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1

Redox Potentials, Laccase Oxidation, and Antilarval Activities of Substituted Phenols  

PubMed Central

Laccases are copper-containing oxidases that are involved in sclerotization of the cuticle of mosquitoes and other insects. Oxidation of exogenous compounds by insect laccases may have the potential to produce reactive species toxic to insects. We investigated two classes of substituted phenolic compounds, halogenated di- and trihydroxybenzenes and substituted di-tert-butylphenols, on redox potential, oxidation by laccase and effects on mosquito larval growth. An inverse correlation between the oxidation potentials and laccase activity of halogenated hydroxybenzenes was found. Substituted di-tert-butylphenols however were found to impact mosquito larval growth and survival. In particular, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl)phenol (15) caused greater than 98% mortality of Anopheles gambiae larvae in a concentration of 180 nM, whereas 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-methylpropanal oxime (13) and 6,8-di-tert-butyl-2,2-dimethyl-3,4-dihydro-2H-chromene (33) caused 93% and 92% mortalities in concentrations of 3.4 and 3.7 ?M, respectively. Larvae treated with di-tert-butylphenolic compounds died just before pupation.

Prasain, Keshar; Nguyen, Thi D. T.; Gorman, Maureen J.; Barrigan, Lydia M.; Peng, Zeyu; Kanost, Michael R.; Syed, Lateef U.; Li, Jun; Zhu, Kun Yan; Hua, Duy H.

2012-01-01

2

Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae  

PubMed Central

Laccase oxidation of phenolic azo dyes was examined with a commercially available laccase from Pyricularia oryzae as the model. Methyl-, methoxy-, chloro-, and nitro-substituted derivatives of 4-(4(prm1)-sulfophenylazo)-phenol were examined as substrates for this laccase. Only the substituents on the phenolic ring were changed. Among the dyes examined, only 2-methyl-, 2-methoxy-, 2,3-dimethyl-, 2,6-dimethyl-, 2,3-dimethoxy-, and 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol served as substrates. Preliminary kinetic studies suggest that 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol is the best substrate. Laccase oxidized the 2,6-dimethyl derivative of 4-(4(prm1)-sulfophenylazo)-phenol to 4-sulfophenylhydroperoxide (SPH) and 2,6-dimethyl-1,4-benzoquinone. The 2-methyl- and 2-methoxy-substituted dyes were oxidized to SPH and either 2-methyl- or 2-methoxy-benzoquinone. Six products were formed from laccase oxidation of the 2,6-dimethoxy-substituted dye. Three of them were identified as SPH, 4-hydroxybenzenesulfonic acid, and 2,6-dimethoxybenzoquinone. A mechanism for the formation of benzoquinone and SPH from laccase oxidation of phenolic azo dyes is proposed. This study suggests that laccase oxidation can result in the detoxification of azo dyes.

Chivukula, M.; Renganathan, V.

1995-01-01

3

Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger  

PubMed Central

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.

2012-01-01

4

Enzymatic oxidative transformation of phenols by Trametes trogii laccases.  

PubMed

The removal of toxic phenolic compounds from industrial wastewater is an important issue to be addressed. Their presence in water and soil has become a great environmental concern, and effective methods for their removal need to be addressed. The feasibility of applying laccases for the degradation of phenolic compounds has received increasing attention. In the present work, the transformation of five phenolic compounds (catechol, hydroxytyrosol, tyrosol, guaiacol and p-coumaric acid), the main constituents of a typical wastewater derived from an olive oil factory, by Trametes trogii laccases was studied at concentrations ranging between 0.2 and 1.6 mM. High-performance liquid chromatography analysis showed high degradation rates of phenolic compounds by T trogii laccases. Independently of the used concentration, a complete transformation of guaiacol, p-coumaric acid, hydroxytyrosol and tyrosol occurred after 1 h of incubation. The transformation of catechol depends on its initial concentration. The liquid chromatography-mass spectrometry analysis showed that laccases catalysed transformation of p-coumaric acid and tyrosol, resulting in the formation of phenolic dimers. No reduction of enzyme activity has been observed during the oxidation of all phenolic compounds. These results suggest that the studied laccases were capable of efficiently removing phenolic compounds, as well as catalysing the production of novel phenolic dimers. PMID:23240190

Chakroun, Hanen; Bouaziz, Mohamed; Dhouib, Abdelhafidh; Sayadi, Sami

2012-09-01

5

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

PubMed Central

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.

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

2012-01-01

6

Enzymatic oxidative transformation of phenols by Trametes trogii laccases  

Microsoft Academic Search

The removal of toxic phenolic compounds from industrial wastewater is an important issue to be addressed. Their presence in water and soil has become a great environmental concern, and effective methods for their removal need to be addressed. The feasibility of applying laccases for the degradation of phenolic compounds has received increasing attention. In the present work, the transformation of

Hanen Chakroun; Mohamed Bouaziz; Abdelhafidh Dhouib; Sami Sayadi

2012-01-01

7

Comparative Study of Substrates and Inhibitors ofAzospirillum lipoferumandPyricularia oryzaeLaccases  

Microsoft Academic Search

In animals, plants, fungi, and eubacteria, the main phenol oxidases (PO) are catechol oxidases (tyrosinases) and laccases. In the presence of molecular oxygen, laccases typically oxidize p- and o-diphenols, whereas catechol oxidases oxidize mono- phenols ando-diphenols (19), and the quinonic products may be polymerized into large molecules, such as melanins (4). Laccases have been detected in many fungi and higher

DENIS FAURE; MARIE-LOUISE BOUILLANT

1995-01-01

8

Laccase-catalyzed oxidative polymerization of phenolic compounds.  

PubMed

Enzymatic polymerization of phenolic compounds (catechol, resorcinol, and hydroquinone) was carried out using laccase. The mechanism of polymerization and the structures of the polymers were evaluated in terms of UV-Vis and Fourier transform infrared spectroscopy. The molecular weights of the produced polyphenols were determined with GPC. The results showed that the phenolic monomers firstly turned into quinone intermediates by laccase catalysis. Through further oxidation, the intermediates formed covalent bonds. Finally, catechol units were linked together with ether bonds, and both resorcinol and hydroquinone units were linked together with C-C bonds. The number-average molecular weights of the polyphenols ranged from 1,000 to 1,400 Da (corresponding to the degree of polymerization that varied from 10 to 12) with a lower polydispersity value of about 1.10, showing selective polymerization of phenolic compounds catalyzed by laccase. PMID:23996120

Sun, Xuejiao; Bai, Rubing; Zhang, Ya; Wang, Qiang; Fan, Xuerong; Yuan, Jiugang; Cui, Li; Wang, Ping

2013-12-01

9

Multicopper Oxidase-3 Is a Laccase Associated with the Peritrophic Matrix of Anopheles gambiae  

PubMed Central

The multicopper oxidase (MCO) family of enzymes includes laccases, which oxidize a broad range of substrates including polyphenols and phenylendiamines; ferroxidases, which oxidize ferrous iron; and several other oxidases with specific substrates such as ascorbate, bilirubin or copper. The genome of Anopheles gambiae, a species of mosquito, encodes five putative multicopper oxidases. Of these five, only AgMCO2 has known enzymatic and physiological functions: it is a highly conserved laccase that functions in cuticle pigmentation and tanning by oxidizing dopamine and dopamine derivatives. AgMCO3 is a mosquito-specific gene that is expressed predominantly in adult midguts and Malpighian tubules. To determine its enzymatic function, we purified recombinant AgMCO3 and analyzed its activity. AgMCO3 oxidized hydroquinone (a p-diphenol), the five o-diphenols tested, 2,2?-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and p-phenylenediamine, but not ferrous iron. The catalytic efficiencies of AgMCO3 were similar to those of cuticular laccases (MCO2 orthologs), except that AgMCO3 oxidized all of the phenolic substrates with similar efficiencies whereas the MCO2 isoforms were less efficient at oxidizing catechol or dopa. These results demonstrate that AgMCO3 can be classified as a laccase and suggest that AgMCO3 has a somewhat broader substrate specificity than MCO2 orthologs. In addition, we observed AgMCO3 immunoreactivity in the peritrophic matrix, which functions as a selective barrier between the blood meal and midgut epithelial cells, protecting the midgut from mechanical damage, pathogens, and toxic molecules. We propose that AgMCO3 may oxidize toxic molecules in the blood meal leading to detoxification or to cross-linking of the molecules to the peritrophic matrix, thus targeting them for excretion.

Lang, Minglin; Kanost, Michael R.; Gorman, Maureen J.

2012-01-01

10

Laccase Catalyzed Synthesis of Iodinated Phenolic Compounds with Antifungal Activity  

PubMed Central

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.

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

2014-01-01

11

Laccase catalyzed synthesis of iodinated phenolic compounds with antifungal activity.  

PubMed

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

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

2014-01-01

12

Characterization of Laccase-like Multicopper Oxidases (LMCOs) in Arabidopsis thaliana.  

National Technical Information Service (NTIS)

Laccase-like multicopper oxidases (LMCOs) have repeatedly been associated with the process of lignification in plants, and previous work suggested that these enzymes might be acting as specific marker for highly localized, small-scale lignification events...

J. F. D. Dean

2008-01-01

13

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

PubMed Central

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.

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

2013-01-01

14

Comparative Study of Substrates and Inhibitors of Azospirillum lipoferum and Pyricularia oryzae Laccases  

PubMed Central

Azospirillum lipoferum and Pyricularia oryzae laccases were compared, using several substrates and inhibitors. Sixteen phenolic or nonphenolic compounds were found to be substrates of both fungal and bacterial laccases. In the presence of different phenol oxidase inhibitors, P. oryzae and A. lipoferum laccase activities had similar properties.

Faure, D.; Bouillant, M.; Bally, R.

1995-01-01

15

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

Microsoft Academic Search

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,

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

2005-01-01

16

Removal of phenols from mixtures by co-immobilized laccase\\/tyrosinase and Polyclar adsorption  

Microsoft Academic Search

  An enzymatic method for removal of phenols from their mixtures was investigated. Phenols in an aqueous solution were removed\\u000a after a two-step treatment with co-immobilized laccase and tyrosinase and Polyclar (polyvinylpolypyrrolidone). A laccase from\\u000a Pyricularia oryzae and mushroom tyrosinase were co-immobilized on Mikroperl in a fixed-bed tubular bioreactor by a rapid and simple method.\\u000a The support immobilized 95% of the

A Krastanov

2000-01-01

17

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

PubMed Central

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.

2014-01-01

18

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

Microsoft Academic Search

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,

Renate Reiss; Julian Ihssen; Linda Thöny-Meyer

2011-01-01

19

Atmospheric N deposition increases bacterial laccase-like multicopper oxidases: implications for organic matter decay.  

