfunctional NADPH oxidase homologs. This appears to be the most reasonable approach, potentially much more efficient than non-selective scavenging of all ROS by the administration of antioxidants. PMID:22405985

  16. Transcriptional regulation of laccase and cellulase genes during growth and fruiting of Lentinula edodes on supplemented sawdust

    Microsoft Academic Search

    Shoji Ohga; Daniel J. Royse

    2001-01-01

    Transcription of laccase and cellulase genes of Lentinula edodes was examined during growth and development under different temperature and moisture levels on a sawdust-based substrate. RNA was extracted from samples of mycelium and fruit bodies at various stages of development and gene expression was determined by competitive RT-PCR. The level of laccase transcripts was maximal during the mycelial growth stage,

  17. Characteristics of murine protoporphyrinogen oxidase.

    PubMed Central

    Proulx, K. L.; Dailey, H. A.

    1992-01-01

    Protoporphyrinogen oxidase (EC 1.3.3.4) (PPO) is the penultimate enzyme of the heme biosynthetic pathway. Mouse PPO has been purified in low yield and kinetically characterized by this laboratory previously. A new more rapid purification procedure is described herein, and with this protein we detect a noncovalently bound flavin moiety. This flavin is present at approximately stoichiometric amounts in the purified enzyme and has been identified by its fluorescence spectrum and high performance liquid chromatography as flavin mononucleotide (FMN). Fluorescence quenching studies on the flavin yielded a Stern-Volmer quenching constant of 12.08 M-1 for iodide and 1.1 M-1 for acrylamide. Quenching of enzyme tryptophan fluorescence resulted in quenching constants of 6 M-1 and 10 M-1 for iodide and acrylamide, respectively. Plasma scans performed on purified enzyme preparations did not reveal the presence of stoichiometric amounts of protein-bound metal ions, and we were unable to detect any protein-associated pyrroloquinoline quinone (PQQ). Data from circular dichroism studies predict a secondary structure of the native protein consisting of 30.5% alpha helix, 40.5% beta sheet, 13.7% turn, and 15.3% random coil. Denaturation of PPO with urea resulted in a biphasic curve when ellipticity is plotted against urea concentration, typical of amphipathic proteins. PMID:1304921

  18. Azide inhibition of urate oxidase.

    PubMed

    Gabison, Laure; Colloc'h, Nathalie; Prangé, Thierry

    2014-07-01

    The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX-UA or UOX-8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site. PMID:25005084

  19. The effect of carbohydrate carbon sources on the production of constitutive and inducible laccases by Botryosphaeria sp.

    PubMed

    Alves da Cunha, Mário A; Barbosa, Aneli M; Giese, Ellen C; Dekker, Robert F H

    2003-01-01

    The influence of carbohydrates: glucose, fructose, galactose, galacturonic acid, xylose, lactose, sucrose, pectin and inulin, were evaluated as sole carbon source for the production of laccases by the ascomycete, Botryosphaeria sp. Veratryl alcohol, a laccase inducer, was added to culture media to study inducible laccase production on the same carbon sources. Inulinase and pectinase were also produced when Botryosphaeria sp. was grown on inulin, and galacturonic acid and pectin, respectively, and their levels were less in the presence of veratryl alcohol. Botryosphaeria sp. produced constitutive laccases on all carbon sources examined, and veratryl alcohol increased the laccase production on most of carbon sources studied except for inulin and galacturonic acid. Evidence is presented that Botryosphaeria sp. is also pectinolytic. PMID:12964181

  20. Effect of inducers and process parameters on laccase production by Streptomyces psammoticus and its application in dye decolourization.

    PubMed

    Niladevi, K N; Prema, P

    2008-07-01

    The process parameters influencing the production of extracellular laccases by Streptomyces psammoticus MTCC 7334 were optimized in submerged fermentation. Coffee pulp and yeast extract were the best substrate and nitrogen source respectively for laccase production by this strain. The optimization studies revealed that the laccase yield was maximum at pH 7.5 and temperature 32 degrees C. Salinity of the medium was also observed to be influencing the enzyme production. An agitation rate of 175 rpm and 15% inoculum were the other optimized conditions for maximum laccase yield (5.9 U/mL). Pyrogallol and para-anisidine proved to be the best inducers for laccase production by this strain and the enzyme yield was enhanced by 50% with these inducers. S. psammoticus was able to decolourize various industrial dyes at different rates and 80% decolourization of Remazol Brilliant Blue R (RBBR) was observed after 10 days of incubation in dye based medium. PMID:17765539

  1. Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin.

    PubMed

    Frasconi, Marco; Favero, Gabriele; Boer, Harry; Koivula, Anu; Mazzei, Franco

    2010-04-01

    The electrochemical studies of laccase-mediator systems are aimed at understanding the mechanism of their redox transformation and their efficiency in both homogeneous and heterogeneous reactions; this topic has paramount application spanning from bleaching of paper pulp and the enzymatic degradation of lignin to the biosensors and biofuel cell development. In this paper four different laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL) were characterized from both biochemical and electrochemical points of view. Two of them (TvL and ThL) are high redox potential and two (RvL and r-MaL) are low redox potential laccases. The outline of this work is focused on the determination of catalytic and bioelectrochemical properties of these four enzymes in homogenous solution as well as immobilized onto electrode surface in the presence of a set of different redox mediators. The results measured in the homogenous reaction system correlated well with those measured with the immobilized enzymes. In addition, they are in good agreement with those reported with reference techniques, suggesting that the electrochemical methods employed in this work can be applied well in place of the traditional techniques commonly used for the kinetic characterization of laccases. These results are also discussed in terms of the known amino acid sequences and three-dimensional (3D) structures of the laccases. PMID:20056172

  2. Activity of Laccase Immobilized on TiO2-Montmorillonite Complexes

    PubMed Central

    Wang, Qingqing; Peng, Lin; Li, Guohui; Zhang, Ping; Li, Dawei; Huang, Fenglin; Wei, Qufu

    2013-01-01

    The TiO2-montmorillonite (TiO2-MMT) complex was prepared by blending TiO2 sol and MMT with certain ratio, and its properties as an enzyme immobilization support were investigated. The pristine MMT and TiO2-MMT calcined at 800 °C (TiO2-MMT800) were used for comparison to better understand the immobilization mechanism. The structures of the pristine MMT, TiO2-MMT, and TiO2-MMT800 were examined by HR-TEM, XRD and BET. SEM was employed to study different morphologies before and after laccase immobilization. Activity and kinetic parameters of the immobilized laccase were also determined. It was found that the TiO2 nanoparticles were successfully introduced into the MMT layer structure, and this intercalation enlarged the “d value” of two adjacent MMT layers and increased the surface area, while the calcination process led to a complete collapse of the MMT layers. SEM results showed that the clays were well coated with adsorbed enzymes. The study of laccase activity revealed that the optimum pH and temperature were pH = 3 and 60 °C, respectively. In addition, the storage stability for the immobilized laccase was satisfactory. The kinetic properties indicated that laccase immobilized on TiO2-MMT complexes had a good affinity to the substrate. It has been proved that TiO2-MMT complex is a good candidate for enzyme immobilization. PMID:23771020

  3. Simple fabrication of polymer-based Trametes versicolor laccase for decolorization of malachite green.

    PubMed

    Zhang, Xiaolin; Zhang, Shujuan; Pan, Bingcai; Hua, Ming; Zhao, Xin

    2012-07-01

    A highly efficient and stable biocatalyst (denoted D201_Lac) was fabricated by encapsulating Trametes versicolor laccase within a macroporous and strongly basic exchange resin D201 through a simple adsorption process. Transmission electron micrographs and Fourier transform infrared spectra of the resultant D201_Lac proved that nanosized laccase clusters were embedded into the inner nano-pores/channels of D201. As compared to the free laccase, D201_Lac showed enhanced resistance in the pH range of 3-7 or at temperature of 30-60°C. Besides, negligible laccase was leached out from the host polymer D201 in solution of pH 3-7 and NaCl concentration up to 0.5M, which might be attributed to the electrostatic attraction and the possible twining between long-chain laccase and the cross-linking host resin. Continuous seven-cycle batch decoloration of malachite green demonstrates that decoloration efficiency of D201_Lac kept constant for more than 320-h operation. PMID:22169216

  4. 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

  5. Contact allergy to 3-methylol phenol, 2,4-dimethylol phenol and 2,6-dimethylol phenol.

    PubMed

    Bruze, M; Zimerson, E

    1985-01-01

    Thirteen patients with contact allergy to phenol-formaldehyde resins (P-F-R) were patch tested with 3-methylol phenol, 2,4-dimethylol phenol and 2,6-dimethylol phenol. Nine patients reacted to at least 1 compound, all giving positive test responses to 2,4-dimethylol phenol. Seven patients reacted simultaneously to 2,6-dimethylol phenol while only 1 patient reacted to 3-methylol phenol. Negative test responses were noted in 20 controls. Chemical investigation by high pressure liquid chromatography indicated that the compounds tested were pure and separable. The 3 reported sensitizers may, theoretically, be generated during the manufacture of P-F-R. 2,4-Dimethylol phenol and 2,6-dimethylol phenol have been demonstrated and there has been chromatographic evidence of 3-methylol phenol in the P-F-R used in the routine test series at the department. PMID:2420124

  6. One-copper laccase-related enzyme from Marasmius sp.: purification, characterization and bleaching of textile dyes.

    PubMed

    Schückel, Julia; Matura, Anke; van Pée, Karl-Heinz

    2011-03-01

    In the culture filtrate of a Marasmius sp. strain isolated in Indonesia during a screening for fungi with the ability to decolorize textile dyes, two laccase-related enzymes (laccase-related enzyme I and II) were detected. Laccase-related enzyme I was purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The native enzyme was shown to have a molecular mass of 53 kDa, an N-terminal amino acid sequence characteristically seen in laccases and an isoelectric point of pH 3.8. The enzyme accepts typical laccase substrates including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), syringaldazine and guaiacol, but has no tyrosinase activity. The pH optimum is at pH 3.0 for ABTS and at 6.0 for syringaldazine and the enzyme is stable up to pH 10. The UV/vis spectrum of the laccase-related enzyme is non-typical for laccases and metal content analysis revealed that the enzyme contains only a single copper atom per enzyme molecule. This suggests that this enzyme could be related to the group of the so-called "white" laccases, however, no zinc or any other metal ion could be detected in this enzyme, suggesting that the enzyme is a unique laccase-related enzyme. Comparison of the bleaching activity of the whole fungus with that of the isolated laccase-related enzyme showed that this enzyme is the major bleaching enzyme produced by this Marasmius sp. strain and was able to bleach violet, red, orange and yellow dyes in addition to a number of blue dyes. PMID:22112912

  7. 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 ?-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. PMID:25563944

  8. Factors affecting the oxidative activity of laccase towards biphenyl derivatives in homogeneous aqueous-organic systems.

    PubMed

    Tominaga, Jo; Michizoe, Junji; Kamiya, Noriho; Ichinose, Hirofumi; Maruyama, Tatsuo; Goto, Masahiro

    2004-01-01

    Catalytic oxidation of biphenyl derivatives was investigated using laccase in a homogeneous aqueous-organic system. A thermostable laccase from Trametes sp. showed the highest catalytic activity for the oxidation of 4-hydroxybiphenyl (4-HB) at a reaction temperature of 60 degrees C when dimethylsulfoxide (DMSO) was employed as a co-solvent. Furthermore, the catalytic performance was successfully enhanced by the incorporation of a laccase mediator system (LMS) into the aqueous-DMSO media. The catalytic performance strongly depended on the type of mediator, and the highest activity was observed with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as mediator, suggesting the importance of the selection of a suitable mediator. It was verified that this mediator system is applicable to the oxidation of several biphenyl derivatives with hydroxyl groups. PMID:16233660

  9. Overproduction of Laccase by the White-Rot Fungus Pleurotus ostreatus Using Apple Pomace as Inducer

    PubMed Central

    Park, Young-Jin; Yoon, Dae-Eun; Kim, Hong-Il; Kwon, O-Chul; Yoo, Young-Bok; Kong, Won-Sik

    2014-01-01

    Laccase activity of Pleurotus ostreatus is significantly increased by the addition of apple pomace. Among various conditions, the best concentration of apple pomace and cultivation time for the production of laccase by P. ostreatus was 2.5% and 9 days, respectively. Reverse transcription polymerase chain reaction analyses of laccase isoenzyme genes, including pox1, pox3, pox4, poxc, poxa3, and poxa1b, revealed a clear effect of apple pomace on transcription induction. Our findings reveal that the use of apple pomace can be a model for the valuable addition of similar wastes and for the development of a solid-state fermenter and commercial production of oyster mushroom P. ostreatus. PMID:25071391

  10. Electroactive nanobiomolecular architectures of laccase and cytochrome c on electrodes: applying silica nanoparticles as artificial matrix.

    PubMed

    Feifel, Sven Christian; Kapp, Andreas; Lisdat, Fred

    2014-05-20

    Fully electroactive multilayer architectures combining the redox protein cytochrome c and the enzyme laccase by the use of silica nanoparticles as artificial matrix have been constructed on gold electrodes capable of direct dioxygen reduction. Laccase form Trametes versicolor and cytochrome c from horse heart were electrostatically coimmobilized by alternate deposition with interlayers of silica nanoparticles in a multilayer fashion. The layer formation has been monitored by quartz crystal microbalance. The electrochemical properties and performance of the nanobiomolecular entities were investigated by cyclic voltammetry, indicating, that a multistep electron transfer cascade, from the electrode via cytochrome c in the layered system toward the enzyme laccase, and here to molecular dioxygen was achieved. The response of the novel architecture is based on direct electron exchange between immobilized proteins and can be tuned by the assembly process. PMID:24804981

  11. Overproduction of Laccase by the White-Rot Fungus Pleurotus ostreatus Using Apple Pomace as Inducer.

    PubMed

    Park, Young-Jin; Yoon, Dae-Eun; Kim, Hong-Il; Kwon, O-Chul; Yoo, Young-Bok; Kong, Won-Sik; Lee, Chang-Soo

    2014-06-01

    Laccase activity of Pleurotus ostreatus is significantly increased by the addition of apple pomace. Among various conditions, the best concentration of apple pomace and cultivation time for the production of laccase by P. ostreatus was 2.5% and 9 days, respectively. Reverse transcription polymerase chain reaction analyses of laccase isoenzyme genes, including pox1, pox3, pox4, poxc, poxa3, and poxa1b, revealed a clear effect of apple pomace on transcription induction. Our findings reveal that the use of apple pomace can be a model for the valuable addition of similar wastes and for the development of a solid-state fermenter and commercial production of oyster mushroom P. ostreatus. PMID:25071391

  12. PEI-coated gold nanoparticles decorated with laccase: a new platform for direct electrochemistry of enzymes and biosensing applications.