PubMed

Anthropogenic release of biologically available nitrogen (N) has increased dramatically over the last 150 years, which can alter the processes controlling carbon (C) storage in terrestrial ecosystems. In a northern hardwood forest ecosystem located in Michigan in the United States, nearly 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This change occurred concomitantly with compositional changes in Basidiomycete fungi and in Actinobacteria, as well as the downregulation of fungal lignocelluloytic genes. Recently, laccase-like multicopper oxidases (LMCOs) have been discovered among bacteria which can oxidize ?-O-4 linkages in phenolic compounds (e.g., lignin and humic compounds), resulting in the production of dissolved organic carbon (DOC). Here, we examined how nearly 2 decades of experimental N deposition has affected the abundance and composition of saprotrophic bacteria possessing LMCO genes. In our experiment, LMCO genes were more abundant in the forest floor under experimental N deposition whereas the abundances of bacteria and fungi were unchanged. Experimental N deposition also led to less-diverse, significantly altered bacterial and LMCO gene assemblages, with taxa implicated in organic matter decay (i.e., Actinobacteria, Proteobacteria) accounting for the majority of compositional changes. These results suggest that experimental N deposition favors bacteria in the forest floor that harbor the LMCO gene and represents a plausible mechanism by which anthropogenic N deposition has reduced decomposition, increased soil C storage, and accelerated phenolic DOC production in our field experiment. Our observations suggest that future rates of atmospheric N deposition could fundamentally alter the physiological potential of soil microbial communities. PMID:24837374

Freedman, Zachary; Zak, Donald R

2014-07-15

20

Methyl syringate: an efficient phenolic mediator for bacterial and fungal laccases.  

PubMed

The aim of the present work is to provide insight into the mechanism of laccase reactions using syringyl-type mediators. We studied the pH dependence and the kinetics of oxidation of syringyl-type phenolics using the low CotA and the high redox potential TvL laccases. Additionally, the efficiency of these compounds as redox mediators for the oxidation of non-phenolic lignin units was tested at different pH values and increasing mediator/non-phenolic ratios. Finally, the intermediates and products of reactions were identified by LC-MS and (1)H NMR. These approaches allow concluding on the (1) mechanism involved in the oxidation of phenolics by bacterial laccases, (2) importance of the chemical nature and properties of phenolic mediators, (3) apparent independence of the enzyme's properties on the yields of non-phenolics conversion, (4) competitive routes involved in the catalytic cycle of the laccase-mediator system with several new C-O coupling type structures being proposed. PMID:22995168

Rosado, Tânia; Bernardo, Pedro; Koci, Kamila; Coelho, Ana V; Robalo, M Paula; Martins, Lígia O

2012-11-01

21

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

PubMed Central

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

Lu, Rong; Miyakoshi, Tetsuo

2012-01-01

22

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

PubMed

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

Kuhar, Francisco; Papinutti, Leandro

2014-01-01

23

[Heterogeneity of molecular forms of phenol oxidase from grape leaves].  

PubMed

The substrate specificity and some kinetic properties of the monomeric (Mr = 26 000--35 000) and dimeric (Mr = 55 000--70 000) forms of phenol oxidase from vine leaves were studied. These forms possess different hydroxylating and o-diphenol oxidase activities. A kinetic analysis demonstrated that the monomeric form of the enzyme possesses a higher affinity for monophenols and can more effectively accomplish the hydroxylation reaction as compared to the dimeric one. During vine vegetation the ratio of molecular forms of phenol oxidase is altered manifesting itself in quantitative and qualitative changes of enzymatic activity. During plant maturation the dimeric fraction is predominant. The maturation process is associated with a sharp rise of the o-phenol oxidase activity, a disappearance of the hydroxylating activity and a substantial deceleration of phenol compounds production. PMID:6412775

Pruidze, G N; Zaprometov, M N; Durmishidze, S V; Kintsurashvili, D F

1983-07-01

24

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

PubMed

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

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

2007-04-15

25

Efficient electrocatalytic oxygen reduction by the 'blue' copper oxidase, laccase, directly attached to chemically modified carbons.  

PubMed

This discussion describes efforts to produce a stable, efficient electrocatalyst for four-electron O2 reduction through the direct attachment of fungal laccase, a 'blue' copper oxidase, to functionalised carbon electrode materials. Commercially available carbons, including fibrous and porous materials, offer important opportunities for achieving high conductivity over high surface areas that can be chemically functionalised. A promising approach for attaching laccase to a carbon surface is to use the diazonium coupling reaction to generate protrusive aromatic functionalities that can bind to hydrophobic residues close to the 'blue' Cu site: this site provides a fast, intramolecular electron relay into the buried trinuclear Cu active site that converts O2 rapidly and cleanly to H2O. This enhancement procedure makes possible the stable, direct electrocatalytic reduction of O2 at high potential with high efficiency in terms of turnover frequency per enzyme active site engaged with the electrode. The absence of electron-transfer mediators and simplicity of electrode system reveals the more inherent characteristics of the electrocatalytic mechanism that are masked in the waveform when a mediator is used. The study includes experiments to assess the effects of methanol and chloride ions on laccase electrocatalysis, complementing studies carried out by other groups, particularly those in which laccase is embedded in an electron-mediating gel. PMID:19213324

Blanford, Christopher F; Foster, Carina E; Heath, Rachel S; Armstrong, Fraser A

2008-01-01

26

A new procedure for the hydrophobization of cellulose fibre using laccase and a hydrophobic phenolic compound.  

PubMed

A new biotechnological procedure using laccase in combination with a hydrophobic phenolic compound (lauryl gallate) for the hydrophobization of cellulose fibres and internal sizing of paper was developed. Cellulose fibres from hardwood kraft pulp were incubated with laccase (Lac), in combination with lauryl gallate (LG). The Lac-LG treatment resulted in the internal sizing of paper, and also in significantly reduced water penetration in the handsheets and wettability of the paper surface. Paper was found not to be effectively rendered hydrophobic by LG alone. SEM images of the fibre network revealed the presence of the sizing agent: a product of the reaction between laccase and lauryl gallate. Binding of lauryl gallate to cellulose fibres was suggested by the increase in kappa number of the pulp and further confirmed by IR spectroscopy. PMID:22440576

Garcia-Ubasart, Jordi; Colom, Josep F; Vila, Carlos; Gómez Hernández, Nuria; Blanca Roncero, M; Vidal, Teresa

2012-05-01

27

Symbiotic fungi produce laccases potentially involved in phenol degradation in fungus combs of fungus-growing termites in Thailand.  

PubMed

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined. PMID:16332742

Taprab, Yaovapa; Johjima, Toru; Maeda, Yoshimasa; Moriya, Shigeharu; Trakulnaleamsai, Savitr; Noparatnaraporn, Napavarn; Ohkuma, Moriya; Kudo, Toshiaki

2005-12-01

28

Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand†  

PubMed Central

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.

Taprab, Yaovapa; Johjima, Toru; Maeda, Yoshimasa; Moriya, Shigeharu; Trakulnaleamsai, Savitr; Noparatnaraporn, Napavarn; Ohkuma, Moriya; Kudo, Toshiaki

2005-01-01

29

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

PubMed

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

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

2014-02-01

30

Comparative characterization of four laccases from Trametes versicolor concerning phenolic C-C coupling and oxidation of PAHs.  

PubMed

The laccase genes lccalpha, lccbeta, lccgamma and lccdelta encoding four isoenzymes from Trametes versicolor have been cloned and expressed in Pichia pastoris. Biochemical characterization allowed classification of these laccases into two distinct groups: Lccalpha and Lccbeta possessed higher thermal stability, but lower catalytic activity towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) compared to Lccgamma and Lccdelta. Activities of the laccases were quite different as well. Laccase Lccdelta showed highest phenolic C-C coupling activity with sinapic acid, but lowest oxidizing activity towards polycyclic aromatic hydrocarbons (PAHs). Highest activity towards PAHs was observed with Lccbeta. After 72h, more than 80% of fluorene, anthracene, acenaphthene and acenaphthylene were oxidized by Lccbeta in the presence of ABTS. Investigation of the structural basis of the different activities of the laccases demonstrated the impact of positions 164 and 265 in the substrate binding site on oxidation of PAHs. PMID:18367094

Koschorreck, Katja; Richter, Sven M; Swierczek, André; Beifuss, Uwe; Schmid, Rolf D; Urlacher, Vlada B

2008-06-01

31

Phenol oxidase activity in secondary transformed peat-moorsh soils  

NASA Astrophysics Data System (ADS)

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.

Sty?a, K.; Szajdak, L.

2009-04-01

32

Loss of cytochrome c oxidase activity and acquisition of resistance to quinone analogs in a laccase-positive variant of Azospirillum lipoferum.  

PubMed

Laccase, a p-diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium, Azospirillum lipoferum. Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome c oxidase of the aa(3) type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the bc(1) complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome c oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity. PMID:10542175

Alexandre, G; Bally, R; Taylor, B L; Zhulin, I B

1999-11-01

33

Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm, Manduca sexta  

PubMed Central

Laccases belong to the group of multicopper oxidases that exhibit wide substrate specificity for polyphenols and aromatic amines. They are found in plants, fungi, bacteria, and insects. In insects the only known role for laccase is in cuticle sclerotization. However, extracting laccase from the insect’s cuticle requires proteolysis, resulting in an enzyme that is missing its amino-terminus. To circumvent this problem, we expressed and purified full-length and amino-terminally truncated recombinant forms of laccase-2 from the tobacco hornworm, Manduca sexta. We also purified the endogenous enzyme from the pharate pupal cuticle and used peptide mass fingerprinting analysis to confirm that it is laccase-2. All three enzymes had pH optima between 5 and 5.5 when using N-acetyldopamine (NADA) or N-?-alanyldopamine (NBAD) as substrates. The laccases exhibited typical Michaelis-Menten kinetics when NADA was used as a substrate, with Km values of 0.46 mM, 0.43 mM, and 0.63 mM, respectively, for the full-length recombinant, truncated recombinant, and cuticular laccases; the apparent kcat values were 100 min?1, 80 min?1, and 290 min?1. The similarity in activity of the two recombinant laccases suggests that laccase-2 is expressed in an active form rather than as a zymogen, as had been previously proposed. This conclusion is consistent with the detection of activity in untanned pupal wing cuticle using the laccase substrate 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Immunoblot analysis of proteins extracted from both tanned and untanned cuticle detected only a single protein of 84 kDa, consistent with the full-length enzyme. With NBAD as substrate, the full-length recombinant and cuticular laccases showed kinetics indicative of substrate inhibition, with Km values of 1.9 mM and 0.47 mM, respectively, and apparent kcat values of 200 min?1 and 180 min?1. These results enhance our understanding of cuticle sclerotization, and may aid in the design of insecticides targeting insect laccases.

Dittmer, Neal T.; Gorman, Maureen J.; Kanost, Michael R.

2009-01-01

34

A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability  

Microsoft Academic Search

A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora hermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7–0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity.

Feng Xu; Woonsup Shin; Stephen H. Brown; Jill A. Wahleithner; Uma M. Sundaram; Edward I. Solomon

1996-01-01

35

Transcriptional analysis of Pleurotus ostreatus laccase genes.  

PubMed

Fungal laccases (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) are multi-copper-containing oxidases that catalyse the oxidation of a great variety of phenolic compounds and aromatic amines through simultaneous reduction of molecular oxygen to water. Fungi generally produce several laccase isoenzymes encoded by complex multi-gene families. The Pleurotus ostreatus genome encodes 11 putative laccase coding genes, and only six different laccase isoenzymes have been isolated and characterised so far. Laccase expression was found to be regulated by culture conditions and developmental stages even if the redundancy of these genes still raises the question about their respective functions in vivo. In this context, laccase transcript profiling analysis has been used to unravel the physiological role played by the different isoforms produced by P. ostreatus. Even if reported results depict a complex picture of the transcriptional responses exhibited by the analysed laccase genes, they were allowed to speculate on the isoform role in vivo. Among the produced laccases, LACC10 (POXC) seems to play a major role during vegetative growth, since its transcription is downregulated when the fungus starts the fructification process. Furthermore, a new tessera has been added to the puzzling mosaic of the heterodimeric laccase LACC2 (POXA3). LACC2 small subunit seems to play an additional physiological role during fructification, beside that of LACC2 complex activation/stabilisation. PMID:22395908

Pezzella, Cinzia; Lettera, Vincenzo; Piscitelli, Alessandra; Giardina, Paola; Sannia, Giovanni

2013-01-01

36

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

NASA Astrophysics Data System (ADS)

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.

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

1993-03-01

37

Laccase from the white-rot fungus Trametes trogii.  

PubMed

The white-rot fungus Trametes trogii excretes a main laccase showing a molecular mass of 70 kDa, acidic isoelectric point and N-terminal sequence homologous to that of several phenol oxidases. The purified enzyme oxidizes a number of phenolic and non-phenolic compounds; recalcitrant molecules may be converted into substrates by introducing, in the correct position, o- or p-orienting ring-activating groups. PMID:9650252

Garzillo, A M; Colao, M C; Caruso, C; Caporale, C; Celletti, D; Buonocore, V

1998-05-01

38

Laccase from the white-rot fungus Trametes trogii  

Microsoft Academic Search

The white-rot fungus Trametes trogii excretes a main laccase showing a molecular mass of 70?kDa, acidic isoelectric point and N-terminal sequence homol-ogous to\\u000a that of several phenol oxidases. The purified enzyme oxidizes a number of phenolic and non-phenolic compounds; recalcitrant\\u000a molecules may be converted into substrates by introducing, in the correct position, o-?or p-orienting ring-activating groups.

A. M. V. Garzillo; M. C. Colao; C. Caruso; C. Caporale; D. Celletti; V. Buonocore

1998-01-01

39

Polymerization of phenolic intermediates of pesticides by a fungal enzyme  

Microsoft Academic Search

Summary The fungusRhizoctonia praticola produces an extracellular phenol oxidase (laccase) which polymerizes phenolic intermediates of various pesticides. The enzyme catalyzes the formation of oligomeric products from halogenated phenolic intermediates of phenoxyalkanoate herbicides and from naphtholic products derived from carbamate insecticides. These findings permit further investigations into the mechanism and role of oxidative coupling leading to the incorporation of xenobiotic compounds

J.-M. Bollag; R. D. Sjoblad; R. D. Minard

1977-01-01

40

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

PubMed Central

• 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.

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

2006-01-01

41

Interfacial behavior and activity of laccase and bilirubin oxidase on bare gold surfaces.  

PubMed

Two blue multicopper oxidases (MCOs) (viz. Trametes hirsuta laccase (ThLc) and Myrothecium verrucaria bilirubin oxidase (MvBOx)) were immobilized on bare polycrystalline gold (Au) surfaces by direct adsorption from both dilute and concentrated enzyme solutions. The adsorption was studied in situ by means of null ellipsometry. Moreover, both enzyme-modified and bare Au electrodes were investigated in detail by atomic force microscopy (AFM) as well as electrochemically. When adsorbed from dilute solutions (0.125 and 0.25 mg mL?¹ in the cases of ThLc and MvBOx, respectively), the amounts of enzyme per unit area were determined to be ca. 1.7 and 4.8 pmol cm?², whereas the protein film thicknesses were determined to be 29 and 30 Å for ThLc and MvBOx, respectively. A well-pronounced bioelectrocatalytic reduction of molecular oxygen (O?) was observed on MvBOx/Au biocathodes, whereas this was not the case for ThLc-modified Au electrodes (i.e., adsorbed ThLc was catalytically inactive). The initially observed apparent k(cat)(app) values for adsorbed MvBOx and the enzyme in solution were found to be very close to each other (viz. 54 and 58 s?¹, respectively (pH 7.4, 25 °C)). However, after 3 h of operation of MvBOx/Au biocathodes, kcatapp dropped to 23 s?¹. On the basis of the experimental results, conformational changes of the enzymes (in all likelihood, their flattening on the Au surface) were suggested to explain the deactivation of MCOs on the bare Au electrodes. PMID:24564218

Pankratov, Dmitry; Sotres, Javier; Barrantes, Alejandro; Arnebrant, Thomas; Shleev, Sergey

2014-03-18

42

Characterization of Laccase-like Multicopper Oxidases (LMCOs) in Arabidopsis thaliana  

SciTech Connect

Laccase-like multicopper oxidases (LMCOs) have repeatedly been associated with the process of lignification in plants, and previous work suggested that these enzymes might be acting as specific marker for highly localized, small-scale lignification events in tissues not typically thought of as lignified. However, plant LMCOs typically occur as members of gene families and different family members can display disparate enzyme activities and overlapping patterns of expression in bulk tissues. This study used reporter genes and knockout mutants to document the involvement of a specific Arabidopsis thaliana LMCO family member (At2g30210 ) in early root development, specifically with development of endodermal tissues. Expression of the gene product was found to be under the control of sucrose levels, but the gene also responded to fluctuations in salt concentrations. The expression patterns of this gene were consistent with its involvement in the formation of suberin in the Casparian strip of root endodermis. An additional LMCO (At5g58910) displayed a more generalized expression in the radicles emergent seedlings. Additional members of the Arabidopsis LMCO family (At2g29130, At5g01190, and At5g05390) were also investigated with reporter gene constructs and knockout mutants. Expression of these LMCOs was associated with lignifying xylem, and the genes had over-lapping expression. Single knockout mutants did not display obvious phenotypes, suggesting that the gene products might have degenerate functionality that could compensate for loss of a single LMCO function.

Jeffrey F.D. Dean

2008-06-09

43

Induction and Transcriptional Regulation of Laccases in Fungi  

PubMed Central

Fungal laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet.

Piscitelli, Alessandra; Giardina, Paola; Lettera, Vincenzo; Pezzella, Cinzia; Sannia, Giovanni; Faraco, Vincenza

2011-01-01

44

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

PubMed

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

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

2014-09-15

45

Engineering Klebsiella sp. 601 multicopper oxidase enhances the catalytic efficiency towards phenolic substrates  

PubMed Central

Background Structural comparison between bacterial CueO and fungal laccases has suggested that a charged residue Glu (E106) in CueO replaces the corresponding residue Phe in fungal laccases at the gate of the tunnel connecting type II copper to the protein surface and an extra ?-helix (L351-G378) near the type I copper site covers the substrate binding pocket and might compromise the electron transfer from substrate to type I copper. To test this hypothesis, several mutants were made in Klebsiella sp. 601 multicopper oxidase, which is highly homologous to E. coli CueO with a similarity of 90% and an identity of 78%. Results The E106F mutant gave smaller Km (2.4-7fold) and kcat (1-4.4 fold) values for all three substrates DMP, ABTS and SGZ as compared with those for the wild-type enzyme. Its slightly larger kcat/Km values for three substrates mainly come from the decreased Km. Deleting ?-helix (L351-G378) resulted in the formation of inactive inclusion body when the mutant ??351-378 was expressed in E. coli. Another mutant ?351-380M was then made via substitution of seven amino acid residues in the ?-helix (L351-G378) region. The ?351-380M mutant was active, and displayed a far-UV CD spectrum markedly different from that for wild-type enzyme. Kinetic studies showed the ?351-380M mutant gave very low Km values for DMP, ABTS and SGZ, 4.5-, 1.9- and 7-fold less than those for the wild type. In addition, kcat/Km values were increased, 9.4-fold for DMP, similar for ABTS and 3-fold for SGZ. Conclusion The Glu residue at position 106 appears not to be the only factor affecting the copper binding, and it may also play a role in maintaining enzyme conformation. The ?-helix (L351-G378) may not only block access to the type I copper site but also play a role in substrate specificities of bacterial MCOs. The ?351-380M mutant catalyzing oxidation of the phenolic substrate DMP effectively would be very useful in green chemistry.

2011-01-01

46

Engineering and Applications of fungal laccases for organic synthesis  

PubMed Central

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.

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

2008-01-01

47

Using planktonic microorganisms to supply the unpurified multi-copper oxidases laccase and copper efflux oxidases at a biofuel cell cathode.  

PubMed

The feasibility to apply crude culture supernatants that contain the multicopper oxidases laccase or copper efflux oxidase (CueO) as oxygen reducing catalysts in a biofuel cell cathode is shown. As enzyme-secreting recombinant planktonic microorganisms, the yeast Yarrowia lipolytica and the bacterium Escherichia coli were investigated. The cultivation and operation conditions (choice of medium, pH) had distinct effects on the electro-catalytic performance. The highest current density of 119±23?Acm(-2) at 0.400V vs. NHE was obtained with the crude culture supernatant of E. coli cells overexpressing CueO and tested at pH 5.0. In comparison, at pH 7.4 the electrode potential at 100?Acm(-2) is 0.25V lower. Laccase-containing supernatants of Y. lipolytica yielded a maximum current density of 6.7±0.4?Acm(-2) at 0.644V vs. NHE. These results open future possibilities to circumvent elaborate enzyme purification procedures and realize cost effective and easy-to-operate enzymatic biofuel cells. PMID:24607459

Sané, Sabine; Richter, Katrin; Rubenwolf, Stefanie; Matschke, Nina Joan; Jolivalt, Claude; Madzak, Catherine; Zengerle, Roland; Gescher, Johannes; Kerzenmacher, Sven

2014-04-01

48

Diversity of Two-Domain Laccase-Like Multicopper Oxidase Genes in Streptomyces spp.: Identification of Genes Potentially Involved in Extracellular Activities and Lignocellulose Degradation during Composting of Agricultural Waste.  