    PubMed

    Brondani, Daniela; de Souza, Bernardo; S Souza, Bruno; Neves, Ademir; C Vieira, Iolanda

    2013-04-15

    This paper describes the synthesis and characterization of PEI-coated gold nanoparticles (PEI-AuNP), which were applied as a new platform in the immobilization of laccase (LAC) originating from Aspergillus oryzae. This material (PEI-AuNP-LAC) was used in the construction of a biosensor based on a glassy carbon electrode coated with a bio-nanostructured film. The occurrence of direct electron transfer (DET) between the electroactive center of LAC and the electrode surface was observed by cyclic voltammetry (CV), suggesting that the presence of AuNP in the film acts as a bridge for electron transfer. In acetate buffer solution (pH 5.0), LAC shows a pair of well-defined redox waves with a formal potential (E?') of 0.226V vs. Ag/AgCl (3M KCl). The biosensor response indicated a surface-controlled process with an apparent electron transfer rate constant (k(s)) of 0.4 s?¹, charge transfer coefficient (?) of 0.5, and surface coverage concentration (?) of 3.45×10?¹? mol cm?². The optimized biosensor showed the following limits of detection (LOD) for the phenolic compounds tested: 0.03 ?M for catechol and guaiacol; 0.14 ?M for pyrogallol and 0.21 ?M for hydroquinone, using square-wave voltammetry (SWV). The proposed biosensor demonstrated high sensitivity, good repeatability and reproducibility, and long-term stability (only 20% decrease in response over 90 days and after 150 measurements by SWV for each film formed). This biosensor was successfully applied to catechol quantification in spiked water samples. Furthermore, this method showed great potential for application in the development of new devices for biosensing. PMID:23208093

  13. Phenolic glucosides from Hasseltia floribunda.

    PubMed

    Dagvadorj, Enkhmaa; Shaker, Kamel H; Windsor, Donald; Schneider, Bernd; Boland, Wilhelm

    2010-11-01

    The leaves of Hasseltia floribunda were examined for their chemical constituents. Twelve phenolic glucosides, namely three hydroxycyclohexenyl acyl glucosides, four acylated salicortin derivatives, and five coumaroyl salicin derivatives, were isolated along with eight known phenolic glycosides, six known flavones, and two known sesquiterpenoid cyclohexenone derivatives. The structures of the isolated compounds were elucidated by NMR spectroscopic and HRMS spectrometric methods and by comparing analytical data with those of related structures. PMID:20822782

  14. Differential Gene Expression in the Laccase Gene Family from Basidiomycete I-62 (CECT 20197)

    PubMed Central

    Mansur, Mariana; Suárez, Teresa; González, Aldo E.

    1998-01-01

    A family of genes encoding laccases has recently been described for the basidiomycete I-62 (CECT 20197). Transcript levels of genes lcc1, lcc2, and lcc3 were analyzed under four different culture conditions to study their expression patterns. Two of the laccase genes were clearly inducible by veratryl alcohol: the lcc1 gene is inducible in early stages of growth, and the lcc2 gene is also inducible but only when the organism reaches the stationary phase. Transcript levels for the third gene, lcc3, were uninduced by veratryl alcohol and repressed by glucose. PMID:16349507

  15. Novel thermotolerant laccases produced by the white-rot fungus Physisporinus rivulosus

    Microsoft Academic Search

    Kristiina Hildén; Terhi K. Hakala; Pekka Maijala; Taina K. Lundell; Annele Hatakka

    2007-01-01

    The white-rot basidiomycete Physisporinus rivulosus strain T241i is highly selective for degradation of softwood lignin, which makes this fungus suitable for biopulping. In\\u000a order to promote laccase production, P. rivulosus was cultivated in nutrient-nitrogen sufficient liquid media containing either charcoal or spruce sawdust as supplements.\\u000a Two laccases with distinct pI values, Lac-3.5 and Lac-4.8, were purified from peptone-spruce sawdust-charcoal cultures

  16. Monoclonal antibodies to the alternative oxidase of higher plant mitochondria

    Microsoft Academic Search

    T. E. Elthon; R. L. Nickels; L. McIntosh

    1989-01-01

    The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M{sub r} of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been

  17. Production of Cellobionate from Cellulose Using an Engineered Neurospora crassa Strain with Laccase and Redox Mediator Addition

    PubMed Central

    Hildebrand, Amanda; Kasuga, Takao; Fan, Zhiliang

    2015-01-01

    We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of Neurospora crassa (F5?ace-1?cre-1?ndvB) was shown to produce cellobionate directly from cellulose without the addition of exogenous cellulases. Specifically, N. crassa produces cellulases, which hydrolyze cellulose to cellobiose, and cellobiose dehydrogenase (CDH), which oxidizes cellobiose to cellobionate. However, the conversion of cellobiose to cellobionate is limited by the slow re-oxidation of CDH by molecular oxygen. By adding low concentrations of laccase and a redox mediator to the fermentation, CDH can be efficiently oxidized by the redox mediator, with in-situ re-oxidation of the redox mediator by laccase. The conversion of cellulose to cellobionate was optimized by evaluating pH, buffer, and laccase and redox mediator addition time on the yield of cellobionate. Mass and material balances were performed, and the use of the native N. crassa laccase in such a conversion system was evaluated against the exogenous Pleurotus ostreatus laccase. This paper describes a working concept of cellobionate production from cellulose using the CDH-ATBS-laccase system in a fermentation system. PMID:25849253

  18. Removal of the insect repellent N,N-diethyl-m-toluamide (DEET) by laccase-mediated systems.

    PubMed

    Tran, Ngoc Han; Hu, Jiangyong; Urase, Taro

    2013-11-01

    Numerous efforts have been made to remove emerging trace organic contaminants, such as pharmaceuticals and personal care products (PPCPs). This study examined the removal of N,N-diethyl-m-toluamide (DEET) by Trametes versicolor laccase and its laccase-mediator systems. Experimental results showed that DEET was poorly removed by laccase alone. The poor removal efficiency of DEET by laccase may be attributed to the presence of strong withdrawing electron group (-CO-N [CH2-CH3]2) in the chemical structure of DEET. Experimental results also indicated that DEET might be indirectly oxidized by laccase-mediator systems. More than 50% initial DEET amount was removed by laccase in the presence of a redox mediator, such as 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS) or 1-hydroxybenzotriazole (HBT). However, laccase activity was considerably decreased in the presence of a redox mediator (ABTS or HBT). Further studies on identification of degradation byproducts and degradation pathways are recommended. PMID:24034986

  19. Improving the performance of a biofuel cell cathode with laccase-containing culture supernatant from Pycnoporus sanguineus.

    PubMed

    Fokina, Oleksandra; Eipper, Jens; Winandy, Lex; Kerzenmacher, Sven; Fischer, Reinhard

    2014-11-01

    Laccases are multicopper oxidoreductases that can be used in biofuel cells to improve cathode performance by cathodic oxygen reduction. Here we present a laccase from the ligninolytic white-rot fungus Pycnoporus sanguineus that, in contrast to the Trametes versicolor laccase, can be produced in the absence of inducers in a standard culture medium. After 7days of cultivation the activity of this laccase in culture supernatant reached 2.5U/ml, which is high enough for direct application of the supernatant in biofuel cells. The highest current density of 115.0±3.5?A/cm(2) at 400mV vs. SCE was obtained at pH 5 with a buckypaper cathode with a laccase-containing culture supernatant. The enzyme also showed electrocatalytic activity at pH 6 and 7. These results not only present a new cost-efficient laccase for improving cathode performance, but also show that new laccases with different catalytic properties can be suitable for biofuel cells. PMID:25459854

  20. 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??M) 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

  1. Laccase-polyacrylonitrile nanofibrous membrane: highly immobilized, stable, reusable, and efficacious for 2,4,6-trichlorophenol removal.

    PubMed

    Xu, Ran; Chi, Chenglong; Li, Fengting; Zhang, Bingru

    2013-12-11

    Increasing attention has been given to nanobiocatalysis for commercial applications. In this study, laccase was immobilized on polyacrylonitrile (PAN) nanofibrous membranes through ethanol/HCl method of amidination reaction and successfully applied for removal of 2,4,6-trichlorophenol (TCP) from water. PAN membranes with fiber diameters from 200 nm to 300 nm were fabricated via electrospinning and provided a large surface area for enzyme immobilization and catalytic reactions. Images of scanning electron microscope demonstrated the enzyme molecules were aggregated on the nanofiber surface. The immobilized laccase exhibited 72% of the free enzyme activity and kept 60% of its initial activity after 10 operation cycles. Moreover, the storage stability of the immobilized laccase was considered excellent because they maintained more than 92% of the initial activity after 18 days of storage, whereas the free laccase retained only 20%. The laccase-PAN nanofibrous membranes exhibited high removal efficiency of TCP under the combined actions of biodegradation and adsorption. More than 85% of the TCP was removed under optimum conditions. Effects of various factors on TCP removal efficiency of the immobilized laccase were analyzed. Results suggest that laccase-PAN nanofibrous membranes can be used in removing TCP from aqueous sources and have potential for use in other commercial applications. PMID:24245853

  2. Efficient secretory production of CotA-laccase and its application in the decolorization and detoxification of industrial textile wastewater.

    PubMed

    Guan, Zheng-Bing; Shui, Yan; Song, Chen-Meng; Zhang, Ning; Cai, Yu-Jie; Liao, Xiang-Ru

    2015-06-01

    Fungal laccases are typically unstable at high pH and temperature conditions, which limit their application in the decolorization of textile wastewater. By contrast, the highly stable bacterial laccases can function within a wider pH range and at high temperatures, thus have significant potential in treatment for textile wastewater. In our previous work, a thermo-alkali-stable CotA-laccase gene was cloned from Bacillus pumilus W3 and overexpressed in Escherichia coli. In this study, the robust CotA-laccase achieved efficient secretory expression in Bacillus subtilis WB600 by screening a suitable signal peptide. A maximum CotA-laccase yield of 373.1 U/mL was obtained at optimum culture conditions in a 3-L fermentor. Furthermore, the decolorization and detoxification of textile industry effluent by the purified recombinant CotA-laccase in the presence and absence of redox mediators were investigated. Among the potential mediators that enhanced effluent decolorization, acetosyringone (ACS) was the most effective. The toxicity of the CotA-laccase-ACS-treated effluent was greatly reduced compared with that of the crude effluent. To the best of our knowledge, this study is the first to report on the heterologous expression of CotA-laccase in B. subtilis. The recombinant strain B. subtilis WB600-5 has a great potential in the industrial production of this bacterial enzyme, and the CotA-laccase-ACS system is a promising candidate for the biological treatment of industrial textile effluents. PMID:25847445

  3. 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

  4. Effect of anti-inflammatory drugs on xanthine oxidase and xanthine oxidase induced depolymerization of hyaluronic acid

    Microsoft Academic Search

    G. Carlin; R. Djursäter; G. Smedegård; B. Gerdin

    1985-01-01

    The inhibitory effect of various anti-inflammatory drugs on the xanthine oxidase derived depolymerization of hyaluronic acid was studied. The depolymerization was assayed by repeated viscosity measurements. By using a low xanthine oxidase activity, the decrease in viscosity with time followed first order reaction kinetics and was therefore suitable for kinetic analysis. The xanthine oxidase activity was monitored by assay of

  5. Flavin Amine Oxidases from the Monoamine Oxidase Structural Family Utilize a Hydride Transfer Mechanism 

    E-print Network

    Henderson Pozzi, Michelle

    2011-08-08

    The amine oxidase family of enzymes has been the center of numerous mechanistic studies because of the medical relevance of the reactions they catalyze. This study describes transient and steady-state kinetic analyses of ...

  6. A theoretical study of the dioxygen activation by glucose oxidase and copper amine oxidase

    Microsoft Academic Search

    Rajeev Prabhakar; Per E. M. Siegbahn; Boris F. Minaev

    2003-01-01

    Glucose oxidase (GO) and copper amine oxidase (CAO) catalyze the reduction of molecular oxygen to hydrogen peroxide. If a closed-shell cofactor (like FADH2 in GO and topaquinone (TPQ) in CAO) is electron donor in dioxygen reduction, the formation of a closed-shell species (H2O2) is a spin forbidden process. Both in GO and CAO, formation of a superoxide ion that leads

  7. Monoamine Oxidase Expression During Development and Aging

    Microsoft Academic Search

    Antonietta Nicotra; Federica Pierucci; Hasan Parvez; Ornella Senatori

    2004-01-01

    Monoamine oxidase (MAO) isoenzymes play a major role in regulating the concentration of several bioactive amines, including serotonin and catecholamines. Both in the nervous system and in peripheral organs, MAOs can potentially modulate all the processes involving these bioactive amines. In the present article, we review some of the most significant articles published so far on changes in MAOs during

  8. Myeloperoxidase-oxidase oxidation of cysteamine.

    PubMed Central

    Svensson, B E; Lindvall, S

    1988-01-01

    Cysteamine oxidation was shown to be catalysed by nanomolar concentrations of myeloperoxidase in a peroxidase-oxidase reaction, i.e. an O2-consuming oxidation of a compound catalysed by peroxidase without H2O2 addition. When auto-oxidation of the thiol was prevented by the metal-ion chelator diethylenetriaminepenta-acetic acid, native, but not heat-inactivated, myeloperoxidase induced changes in the u.v.-light-absorption spectrum of cysteamine. These changes were consistent with disulphide (cystamine) formation. Concomitantly, O2 was consumed and superoxide radical anion formation could be detected by Nitro Blue Tetrazolium reduction. Both superoxide dismutase and catalase inhibited the reaction, whereas the hydroxyl-radical scavengers mannitol and ethanol did not. O2 consumption increased with increasing pH (between pH 6.0 and 8.0), and 50% inhibition was exhibited by about 3 mM-NaCl at pH 7.0 and by about 100 mM-NaCl at pH 8.0. Cysteamine was about 5 times as active (in terms of increased O2 consumption at pH 7.5) as the previously reported peroxidase-oxidase substrates NADPH, dihydroxyfumaric acid and indol-3-ylacetic acid. A possible reaction pathway for the myeloperoxidase-oxidase oxidation of cysteamine is discussed. These results indicate that cysteamine is a very useful substrate for studies on myeloperoxidase-oxidase activity. PMID:2829860

  9. SAR of Sponge-Inspired Hemibastadin Congeners Inhibiting Blue Mussel PhenolOxidase.

    PubMed

    Niemann, Hendrik; Hagenow, Jens; Chung, Mi-Young; Hellio, Claire; Weber, Horst; Proksch, Peter

    2015-05-01

    Hemibastadin derivatives, including the synthetically-derived 5,5'-dibromohemibastadin-1 (DBHB), are potent inhibitors of blue mussel phenoloxidase (PO), which is a key enzyme involved in the firm attachment of this invertebrate to substrates and, thus, a promising molecular target for anti-fouling research. For a systematic investigation of the enzyme inhibitory activity of hemibastadin derivatives, we have synthesized nine new congeners, which feature structural variations of the DBHB core structure. These structural modifications include, e.g., different halogen substituents present at the aromatic rings, different amine moieties linked to the (E)-2-(hydroxyimino)-3-(4-hydroxyphenyl)propionic acid, the presence of free vs. substituted aromatic hydroxyl groups and a free vs. methylated oxime group. All compounds were tested for their inhibitory activity towards the target enzyme in vitro, and IC50 values were calculated. Derivatives, which structurally closely resemble sponge-derived hemibastadins, revealed superior enzyme inhibitory properties vs. congeners featuring structural moieties that are absent in the respective natural products. This study suggests that natural selection has yielded structurally-optimized antifouling compounds. PMID:25988522

  10. Detection of xanthine oxidase in human plasma.

    PubMed

    Newaz, M A; Adeeb, N N

    1998-03-01

    Xanthine oxidase is a highly versatile enzyme which is widely distributed among various species. Though the presence of the enzyme in serum is not yet established, high antibody titre of this enzyme has been reported. Xanthine oxidase is thought to be the principal source of free radical generation via degradation of nucleotides to the end product, uric acid. The aim of this study was to detect xanthine oxidase activity in human plasma and report any significant relationships found between its activity and variables such as race, age and sex for the sample size studied. Forty six normal healthy individuals (14 males and 32 females) were studied. The enzyme activity was measured by a spectrophotometric method whereby the reduction of ferricytochrome c by free radicals was calculated and expressed as nmol O2 production/ml/min. Results obtained showed that there was a positive relationship between xanthine oxidase activity with age (r = 0.415, p < 0.05) and weight (r = 0.369, p < 0.05) in the normal individual. For the age group 30-39 yrs (n = 11), a higher enzyme activity was observed in males (2.71 +/- 1.44) as compared to females (2.34 +/- 1.23) but it was not significant (p = 0.53). For racial distribution, the Malays [M] have a higher enzyme activity (2.65 +/- 0.86, N = 32) than their Indian [I] (2.27 +/- 0.58; N = 7) and Chinese counterparts [C] (1.44 +/- 1.22; N = 7) but this was also not statistically significant (M vs I: p = 0.39; M vs C: p = 0.07; I vs C: p = 0.16). In conclusion this study showed that there is a measurable amount of xanthine oxidase activity in the human plasma. PMID:10968141

  11. Cloning and characterization of three laccase genes from the white-rot basidiomycete Trametes villosa: genomic organization of the laccase gene family.