PubMed

Traditional three-domain fungal and bacterial laccases have been extensively studied for their significance in various biotechnological applications. Growing molecular evidence points to a wide occurrence of more recently recognized two-domain laccase-like multicopper oxidase (LMCO) genes in Streptomyces spp. However, the current knowledge about their ecological role and distribution in natural or artificial ecosystems is insufficient. The aim of this study was to investigate the diversity and composition of Streptomyces two-domain LMCO genes in agricultural waste composting, which will contribute to the understanding of the ecological function of Streptomyces two-domain LMCOs with potential extracellular activity and ligninolytic capacity. A new specific PCR primer pair was designed to target the two conserved copper binding regions of Streptomyces two-domain LMCO genes. The obtained sequences mainly clustered with Streptomyces coelicolor, Streptomyces violaceusniger, and Streptomyces griseus. Gene libraries retrieved from six composting samples revealed high diversity and a rapid succession of Streptomyces two-domain LMCO genes during composting. The obtained sequence types cluster in 8 distinct clades, most of which are homologous with Streptomyces two-domain LMCO genes, but the sequences of clades III and VIII do not match with any reference sequence of known streptomycetes. Both lignocellulose degradation rates and phenol oxidase activity at pH 8.0 in the composting process were found to be positively associated with the abundance of Streptomyces two-domain LMCO genes. These observations provide important clues that Streptomyces two-domain LMCOs are potentially involved in bacterial extracellular phenol oxidase activities and lignocellulose breakdown during agricultural waste composting. PMID:24657870

Lu, Lunhui; Zeng, Guangming; Fan, Changzheng; Zhang, Jiachao; Chen, Anwei; Chen, Ming; Jiang, Min; Yuan, Yujie; Wu, Haipeng; Lai, Mingyong; He, Yibin

2014-06-01

49

Enhanced enzymatic hydrolysis of rice straw by removal of phenolic compounds using a novel laccase from yeast Yarrowia lipolytica.  

PubMed

An extracellular laccase-producing yeast was isolated from soil and identified as Yarrowia lipolytica by its morphology and by comparison of its internal transcribed spacer rDNA gene sequence. Extracellular laccase (YlLac) from Y. lipolytica was purified to homogeneity by anion-exchange and gel filtration chromatography. YlLac is a monomeric glycoprotein with 14% carbohydrate content and a molecular mass of 67kDa. It showed a higher catalytic efficiency towards 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (k(cat)/K(m)=19.3s(-1)?M(-1)) and 2,6-dimethoxyphenol (k(cat)/K(m)=13s(-1)?M(-1)) than any other reported laccase. This enzyme was able to oxidize phenolic compounds present in pretreated rice straw. Several parameters (temperature, enzyme concentration, and mediator compounds) to enhance removal of phenolic compounds from pretreated rice straw were optimized using response surface methodology. The use of YlLac for the removal of cellulase inhibitory compounds from biomass slurries was found to be a promising approach for improving the efficiency of biorefineries. PMID:22960123

Lee, Kyoung-Mi; Kalyani, Dayanand; Tiwari, Manish Kumar; Kim, Tae-Su; Dhiman, Saurabh Sudha; Lee, Jung-Kul; Kim, In-Won

2012-11-01

50

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

PubMed

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

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

2014-04-01

51

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

Microsoft Academic Search

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

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

2008-01-01

52

Phenol oxidase is a necessary enzyme for the silkworm molting which is regulated by molting hormone.  

PubMed

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. The molting process includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis excoriation etc. Polyphenol oxidases (PPOs), dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the phenol oxidase was considered as an enzyme for epidermal layer's tanning and melanization. This work suggested that polyphenol oxidases are one set of the key enzymes in molting, which closely related with the role of ecdysone in regulation of molting processes. The data showed that the expression peak of phenol oxidase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the phenol oxidase expression was significantly elevated compared to the control. PPO1 RNAi induced phenol oxidase expression obviously declined in the silkworm larvae, and caused the pupae incomplete pupation. Overall, the results described that the phenol oxidase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting. PMID:23275200

Wang, Mei-xian; Lu, Yan; Cai, Zi-zheng; Liang, Shuang; Niu, Yan-shan; Miao, Yun-gen

2013-05-01

53

Oxidative biodegradation of phosphorothiolates by fungal laccase  

Microsoft Academic Search

Organophosphorus (OP) insecticides and nerve agents that contain P-S bond are relatively more resistant to enzymatic hydrolysis. Purified phenol oxidase (laccase) from the white rot fungus Pleurotus ostreatus (Po) together with the mediator 2,2?-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) displayed complete and rapid oxidative degradation of the nerve agents VX and Russian VX (RVX) and the insecticide analog diisopropyl-Amiton with specific activity: ksp=2200, 667

G Amitai; R Adani; G Sod-Moriah; I Rabinovitz; A Vincze; H Leader; B Chefetz; L Leibovitz-Persky; D Friesem; Y Hadar

1998-01-01

54

[Thermostabilities of plant phenol oxidase and peroxidase, determining the technology of their use in food industry].  

PubMed

Stabilities of phenol oxidase and peroxidase from tea plant (Camellia sinensis L.) clone Kolkhida leaves, apple (Malus domestica L.) cultivar Kekhura fruits, walnut (Juglans regia L.) green pericarp, and horseradish (Armoracia lapathifolia Gilib) roots were studied using different storage temperature modes and storage duration. It was demonstrated that both enzymes retained residual activities (approximately 10%) upon 20-min incubation at 80 degrees C. Phenol oxidases from tea, walnut, and, especially, apple, as well as tea peroxidase were stable during storage. A technology for treatment of plant oxidases was proposed, based on the use of a natural inhibitor phenol oxidase and peroxidase, isolated from tea leaves, which solving the problem of residual activities of these enzymes, arising during pasteurization and storage of beverages and juices. It was demonstrated that browning of apple juice during pasteurization and beer turbidity during storage could be efficiently prevented using the natural inhibitor of these enzymes. PMID:15859458

Mchedlishvili, N I; Omiadze, N T; Gulua, L K; Sadunishvili, T A; Zamtaradze, R K; Abutidze, M O; Bendeliani, E G; Kvesitadze, G I

2005-01-01

55

Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes.  

PubMed

A new full enzymatic fuel cell was built and characterized. Both enzymatic electrodes were molecularly oriented to enhance the direct electron transfer between the enzyme active site and the electrode surface. The anode consisted in immobilized alcohol oxidase on functionalized carbon nanotubes with 4-azidoaniline, which acts as active-site ligand to orientate the enzyme molecule. The cathode consisted of immobilized laccase on functionalized graphite electrode with 4-(2-aminoethyl) benzoic acid. The enzymatic fuel cell reaches 0.5V at open circuit voltage with both, ethanol and methanol, while in short circuit the highest current intensity of 250?Acm(-2) was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60?Wcm(-2), while with ethanol 40?Wcm(-2) was obtained. PMID:24953844

Arrocha, Andrés A; Cano-Castillo, Ulises; Aguila, Sergio A; Vazquez-Duhalt, Rafael

2014-11-15

56

Laccases: blue enzymes for green chemistry.  

PubMed

Laccases are oxidoreductases belonging to the multinuclear copper-containing oxidases; they catalyse the monoelectronic oxidation of substrates at the expense of molecular oxygen. Interest in these essentially "eco-friendly" enzymes--they work with air and produce water as the only by-product--has grown significantly in recent years: their uses span from the textile to the pulp and paper industries, and from food applications to bioremediation processes. Laccases also have uses in organic synthesis, where their typical substrates are phenols and amines, and the reaction products are dimers and oligomers derived from the coupling of reactive radical intermediates. Here, we provide a brief discussion of this interesting group of enzymes, increased knowledge of which will promote laccase-based industrial processes in the future. PMID:16574262

Riva, Sergio

2006-05-01

57

Synthesis and characterization of combined cross-linked laccase and tyrosinase aggregates transforming acetaminophen as a model phenolic compound in wastewaters.  

PubMed

Laccase (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1) are ubiquitous enzymes present in nature as they are known to originate from bacteria, fungi, plants, etc. Both laccase and tyrosinase are copper-containing phenoloxidases requiring readily available O2 without auxiliary cofactor for their catalytic transformation of numerous phenolic substrates. In the present study, laccase and tyrosinase have been insolubilized as combined crosslinked enzyme aggregates (combi-CLEA) using chitosan, a renewable and biodegradable polymer, as crosslinker. The combi-CLEA, with specific activity of 12.3U/g for laccase and 167.4U/g for tyrosinase, exhibited high enzymatic activity at pH5-8 and temperature at 5-30°C, significant resistance to denaturation and no diffusional restriction to its active site based upon the Michaelis-Menten kinetic parameters. Subsequently, the combi-CLEA was applied to the transformation of acetaminophen as a model phenolic compound in samples of real wastewaters in order to evaluate the potential efficiency of the biocatalyst. In batch mode the combi-CLEA transformed more than 80% to nearly 100% of acetaminophen from the municipal wastewater and more than 90% from the hospital wastewater. UPLC-MS analysis of acetaminophen metabolites showed the formation of its oligomers as dimers, trimers and tetramers due to the laccase and 3-hydroxyacetaminophen due to the tyrosinase. PMID:24867811

Ba, Sidy; Haroune, Lounes; Cruz-Morató, Carles; Jacquet, Chloé; Touahar, Imad E; Bellenger, Jean-Phillipe; Legault, Claude Y; Jones, J Peter; Cabana, Hubert

2014-07-15

58

An artificial di-iron oxo-protein with phenol oxidase activity.  

PubMed

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site. PMID:19915535

Faiella, Marina; Andreozzi, Concetta; de Rosales, Rafael Torres Martin; Pavone, Vincenzo; Maglio, Ornella; Nastri, Flavia; DeGrado, William F; Lombardi, Angela

2009-12-01

59

An artificial di-iron oxo-protein with phenol oxidase activity  

PubMed Central

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.

Faiella, Marina; Andreozzi, Concetta; de Rosales, Rafael Torres Martin; Pavone, Vincenzo; Maglio, Ornella; Nastri, Flavia; DeGrado, William F; Lombardi, Angela

2013-01-01

60

Phenol oxidases production and wood degradation by a thermophilic fungus Thermoascus aurantiacus  

SciTech Connect

The ability of a Brazilian strain of Thermoascus aurantiacus, a thermophilic fungus, to produce extracellular phenol oxidases and to degrade Eucalyptus grandis sawdust was studied. T. aurantiacus was capable of good growth in liquid culture containing 1.5% (w/v) of various lignocellulosic substrates (sugar cane bagasse, rice hulls, and chips and sawdust of E. grandis) plus 5 mg/mL of glucose. When lignocellulosic substrates were used, enzymes involved in cellulose and hemicellulose metabolism were stimulated in T. aurantiacus. It was also found that these substrates have an inductive effect on phenol oxidase production. The most effective inducer of phenol oxidase activity was E. grandis sawdust, which led to the production of 0.80 U/mL (o-dianisidine oxidation) on day 12. Low phenol oxidase activity was observed at cultures when only glucose was used. Cultures of T. aurantiacus also exhibited cellobiose-quinone oxidoreductase activity when lignocellulosic materials were used as substrate. However, under the experimental conditions, lignin peroxidase activity was not detected. E. grandis sawdust supplemented with 5 mg/mL of glucose suffered a total weight loss of 6.7% accompanied by 15% lignin loss and 64.4% extractive loss after 21 d incubation with T. aurantiacus. 31 refs., 1 fig., 3 tabs.