    PubMed

    Yaver, D S; Golightly, E J

    1996-11-28

    Three laccase genes were isolated from the white-rot basidiomycete Trametes villosa (Tv). The predicted protein products have 63-71% identity to the previously cloned Tv laccase genes lcc1 and lcc2. The genes lcc3, lcc4 and lcc5 contain 12, 10 and 11 introns, respectively. The position of several of the introns is conserved among all 5 genes. The 5 genes appear to be differentially regulated, and message has only been detected for lcc1 and lcc2. The karyotype of Tv was determined by CHEF, and 8 bands ranging in size from approximately 5.7 to 2.2 Mb were resolved of which 2 appear to be doublets. The 5 laccase genes have been mapped to specific bands resolved by CHEF. The lcc1 and lcc2 genes hybridize to a band of approximately 5.7 Mb. The lcc4 and lcc5 genes are on a chromosome of approximately 3.7 Mb, and lcc3 is on a chromosome of approximately 2.8 Mb. PMID:8973314

  12. Laccases Direct Lignification in the Discrete Secondary Cell Wall Domains of Protoxylem1[W][OPEN

    PubMed Central

    Schuetz, Mathias; Benske, Anika; Smith, Rebecca A.; Watanabe, Yoichiro; Tobimatsu, Yuki; Ralph, John; Demura, Taku; Ellis, Brian; Samuels, A. Lacey

    2014-01-01

    Plants precisely control lignin deposition in spiral or annular secondary cell wall domains during protoxylem tracheary element (TE) development. Because protoxylem TEs function to transport water within rapidly elongating tissues, it is important that lignin deposition is restricted to the secondary cell walls in order to preserve the plasticity of adjacent primary wall domains. The Arabidopsis (Arabidopsis thaliana) inducible VASCULAR NAC DOMAIN7 (VND7) protoxylem TE differentiation system permits the use of mutant backgrounds, fluorescent protein tagging, and high-resolution live-cell imaging of xylem cells during secondary cell wall development. Enzymes synthesizing monolignols, as well as putative monolignol transporters, showed a uniform distribution during protoxylem TE differentiation. By contrast, the oxidative enzymes LACCASE4 (LAC4) and LAC17 were spatially localized to secondary cell walls throughout protoxylem TE differentiation. These data support the hypothesis that precise delivery of oxidative enzymes determines the pattern of cell wall lignification. This view was supported by lac4lac17 mutant analysis demonstrating that laccases are necessary for protoxylem TE lignification. Overexpression studies showed that laccases are sufficient to catalyze ectopic lignin polymerization in primary cell walls when exogenous monolignols are supplied. Our data support a model of protoxylem TE lignification in which monolignols are highly mobile once exported to the cell wall, and in which precise targeting of laccases to secondary cell wall domains directs lignin deposition. PMID:25157028

  13. New uses of food waste: application to laccase production by Trametes hirsuta

    Microsoft Academic Search

    E. Rosales; S. Rodríguez Couto; A. Sanromán

    2002-01-01

    Diverse food wastes, apple, orange and potato, were screened for laccase production, under solid-state fermentation conditions, by the white-rot fungus Trametes hirsuta. Potato peelings gave the highest activity, reaching about 5000 U l-1 within 8 days. These values are higher than those reported to date.

  14. Laccase-catalyzed bisphenol A oxidation in the presence of 10-propyl sulfonic acid phenoxazine.

    PubMed

    Ivanec-Goranina, R?ta; Kulys, Juozas; Bachmatova, Irina; Marcinkevi?ien?, Liucija; Meškys, Rolandas

    2015-04-01

    The kinetics of the Coriolopsis byrsina laccase-catalyzed bisphenol A (BisA) oxidation was investigated in the absence and presence of electron-transfer mediator 3-phenoxazin-10-yl-propane-1-sulfonic acid (PPSA) at pH5.5 and 25°C. It was shown that oxidation rate of the hardly degrading compound BisA increased in the presence of the highly reactive substrate PPSA. The increase of reaction rate depends on PPSA and BisA concentrations as well on their ratio, e.g., at 0.2mmol/L of BisA and 2?mol/L of PPSA the rate increased 2 times. The kinetic data were analyzed using a scheme of synergistic laccase-catalyzed BisA oxidation. The calculated constant, characterizing reactivity of PPSA with laccase, is almost 1000 times higher than the constant, characterizing reactivity of BisA with laccase. This means that mediator-assisted BisA oxidation rate can be 1000 times higher in comparison to non-mediator reaction if compounds concentration is equal but very low. PMID:25872719

  15. 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. PMID:18506632

  16. Oxidation of Anthracene and Benzo[a]pyrene by Laccases from Trametes versicolor

    PubMed Central

    Collins, P. J.; Kotterman, M.; Field, J. A.; Dobson, A.

    1996-01-01

    The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of <=7.45 eV, is catalyzed by laccases from Trametes versicolor. Crude laccase preparations were able to oxidize both anthracene and the potent carcinogen benzo[a]pyrene. Oxidation of benzo[a]pyrene was enhanced by the addition of the cooxidant 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), while an increased anthracene oxidizing ability was observed in the presence of the low-molecular-weight culture fluid ultrafiltrate. Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. Oxidation of anthracene by the purified isozymes was enhanced in the presence of ABTS, while ABTS was essential for the oxidation of benzo[a]pyrene. In all cases anthraquinone was identified as the major end product of anthracene oxidation. These findings indicate that laccases may have a role in the oxidation of polycyclic aromatic hydrocarbons by white rot fungi. PMID:16535468

  17. Enzyme-Catalyzed Oxidation of 17?-Estradiol Using Immobilized Laccase from Trametes versicolor

    PubMed Central

    Cardinal-Watkins, Chantale; Nicell, Jim A.

    2011-01-01

    Many natural and synthetic estrogens are amenable to oxidation through the catalytic action of oxidative enzymes such as the fungal laccase Trametes versicolor. This study focused on characterizing the conversion of estradiol (E2) using laccase that had been immobilized by covalent bonding onto silica beads contained in a bench-scale continuous-flow packed bed reactor. Conversion of E2 accomplished in the reactor declined when the temperature of the system was changed from room temperature to just above freezing at pH 5 as a result of a reduced rate of reaction rather than inactivation of the enzyme. Similarly, conversion increased when the system was brought to warmer temperatures. E2 conversion increased when the pH of the influent to the immobilized laccase reactor was changed from pH 7 to pH 5, but longer-term experiments showed that the enzyme is more stable at pH 7. Results also showed that the immobilized laccase maintained its activity when treating a constant supply of aqueous E2 at a low mean residence time over a 12-hour period and when treating a constant supply of aqueous E2 at a high mean residence time over a period of 9 days. PMID:21869925

  18. Application parameters of laccase-mediator systems for treatment of sulfonamide antibiotics.

    PubMed

    Weng, Shin-Sian; Liu, Shiu-Mei; Lai, Hong-Thih

    2013-08-01

    This is the first study to examine the application parameters for oxidation of two sulfonamide antibiotics (SAs), sulfadimethoxine (SDM) and sulfamonomethoxine (SMM), using a novel laccase and mediators. The optimal conditions in the laccase-mediator system (LMS) were pH 4, 50-60 °C, and 1 mM for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); pH 4, 40-60 °C, and 1 mM for violuric acid (VLA); pH 6, 50 °C, and 2 mM for syringaldehyde (SIR). Additionally, the conditions of the 4-hydroxybenzyl alcohol (HBA) mediator were pH 4, 30 °C, and 2 mM in the oxidation of SDM; and the temperature increased to 60 °C for SMM. The laccase coupled with VLA and HBA resulted in the lowest toxicity of the SA solutions during processing, whereas treatments with ABTS and SIR resulted in higher toxicities. Furthermore, the laccase used in this study was stable and resistant to dialysis, thus can be reused for oxidation process. PMID:23561949

  19. Crystallization and preliminary structure analysis of the blue laccase from the ligninolytic fungus Panus tigrinus

    PubMed Central

    Ferraroni, Marta; Duchi, Ilaria; Myasoedova, Nina M.; Leontievsky, Alexey A.; Golovleva, Ludmila A.; Scozzafava, Andrea; Briganti, Fabrizio

    2005-01-01

    The blue laccase from the white-rot basidiomycete fungus Panus tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. P. tigrinus laccase crystals grew within one week at 296?K using the sitting-drop vapour-diffusion method in 22%(w/v) PEG 4000, 0.2?M CaCl2, 100?mM Tris–HCl pH 7.5. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 54.2, b = 111.6, c = 97.1, ? = 97.7°, and contain 46% solvent. A complete native data set was collected to 1.4?Å resolution at the copper edge. Molecular replacement using the Coprinus cinereus laccase structure (PDB code ) as a starting model was performed and initial electron-density maps revealed the presence of a full complement of copper ions. Model refinement is in progress. The P. tigrinus laccase structural model exhibits the highest resolution available to date and will assist in further elucidation of the catalytic mechanism and electron-transfer processes for this class of enzymes. PMID:16510995

  20. 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

  1. Cloning and Expression of Laccase from Trametes versicolor in Saccharomyces cerevisiae using a Novel Vector System

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The long-term goal of this research is to increase efficiency and decrease cost of ethanol fermentation of lignocellulosic feedstocks by combining pre-treatment using laccase enzyme and subsequent fermentation to ethanol through simultaneous saccharification and fermentation paradigms. The first st...

  2. Cloning and characterization of a second laccase gene from the lignin-degrading basidiomycete Pycnoporus cinnabarinus.

    PubMed

    Temp, U; Zierold, U; Eggert, C

    1999-08-01

    The gene lcc3-2 encoding a second laccase of the white-rot fungus Pycnoporus cinnabarinus has been cloned, sequenced, and characterized. The isolated gene consists of 2840bp, with the coding region interrupted by ten introns and flanked by an upstream region in which putative CAAT and TATA boxes were identified. The cDNA of lcc3-2 contains an open reading frame of 1563bp. The deduced mature laccase protein consisted of 498 amino acids and was preceded by a signal peptide of 23 amino acids. The sequence of lcc3-2 reveals 73% similarity on the protein level to the previously characterized lcc3-1. The new laccase gene shares highest similarity to lcc1 from Trametes villosa (75%), and lcc2 from the unidentified basidiomycete CECT 20197 (75%). The calculated isoelectric point (pI) of 6.1 for the gene product LCC3-2 was in good agreement with the experimentally determined pI of a laccase secreted by P. cinnabarinus grown on cellulose. Transcription analysis using competitive reverse transcription (RT)-PCR showed that lcc3-2 was expressed in glucose and cellulose containing cultures. However, in contrast to lcc3-1, lcc3-2 transcription was not increased in response to 2,5-xylidine. PMID:10433978

  3. Molecular Analysis of a Laccase Gene from the White Rot Fungus Pycnoporus cinnabarinus

    PubMed Central

    Eggert, Claudia; LaFayette, Peter R.; Temp, Ulrike; Eriksson, Karl-Erik L.; Dean, Jeffrey F. D.

    1998-01-01

    It was recently shown that the white rot basidiomycete Pycnoporus cinnabarinus secretes an unusual set of phenoloxidases when it is grown under conditions that stimulate ligninolysis (C. Eggert, U. Temp, and K.-E. L. Eriksson, Appl. Environ. Microbiol. 62:1151–1158, 1996). In this report we describe the results of a cloning and structural analysis of the laccase-encoding gene (lcc3-1) expressed by P. cinnabarinus during growth under xylidine-induced conditions. The coding region of the genomic laccase sequence, which is preceded by the eukaryotic promoter elements TATA and CAATA, spans more than 2,390 bp. The corresponding laccase cDNA was identical to the genomic sequence except for 10 introns that were 50 to 60 bp long. A sequence analysis indicated that the P. cinnabarinus lcc3-1 product has a Phe residue at a position likely to influence the reduction-oxidation potential of the enzyme’s type 1 copper center. The P. cinnabarinus lcc3-1 sequence was most similar to the sequence encoding a laccase from Coriolus hirsutus (level of similarity, 84%). PMID:9572949

  4. Total monoamine oxidase (MAO) inhibition by chestnut honey, pollen and propolis.

    PubMed

    Yildiz, O; Karahalil, F; Can, Z; Sahin, H; Kolayli, S

    2014-10-01

    Monoamine oxidase (MAO) inhibitors are generally used in the treatment of depressive disorders and some neurodegenerative illnesses, such as Parkinson's disease and Alzheimer's disease. The aim of this preliminary study was to investigate the MAO [MAO (E.C.1.4.3.4)] inhibiting effect of various apitherapeutic products, such as chestnut honey, pollen and propolis. Extracts' MAO inhibition was measured using peroxidase-linked spectrophotometric assay in enzyme isolated from rat liver microsomes, and the values are expressed as the inhibition concentration (IC50) causing 50% inhibition of MAO. The antioxidant activity of the bee products was also determined in terms of total phenolic content (TPC) and ferric reducing/antioxidant power in aquatic extracts. All samples exhibited substantial inhibition of MAO, propolis having the highest. Inhibition was related to samples' TPCs and antioxidant capacities. These results show that bee products possess a sedative effect and may be effective in protecting humans against depression and similar diseases. PMID:24156742

  5. Phenolic compounds from Hypericum perforatum.

    PubMed

    Jürgenliemk, Guido; Nahrstedt, Adolf

    2002-01-01

    During a re-investigation of phenolic compounds from the dried crude drug material of St. John's wort (Hypericum perforatum L.) 22 phenolic compounds were detected by HPLC; 14 of them were quantified using the same system. Twelve phenolic compounds were isolated from the plant material and their structures identified mainly by spectroscopic methods, among them quercetin-3-O-(2"-O-acetyl)-beta-D-galactoside as a new natural product. Cryptochlorogenic acid, protocatechuic acid, 3-O-[Z]-p-coumaroylquinic acid, isoorientin, cyanidin-3-O-alpha-L-rhamnoside, and astilbin were obtained for the first time from this source; the earlier suspected neochlorogenic acid, 3-O-[E]-p-coumaroylquinic acid, mangiferin, miquelianin and guaijaverin were confirmed. PMID:11842341

  6. 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.