Machuca, A.; Duran, N. (Universidade Estadual de Campinas (Brazil))

1993-10-01

61

Fungal laccases and their applications in bioremediation.  

PubMed

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

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

2014-01-01

62

Fungal Laccases and Their Applications in Bioremediation  

PubMed Central

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.

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

2014-01-01

63

New oxidase from Bjerkandera arthroconidial anamorph that oxidizes both phenolic and nonphenolic benzyl alcohols.  

PubMed

A new flavooxidase is described from a Bjerkandera arthroconidial anamorph. Its physicochemical characteristics, a monomeric enzyme containing non-covalently bound flavin adenine dinucleotide (FAD), and several catalytic properties, such as oxidation of aromatic and polyunsaturated aliphatic primary alcohols, are similar to those of Pleurotus eryngii aryl-alcohol oxidase (AAO). However, it also efficiently oxidizes phenolic benzyl and cinnamyl alcohols that are typical substrates of vanillyl-alcohol oxidase (VAO), a flavooxidase from a different family, characterized by its multimeric nature and presence of covalently-bound FAD. The enzyme also differs from P. eryngii AAO by having extremely high efficiency oxidizing chlorinated benzyl alcohols (1000-1500 s(-1) mM(-1)), a feature related to the different alcohol metabolites secreted by the Pleurotus and Bjerkandera species including chloroaromatics, and higher activity on aromatic aldehydes. What is even more intriguing is the fact that, the new oxidase is optimally active at pH 6.0 on both p-anisyl and vanillyl alcohols, suggesting a mechanism for phenolic benzyl alcohol oxidation that is different from that described in VAO, which proceeds via the substrate phenolate anion formed at basic pH. Based on the above properties, and its ADP-binding motif, partially detected after N-terminus sequencing, the new enzyme is classified as a member of the GMC (glucose-methanol-choline oxidase) oxidoreductase family oxidizing both AAO and VAO substrates. PMID:19110079

Romero, Elvira; Ferreira, Patricia; Martínez, Angel T; Martínez, María Jesús

2009-04-01

64

Thermotolerant and thermostable laccases.  

PubMed

Laccases are phenol-oxidizing, usually four-copper containing metalloenzymes. For industrial and biotechnological purposes, laccases were among the first fungal oxidoreductases providing larger-scale applications such as removal of polyphenols in wine and beverages, conversion of toxic compounds and textile dyes in waste waters, and in bleaching and removal of lignin from wood and non-wood fibres. In order to facilitate novel and more efficient bio-catalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability and thermotolerance. This review gives a current overview on the sources and characteristics of such laccases, with particular emphasis on the fungal enzymes. PMID:19360388

Hildén, Kristiina; Hakala, Terhi K; Lundell, Taina

2009-08-01

65

Changes in phenol oxidase and peroxidase levels in cocoyam tubers of different postharvest ages infected by Sclerotium rolfsii sacc.  

PubMed

Crude aqueous extracts from the peripheral rot zone of cocoyam tubers infected by Sclerotium rolfsii sacc were shown to be inhibitory to dialysed in vivo polygalacturonase (PG) of the pathogen. The PG inhibitory action, phenol oxidase and peroxidase activities were higher in cocoyam tubers of the Xanthosoma sagittifolium varieties than in those of the Coolocasia esculenta varieties. The levels of phenol oxidase, peroxidase and PG inhibitory activities also decreased as the postharvest age of the tubers increased. PMID:8975142

Ohazurike, N C; Arinze, A E

1996-02-01

66

Study of enzymatic properties of phenol oxidase from nitrogen-fixing azotobacter chroococcum  

Microsoft Academic Search

Azotobacter chroococcum is a widespread free-living soil bacterium within the genus of Azotobacter known for assimilation of atmospheric nitrogen and subsequent conversion into nitrogenous compounds, which henceforth enrich\\u000a the nitrogen content of soils. A. chroococcum SBUG 1484, isolated from composted earth, exhibits phenol oxidase (PO) activity when growing under nitrogen-fixing conditions.\\u000a In the present study we provide incipient analysis of

Susanne Herter; Marlen Schmidt; Mark L Thompson; Annett Mikolasch; Frieder Schauer

2011-01-01

67

Polyphenol oxidase activity, phenolic acid composition and browning in cashew apple ( Anacardium occidentale, L.) after processing  

Microsoft Academic Search

This study describes the extraction and characterisation of cashew apple polyphenol oxidase (PPO) and the effect of wounding on cashew apple phenolic acid composition, PPO activity and fruit browning. Purification factor was 59 at 95% (NH4)2SO4 saturation. For PPO activity, the optimal substrate was catechol and the optimum pH was 6.5. PPO Km and Vmax values were 18.8mM and 13.6Umin?1ml?1,

Christiane Queiroz; Antonio Jorge Ribeiro da Silva; Maria Lúcia Mendes Lopes; Eliane Fialho; Vera Lúcia Valente-Mesquita

2011-01-01

68

Thermostabilities of plant phenol oxidase and peroxidase determining the technology of their use in the food industry  

Microsoft Academic Search

Stabilities of phenol oxidase and peroxidase from tea plant (Camellia sinensis L.) clone Kolkhida leaves, apple (Mallus domestica L.) cultivar Kekhura fruits, walnut (Juglans regia L.) green pericarp, and horseradish (Armoracia lapathifolia Gilib) roots were studied using different storage temperature modes and storage duration. It was demonstrated that both enzymes retained residual activities (~10%) upon 20-min incubation at 8°C. Phenol

N. I. Mchedlishvili; N. T. Omiadze; L. K. Gulua; T. A. Sadunishvili; R. K. Zamtaradze; M. O. Abutidze; E. G. Bendeliani; G. I. Kvesitadze

2005-01-01

69

Structure of native laccase B from Trametes sp. AH28-2  

PubMed Central

Fungal laccases are oxidoreductases that belong to the multinuclear copper-containing oxidases. They are able to oxidize a wide range of substrates, preferably phenolic compounds, which makes them suitable for employment in the bioremediation of soil and water as well as in other biotechnological applications. Here, the structural analysis of natural laccase B (LacB) from Trametes sp. AH28-2 is presented. This structure provides the opportunity to study the natural post-translational modifications of the enzyme. The overall fold shows a high homology to those of previously analyzed laccases with known three-dimensional structure. However, LacB contains a new structural element, a protruding loop near the substrate-binding site, compared with the previously reported laccase structures. This unique structural feature may be involved in modulation of the substrate recognition of LacB.

Ge, Honghua; Gao, Yongxiang; Hong, Yuzhi; Zhang, Min; Xiao, Yazhong; Teng, Maikun; Niu, Liwen

2010-01-01

70

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

PubMed Central

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.

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

2007-01-01

71

Characterization of combined cross-linked enzyme aggregates from laccase, versatile peroxidase and glucose oxidase, and their utilization for the elimination of pharmaceuticals.  

PubMed

In order to transform a wide range of pharmaceutically active compounds (PhACs), the three oxidative enzymes laccase (Lac) from Trametes versicolor, versatile peroxidase (VP) from Bjerkandera adusta and glucose oxidase (GOD) from Aspergillus niger were concomitantly cross-linked after aggregation, thus, making a combined cross-linked enzyme aggregate (combi-CLEA) that was versatile and involved in an enzymatic cascade reaction. From the initial enzymes about 30% of initial laccase activity was recovered along with 40% for each of VP and GOD. The combi-CLEA showed good results in conditions close to those of real wastewater (neutral pH and medium temperature) as well as a good ability to resist to denaturing conditions such as high temperature (60°C) and low pH (3). Batch experiments were realized to test the free enzyme's ability to degrade, a PhACs cocktail, mainly in a synthetic wastewater containing acetaminophen, naproxen, mefenamic acid, indometacin, diclofenac, ketoprofen, caffeine, diazepam, ciprofloxacin, trimethoprim, fenofibrate and bezafibrate, carbamazepine and its by-product 10-11 epoxy-carbamazepine. High removal was achieved (more than 80%) for the five first compounds. Then, the elimination ability of the combi-CLEA with or without hydrogen peroxide, glucose or manganese sulfate was determined. Globally, our results demonstrated that VP has a wider removal spectrum than Lac. These removal features are enhanced under more specific conditions, whereas the combi-CLEA combined advantages of both VP and laccase. Finally, the elimination of PhACs in a municipal wastewater treatment plant effluent using the combi-CLEA was marginally investigated. Concentrations of most of the selected PhACs were below the limit of quantification (lower than 20ng/L) except for acetaminophen. Its combi-CLEA-mediated removal reached up to 25%. PMID:24589758

Touahar, Imad E; Haroune, Lounès; Ba, Sidy; Bellenger, Jean-Phillipe; Cabana, Hubert

2014-05-15

72

[Nature of the brown pigment and the composition of the phenol oxidases of Streptomyces galbus].  

PubMed

The culture of Streptomyces galbus ISP-5089 has a yellow-green colour caused by the accumulation of actinomycin X when it is grown in synthetic media at 28 degrees C; the colour turns dark-brown at 42 degrees C due to the synthesis of melanoid pigments. The population composition does not undergo any noticeable changes in that case, an no specific melanin-synthesizing mutants appear as a result of autoselection . The biosynthesis of actinomycin X (at 28 degrees C) and melanoid pigments (at 42 degrees C) is regulated by temperature. At 42 degrees C, L-DOPA oxidase is synthesized and laccase is activated; these two enzymes are involved in the synthesis of melanoid pigments. The organism does not has tyrosinase. The synthesis of melanoid pigments, when the mesophilic culture of S. galbus ISP-5089 is grown in the regime of superoptimal temperatures (42 to 47 degrees C), may be considered as a protective ecological reaction of the organism to unfavourable conditions of the environment. PMID:6204187

Kuznetsov, V D; Filippova, S N; Rybakova, A M

1984-01-01

73

A comparison of glucose oxidase and aldose dehydrogenase as mediated anodes in printed glucose/oxygen enzymatic fuel cells using ABTS/laccase cathodes.  

PubMed

Current generation by mediated enzyme electron transfer at electrode surfaces can be harnessed to provide biosensors and redox reactions in enzymatic fuel cells. A glucose/oxygen enzymatic fuel cell can provide power for portable and implantable electronic devices. High volume production of enzymatic fuel cell prototypes will likely require printing of electrode and catalytic materials. Here we report on preparation and performance of, completely enzymatic, printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks, enzyme and mediator. A comparison of cell performance using a range of mediators for either glucose oxidase (GOx) or aldose dehydrogenase (ALDH) oxidation of glucose at the anode and ABTS and a fungal laccase, for reduction of oxygen at the cathode, is reported. Highest power output, although of limited stability, is observed for ALDH anodes mediated by an osmium complex, providing a maximum power density of 3.5 ?W cm(-2) at 0.34 V, when coupled to a laccase/ABTS cathode. The stability of cell voltage in a biobattery format, above a threshold of 200 mV under a moderate 75 k? load, is used to benchmark printed fuel cell performance. Highest stability is obtained for printed fuel cells using ALDH, providing cell voltages over the threshold for up to 74 h, compared to only 2 h for cells with anodes using GOx. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells. PMID:22200380

Jenkins, Peter; Tuurala, Saara; Vaari, Anu; Valkiainen, Matti; Smolander, Maria; Leech, Dónal

2012-10-01

74

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

Microsoft Academic Search

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. MnII was not required for catalysis by TAP. The optimum pH for

T. Johjima; M. Ohkuma; T. Kudo

2003-01-01

75

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

PubMed Central

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.

2013-01-01

76

On the reactions of two fungal laccases differing in their redox potential with lignin model compounds: products and their rate of formation.  

PubMed

Laccases (EC 1.10.3.2) are multicopper oxidases able to oxidize phenolic compounds such as lignin-related polyphenols. Since the discovery that so-called mediators effectively extend the family of laccase substrates, direct interactions between lignin-like materials and laccase have gained much less attention. In this work, the aim was to characterize oxidation products formed in direct laccase-catalyzed oxidation of different guaiacylic and syringylic lignin model compounds with two different laccases: a low redox potential Melanocarpus albomyces laccase and a high redox potential Trametes hirsuta laccase. By following the formation of different, mainly biphenylic (5-5) and benzylic oxidation products, it was found that although both of these enzymes generated practically the same pattern of products with particular types of syringyl and guaiacyl compounds, in some cases a clear difference in the rates of their formation was observed. The results also confirm further to the suggestions that syringylic compounds are able to act as mediators in their own oxidation reactions and also that in some instances acetylation of phenolic material may produce altered, unexpected structures. PMID:19702333

Lahtinen, Maarit; Kruus, Kristiina; Heinonen, Petri; Sipilä, Jussi

2009-09-23

77

Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of native and type-2-copper-depleted Vietnamese-lacquer-tree and Japanese-lacquer-tree laccases.  

PubMed Central

The interactions of one-electron reduced metronidazole (ArNO2.-) and O2.- with native and Type-2-copper-depleted Vietnamese- and Japanese-lacquer-tree laccases were studied in aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. On reaction with ArNO2.-, in the absence of O2, the holo- and the Type-2-copper-depleted proteins accept, with reduction of Type 1 copper, 2 and 1 reducing equivalents respectively. On reaction with O2.- of both holo- and Type-2-copper-depleted Vietnamese-lacquer-tree laccase, almost complete reduction of Type 1 copper was observed and, after completion of the reaction, some (less than 20%) reoxidation of Type 1 copper occurs. Reduction of Type 1 copper of the laccases by these one-electron donors occurs via a bimolecular step; however, the rate of reduction of Vietnamese-lacquer-tree laccase is over 10 times that of Japanese-lacquer-tree laccase. It is inferred that electrons enter the protein via Type 1 copper with, in the case of the holoprotein, subsequent rapid intramolecular transfer of 1 reducing equivalent within the protein. Furthermore it is suggested that intra-molecular electron transfer to Type 3 copper atoms is slow and, in the case of Type-2-copper-depleted protein, may not occur. This slow process may partially account for the variation of the catalytic activities of 'blue' oxidases.

O'Neill, P; Fielden, E M; Morpurgo, L; Agostinelli, E

1984-01-01

78

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

PubMed Central

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.

2011-01-01

79

Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae  

PubMed Central

Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae. We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, N-acetyldopamine (NADA), N-?-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone) and one non-phenolic substrate (2,2?-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 – 550 min?1 mM?1. These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 – 30 min?1 mM?1; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min?1 mM?1. We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo, and additional experiments will be required to discover any isoform-specific functions.

Gorman, Maureen J.; Sullivan, Lucinda I.; Nguyen, Thi D. T.; Dai, Huaien; Arakane, Yasuyuki; Dittmer, Neal T.; Syed, Lateef U.; Li, Jun; Hua, Duy H.; Kanost, Michael R.

2011-01-01

80

Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris.  

PubMed

Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae alpha-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated. PMID:10712591

Otterbein, L; Record, E; Longhi, S; Asther, M; Moukha, S

2000-03-01

81

Study of enzymatic properties of phenol oxidase from nitrogen-fixing Azotobacter chroococcum  

PubMed Central

Azotobacter chroococcum is a widespread free-living soil bacterium within the genus of Azotobacter known for assimilation of atmospheric nitrogen and subsequent conversion into nitrogenous compounds, which henceforth enrich the nitrogen content of soils. A. chroococcum SBUG 1484, isolated from composted earth, exhibits phenol oxidase (PO) activity when growing under nitrogen-fixing conditions. In the present study we provide incipient analysis of the crude PO activity expressed by A. chroococcum SBUG 1484 within comparative analysis to fungal crude PO from the white-rot fungus Pycnoporus cinnabarinus SBUG-M 1044 and tyrosinase (PPO) from the mushroom Agaricus bisporus in an attempt to reveal desirable properties for exploitation with future recombinant expression of this enzyme. Catalytic activity increased with pre-incubation at 35°C; however 70% of activity remained after pre-treatment at 50°C. Native A. chroococcum crude PO exhibited not only strong preference for 2,6-dimethoxyphenol, but also towards related methoxy-activated substrates as well as substituted ortho-benzenediols from over 40 substrates tested. Presence of CuSO4 enhanced crude phenol oxidase activity up to 30%, whereas NaN3 (0.1 mM) was identified as the most inhibiting substance of all inhibitors tested. Lowest inhibition of crude PO activity occurred after 60 minutes of incubation in presence of 15% methanol and ethanol with 63% and 77% remaining activities respectively, and presence of DMSO even led to increasing oxidizing activities. Substrate scope and inhibitor spectrum strongly differentiated A. chroococcum PO activity comprised in crude extracts from those of PPO and confirmed distinct similarities to fungal PO.

2011-01-01

82

Laccases from Aureobasidium pullulans.  

PubMed

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

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

2013-06-10

83

An evidence of laccases in archaea  

Microsoft Academic Search

Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are a diverse group of multicopper oxidases that catalyze the oxidation\\u000a of a variety of aromatic compounds. Here we present evidence for distribution of laccases among archaea and their probable\\u000a functions. Putative laccase genes have been found in different archaeal groups that might have branched off early during evolution,\\u000a e.g. Haloarcula marismortui ATCC 43049, Natronomonas

Krishna Kant Sharma; Ramesh Chander Kuhad

2009-01-01

84

A highly stable biosensor for phenols prepared by immobilizing polyphenol oxidase into polyaniline–polyacrylonitrile composite matrix  

Microsoft Academic Search

A novel biosensor for phenols was constructed by immobilizing polyphenol oxidase (PPO) into polyaniline–polyacrylonitrile composite matrix. The sensing film was prepared by electropolymerization of aniline into polyacrylonitrile (PAN)-coated platinum electrode in the presence of PPO. The scanning electron micrographs (SEM) showed that PAN had microporous structure and polyaniline and the enzyme could co-intercalated into PAN matrix. The obtained biosensor exhibited

Huaiguo Xue; Zhiquan Shen

2002-01-01

85

Low-Density Lipoprotein Antioxidant Activity of Phenolic Compounds and Polyphenol Oxidase Activity in Selected Clingstone Peach Cultivars  

Microsoft Academic Search

The antioxidant potential of eight clingstone peach cultivars was investigated by determining phenolic compounds and inhibition of low-density lipoprotein (LDL) oxidation. Cultivars low in polyphenol oxidase (PPO) were also selected to minimize enzymatic browning. Inhibition of LDL oxidation varied from 17.0 to 37.1% in peach flesh extract, from 15.2 to 49.8% in whole peach extract, and from 18.2 to 48.1%

Susan Chang; Christine Tan; Edwin N. Frankel; Diane M. Barrett

2000-01-01

86

Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin.  

PubMed Central

Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.

Kawabata, T; Yasuhara, Y; Ochiai, M; Matsuura, S; Ashida, M

1995-01-01

87

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

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

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

2012-09-10

88

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

PubMed

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

Feng, Bao Zhen; Li, Peiqian

2014-01-01

89

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

PubMed Central

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.

Feng, Bao Zhen; Li, Peiqian

2014-01-01

90

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

PubMed

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

Johjima, T; Ohkuma, M; Kudo, T

2003-05-01

91

Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilum  

PubMed Central

Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8?Å resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P21 and contained one tetramer per asymmetric unit.

Sutay Kocabas, Didem; Pearson, Arwen R.; Phillips, Simon E. V.; Bakir, Ufuk; Ogel, Zumrut B.; McPherson, Michael J.; Trinh, Chi H.

2009-01-01

92

Nucleotide sequence of the cDNA encoding the proenzyme of phenol oxidase A1 of Drosophila melanogaster.  

PubMed Central

Clones encoding pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] A1 were isolated from a lambda gt10 library that originated from Drosophila melanogaster strain Oregon-R male adults. The 2294 bp of the cDNA included a 13-bp 5'-noncoding region, a 2070-bp encoding open reading frame of 690 amino acids, and a 211-bp 3'-noncoding region. A hydrophobic NH2-terminal sequence for a signal peptide is absent in the protein. Furthermore, there are six potential N-glycosylation sites in the sequence, but no amino sugar was detected in the purified protein by amino acid analysis, indicating the lack of an N-linked sugar chain. The potential copper-binding sites, amino acids 200-248 and 359-414, are highly homologous to the corresponding sites of hemocyanin of the tarantula Eurypelma californicum, the horseshoe crab Limulus polyphemus, and the spiny lobster Panulirus interruptus. On the basis of the phylogenetic tree constructed by the neighbor-joining method, vertebrate tyrosinases and molluscan hemocyanins constitute one family, whereas pro-POs and arthropod hemocyanins group with another family. It seems, therefore, likely that pro-PO originates from a common ancestor with arthropod hemocyanins, independently to the vertebrate and microbial tyrosinases.

Fujimoto, K; Okino, N; Kawabata, S; Iwanaga, S; Ohnishi, E

1995-01-01

93

Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning.  