  7. Biodegradation of 2,4-dinitrophenol with laccase immobilized on nano-porous silica beads

    PubMed Central

    2013-01-01

    Many organic hazardous pollutants, including 2,4-dinitrophenol (2,4-DNP), which are water soluble, toxic, and not easily biodegradable make concerns for environmental pollution worldwide. In the present study, degradation of nitrophenols-contained effluents by using laccase immobilized on the nano-porous silica beads was evaluated. 2,4-DNP was selected as the main constituent of industrial effluents containing nitrophenols. The performance of the system was characterized as a function of pH, contact time, temperature, pollutant, and mediator concentrations. The laccase-silica beads were employed in a mixed-batch reactor to determine the degradation efficiency after 12?h of enzyme treatment. The obtained data showed that the immobilized laccase degraded more than 90% of 2,4-DNP within 12?h treatment. The immobilization process improved the activity and sustainability of laccase for degradation of the pollutant. Temperatures more than 50°C reduced the enzyme activity to about 60%. However, pH and the mediator concentration could not affect the enzyme activity. The degradation kinetic was in accordance with a Michaelis–Menten equation with Vmax and Km obtained as 0.25–0.38 ?moles/min and 0.13–0.017?mM, respectively. The stability of the immobilized enzyme was maintained for more than 85% of its initial activity after 30?days. Based on the results, it can be concluded that high resistibility and reusability of immobilized laccase on CPC-silica beads make it considerable choice for wastewater treatment. PMID:23547870

  8. Biodegradation of 2,4-dinitrophenol with laccase immobilized on nano-porous silica beads.

    PubMed

    Dehghanifard, Emad; Jonidi Jafari, Ahmad; Rezaei Kalantary, Roshanak; Mahvi, Amir Hosein; Faramarzi, Mohammad Ali; Esrafili, Ali

    2013-01-01

    Many organic hazardous pollutants, including 2,4-dinitrophenol (2,4-DNP), which are water soluble, toxic, and not easily biodegradable make concerns for environmental pollution worldwide. In the present study, degradation of nitrophenols-contained effluents by using laccase immobilized on the nano-porous silica beads was evaluated. 2,4-DNP was selected as the main constituent of industrial effluents containing nitrophenols. The performance of the system was characterized as a function of pH, contact time, temperature, pollutant, and mediator concentrations. The laccase-silica beads were employed in a mixed-batch reactor to determine the degradation efficiency after 12?h of enzyme treatment. The obtained data showed that the immobilized laccase degraded more than 90% of 2,4-DNP within 12?h treatment. The immobilization process improved the activity and sustainability of laccase for degradation of the pollutant. Temperatures more than 50°C reduced the enzyme activity to about 60%. However, pH and the mediator concentration could not affect the enzyme activity. The degradation kinetic was in accordance with a Michaelis-Menten equation with Vmax and Km obtained as 0.25-0.38 ?moles/min and 0.13-0.017?mM, respectively. The stability of the immobilized enzyme was maintained for more than 85% of its initial activity after 30?days. Based on the results, it can be concluded that high resistibility and reusability of immobilized laccase on CPC-silica beads make it considerable choice for wastewater treatment. PMID:23547870

  9. Recovery of phenol from phenolic resin plant effluents by emulsion liquid membranes

    Microsoft Academic Search

    Paulo F. M. M Correia; Jorge M. R de Carvalho

    2003-01-01

    In this work, the recovery of phenol from a phenolic resin plant effluent by emulsion liquid membranes has been investigated. The selected membrane phase allowed a selective recovery of phenol towards formaldehyde by this technology. The effect on phenol permeation of relevant variables such as volume phase ratio between the effluent and the internal phase (in the 10–42 range), stirring

  10. Induction of laccase activity in the white rot fungus Pleurotus ostreatus using water polluted with wheat straw extracts.

    PubMed

    Parenti, Alejandra; Muguerza, Elaia; Iroz, Amaia Redin; Omarini, Alejandra; Conde, Enma; Alfaro, Manuel; Castanera, Raúl; Santoyo, Francisco; Ramírez, Lucía; Pisabarro, Antonio G

    2013-04-01

    The purpose of this work was to explore the use of polluted water effluents from wheat straw using industries as inducers of lignocellulolytic enzymatic activities in cultures of white rot basidiomycetes. For this purpose, we studied the effect of a wheat straw water extract on the evolution of the laccase activity recovered from submerged cultures of Pleurotus ostreatus made in different media and under various culture conditions. Our results demonstrated an accumulative induction effect in all the cultures and conditions tested. This induction is parallel to changes in the laccase electrophoretic profiles recovered from the culture supernatants. The isoenzyme that appeared to be mainly responsible for the laccase activity under these conditions was laccase 10, as confirmed by sequencing the induced protein. These results support the idea of using wheat straw effluents as inducers in liquid cultures of P. ostreatus mycelia for the production of ligninolytic enzymatic cocktails. PMID:23425584

  11. Enhanced lignin biodegradation by a laccase-overexpressed white-rot fungus Polyporus brumalis in the pretreatment of wood chips.

    PubMed

    Ryu, Sun-Hwa; Cho, Myung-Kil; Kim, Myungkil; Jung, Sang-Min; Seo, Jin-Ho

    2013-11-01

    The laccase gene of Polyporus brumalis was genetically transformed to overexpress its laccase. The transformants exhibited increased laccase activity and effective decolorization of the dye Remazol Brilliant Blue R than the wild type. When the transformants were pretreated with wood chips from a red pine (softwood) and a tulip tree (hardwood) for 15 and 45 days, they showed higher lignin-degradation activity as well as higher wood-chip weight loss than the wild type. When the wood chips treated with the transformant were enzymatically saccharified, the highest sugar yields were found to be 32.5 % for the red pine wood and 29.5 % for the tulip tree wood, on the basis of the dried wood weights, which were 1.6-folds higher than those for the wild type. These results suggested that overexpression of the laccase gene from P. brumalis significantly contributed to the pretreatment of lignocellulose for increasing sugar yields. PMID:23975277

  12. Modifications of amino acids during ferulic acid-mediated, laccase-catalysed cross-linking of peptides.

    PubMed

    Steffensen, Charlotte L; Stensballe, Allan; Kidmose, Ulla; Degn, Peter E; Andersen, Mogens L; Nielsen, Jacob H

    2009-12-01

    Mass spectral analysis demonstrated oligomerization of peptides that had been subjected to oxidation catalysed by Trametes (Coriolus) versicolor laccase. Peptide oligomerization occurred only when cysteines or tyrosines were present in the peptides. MS/MS confirmed the cross-linking in tyrosine-containing peptides to be located between tyrosine residues. Ferulic acid mediated oligomerization of cysteine-containing peptides, but prevented cross-linking of tyrosines when used in the same concentration as the peptides. This suggests an antioxidative effect of ferulic acid in relation to tyrosine oxidation, although incorporation of ferulic acid into peptide oligomers was found in some of the tyrosine-containing peptides. No other modifications to amino acid residues by laccase-catalysed oxidation were observed by mass spectroscopy. Thus, it is suggested that oxidative modifications of other amino acids observed in proteins oxidized by laccase are not major reaction products of laccase-catalysed oxidation. PMID:19905979

  13. Phenol degradation in microbial fuel cells

    Microsoft Academic Search

    Haiping Luo; Guangli Liu; Renduo Zhang; Song Jin

    2009-01-01

    Microbial fuel cell (MFC) has gained a great attention attributable to its ability in generating electricity directly from and potentially enhancing biodegradation of contaminants. In this study, MFCs using phenol or glucose–phenol mixture as the substrate (fuel) were designed to investigate the biodegradation of phenol. In an aqueous air cathode MFC using phenol (400mg\\/L) as the sole fuel, electricity was

  14. Assay of H 2O 2 by HRP catalysed co-oxidation of phenol-4-sulphonic acid and 4-aminoantipyrine: characterisation and optimisation

    Microsoft Academic Search

    V Vojinovi?; A. M Azevedo; V. C. B Martins; J. M. S Cabral; T. D Gibson; L. P Fonseca

    2004-01-01

    A colorimetric system involving horseradish peroxidase (HRP), phenol-4-sulfonic acid (PSA) and 4-aminoantipyrine (4-AAP) for the determination of hydrogen peroxide concentration and peroxidase activity, and possible coupling with oxidases for other analytical purposes, was developed and optimised. Proposed reagents concentrations are 25mM PSA, 0.4mM 4-AAP, and 100mM phosphate buffer pH 7. The absorptivity of the product calculated to the added hydrogen

  15. Transformation of N ??, N ??-dimethyl- N ?-(hydroxyphenyl)ureas by laccase from the white rot fungus Trametes versicolor

    Microsoft Academic Search

    C. Jolivalt; A. Raynal; E. Caminade; B. Kokel; F. Le Goffic; C. Mougin

    1999-01-01

    Transformation of N?,N?-dimethyl-N-(hydroxyphenyl)ureas was assayed in the presence of purified laccase produced by the fungus Trametes versicolor. The para- and ortho-hydroxyphenyl derivatives were enzymatically transformed, whereas the meta derivative was not. The performance of laccase-mediated transformation depended on the pH, with an optimum for the para-derivative degradation rate at pH 5. The pH also influenced the nature of the reaction

  16. Reutilisation of food processing wastes for production of relevant metabolites: application to laccase production by Trametes hirsuta

    Microsoft Academic Search

    Emilio Rosales; Susana Rodr??guez Couto; M Ángeles Sanromán

    2005-01-01

    The aim of the present study was to investigate the feasibility of kiwi fruit wastes as a support-substrate for laccase production by the white-rot fungus Trametes hirsuta under solid-state conditions. Different experiments were conducted in order to select the variables that allow obtaining high levels of laccase activity. In particular, the effect of the initial ammonium concentration, the amount of

  17. Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

    PubMed

    Yaver, D S; Xu, F; Golightly, E J; Brown, K M; Brown, S H; Rey, M W; Schneider, P; Halkier, T; Mondorf, K; Dalboge, H

    1996-03-01

    Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein. PMID:8975613

  18. Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

    PubMed Central

    Yaver, D S; Xu, F; Golightly, E J; Brown, K M; Brown, S H; Rey, M W; Schneider, P; Halkier, T; Mondorf, K; Dalboge, H

    1996-01-01

    Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein. PMID:8975613

  19. Novel laccases of Ganoderma sp. KU-Alk4, regulated by different glucose concentration in alkaline media

    Microsoft Academic Search

    Churapa Teerapatsakul; Naoki Abe; Christopher Bucke; Ngampong Kongkathip; Saeree Jareonkitmongkol; Lerluck Chitradon

    2007-01-01

    Physiological regulation of laccase production from Ganoderma sp. KU-Alk4, isolated in Thailand, was controlled by the initial glucose concentration in liquid culture. Different laccase\\u000a isozymes were produced using different starting concentrations of glucose. With 1% glucose, two isozymes, KULac 1 and 2 were\\u000a produced, while with 4% glucose, three different isozymes, KULac 3, 4 and 5, were produced. The KULacs

  20. Homologous cloning, expression, and characterisation of a laccase from Streptomyces coelicolor and enzymatic decolourisation of an indigo dye

    Microsoft Academic Search

    Etienne Dubé; François Shareck; Yves Hurtubise; Claude Daneault; Marc Beauregard

    2008-01-01

    The lack of a commercially available robust and inexpensive laccase is a major barrier to the widespread application of this\\u000a enzyme in various industrial sectors. By using an efficient system developed in Streptomyces lividans, we have produced by homologous expression 350 mg L?1 of a bacterial laccase with a high purity and without any extensive purification. This is the highest production

  1. Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus

    Microsoft Academic Search

    Venelina Sugareva; Albert Härtl; Matthias Brock; Katrin Hübner; Manfred Rohde; Thorsten Heinekamp; Axel A. Brakhage

    2006-01-01

    Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent

  2. Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes , and decolorization of chemically different dyes

    Microsoft Academic Search

    M. Nagai; T. Sato; H. Watanabe; K. Saito; M. Kawata; H. Enei

    2002-01-01

    A laccase (EC 1.10.3.2) was isolated from the culture filtrate of Lentinula edodes. The enzyme was purified to a homogeneous preparation using hydrophobic, anion-exchange, and size-exclusion chromatographies. SDS-PAGE analysis showed the purified laccase, Lcc 1, to be a monomeric protein of 72.2 kDa. The enzyme had an isoelectric point of around pH 3.0. The optimum pH for enzyme activity was

  3. Phenolic problems solved with hydrogen peroxide oxidation

    Microsoft Academic Search

    E. J. Keating; R. A. Brown; E. S. Greenberg

    1978-01-01

    Major industrial sources of phenolic waste discharges are: insulation fiberglass manufacturing, petroleum refineries, smelting and slag operations, organic products manufacture, synthetic resin manufacture, textile mills, steel-making, paint stripping, plywood, hardboard, and wood preserving. Phenolic discharges create problems in three areas: toxicity to marine life, taste and odor disturbances, and oxygen depletion of the receiving water. Methods for analyzing phenols are

  4. Advances in addition-cure phenolic resins

    Microsoft Academic Search

    C. P. Reghunadhan Nair

    2004-01-01

    Recent developments in the area of addition curable phenolic resins are reviewed. The article highlights the chemistry of addition-cure phenolic resins and discusses the different strategies involved in their molecular design. Structural modification through incorporation of thermally stable, addition curable groups on the novolac backbone is one strategy. The transformation of phenolic hydroxyl groups to addition curable functions forms an

  5. Lysyl oxidase expression and inhibition in uveal melanoma.

    PubMed

    Abourbih, Daniel A; Di Cesare, Sebastian; Orellana, Maria E; Antecka, Emilia; Martins, Claudia; Petruccelli, Luca A; Burnier, Miguel N

    2010-04-01

    Lysyl oxidase is a marker of poor prognosis in several malignancies and is hypothesized to promote a migratory phenotype in hypoxic breast carcinomas. This study aims to characterize the expression of the lysyl oxidase and lysyl oxidase-like proteins in human uveal melanoma cell lines and archival choroidal melanomas using immunohistochemistry. The transcriptional control of lysyl oxidase will also be investigated under simulated hypoxic conditions using cobalt chloride. Lastly, changes in cellular proliferation and invasion will be assessed after the treatment of cell lines with beta-aminopropionitrile, a lysyl oxidase catalytic inhibitor. Retrospective analysis of lysyl oxidase expression in primary human uveal melanoma showed 82% (27 of 33) of tumors being stained positive. High lysyl oxidase expression correlated with the aggressive epithelioid cell type and was associated with shorter metastasis-free survival. Simulated hypoxia resulted in a significant increase in lysyl oxidase mRNA expression. Inhibiting lysyl oxidase's catalytic activity significantly reduced cellular invasion but had no effect on cell proliferation. Our study is the first to show lysyl oxidase expression in primary choroidal melanomas. This protein may represent a potential therapeutic target that warrants further study in this malignancy. PMID:20179655

  6. Bioactive Compounds, Antioxidant, Xanthine Oxidase Inhibitory, Tyrosinase Inhibitory and Anti-Inflammatory Activities of Selected Agro-Industrial By-products

    PubMed Central

    Oskoueian, Ehsan; Abdullah, Norhani; Hendra, Rudi; Karimi, Ehsan

    2011-01-01

    Evaluation of abundantly available agro-industrial by-products for their bioactive compounds and biological activities is beneficial in particular for the food and pharmaceutical industries. In this study, rapeseed meal, cottonseed meal and soybean meal were investigated for the presence of bioactive compounds and antioxidant, anti-inflammatory, xanthine oxidase and tyrosinase inhibitory activities. Methanolic extracts of rapeseed meal showed significantly (P < 0.01) higher phenolics and flavonoids contents; and significantly (P < 0.01) higher DPPH and nitric oxide free radical scavenging activities when compared to that of cottonseed meal and soybean meal extracts. Ferric thiocyanate and thiobarbituric acid tests results showed rapeseed meal with the highest antioxidant activity (P < 0.01) followed by BHT, cotton seed meal and soybean meal. Rapeseed meal extract in xanthine oxidase and tyrosinase inhibitory assays showed the lowest IC50 values followed by cottonseed and soybean meals. Anti-inflammatory assay using IFN-?/LPS stimulated RAW 264.7 cells indicated rapeseed meal is a potent source of anti-inflammatory agent. Correlation analysis showed that phenolics and flavonoids were highly correlated to both antioxidant and anti-inflammatory activities. Rapeseed meal was found to be promising as a natural source of bioactive compounds with high antioxidant, anti-inflammatory, xanthine oxidase and tyrosinase inhibitory activities in contrast to cotton and soybean meals. PMID:22272095

  7. Laccase biosensor based on phytic acid modification of nanostructured SiO? surface for sensitive detection of dopamine.

    PubMed

    Zhao, Wenbo; Wang, Kuai; Wei, Yuan; Ma, Yinghui; Liu, Lingling; Huang, Xiaohua

    2014-09-23

    In this work, three kinds of nanostructured silica-phytic acid (SiO2-PA) materials with diverse morphologies including spherical SiO2-PA (s-SiO2-PA), rod-like (r-SiO2-PA), and helical SiO2-PA (h-SiO2-PA) were prepared with the help of electrostatic interaction. The SiO2-PA nanomaterials with different morphologies were characterized by using transmission electron microscopy (TEM), Fourier transform infrared (FTIR), electrochemical impedance spectroscopy (EIS), and circular dichroism spectrum (CD). Diverse morphologies of SiO2-PA were used as electrode decorated materials to achieve a high efficiency for electrochemical dopamine (DA) detection. The laccase biosensors were fabricated by immobilizing different morphologies of SiO2-PA nanomaterials and laccase onto the glassy carbon electrode (GCE) surface, successively. Then the electrochemical responses of the different morphologies of nanostructured SiO2-PA nanomaterials to laccase were discussed. Results indicated that compared to laccase/s-SiO2-PA and laccase/r-SiO2-PA, the laccase/h-SiO2-PA-modified electrode showed the best electrochemical performances. PMID:25110941

  8. Improved production of laccase by Daedalea flavida: consideration of evolutionary process optimization and batch-fed culture.

    PubMed

    Singha, Siddhartha; Panda, Tapobrata

    2014-03-01

    The genetic algorithm was used effectively to find the optimal values of eight process variables for the maximum laccase production by Daedalea flavida in a stationary culture. The algorithm was modified suitably to improve laccase production with 18 parallel experiments in 4 generations. A high enzyme titer of 65 % was achieved after the optimization and compared to the titer obtained before optimization. To study the effect of the surface immobilized growth on the enzyme production, the fungus was grown on three solid carriers. When cultured on polymer composite fibers, polyurethane foam, or steel wool, at least 2.5 times more biomass was produced, compared to the biomass produced in support-free growth. On the contrary, the mycelia grown on solid support produced much less laccase than non-adhering mycelia. Four parallel runs of batch-fed cultures were done, using the cell mass of D. flavida to evaluate the influence of four different volumes of medium exchanged on laccase production. For sustainable production of the enzyme, complete exchange of medium was favorable, where the laccase activity increased continuously in six consecutive cycles, though, 50 % exchange of medium produced the maximum laccase in terms of mean enzyme activity obtained in six cycles. PMID:23903597

  9. A mechanism for NaCl inhibition of Reactive Blue 19 decolorization and ABTS oxidation by laccase.

    PubMed

    Champagne, P-P; Nesheim, M E; Ramsay, J A

    2013-07-01

    Laccases produced by white rot fungi have been extensively evaluated for their potential to decolorize textile wastewaters which contain salts like sodium chloride and sodium sulfate. The effect of sodium chloride and sodium sulfate on Trametes versicolor laccase during the decolorization of an anthraquinone dye (Reactive Blue 19) and the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by steady-state kinetic analysis. The results showed that, while sodium sulfate did not affect laccase activity, sodium chloride inhibited both ABTS oxidation and dye decolorization. However, the type of inhibition was substrate-dependent: it was hyperbolic, noncompetitive with ABTS and parabolic, noncompetitive with Reactive Blue 19. Furthermore, the results suggested that two chlorides may bind to laccase in the presence of the dye unlike recent inhibition models which suggest that there is only one inhibition site. This investigation is the first to provide evidence for and to propose a two-site model of laccase inhibition, providing new insight into NaCl inhibition of laccase. The proposed model is also useful to predict decolorization rates in the presence of sodium chloride and to determine operating conditions that will minimize inhibition. PMID:23129183

  10. Differential effects of NADPH oxidase and xanthine oxidase inhibition on sympathetic reinnervation in postinfarct rat hearts.

    PubMed

    Lee, Tsung-Ming; Chen, Chien-Chang; Hsu, Yu-Jung

    2011-06-01

    Superoxide has been shown to play a major role in ventricular remodeling and arrhythmias after myocardial infarction. However, the source of increased myocardial superoxide production and the role of superoxide in sympathetic innervation remain to be further characterized. Male Wistar rats, after coronary artery ligation, were randomized to vehicle, allopurinol, or apocynin for 4weeks. To determine the role of peroxynitrite in sympathetic reinnervation, we also used 3-morpholinosydnonimine (a peroxynitrite generator). The postinfarction period was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine, xanthine oxidase activity, NADPH oxidase activity, and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Sympathetic hyperinnervation was blunted after administration of allopurinol. Arrhythmic scores in the allopurinol-treated infarcted rats were significantly lower than those in vehicle. For similar levels of ventricular remodeling, apocynin had no beneficial effects on oxidative stress, sympathetic hyperinnervation, or arrhythmia vulnerability. Allopurinol-treated hearts had significantly decreased nerve growth factor expression, which was substantially increased after coadministration of 3-morpholinosydnonimine. These results indicate that xanthine oxidase but not NADPH oxidase largely mediates superoxide production after myocardial infarction. Xanthine oxidase inhibition ameliorates sympathetic innervation and arrhythmias possibly via inhibition of the peroxynitrite-mediated nerve growth factor pathway. PMID:21295134

  11. Xanthine oxidase biosensor for monitoring meat spoilage

    NASA Astrophysics Data System (ADS)

    Vanegas, D. C.; Gomes, C.; McLamore, E. S.

    2014-05-01

    In this study, we have designed an electrochemical biosensor for real-time detection of specific biomarkers of bacterial metabolism related to meat spoilage (hypoxanthine and xanthine). The selective biosensor was developed by assembling a `sandwich' of nanomaterials and enzymes on a platinum-iridium electrode (1.6 mm tip diameter). The materials deposited on the sensor tip include amorphous platinum nanoclusters (i.e. Pt black), reduced graphene oxide, nanoceria, and xanthine oxidase. Xanthine oxidase was encapsulated in laponite hydrogel and used for the biorecognition of hypoxanthine and xanthine (two molecules involved in the rotting of meat by spoilage microorganisms). The developed biosensor demonstrated good electrochemical performance toward xanthine with sensitivity of 2.14 +/- 1.48 ?A/mM, response time of 5.2 +/- 1.5 sec, lower detection limit of 150 +/- 39 nM, and retained at least 88% of its activity after 7 days of continuous use.

  12. Natural Compounds as Modulators of NADPH Oxidases

    PubMed Central

    2013-01-01

    Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols. PMID:24381714

  13. Cytokinin Oxidase Regulates Rice Grain Production

    Microsoft Academic Search

    Motoyuki Ashikari; Hitoshi Sakakibara; Shaoyang Lin; Toshio Yamamoto; Tomonori Takashi; Asuka Nishimura; Enrique R. Angeles; Qian Qian; Hidemi Kitano; Makoto Matsuoka

    2005-01-01

    Most agriculturally important traits are regulated by genes known as quantitative trait loci (QTLs) derived from natural allelic variations. We here show that a QTL that increases grain productivity in rice, Gn1a, is a gene for cytokinin oxidase\\/dehydrogenase (OsCKX2), an enzyme that degrades the phytohormone cytokinin. Reduced expression of OsCKX2 causes cytokinin accumulation in inflorescence meristems and increases the number

  14. Imaging Monoamine Oxidase in the Human Brain

    SciTech Connect

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-11-10

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets.

  15. Defensive Roles of Polyphenol Oxidase in Plants

    Microsoft Academic Search

    C. Peter Constabel; Raymond Barbehenn

    Plant polyphenol oxidases (PPOs) are widely distributed and well-studied oxidative enzymes, and their effects on discoloration in damaged and diseased plant tissues have been known for many years. The discovery in C.A. Ryan's laboratory in the mid-1990s that tomato PPO is induced by the herbivore defense signals systemin and jasmonate, together with seminal work on PPO's possible effects on herbiv-

  16. Deactivation of cellulases by phenols

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pretreatment of lignocellulosic materials may result in the release of inhibitors and deactivators of cellulose enzyme hydrolysis. We report the identification of phenols with major inhibition and/or deactivation effect on enzymes used for conversion of cellulose to ethanol. The inhibition effects w...

  17. [Cloning and sequencing of ACC oxidase gene from sugarcane].

    PubMed

    Wang, Zi-Zhang; Li, Yang-Rui; Zhang, Shu-Zhen; Lin, Jun-Fang; Guo, Li-Qiong

    2003-01-01

    The plant hormone ethylene is not only responsible for the initiation of fruit ripening, senescence and dormancy but also for regulating many other plant developmental processes, such as seed germination, root initiation, growth, floral differentiation, sex differentiation and responding to environment stresses. One of the rate-limiting steps for ethylene biosynthesizing in plant is catalyzed by 1-aminocyclopropane-1-carboxylate (ACC) oxidase. Understanding of ethylene expressive pattern in plant is an entrance to understand the roles of ethylene on plant. In this paper, two degenerate oligonucleotide primers were designed, coding for two conservative amino acid regions in ACC oxidase protein family, the sequences of the two primers were TAGAGCTCGATGC[TA]TG [CT]GA[GA]AA[AC]TGGGG and CGTCTAGAGCTTC[GA]AATCTTGGCTCCTT respectively. A PCR amplification was performed on sugarcane (Saccharum L. Hybrid cv. ROC16) DNA template, and produced a fragment of 940 bp. By using the program of BLAST on NCBI GenBank database, the sequence presented a very high match with the ACC oxidase genes from other plants, 63 searched out sequences were all ACC oxidase genes. After alignment on PCgene program, the identities of the cloned fragment with ACC oxidase genes from rice and bamboo were both reaching about 88%. So we can concluded that the cloned sequence was a member of ACC oxidase genes fragment from sugarcane. The sequence has been submitted to the GenBank database, the accession number is AF442821. According to the ACC oxidase protein family, a 'intron' of 103 bp was excluded and the sequence coded 279 amino acids, which spanned 88% of the putative whole sequence in length. Alignment and phylogenetic analysis of the amino acid sequence deduced from this fragment and the ACC oxidase sequences of other plants retrieved from GenBank were carried out by using PCgene program. The putative amino acid sequence shared a homology of 86% with the ACC oxidases of bamboo and rice, 74.6% with banana, 70% with tomato and potato and 68% with melon and carnation, which showed that the homology of sugarcane ACC oxidase with monocot was higher than with dicot. The results of phylogenetic analysis showed that ACC oxidase from sugarcane and ACC oxidases from rice clustered together firstly, and then came those from banana, ACC oxidases of dicot from potato, tomato, petunia, melon, Arabidopsis thaliana and carnation came subsequently. It indicated that sugarcane ACC oxidase had a closer phylogenetic affinities to the monocot ACC oxidase sequences than to the dicot ACC oxidases sequences. The clustering results of ACC oxidase molecules accorded with morphological classification system. PMID:12812078

  18. Effect of enzymatic orientation through the use of syringaldazine molecules on multiple multi-copper oxidase enzymes.

    PubMed

    Ulyanova, Yevgenia; Babanova, Sofia; Pinchon, Erica; Matanovic, Ivana; Singhal, Sameer; Atanassov, Plamen

    2014-07-14

    The effect of proper enzyme orientation at the electrode surface was explored for two multi-copper oxygen reducing enzymes: Bilirubin Oxidase (BOx) and Laccase (Lac). Simultaneous utilization of "tethering" agent (1-pyrenebutanoic acid, succinimidyl ester; PBSE), for stable enzyme immobilization, and syringaldazine (Syr), for enzyme orientation, of both Lac and BOx led to a notable enhancement of the electrode performance. For Lac cathodes tested in solution it was established that PBSE-Lac and PBSE-Syr-Lac modified cathodes demonstrated approximately 6 and 9 times increase in current density, respectively, compared to physically adsorbed and randomly oriented Lac cathodes. Further testing in solution utilizing BOx showed an even higher increase in achievable current densities, thus BOx was chosen for additional testing in air-breathing mode. In subsequent air-breathing experiments the incorporation of PBSE and Syr with BOx resulted in current densities of 0.65 ± 0.1 mA cm(-2); 2.5 times higher when compared to an unmodified BOx cathode. A fully tethered/oriented BOx cathode was combined with a NAD-dependent Glucose Dehydrogenase anode for the fabrication of a complete enzymatic membraneless fuel cell. A maximum power of 1.03 ± 0.06 mW cm(-2) was recorded for the complete fuel cell. The observed significant enhancement in the performance of "oriented" cathodes was a result of proper enzyme orientation, leading to facilitated enzyme/electrode interface interactions. PMID:24875125

  19. Induction of human monocyte motility by lysyl oxidase.

    PubMed

    Lazarus, H M; Cruikshank, W W; Narasimhan, N; Kagan, H M; Center, D M

    1995-12-01

    Lysyl oxidase highly purified from calf aorta was found to be a potent chemotactic agent for unstimulated human peripheral blood mononuclear cells, determined in in vitro assays in Boyden chambers. A typical chemotactic bell-shaped curve was observed, with a maximal migratory response of 237% of control occurring at 10(-10) M lysyl oxidase. The chemotactic response was prevented by prior heat inactivation of the enzyme, by treatment of the enzyme with beta-aminopropionitrile or ethylenediamine, which are active site-directed inhibitors of lysyl oxidase, and by a competing, lysine-containing peptide substrate of lysyl oxidase. The chemoattractant response to lysyl oxidases was characterized by both chemokinetic and chemotactic components. These results raise the possibility that extracellular lysyl oxidase may have important roles to play in biology in addition to its established function in the crosslinking of elastin and collagen. PMID:8785587

  20. Hyperuricemia and urate nephropathy in urate oxidase-deficient mice.

    PubMed

    Wu, X; Wakamiya, M; Vaishnav, S; Geske, R; Montgomery, C; Jones, P; Bradley, A; Caskey, C T

    1994-01-18

    Urate oxidase, or uricase (EC 1.7.3.3), is a purine metabolic enzyme that catalyzes the conversion of uric acid to allantoin in most mammals except humans and certain other primates. The loss of urate oxidase in the human during primate evolution predisposes man to hyperuricemia, a metabolic disturbance that can lead to gouty arthritis and renal stones. To create a mouse model for hyperuricemia and gout, and to address the question of whether urate oxidase is essential in lower mammalian species, we have disrupted the urate oxidase gene in the mouse by homologous recombination in embryonic stem cells. Unlike the human situation, urate oxidase deficiency in mice causes pronounced hyperuricemia and urate nephropathy. More than half of the mutant mice died before 4 weeks of age, indicating that urate oxidase is essential in mice. These mutant mice may also serve as animal models for hyperuricemia and its related nephropathy in humans. PMID:8290593

  1. Polyphenol oxidase activity and antioxidant properties of Yomra apple (Malus communis L.) from Turkey.

    PubMed

    Can, Zehra; Dincer, Barbaros; Sahin, Huseyin; Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

    2014-12-01

    In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors. PMID:24246090

  2. Regulation of Superoxide?Producing NADPH Oxidases in Nonphagocytic Cells

    Microsoft Academic Search

    Ryu Takeya; Noriko Ueno; Hideki Sumimoto

    2006-01-01

    The membrane?integrated protein gp91phox functions as the catalytic center of the superoxide?producing phagocyte NADPH oxidase. Recent studies have identified homologs of gp91phox in nonphagocytic cells, which constitute the NADPH oxidase (Nox) family. Activation of the Nox oxidases leads to production of reactive oxygen species (ROS), thereby participating in a variety of biological events, such as host defense, hormone biosynthesis, and

  3. Comparison of the Inhibition of Monoamine Oxidase and Butyrylcholinesterase Activities by Infusions from Green Tea and Some Citrus Peels

    PubMed Central

    Ademosun, Ayokunle O.