PubMed Central

Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two bands with an apparent M(r) of approximately 100,000 on SDS/PAGE, as well as size-exclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of approximately 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. Sexta pro-PO, together with other arthropod pro-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4. Images Fig. 2

Hall, M; Scott, T; Sugumaran, M; Soderhall, K; Law, J H

1995-01-01

94

Optimization of recombinant fungal laccase production with strains of the yeast Kluyveromyces lactis from the pyruvate decarboxylase promoter.  

PubMed

Laccases are multicopper oxidases of wide specificity that catalyze the oxidation of phenolic and related compounds using molecular oxygen as the electron acceptor. Here, we report the production of the Lcc1 laccase of the fungus Trametes trogii in strains of the yeast Kluyveromyces lactis, using the pyruvate decarboxylase promoter (KlPDC1) as an expression system. We assayed laccase production in various strains, with replicative and integrative transformants and with different cultivation parameters. A comparison with Lcc1 enzymes from other yeasts and from the original organism was also performed. The best production conditions were obtained with integrative transformants of an individual strain, whereas cultivation conditions were less stringent than the use of the regulated KlPDC1 promoter could anticipate. The secreted recombinant laccase showed better enzyme properties than the native enzyme or recombinant enzyme from other yeasts. We conclude that selected K. lactis strains, with opportune physiological properties and transcription regulation of the heterologous gene, could be optimal hosts for laccase isoenzyme production. PMID:19527303

Ranieri, Danilo; Colao, Maria Chiara; Ruzzi, Maurizio; Romagnoli, Gabriele; Bianchi, Michele M

2009-09-01

95

Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii.  

PubMed

A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXAlb) whose sequences are known. PMID:11565899

Garzillo, A M; Colao, M C; Buonocore, V; Oliva, R; Falcigno, L; Saviano, M; Santoro, A M; Zappala, R; Bonomo, R P; Bianco, C; Giardina, P; Palmieri, G; Sannia, G

2001-04-01

96

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

PubMed

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

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

2014-01-01

97

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

PubMed Central

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.

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

2014-01-01

98

Phenylalanine ammonia-lyase, polyphenol oxidase, and phenol concentration in fruits of Olea europaea L. cv. Picual, Verdial, Arbequina, and Frantoio during ripening.  

PubMed

The kinetics and protein-expression level of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) in fruits of olive trees (Olea europaea) cv. Picual, Verdial, Arbequina, and Frantoio have been studied in relation to the concentration of total phenolic compounds, oleuropein, hydroxytyrosol, and tyrosol during fruit ripening. Frantoio was the variety that showed the highest total phenol concentration, the highest PAL activity, the lowest PPO activity, and the lowest protein levels. In contrast, Verdial was the variety that showed the lowest total phenol concentration, the least PAL activity, the greatest PPO activity, and the highest protein levels. Arbequina and Picual showed intermediate levels. These results suggest the existence of a coordinated response between PAL, PPO, and the concentration of total phenols over ripening in the four varieties. The concentration of total and specific phenols differed between varieties and specifically changed over ripening. PMID:19813730

Ortega-García, Francisca; Peragón, Juan

2009-11-11

99

Electrochemical and spectroscopic effects of mixed substituents in bis(phenolate)-copper(II) galactose oxidase model complexes  

PubMed Central

Non-symmetric substitution of salen (1R1,R2) and reduced salen (2R1,R2) CuII-phenoxyl complexes with a combination of -tBu, -SiPr, and -OMe substituents leads to dramatic differences in their redox and spectroscopic properties, providing insight into the influence of the cysteine-modified tyrosine cofactor in the enzyme galactose oxidase (GO). Using a modified Marcus-Hush analysis, the oxidized copper complexes are characterized as Class II mixed-valent due to the electronic differentiation between the two substituted phenolates. Sulfur K-edge X-ray absorption spectroscopy (XAS) assesses the degree of radical delocalization onto the single sulfur atom of non-symmetric [1tBu,SMe]+ at 7%, consistent with other spectroscopic and electrochemical results that suggest preferential oxidation of the -SMe bearing phenolate. Estimates of the thermodynamic free-energy difference between the two localized states (?G?) and reorganizational energies (?R1R2) of [1R1,R2]+ and [2R1,R2]+ leads to accurate predictions of the spectroscopically observed IVCT transition energies. Application of the modified Marcus-Hush analysis to GO using parameters determined for [2R1,R2]+ predicts a ?max of ~ 13600 cm?1, well within the energy range of the broad Vis-NIR band displayed by the enzyme.

Pratt, Russell C.; Lyons, Christopher T.; Wasinger, Erik C.; Stack, T. Daniel. P.

2012-01-01

100

A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element  

Microsoft Academic Search

Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold

Fabio Vianello; Antonio Cambria; Santa Ragusa; Maria Teresa Cambria; Lucio Zennaro; Adelio Rigo

2004-01-01

101

Phenols  

NASA Astrophysics Data System (ADS)

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.

Weber, Manfred; Weber, Markus

102

Laccase production by the white-rot fungus Termitomyces clypeatus.  

PubMed

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

Bose, Shilpi; Mazumder, Sharmishtha; Mukherjee, Mina

2007-04-01

103

On the factors affecting product distribution in laccase-catalyzed oxidation of a lignin model compound vanillyl alcohol: experimental and computational evaluation.  

PubMed

Laccases (EC 1.10.3.2) are multicopper oxidases, which can oxidize phenolic substrates by the concomitant reduction of oxygen to water. The phenolic substructures of lignin are also oxidized by laccases, resulting mainly in various polymerized products. Several model compound studies indicate that variations in the reaction media, such as the pH and the enzyme dosage used, have an impact on the observed product distribution of laccase promoted oxidation, but no detailed study has been reported to explain these results. In the present study, a monomeric lignin model compound, vanillyl alcohol, was oxidized in laccase-catalyzed reactions by varying the pH, enzyme dosage and temperature. The energies of all the observed products and potential intermediates were calculated by applying density functional theory (DFT) and the polarizable continuum solvation model (PCM). The observed predominant product at pH 4.5 to 7.5 was clearly the 5-5' dimer, although the thermodynamic product according to the calculated free energies was vanillin, the difference being 5.6 kcal mol(-1). The hydrogen bonding is shown to give an additional stabilizing effect on the transition state leading to the 5-5' dimer, but also a kinetic barrier reduces the formation of vanillin. Based on the calculated pKa-values of the proposed intermediates we suggest that the rearomatization reactions of the quinones formed in the radical reactions under mildly acidic and neutral conditions would preferentially occur through deprotonation rather than through protonation. PMID:23851662

Lahtinen, Maarit; Heinonen, Petri; Oivanen, Mikko; Karhunen, Pirkko; Kruus, Kristiina; Sipilä, Jussi

2013-09-01

104

A high sensitivity amperometric biosensor using laccase as biorecognition element  

Microsoft Academic Search

An amperometric flow biosensor, using laccase from Rigidoporus lignosus as bioelement was developed. The laccase was kinetically characterized towards various phenolics both in solution and immobilized to a hydrophilic matrix by carbodiimide chemistry. A bioreactor connected to an amperometric flow cell by a FIA system was filled with the immobilized enzyme and the operational conditions of this biosensor were optimized

Fabio Vianello; Santa Ragusa; Maria Teresa Cambria; Adelio Rigo

2006-01-01

105

A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity  

PubMed Central

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

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

2003-01-01

106

Removal of monomer delignification products by laccase from Trametes versicolor.  

PubMed

The influence of a laccase from Trametes versicolor on the removal of phenolic monomers in liquid hot water pretreated wheat straw supernatants (LHW-S) was examined. Beside the total phenol content derived by Folin-Ciocalteu (FC-) assay, phenolic monomers were measured via headspace-solid phase micro-extraction (HS-SPME)/GC-MS. A notable decrease of the phenols was achieved using 0.2 and 0.5 U/mL laccase whilst higher dosage showed no improvement. Nearly all kind of monomer phenolic compounds identified in the LHW-S were found to be removed after 24h. However, acetophenone and 4-hydroxybenzaldehyde (HBA) were obviously not affected by laccase. Summarizing, three laccase reaction groups (LRG) of phenolic monomers could be classified: immediate removal (LRG-A), degradation after 1 day (LRG-B), no effect of laccase (LRG-C). Additionally, HS-SPME/GC was found to be a powerful tool to study the reaction of laccase and phenolic monomers in complex lignocellulose derived solutions. PMID:22176974

Kolb, Michaela; Sieber, Volker; Amann, Manfred; Faulstich, Martin; Schieder, Doris

2012-01-01

107

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

PubMed Central

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.

Yang, Han-Yu; Chen, Carton W.

2009-01-01

108

Preparation of biosensors by immobilization of polyphenol oxidase in conducting copolymers and their use in determination of phenolic compounds in red wine  

Microsoft Academic Search

Electrochemically produced graft copolymers of thiophene capped polytetrahydofuran (TPTHF1 and TPTHF2) and pyrrole were achieved by constant potential electrolysis using sodium dodecylsulfate (SDS) as the supporting electrolyte. Characterizations were based on Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Electrical conductivities were measured by the four-probe technique.Novel biosensors for phenolic compounds were constructed by immobilizing polyphenol oxidase (PPO)

A. Elif Böyükbayram; Senem K?ralp; Levent Toppare; Yusuf Ya?c?

2006-01-01

109

Heterologous laccase production and its role in industrial applications  

PubMed Central

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

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

2010-01-01

110

Phenols  

NSDL National Science Digital Library

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.

Reich, Ieva

2007-12-24

111

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

Microsoft Academic Search

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

CHRISTIAN JOHANNES; ANDRZEJ MAJCHERCZYK

2000-01-01

112

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

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

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

2012-10-15

113

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

PubMed

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 on the efficiency of detoxification. A reduction of the toxic effect of phenolic compounds by laccase polymerization of free phenols was demonstrated. Laccase treatment of steam-exploded wheat straw reduced sugar recovery after enzymatic hydrolysis, and it should be better performed after hydrolysis with cellulases. The fermentability of hydrolysates was greatly improved by the laccase treatment in all the samples. Our results demonstrate the action of phenolic compounds as fermentation inhibitors, and the advantages of a laccase treatment to increase the ethanol production from steam-exploded wheat straw. PMID:19683434

Jurado, Miguel; Prieto, Alicia; Martínez-Alcalá, Angeles; Martínez, Angel T; Martínez, María Jesús

2009-12-01

114

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

PubMed

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 20gL(-1), an extracellular laccase activity of 633.3UL(-1) was produced after a culture period of only 5days. 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

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

2014-08-01

115

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

PubMed

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

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

2013-10-01

116

Screening and assessment of laccase producing fungi isolated from different environmental samples  

Microsoft Academic Search

Laccase is a copper-containing polyphenol oxidase that acts on a wide range of substrates. This enzyme is found in many plant species and is widely distributed in fungi including wood-rotting fungi where it is often associated with lignin peroxidase, manganese dependent peroxidase, or both. Because of its importance in bioremediation, fungal cultures were screened for laccase positive production by plate

Buddolla Viswanath; M. Subhosh Chandra; H. Pallavi; B. Rajasekhar Reddy

2008-01-01

117

Prediction model based on decision tree analysis for laccase mediators.  