    2014-01-01

    This study sought to investigate the effect of infusions from green tea (Camellia sinensis) and some citrus peels [shaddock (Citrus maxima), grapefruit (Citrus paradisi), and orange (Citrus sinensis)] on key enzymes relevant to the management of neurodegenerative conditions [monoamine oxidase (MAO) and butyrylcholinesterase (BChE)]. The total phenol contents and antioxidant activities as typified by their 2,2?-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals scavenging abilities, ferric reducing antioxidant properties, and Fe2+ chelating abilities were also investigated. Green tea had the highest total phenol (43.3?mg/g) and total flavonoid (16.4?mg/g) contents, when compared to orange [total phenol (19.6?mg/g), total flavonoid (6.5?mg/g)], shaddock [total phenol (16.3?mg/g), total flavonoid (5.2?mg/g)], and grapefruit [total phenol (17.7?mg/g), total flavonoid (5.9?mg/g)]. Orange (EC50 = 1.78?mg/mL) had the highest MAO inhibitory ability, while green tea had the least MAO inhibitory ability (EC50 = 2.56?mg/mL). Similarly, green tea had the least BChE inhibitory ability (EC50 = 5.43?mg/mL) when compared to the citrus peels' infusions. However, green tea infusions had the strongest highest ABTS radical scavenging ability, reducing power, and Fe2+ chelating ability. The inhibition of MAO and BChE activities by the green tea and citrus peels infusions could make them good dietary means for the prevention/management of neurodegenerative conditions. PMID:25243093

  4. A study on the thermostability of microencapsulated glucose oxidase.

    PubMed

    Hoshino, K; Muramatsu, N; Kondo, T

    1989-01-01

    Glucose oxidase was microencapsulated within polyurea membranes by the interfacial polymerization method and the stability to heat of the encapsulated enzyme was examined. Thermostability of microencapsulated glucose oxidase was prominent and increased with increase in the amount of glucose oxidase entrapped. This stability, however, could not be ascribed to the peculiar properties of microcapsules but was suggested to be caused by the incorporation of glucose oxidase molecules in the membranes through chemical bonding. This stability revealed that the enzyme molecules in the microcapsules could not always exist in the dissolved form but a fairly large portion of the molecules participated in the polymerization reaction and changed their enzymatic properties. PMID:2723964

  5. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase screening test for gonorrhea. (a) Identification. An...

  6. Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene

    Microsoft Academic Search

    C. Martin; M. Pecyna; H. Kellner; N. Jehmlich; C. Junghanns; D. Benndorf; M. von Bergen; D. Schlosser

    2007-01-01

    Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric,\\u000a and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption\\/ionization-time\\u000a of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO4 and vanillic acid amended liquid fungal cultures grown in malt extract medium. The

  7. Biodegradability of kraft mill TCF biobleaching effluents: application of enzymatic laccase-mediator system.

    PubMed

    Monje, P G; González-García, S; Moldes, D; Vidal, T; Romero, J; Moreira, M T; Feijoo, G

    2010-04-01

    The great amount of pollutants released from kraft pulp processes, mainly from cooking and bleaching stages, is one of the most relevant environmental problems in this type of industry. New bleaching sequences are being studied based on the use of oxidative enzymes from fungal cultures. In this study, the bleaching systems consisting of Laccase and different mediators such as 1-hydroxybenzotriazole, violuric acid, syringaldehyde and methyl syringate in the bleaching sequence of Eucalyptus globulus kraft pulp were applied. The main objective of this study is to evaluate the aerobic and anaerobic biodegradability and toxicity to Vibrium fischeri of generated L-stage and total bleaching sequence effluents. The highest levels of aerobic and anaerobic degradation of the generated effluents were achieved for treatments with laccase plus violuric acid, with 80% of aerobic degradation and 68% of anaerobic biodegradation. V. fischeri toxicity was remarkably reduced for all the effluents after aerobic degradation. PMID:20097403

  8. Production of laccase by immobilized cells of Agaricus sp.: induction effect of xylan and lignin derivatives.

    PubMed

    Kaluskar, V M; Kapadnis, B P; Jaspers, C; Penninckx, M J

    1999-03-01

    Laccase was produced in the supernatant of culture of a local isolate of Agaricus sp. obtained from decaying Ficus religiosa wood. The enzyme was produced at a constitutive level when growing the fungus in a nitrogen-limited medium supplemented with either glycerol, glucose, fructose, mannitol, arabinose, maltose, saccharose, cellulose, or cellobiose. A two- to sixfold increase in enzyme specific activity was observed when growing the strain in the presence of straw, xylan, xylose, lignosulfonate, veratryl alcohol, and ferulic and veratric acid. Experiments are consistent with the existence of an induction control on laccase and the absence of a form of carbon catabolite repression mediated by noninducing carbon sources. Immobilization of the Agaricus sp. on several supports, including polyurethane foam, textile strips, and straw, resulted in an increase of enzyme production as compared to cultivation in liquid medium. PMID:15304726

  9. 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. PMID:19763900

  10. Three-dimensional graphene networks as a new substrate for immobilization of laccase and dopamine and its application in glucose/O2 biofuel cell.

    PubMed

    Zhang, Yijia; Chu, Mi; Yang, Lu; Tan, Yueming; Deng, Wenfang; Ma, Ming; Su, Xiaoli; Xie, Qingji

    2014-08-13

    We report here three-dimensional graphene networks (3D-GNs) as a novel substrate for the immobilization of laccase (Lac) and dopamine (DA) and its application in glucose/O2 biofuel cell. 3D-GNs were synthesized with an Ni(2+)-exchange/KOH activation combination method using a 732-type sulfonic acid ion-exchange resin as the carbon precursor. The 3D-GNs exhibited an interconnected network structure and a high specific surface area. DA was noncovalently functionalized on the surface of 3D-GNs with 3,4,9,10-perylene tetracarboxylic acid (PTCA) as a bridge and used as a novel immobilized mediating system for Lac-based bioelectrocatalytic reduction of oxygen. The 3D-GNs-PTCA-DA nanocomposite modified glassy carbon electrode (GCE) showed stable and well-defined redox current peaks for the catechol/o-quinone redox couple. Due to the mediated electron transfer by the 3D-GNs-PTCA-DA nanocomposite, the Nafion/Lac/3D-GNs-PTCA-DA/GCE exhibited high catalytic activity for oxygen reduction. The 3D-GNs are proven to be a better substrate for Lac and its mediator immobilization than 2D graphene nanosheets (2D-GNs) due to the interconnected network structure and high specific surface area of 3D-GNs. A glucose/O2 fuel cell using Nafion/Lac/3D-GNs-PTCA-DA/GCE as the cathode and Nafion/glucose oxidase/ferrocence/3D-GNs/GCE as the anode can output a maximum power density of 112 ?W cm(-2) and a short-circuit current density of 0.96 mA cm(-2). This work may be helpful for exploiting the popular 3D-GNs as an efficient electrode material for many other biotechnology applications. PMID:25019407

  11. 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. PMID:24315640

  12. Complete genome sequence of Bacillus pumilus W3: A strain exhibiting high laccase activity.

    PubMed

    Guan, Zheng-Bing; Cai, Yu-Jie; Zhang, Yan-Zhou; Zhao, Hong; Liao, Xiang-Ru

    2015-08-10

    Here we report the full genome sequence of Bacillus pumilus W3, which was isolated from raw gallnut honey in Nandan County, Guangxi Province of China, showing high CotA-laccase activity. The W3 strain contains 3,745,123bp with GC content of 41.39%, and contains 3695 protein-coding genes, 21 rRNAs and 70 tRNAs. PMID:25957807

  13. Purification, crystallization and preliminary X-ray structure analysis of the laccase from Ganoderma lucidum

    PubMed Central

    Lyashenko, Andrey V.; Belova, Oksana; Gabdulkhakov, Azat G.; Lashkov, Alexander A.; Lisov, Alexandr V.; Leontievsky, Alexey A.; Mikhailov, Al’bert M.

    2011-01-01

    The ligninolytic enzymes of the basidiomycetes play a key role in the global carbon cycle. A characteristic property of these enzymes is their broad substrate specificity, which has led to their use in various biotechnologies, thus stimulating research into the three-dimensional structures of ligninolytic enzymes. This paper presents the purification, crystallization and preliminary X-ray analysis of the laccase from the ligninolytic basidiomycete Ganoderma lucidum. PMID:21821897

  14. Pycnoporus laccase-mediated bioconversion of rutin to oligomers suitable for biotechnology applications

    Microsoft Academic Search

    Eva Uzan; Bénédicte Portet; Christian Lubrano; Sandrine Milesi; Anne Favel; Laurence Lesage-Meessen; Anne Lomascolo

    2011-01-01

    The Pycnoporus fungi are white-rot basidiomycetes listed as food- and cosmetic-grade microorganisms. Three high redox potential laccases\\u000a from Pycnoporus coccineus and Pycnoporus sanguineus were tested and compared, with the commercial Suberase® as reference, for their ability to synthesise natural active oligomers\\u000a from rutin (quercetin-3-rutinoside, one of the best-known naturally occurring flavonoid glycosides). The aim of this work\\u000a was to develop

  15. 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. PMID:23831273

  16. Enzymatic deinking of secondary fibers: cellulases/hemicellulases versus laccase-mediator system.

    PubMed

    Ibarra, David; Concepción Monte, M; Blanco, Angeles; Martínez, Angel T; Martínez, María J

    2012-01-01

    The use of enzymes has been suggested as an environmentally friendly alternative to complement conventional chemical deinking in the recycling of recovered paper. This study compares the use of cellulases/hemicellulases versus the laccase-mediator system for deinking printed fibers from newspapers and magazines. For this purpose, two commercial enzyme preparations with endoglucanase and endoxylanase activities (Viscozyme Wheat from Aspergillus oryzae and Ultraflo L from Humicola insolens, Novozymes) and a commercial laccase (NS51002 from Trametes villosa, Novozymes), the latter in the presence of synthetic or natural (lignin-related) mediators, were evaluated. The enzymatic treatments were studied at the laboratory scale using a standard chemical deinking sequence consisting of a pulping stage; an alkaline stage using NaOH, sodium silicate and fatty acid soap; and a bleaching stage using hydrogen peroxide. The handsheets were then prepared and their brightness, residual ink concentration, and strength properties were measured. Among the different enzymatic treatments assayed, both carbohydrate hydrolases were found to deink the secondary fibers more efficiently. Brightness increased up to 3-4% ISO on newspaper fibers, being Ultraflo 20% more efficient in the ink removal. Up to 2.5% ISO brightness increase was obtained when magazine fibers were used, being Viscozyme 9% more efficient in the ink removal. Regarding the laccase-mediator system, alone or in combination with carbohydrate hydrolases, it was ineffective in deinking both newspaper and magazine fibers, resulting in pulps with worse brightness and residual ink concentration values. However, pulp deinking by the laccase-mediator system was displayed when secondary fibers from printed cardboard were used, obtaining up to 3% ISO brightness increase and lower residual ink concentrations. PMID:21643708

  17. Oxidation of Polycyclic Aromatic Hydrocarbons (PAH) by Laccase of Trametes Versicolor

    Microsoft Academic Search

    Andrzej Majcherczyk; Christian Johannes; Aloys Hüttermann

    1998-01-01

    Laccase of Trametes versicolor was able to oxidize in vitro most of the 14 polycyclic aromatic hydrocarbons (PAH) tested. Acenaphthylene was removed by 37% followed by anthracene and benzo[a]pyrene which were oxidized by 18 and 19%, respectively. Lower but significant oxidation of about 10% was found for eight additional PAH: acenaphthene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, and perylene. Naphthalene,

  18. Fungal laccases as tools for the synthesis of new hybrid molecules and biomaterials

    Microsoft Academic Search

    Annett Mikolasch; Frieder Schauer

    2009-01-01

    Laccase is a ligninolytic enzyme widely distributed in wood-rotting fungi and which is also found in a variety of molds and\\u000a insects as well as some plants and bacteria. Its biological roles range from depolmerization of lignin, coal and humic acids\\u000a via the oxidation of various mono- and diaromatic structures, to polymerization reactions and pigment formation in microbial\\u000a cells or

  19. Laccase-catalysed protein–flavonoid conjugates for flax fibre modification

    Microsoft Academic Search

    Suyeon Kim; Artur Cavaco-Paulo

    The introduction of flavonoid compounds into proteins can improve the natural properties of proteins, being promising products\\u000a which essentially require antioxidant property. The oxidative conjugation of protein–flavonoids was processed by laccase catalysis\\u000a resulting in the synthesis of biologically functional polymers. The new reaction products were detected in terms of sodium\\u000a dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption\\/ionisation-time of

  20. Electrochemical analysis of the interactions of laccase mediators with lignin model compounds

    Microsoft Academic Search

    Robert Bourbonnais; Dónal Leech; Michael G. Paice

    1998-01-01

    The mechanism of oxidation of lignin by laccase and mediator has been investigated by cyclic voltammetry and bulk electrolysis. Electrochemical properties and reactivities of the two mediators 2,2?-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT) were studied and their intermediates responsible for lignin oxidation were characterized. ABTS was oxidized first to the cation radical (ABTS+?) and then to the dication (ABTS2+). The two

  1. Lentinula edodes produces a multicomponent protein complex containing manganese (II)-dependent peroxidase, laccase and ?-glucosidase

    Microsoft Academic Search

    Randhir S. Makkar; Akihiko Tsuneda; Ken Tokuyasu; Yutaka Mori

    2001-01-01

    A multicomponent protein complex containing manganese (II)-dependent peroxidase, laccase and ?-glucosidase was isolated from culture extracts of the white rot basidiomycete Lentinula edodes. This protein complex showed a single protein band on native polyacrylamide gel electrophoresis (PAGE). On sodium dodecyl sulfate (SDS)–PAGE, however, it displayed three major bands and several additional minor bands ranging in size from 60 kDa to

  2. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox?/?) mice which had resolved in wild-type mice by day 5 but progressed in p47phox?/? mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox?/? mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  3. High performance phenolic pultrusion resin

    SciTech Connect

    Qureshi, S.P.; Ingram, W.H. [Georgia-Pacific Resins, Inc., Decatur, GA (United States); Smith, C. [Morrison Molded Fiber Glass, Bristol, VA (United States)

    1996-11-01

    Today, Phenol-Formaldehyde (PF) resins are the materials of choice for aerospace interior applications, primarily due to low FST (flame, smoke and toxicity). Since 1990, growth of PF resins has been steadily increasing in non-aerospace applications (which include mass transit, construction, marine, mine ducting and offshore oil) due to low FST and reasonable cost. This paper describes one component phenol-formaldehyde resin that was jointly developed with Morrison Molded Fiber Glass for their pultrusion process. Physical properties of the resin with flame/smoke/toxicity, chemical resistance and mechanical performance of the pultruded RP are discussed. Neat resin screening tests to identify high-temperature formulations are explored. Research continues at Georgia-Pacific to investigate the effect of formulation variables on processing and mechanical properties.

  4. Phenolic compounds in Catharanthus roseus

    Microsoft Academic Search

    Natali Rianika Mustafa; Robert Verpoorte

    2007-01-01

    Besides alkaloids Catharanthus roseus produces a wide spectrum of phenolic compounds, this includes C6C1 compounds such as 2,3-dihydoxybenzoic acid, as well as\\u000a phenylpropanoids such as cinnamic acid derivatives, flavonoids and anthocyanins. The occurrence of these compounds in C. roseus is reviewed as well as their biosynthesis and the regulation of the pathways. Both types of compounds compete with the indole

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

    PubMed

    Fi?ig?u, Ioni?a Firu?a; 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. PMID:24432339

  6. An electrodeposited redox polymer-laccase composite film for highly efficient four-electron oxygen reduction

    NASA Astrophysics Data System (ADS)

    Shen, Wei; Deng, Huimin; Teo, Alan Kay Liang; Gao, Zhiqiang

    2013-03-01

    In this report, it is shown that novel thin films of Os(dcbpy)2 (dcbpy = 4,4?-dicarboxylic acid-2,2?-bipyridine)-based redox polymer-laccase composite can be electrodeposited onto carbon electrodes under mild conditions. In a nutshell, the exchange of the inner-sphere Cl- of the Os(dcbpy)2Cl+/2+ complex tethered to partially quaternized poly (4-vinylpyridine) (PVP) by a pyridine ligand of a second PVP chain leads to cross-linking and deposition of the redox polymer. Laccase, which has coordinatively linkable functions of amines and histidines, is readily incorporated in the electrodeposited redox polymer. Because the reaction centers of the co-deposited laccase are electrically connected to the electrode through the deposited redox polymer, the electrodeposited film can catalyze the electroreduction of O2 at 0.58 V (vs. Ag/AgCl) - the least reducing potential for highly efficient four-electron reduction of O2 in pH 5.5 0.10 M phosphate buffer solution. Furthermore, the electroreduction of O2 is found to be O2 transport-limited when the reduction potential is poised at ?120 mV more reducing than that of the reversible O2/H2O couple. This composite film could be an excellent candidate for uses as cathode in enzymatic biofuel cells.

  7. Synthesis and effect of modification on methacylate - acrylate microspheres for Trametes versicolor laccase enzyme immobilization

    NASA Astrophysics Data System (ADS)

    Mazlan, Siti Zulaikha; Hanifah, Sharina Abu

    2014-09-01

    Immobilization of laccase on the modified copolymer methacrylate-acrylate microspheres was studied. A poly (glycidyl methacrylate-co-n-butyl acrylate) microsphere consists of epoxy groups were synthesized using suspension photocuring technique. The epoxy group in poly (GMA-nBA) microspheres were converted into amino groups with aldehyde group. Laccase immobilization is based on having the amino groups on the enzyme surface and aldehyde group on the microspheres via covalent binding. Fourier transform infrared spectroscopy (FT-IR) analysis proved the successful surface modification on microspheres. The FTIR spectrum shows the characteristic peaks at 1646 cm-1 assigned to the conformation of the polymerization that took place between monomer GMA and nBA respectively. In addition, after modification, FTIR peaks that assigned to the epoxy ring (844 cm-1 and 904 cm-1) were decreased. The results obtained from FTIR method signify good agreement with the epoxy content method. Hence, the activity of the laccase-immobilized microspheres increased upon increasing the epoxy content. Furthermore, poly (GMA-nBA) exhibited uniform microspheres with below 2 ?m surface. Immobilized enzyme showed a broader pH profile and higher temperature compared native enzyme.

  8. Reduction in Acute Ecotoxicity of Paper Mill Effluent by Sequential Application of Xylanase and Laccase

    PubMed Central

    Sharma, Jitender; Kalia, Vipin C.; Kang, Yun Chan; Lee, Jung-Kul

    2014-01-01

    In order to reduce the ecotoxicity of paper mill, four different enzymatic pretreatment strategies were investigated in comparison to conventional chemical based processes. In strategy I, xylanase-aided pretreatment of pulp was carried out, and in strategy II, xylanase and laccase-mediator systems were used sequentially. Moreover, to compare the efficiency of Bacillus stearothermophilus xylanase and Ceriporiopsis subvermispora laccase in the reduction of ecotoxicity and pollution, parallel strategies (III and IV) were implemented using commercial enzymes. Conventional CDEOPD1D2 (CD, Cl2 with ClO2; EOP, H2O2 extraction; D1 and D2, ClO2) and X/XLCDEOPD1D2 (X, xylanase; L, laccase) sequences were employed with non-enzymatic and enzymatic strategies, respectively. Acute toxicity was determined by the extent of inhibition of bioluminescence of Vibrio fischeri with different dilutions of the effluent. Two-fold increase was observed in EC50 values for strategy I compared to the control process. On the other hand, sequential application of commercial enzymes resulted in higher acute toxicity compared to lab enzymes. In comparison to the control process, strategy II was the most efficient and successfully reduced 60.1 and 25.8% of biological oxygen demand (BOD) and color of effluents, respectively. We report for the first time the comparative analysis of the ecotoxicity of industrial effluents. PMID:25058160

  9. Reduction in acute ecotoxicity of paper mill effluent by sequential application of xylanase and laccase.

    PubMed

    Dhiman, Saurabh Sudha; Garg, Gaurav; Sharma, Jitender; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

    2014-01-01

    In order to reduce the ecotoxicity of paper mill, four different enzymatic pretreatment strategies were investigated in comparison to conventional chemical based processes. In strategy I, xylanase-aided pretreatment of pulp was carried out, and in strategy II, xylanase and laccase-mediator systems were used sequentially. Moreover, to compare the efficiency of Bacillus stearothermophilus xylanase and Ceriporiopsis subvermispora laccase in the reduction of ecotoxicity and pollution, parallel strategies (III and IV) were implemented using commercial enzymes. Conventional C(D)E(OP)D(1)D(2) (C(D), Cl(2) with ClO2; EOP, H2O2 extraction; D1 and D2, ClO2) and X/XLC(D)E(OP)D(1)D(2) (X, xylanase; L, laccase) sequences were employed with non-enzymatic and enzymatic strategies, respectively. Acute toxicity was determined by the extent of inhibition of bioluminescence of Vibrio fischeri with different dilutions of the effluent. Two-fold increase was observed in EC50 values for strategy I compared to the control process. On the other hand, sequential application of commercial enzymes resulted in higher acute toxicity compared to lab enzymes. In comparison to the control process, strategy II was the most efficient and successfully reduced 60.1 and 25.8% of biological oxygen demand (BOD) and color of effluents, respectively. We report for the first time the comparative analysis of the ecotoxicity of industrial effluents. PMID:25058160

  10. Polyoxometalate/laccase-mediated oxidative polymerization of catechol for textile dyeing.

    PubMed

    Kim, Suyeon; Silva, Carla; Evtuguin, Dmitry V; Gamelas, José A F; Cavaco-Paulo, Artur

    2011-02-01

    The synergistic effect between polyoxometalates (POMs), namely K(5)[SiW(11)V(V)O(40)]·11H(2)O and H(5)[PMo(10)V(V) (2)O(40)]·13H(2)O and laccase from ascomycete Myceliophthora thermophila has been employed for the first time in oxidative polymerization of catechol. Such a laccase-mediator system allowed the formation of a relatively high molecular weight polycatechol as confirmed by size exclusion chromatography and electrospray ionization mass spectrometry (ESI-MS) (3990 Da when using K(5)[SiW(11)V(V)O(40)]·11H(2)O and 3600 Da with H(5)[PMo(10)V(V) (2)O(40)]·13H(2)O). The synthesized polymers were applied as dyes for the dyeing of flax fabrics. The color intensity of flax fabrics colored with polymer solutions was evaluated by diffuse reflectance spectrophotometry via k/s measurements (+10% of fixation ratio). A new synthetic process allowed a dyeing polymer, provided upon flax coloration, better color fixation and color resistance when compared to that obtained by conventional synthesis with laccase solely or with addition of organic mediator (1-hydroxybenzotriazole). PMID:20953600

  11. 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. PMID:26020270

  12. Degradation of lindane and endosulfan by fungi, fungal and bacterial laccases.

    PubMed

    Ul?nik, A; Kralj Cigi?, 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. PMID:23736895

  13. 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][CuCl?]) 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][CuCl?], was exclusively effective on the refolding process of a copper-containing protein. PMID:23975279

  14. Can the laccase mediator system affect the chemical and refining properties of the eucalyptus pulp?

    PubMed

    Cadena, Edith M; Vidal, Teresa; Torres, Antonio L

    2010-11-01

    Application of a laccase mediator system (an L stage) to TCF and ECF bleached pulp from Eucalyptus globulus with low residual lignin content (KN(lig)1.0) provides useful information about its effects on hexenuronic acids, functional groups (carboxyl and carbonyl) and electrokinetic properties such as zeta potential and surface charge. The use of laccase from Trametes villosa in combination with the mediator 1-hydroxybenzotriazole (HBT) was found to oxidize cellulose to carbonyl groups and reduce the amount of carboxyl groups present in TCF pulp by effect of its partially removing hexenuronic acids from it. This result may open up new prospects for improving brightness stability in pulp. In addition, the laccase mediator system modifies the surface charge and zeta potential in the fibre suspension for the removal of ionizables groups in TCF pulp. This result has no adverse effect on the pulp refining efficiency. L treatment requires less mechanical energy than conventionally refined pulp to obtain an optimal tensile and tear index in handsheets. This behavior may be attributable to the modification of the electrokinetic properties. PMID:20561782

  15. Laccase-mediated coupling of nonpolar chains for the hydrophobization of lignocellulose.

    PubMed

    Garcia-Ubasart, Jordi; Vidal, Teresa; Torres, Antonio L; Rojas, Orlando J

    2013-05-13

    We investigate the use of laccase enzymes to couple short nonpolar chains containing aromatic groups onto flax fibers and nanofibrillated cellulose (NFC) with different lignin contents. Trametes villosa , Pycnoporus cinnabarinus , and Myceliophthora thermophila were used to facilitate surface coupling and to produce materials with different levels of hydrophobicity. Heat treatment of fiber webs after lacccase-mediated coupling markedly increased the resistance to water absorption. The highest hydrophobization levels of flax fibers was achieved by coupling dodecyl 3,4,5-trihydroxybenzoate (HB-C12), which yielded water contact angles (WCAs) of 80-96 degrees and water absorption times (drop tests) of ca. 73 min. The results from apparent aromatic content and FTIR analyses confirmed the laccase-mediated coupling of HB-C12 onto the cellulose fibers. Ultrathin films of NFC were also used as substrates for enzyme-mediated hydrophobization with HB-C12. In these cases, WCAs in the range of 87-104 degrees were achieved, depending on the conditions. Quartz crystal microgravimetry (QCM) was used to study the dynamics and the extent of the coupling process onto cellulose. The results help to better understand the mechanisms involved in laccase-mediated hydrophobization and provide a proof of a biotechnological platform for the development of value-added fiber products. PMID:23570533

  16. Stability and decolourization ability of Trametes villosa laccase in liquid ultrasonic fields.

    PubMed

    Basto, Carlos; Silva, Carla Joana; Gübitz, Georg; Cavaco-Paulo, Artur

    2007-03-01

    We report in this study that the sonication of laccase from Trametes villosa and bovine serum albumin promotes the formation of protein aggregates with high molecular weight. The formation of aggregates leads to the deactivation of the enzyme, fact that was confirmed by the analysis of the enzyme stability (half-life time) upon ultrasound treatment. This inactivation was mainly caused by the radicals formed by the cavitation phenomenon. It was verified that the addition of polyvinyl alcohol to laccase had a protecting effect against enzyme inactivation. The performance of laccase in the decolourization of indigo carmine was studied. It was observed that the best results were attained when the dye solution was treated with ultrasound and enzyme stabilized with polyvinyl alcohol, where more than 65% of decolourization was achieved. This value is remarkably higher than that attained for the enzyme alone, which was only able to decolourize 20% of the dye solution within 1h of treatment. These results have important implications for the exploitation of sonication in textile industry, where the pollution caused by the release of dyes into effluents is one of the major concerns. PMID:16987690

  17. Highly stable laccase from repeated-batch culture of Funalia trogii ATCC 200800.

    PubMed

    Yesilada, O; Birhanli, E; Ozmen, N; Ercan, S

    2014-01-01

    The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55-90 degrees C, which was maximal at 80-95 degrees C. It was highly stable within the temperature intervals 20-50 degrees C. The half life of the enzyme was about 2 h and 5 min at 60 degrees C and 70 degrees C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0-9.0. NaN3 and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications. PMID:25272754

  18. Screening of lignocellulose-degrading superior mushroom strains and determination of their CMCase and laccase activity.

    PubMed

    Fen, Li; Xuwei, Zhu; Nanyi, Li; Puyu, Zhang; Shuang, Zhang; Xue, Zhao; Pengju, Li; Qichao, Zhu; Haiping, Lin

    2014-01-01

    In order to screen lignocellulose-degrading superior mushroom strains ten strains of mushrooms (Lentinus edodes939, Pholiota nameko, Lentinus edodes868, Coprinus comatus, Macrolepiota procera, Auricularia auricula, Hericium erinaceus, Grifola frondosa, Pleurotus nebrodensis, and Shiraia bambusicola) were inoculated onto carboxymethylcellulose agar-Congo red plates to evaluate their ability to produce carbomethyl cellulase (CMCase). The results showed that the ratio of transparent circle to mycelium circle of Hericium erinaceus was 8.16 (P < 0.01) higher than other strains. The filter paper culture screening test showed that Hericium erinaceus and Macrolepiota procera grew well and showed extreme decomposition of the filter paper. When cultivated in guaiacol culture medium to detect their abilities to secrete laccase, Hericium erinaceus showed the highest ability with the largest reddish brown circles of 4.330 cm. CMCase activity determination indicated that Coprinus comatus and Hericium erinaceus had the ability to produce CMCase with 33.92 U/L on the 9th day and 22.58 U/L on the 10th day, respectively, while Coprinus comatus and Pleurotus nebrodensis had the ability to produce laccase with 496.67 U/L and 489.17 U/L on the 16th day and 18th day. Based on the results, Coprinus comatus might be the most promising lignocellulose-degrading strain to produce both CMCase and laccase at high levels. PMID:24693246

  19. Purification and characterization of a novel laccase from Fomitopsis pinicola mycelia.

    PubMed

    Park, Naomi; Park, Sang-Shin

    2014-09-01

    A novel laccase was isolated from the culture filtrate of the brown-rot fungus, Fomitopsis pinicola. Enzyme production reached its highest level after cultivation for 8 days at 25°C. The enzyme was purified by ultrafiltration, ion exchange chromatography, gelfiltration chromatography, and hydrophobic interaction chromatography. Zymography analysis of the purified enzyme showed a laccase band with a molecular mass of 92 kDa. The molecular weight of the enzyme was 92 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The enzyme also had an isoelectric point of 3.8. The optimum temperature and pH for enzyme activity were 80°C and 3.0, respectively. Enzyme activity was relatively stable in the pH range from 1.5 to 11.0 and at temperatures below 40°C. The N-terminal amino acid sequence of the enzyme was DTHKAEIACRFKDLG. Enzyme activity was potently inhibited by NaN3 and SDS. The enzyme showed the highest specific activity for 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) as a substrate. The Km value of the enzyme for ABTS substrate was 0.28 mM with a Vmax value of 4.5 U/min. The enzyme degraded several recalcitrant dyes at different time intervals during decolorization. Therefore, the novel laccase from F. pinicola may be potentially useful in industry. PMID:25083593

  20. 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

  1. Synthesis of improved phenolic resins

    NASA Technical Reports Server (NTRS)

    Delano, C. B.; Mcleod, A. H.

    1979-01-01

    Twenty seven addition cured phenolic resin compositions were prepared and tested for their ability to give char residues comparable to state-of-the-art phenolic resins. Cyanate, epoxy, allyl, acrylate, methacrylate and ethynyl derivatized phenolic oligomers were investigated. The novolac-cyanate and propargyl-novolac resins provided anaerobic char yields at 800 C of 58 percent. A 59 percent char yield was obtained from modified epoxy novolacs. A phosphonitrilic derivative was found to be effective as an additive for increasing char yields. The novolac-cyanate, epoxy-novolac and methacrylate-epoxy-novolac systems were investigated as composite matrices with Thornel 300 graphite fiber. All three resins showed good potential as composite matrices. The free radical cured methacrylate-epoxy-novolac graphite composite provided short beam shear strengths at room temperature of 93.3 MPa (13.5 ksi). The novolac-cyanate graphite composite produced a short beam shear strength of 74 MPa (10.7 ksi) and flexural strength of 1302 MPa (189 ksi) at 177 C. Air heat aging of the novolac-cyanate and epoxy novolac based composites for 12 weeks at 204 C showed good property retention.

  2. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between Noxes and the ER may provide relevant insights in Nox-related (patho)physiology. Antioxid. Redox Signal. 20, 2755–2775. PMID:24386930

  3. The interaction of arsenite with xanthine oxidase.

    PubMed

    Hille, R; Stewart, R C; Fee, J A; Massey, V

    1983-04-25

    The binding of arsenite to the molybdenum center of milk xanthine oxidase is re-examined. The Kd for the arsenite complex has been determined to be 24 microM from equilibrium binding studies and this value has been confirmed by determination of the association and dissociation rate constants for the interaction of arsenite with xanthine oxidase. Formation of the complex is not prevented by prior reaction of the enzyme with thiol reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) or methyl methanethiosulfonate. Binding of arsenite to the enzyme perturbs both the oxidation-reduction potentials and the electron paramagnetic resonance signal of the molybdenum center observed after partial reduction of the enzyme with sodium dithionite. The EPR signal of the partially reduced arsenite-complexed enzyme is further modified in two different ways by the addition of xanthine or salicylate. Other purine and pteridine substrates and products for the enzyme yield EPR signals indistinguishable from that generated by xanthine, whereas aromatic aldehydes and carboxylic acids give signals similar to that observed in the presence of salicylate. It is thus clear that while arsenite prevents enzyme turnover, it does not preclude binding of substrate and product molecules. Binding of arsenite at the molybdenum center of xanthine oxidase does not disturb the oxidation-reduction potentials of the iron-sulfur centers of the enzyme, but evidence is presented to suggest that the midpoint potential of the FAD site is decreased by approximately 15 mV. A structure for the arsenite complex is proposed to provide a framework in which to interpret the EPR signals in a quantitative fashion. PMID:6300101

  4. Undesirable Enzymatic Browning in Crustaceans: Causative Effects and Its Inhibition by Phenolic Compounds.

    PubMed

    Nirmal, Nilesh Prakash; Benjakul, Soottawat; Ahmad, Mehraj; Arfat, Yasir Ali; Panichayupakaranant, Pharkphoom

    2015-12-01

    Undesirable enzymatic browning mediated by polyphenol oxidase (E.C. 1.14.18.1) on the surface of seafood from crustaceans have been a great concern to food processors, causing quality losses of seafood products. Seafoods especially from crustaceans are worldwide consumed due to their delicacy and nutritional value. However, black spot formation (melanosis) is the major problem occurring in crustaceans during postmortem handling and refrigerated storage induce deleterious changes in organoleptic properties and, therefore, decreases commercial value. Polyphenoloxidase (PPO), the copper-containing metalloprotein involved in oxidation of phenol to quinone is the major biochemical reaction of melanosis formation. This enzymatic mechanism causes unappealing blackening in postharvest crustaceans. To alleviate the melanosis formation in crustaceans, use of phenolic compounds from plant extract can serve as antimelanotics and appears to be a good alternative to the conventional sulfites which are associated with health-related disorders. In this review, we focuses on the unique features about the structure, distribution, and properties of PPO as well as mechanism of melanosis formation and provide a comprehensive deeper insight on the factors affecting melanosis formation and its inhibition by various antimelanotics including newly discovered plant phenolic compounds. PMID:25584522

  5. Optical biosensor with poly[N-nonyl-3,6-bis(ethylenedioxythiophene)carbazole] matrix for monitoring of phenol derivatives

    NASA Astrophysics Data System (ADS)

    Jedrychowska, Agnieszka; Malecha, Karol; Cabaj, Joanna; So?oducho, Jadwiga

    2014-08-01

    The aim of the research was to develop an enzymatic, optical biosensor which provides quick and convenient determination of phenolic compounds in aqueous solutions. The biosensing strategy concerns design, fabrication and testing of a miniature ceramic-based biosensor which is destined for in-situ substrate monitoring. The base of the measuring system was fabricated using low temperature co-fired ceramics (LTCC) technology. The biocatalyst - laccase- was immobilized on the thin film of poly[N-nonyl-3,6-bis(ethylenedioxythiophene)carbazole] which provided good binding of the enzyme to the substrate and positively affected on the catalytic activity of the protein. In order to evaluate properties of the designed biosensor, its response for various concentrations of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamonnium sal (ABTS) was measured. The optical biosensor produced by presented method could find applications in many fields, i.e. for detection of phenolic compounds in food products and beverages, in industry for control of technological processes or for environmental monitoring

  6. Purification of Xanthine Dehydrogenase and Sulfite Oxidase from Chicken Liver

    Microsoft Academic Search

    Kapila Ratnam; Michael S. Brody; Russ Hille

    1996-01-01

    Xanthine dehydrogenase and sulfite oxidase from chicken liver are oxomolybdenum enzymes which catalyze the oxidation of xanthine to uric acid and sulfite to sulfate, respectively. Independent purification protocols have been previously described for both enzymes. Here we describe a procedure by which xanthine dehydrogenase and sulfite oxidase are purified simultaneously from the same batch of fresh chicken liver. Also, unlike

  7. Molecular evolution of cytochrome bd oxidases across proteobacterial genomes.

    PubMed

    Degli Esposti, Mauro; Rosas-Pérez, Tania; Servín-Garcidueñas, Luis Eduardo; Bolaños, Luis Manuel; Rosenblueth, Monica; Martínez-Romero, Esperanza

    2015-03-01

    This work is aimed to resolve the complex molecular evolution of cytochrome bd ubiquinol oxidase, a nearly ubiquitous bacterial enzyme that is involved in redox balance and bioenergetics. Previous studies have created an unclear picture of bd oxidases phylogenesis without considering the existence of diverse types of bd oxidases. Integrated approaches of genomic and protein analysis focused on proteobacteria have generated a molecular classification of diverse types of bd oxidases, which produces a new scenario for interpreting their evolution. A duplication of the original gene cluster of bd oxidase might have occurred in the ancestors of extant ?-proteobacteria of the Rhodospirillales order, such as Acidocella, from which the bd-I type of the oxidase might have diffused to other proteobacterial lineages. In contrast, the Cyanide-Insensitive Oxidase type may have differentiated into recognizable subtypes after another gene cluster duplication. These subtypes are widespread in the genomes of ?-, ?-, and ?-proteobacteria, with occasional instances of lateral gene transfer. In resolving the evolutionary pattern of proteobacterial bd oxidases, this work sheds new light on the basal taxa of ?-proteobacteria from which the ?-proteobacterial lineage probably emerged. PMID:25688108

  8. Kinetic properties of glycerophosphate oxidase isolated from dry baker's yeast

    Microsoft Academic Search

    Luciana Amade Camargo; Maria Henriques Lourenço Ribeiro; Maristela de Freitas Sanches Peres; Edwil Aparecida de Lucca Gattás

    2008-01-01

    The glycerophosphate oxidase is a flavoprotein responsible for the catalysis of the oxidation of the glycerophosphate to dihydroxyacetone phosphate, through the reduction of the oxygen to hydrogen peroxide. The glycerophosphate oxidase from baker's yeast was specific for l-?-glycerol phosphate. It was estimated by monitoring the consumption of oxygen with an oxygraph. An increase of 32% in consumption of oxygen was

  9. Methods and approaches to study plant mitochondrial alternative oxidase

    Microsoft Academic Search

    Allison E. McDonald; Stephen M. Sieger; Greg C. Vanlerberghe

    2002-01-01

    The alternative oxidase is a non-proton motive 'alternative' to electron transport through the cytochrome pathway. Despite its wasteful nature in terms of energy conservation, the path- way is likely present throughout the plant kingdom and ap- pears to be expressed in most plant tissues. A small alterna- tive oxidase gene family exists, the members of which are differentially expressed in

  10. A prokaryotic alternative oxidase present in the bacterium Novosphingobium aromaticivorans

    Microsoft Academic Search

    Pål Stenmark; Pär Nordlund

    2003-01-01

    The alternative oxidase (AOX) is a terminal oxidase present in the respiratory chain of all plants as well as some yeasts and trypanosomes, but has not previously been found in a prokaryote. We have identified an AOX homologue in Novosphingobium aromaticivorans, the first AOX found in a prokaryote. We have cloned the gene for the N. aromaticivorans AOX and showed

  11. A study on heat-resistance of microencapsulated glucose oxidase.

    PubMed

    Komori, T; Muramatsu, N; Kondo, T

    1986-01-01

    Polyurea microcapsules containing glucose oxidase were prepared and their thermodurability was examined. Microencapsulated glucose oxidase was found to be more stable to heat than the enzyme in free solution. This stability was enhanced with an increase in the amount of enzyme entrapped in the microcapsules. PMID:3508188

  12. Plant phenolics affect oxidation of tryptophan.

    PubMed

    Salminen, Hanna; Heinonen, Marina

    2008-08-27

    The effect of berry phenolics such as anthocyanins, ellagitannins, and proanthocyanidins from raspberry (Rubus idaeus), black currant (Ribes nigrum), and cranberry (Vaccinium oxycoccus) and byproducts of deoiling processes rich in phenolics such as rapeseed (Brassica rapa L.), camelina (Camelina sativa), and soy (Glycine max L.) as well as scots pine bark (Pinus sylvestris) was investigated in an H2O2-oxidized tryptophan (Trp) solution. The oxidation of Trp was analyzed with high-performance liquid chromatography using both fluorescence and diode array detection of Trp and its oxidation products. Mechanisms of antioxidative action of the phenolic compounds toward the oxidation of Trp were different as the pattern of Trp oxidation products varied with different phenolic compounds. The antioxidant protection toward oxidation of Trp was best provided with pine bark phenolics, black currant anthocyanins, and camelina meal phenolics as well as cranberry proanthocyanidins. PMID:18646765

  13. Reactions of polyfluoroalkylsulfenyl chlorides with phenols

    Microsoft Academic Search

    A. Yu. Sizov; A. F. Kolomiets; A. V. Fokin

    1991-01-01

    Polyfluoroalkylsulfenyl chlorides thiolate phenol and its ortho- and meta-substituted derivatives regiospecifically at the para-position in the absence of a catalyst and of a hydrogen chloride acceptor. Ortho thiolation occurs with significantly greater difficulty in the para-substituted phenols, and is only possible with the strong electron-donor properties of the substituent. Polyfluoroalkylthiolation of phenols is rendered more difficult with the increase in

  14. Understanding pulp delignification by laccase-mediator systems through isolation and characterization of lignin-carbohydrate complexes.

    PubMed

    Du, Xueyu; Li, Jiebing; Gellerstedt, Göran; Rencoret, Jorge; Del Río, José C; Martínez, Angel T; Gutiérrez, Ana

    2013-09-01

    The effects and mechanism of pulp delignification by laccases in the presence of redox mediators have been investigated on unbleached eucalyptus kraft pulp treated with laccases from Pycnoporus cinnabarinus (PcL) and Myceliophthora thermophila (MtL) and 1-hydroxybenzotriazole (HBT) and methyl syringate (MeS) as mediators, respectively. Determination of the corrected ? number in eucalyptus pulps after the enzymatic treatments revealed that the PcL-HBT system exhibited a more remarkable delignification effect than the MtL-MeS system. To obtain further insight, lignin-carbohydrate complexes were fractionated and subsequently characterized by nuclear magnetic resonance, thioacidolysis (followed by gas chromatography and size exclusion chromatography), and pyrolysis-gas chromatography-mass spectrometry (pyrolysis-GC-MS) analyses before and after the enzymatic treatments and their controls. We can conclude that the laccase-mediator treatments altered the lignin structures in such a way that more lignin was recovered in the xylan-lignin fractions, as shown by Klason lignin estimation, with smaller amounts of both syringyl (S) and guaiacyl (G) uncondensed units, as shown by thioacidolysis and gas chromatography, especially after the PcL-HBT treatment. The laccase-mediator treatment produced oxidation at C? and cleavage of C? and C? bonds in pulp lignin, as shown by pyrolysis-GC-MS. The general mechanism of residual lignin degradation in the pulp by laccase-mediator treatments is discussed in light of the results obtained. PMID:23841747

  15. Genome-Wide Identification and Characterization of Novel Laccase Genes in the White-Rot Fungus Flammulina velutipes.

    PubMed

    Kim, Hong-Il; Kwon, O-Chul; Kong, Won-Sik; Lee, Chang-Soo; Park, Young-Jin

    2014-12-01

    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

  16. 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??M, 1.7??M, and 1.25??M, respectively, signifying that it is an antipathogenic protein. PMID:23093860

  17. 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

  18. Isolation and identification of novel high strength phenol degrading bacterial strains from phenol-formaldehyde resin manufacturing industrial wastewater

    Microsoft Academic Search

    V. Arutchelvan; V. Kanakasabai; S. Nagarajan; V. Muralikrishnan

    2005-01-01

    Phenols are toxic to all types of organisms. Two bacterial strains capable of utilizing phenol as a sole carbon source were isolated from the phenol bearing industrial wastewater. Based on the biochemical test results the organisms were identified as Pseudomonas cepacia and Bacillus brevis. The organisms were very efficient in phenol degradation, the lag phase increased with increase in phenol

  19. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    PubMed Central

    2012-01-01

    Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD) was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ) anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG) 25 and diazo-dye Acid Red (AR) 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1?g/l) with relative decolorization values of 91.2% (3?h) and 97.1% (18?h), as well as high activity to AR18 (1?g/l) by 80.5% (3?h) and 89.0% (18?h), was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1?g/l). No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved via a subsequent 4-h cell culturing. Conclusions This study demonstrates, for the first time, the methodology by which the engineered P. putida with surface-immobilized laccase was successfully used as regenerable biocatalyst for biodegrading synthetic dyes, thereby opening new perspectives in the use of biocatalysis in industrial dye biotreatment. PMID:22686507

  20. 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. PMID:24448164