PubMed

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

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

2013-01-10

118

An evidence of laccases in archaea.  

PubMed

Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are a diverse group of multicopper oxidases that catalyze the oxidation of a variety of aromatic compounds. Here we present evidence for distribution of laccases among archaea and their probable functions. Putative laccase genes have been found in different archaeal groups that might have branched off early during evolution, e.g. Haloarcula marismortui ATCC 43049, Natronomonas pharaonis DSM2160, Pyrobaculum aerophilum IM2, Candidatus Nitrosopumilus maritimus SCM1, Halorubrum lacusprofundi ATCC 49239. Most of the archaeal multicopper oxidases reported here are of Type 1 and Type 2 whereas type 3 copper-binding domain could be found in Pyrobaculum aerophilum IM2 and Halorubrum lacusprofundi ATCC49239. An analysis of the genome sequence database revealed the presence of novel types of two-domain laccases in archaea. ed using this method. CyMVin the positive samples of Phalaenopsis sp. and Arachnis sp. was confirmed by DNA sequencing and cp gene homeology blast. The results showed that CyMV extracted from the leaves of orchid in Hangzhou, Zhejiang Province, China, could be derived from Kunming city (KM), Yunnan Province, China. This method characterized by high sensitivity, specificity, and precision is suitable for early diagnosis and quantitative detection of CyMV. PMID:23100763

Sharma, Krishna Kant; Kuhad, Ramesh Chander

2009-06-01

119

Biochemical characteristics of a textile dye degrading extracellular laccase from a Bacillus sp. ADR  

Microsoft Academic Search

Bacillus sp. ADR secretes an extracellular laccase in nutrient broth, and this enzyme was purified up to 56-fold using acetone precipitation and DEAE-cellulose anion exchange chromatography. The molecular weight of purified laccase was estimated to be 66kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified laccase oxidized 2,6-dimethoxy phenol, o-tolidine, hydroquinone, l-DOPA and guaiacol. The optimum pH for oxidation

Amar A. Telke; Gajanan S. Ghodake; Dayanand C. Kalyani; Rhishikesh S. Dhanve; Sanjay P. Govindwar

2011-01-01

120

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

Microsoft Academic Search

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,

ELIAS ABADULLA; TZANKO TZANOV; SILGIA COSTA; KARL-HEINZ ROBRA; ARTUR CAVACO-PAULO; GEORG M. GUBITZ

2000-01-01

121

Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris.  

PubMed

Laccases are strong oxidizing enzymes that oxidize chlorinated phenols, synthetic dyes, pesticides, polycyclic aromatic hydrocarbons as well as a very wide range of other compounds with high redox potential. Based on the bias of genetic codons between fungus and yeast, we synthesized a laccase gene GlLCCI, originated from Ganoderma lucidum using optimized codons and a PCR-based two-step DNA synthesis method. The recombinant laccase, GlLCCI was successfully over-expressed in yeast, Pichia pastoris, with an alcohol oxidase1 promoter. The recombinant GlLCCI has a molecular mass of approximately 58 kDa. The K (m) values of GlLCCI for 2-2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol were 0.9665, and 1.1122 mM, respectively. The V (max) of GlLCCI for both substrates was 3,024 and 82.13 ?M mg(-1 )min(-1). When ABTS was used as a substrate, the enzyme had an optimal temperature of approximately 55°C. The enzyme was detected over pH values from 2 to 8. The enzyme was strongly activated by K(+), Na(+), Cu(2+) and mannitol. Six amino acids (alanine, histidine, glycine, arginine, aspartate and phenylalanine) increased the catalytic ability of the enzyme. The activity of laccase was obviously inhibited by Fe(2+), Fe(3+), sodium hydrosulphite, and sodium azide. Additionally, under optimal conditions, GlLCCI decolorized 37.62 mg l(-1) of azo dye methyl orange (MO) in cultural medium. With a high MO degradation ability, GlLCCI may have potential in the treatment of industrial effluent containing azo dye MO. PMID:21755293

Sun, Jing; Peng, Ri-He; Xiong, Ai-Sheng; Tian, Yongsheng; Zhao, Wei; Xu, Hu; Liu, Da-Tong; Chen, Jian-Min; Yao, Quan-Hong

2012-04-01

122

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

NASA Astrophysics Data System (ADS)

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.

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

2007-09-01

123

Design of Carbon Nanotube-Based Gas-Diffusion Cathode for O2 Reduction by Multicopper Oxidases (Postprint).  

National Technical Information Service (NTIS)

Multicopper oxidases, such as laccase or bilirubin oxidase, are known to reduce molecular oxygen at very high redox potentials, which makes them attractive biocatalysts for enzymatic cathodes in biological fuel cells. By designing an enzymatic gas-diffusi...

C. Lau E. R. Adkins H. R. Luckarift P. Atanassov R. P. Ramasamy

2011-01-01

124

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

PubMed Central

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.

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

2013-01-01

125

In silico study of structural determinants modulating the redox potential of Rigidoporus lignosus and other fungal laccases  

Microsoft Academic Search

Laccases are multicopper oxidases in which substrate oxidation takes place at the type-1 (T1) copper site. The redox potential (E ) significantly varies amongst members of the family and is a key parameter for substrate specificity. Despite sharing highly conserved features at the T1 copper site, laccases span a large range of E , suggesting that the influence of the

Maria Teresa Cambria; Danilo Gullotto; Silvia Garavaglia; Antonio Cambria

2012-01-01

126

Effects of laccase-natural mediator systems on kenaf pulp.  

PubMed

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

Andreu, Glòria; Vidal, Teresa

2011-05-01

127

Olea europaea leaf (Ph.Eur.) extract as well as several of its isolated phenolics inhibit the gout-related enzyme xanthine oxidase.  

PubMed

In Mediterranean folk medicine Olea europaea L. leaf (Ph.Eur.) preparations are used as a common remedy for gout. In this in vitro study kinetic measurements were performed on both an 80% ethanolic (v/v) Olea europaea leaf dry extract (OLE) as well as on nine of its typical phenolic constituents in order to investigate its possible inhibitory effects on xanthine oxidase (XO), an enzyme well known to contribute significantly to this pathological process. Dixon and Lineweaver-Burk plot analysis were used to determine K(i) values and the inhibition mode for the isolated phenolics, which were analysed by RP-HPLC for standardisation of OLE. The standardised OLE as well as some of the tested phenolics significantly inhibited the activity of XO. Among these, the flavone aglycone apigenin exhibited by far the strongest effect on XO with a K(i) value of 0.52 ?M. In comparison, the known synthetic XO inhibitor allopurinol, used as a reference standard, showed a K(i) of 7.3 ?M. Although the phenolic secoiridoid oleuropein, the main ingredient of the extract (24.8%), had a considerable higher K(i) value of 53.0 ?M, it still displayed a significant inhibition of XO. Furthermore, caffeic acid (K(i) of 11.5 ?M; 1.89% of the extract), luteolin-7-O-?-D-glucoside (K(i) of 15.0 ?M; 0.86%) and luteolin (K(i) of 2.9 ?M; 0.086%) also contributed significantly to the XO inhibiting effect of OLE. For oleuropein, a competitive mode of inhibition was found, while all other active substances displayed a mixed mode of inhibition. Tyrosol, hydroxytyrosol, verbascoside, and apigenin-7-O-?-D-glucoside, which makes up for 0.3% of the extract, were inactive in all tested concentrations. Regarding the pharmacological in vitro effect of apigenin-7-O-?-D-glucoside, it has to be considered that it is transformed into the active apigenin aglycone in the mammalian body, thus also contributing substantially to the anti-gout activity of olive leaves. For the first time, this study provides a rational basis for the traditional use of olive leaves against gout in Mediterranean folk medicine. PMID:21144719

Flemmig, J; Kuchta, K; Arnhold, J; Rauwald, H W

2011-05-15

128

Modeling the 3-D Structure of a Recombinant Laccase from Trametes trogii Active at a pH Close to Neutrality  

Microsoft Academic Search

A cDNA encoding a novel laccase from the white-rot fungus Trametes trogii was cloned and expressed in Pichia pastoris. The recombinant protein (Lcc2) exhibited kinetic parameters for both phenolic and non phenolic substrates that were different\\u000a from the previously described Lcc1, the main laccase isoform expressed by T. trogii; in addition, the pH\\/activity profiles for phenolic substrates of Lcc2 were

Maria Chiara Colao; Carlo Caporale; Federica Silvestri; Maurizio Ruzzi; Vincenzo Buonocore

2009-01-01

129

LccA, an Archaeal Laccase Secreted as a Highly Stable Glycoprotein into the Extracellular Medium by Haloferax volcanii? †  

PubMed Central

Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic archaeon Haloferax volcanii. LccA was secreted at high levels into the culture supernatant of a recombinant H. volcanii strain, with peak activity (170 ± 10 mU·ml?1) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U·mg?1 with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO4. Optimal oxidation of ABTS and SGZ was at 45°C and pH 6 and pH 8.4, respectively. The apparent Km values for SGZ, bilirubin, and ABTS were 35, 236, and 670 ?M, with corresponding kcat values of 22, 29, and 10 s?1, respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50°C of 31.5 h.

Uthandi, Sivakumar; Saad, Boutaiba; Humbard, Matthew A.; Maupin-Furlow, Julie A.

2010-01-01

130

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

SciTech Connect

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.

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

1999-12-01

131

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

PubMed Central

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.

Munoz, C; Guillen, F; Martinez, A T; Martinez, M J

1997-01-01

132

Protection of wood from microorganisms by laccase-catalyzed iodination.  

PubMed

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 (I(2)) 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

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

2012-10-01

133

Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production.  

PubMed

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of "alpeorujo" (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues. PMID:20607234

Díaz, Rosario; Saparrat, Mario C N; Jurado, Miguel; García-Romera, Inmaculada; Ocampo, Juan Antonio; Martínez, María Jesús

2010-09-01

134

Response of recalcitrant soil substances to reduced N deposition in a spruce forest soil: integrating laccase-encoding genes and lignin decomposition.  

PubMed

A long-term field experiment conducted in a Norway spruce forest at Solling, Central Germany, was used to verify and compare the response of lignin-decomposing fungal communities in soils receiving current and preindustrial atmospheric nitrogen (N) input for 14.5 years. Therefore, we investigated the decomposition of lignin compounds in relation to phenol oxidase activity and the diversity of basidiomycetes containing laccase genes in organic and mineral horizons. Lignin-derived CuO oxidation products and enzyme activity decreased with soil depth, while the degree of oxidative transformation of lignin increased. These patterns did not change with reduced atmospheric N input, likely reflecting a lasting saturation in available N. The laccase gene diversity decreased with soil depth in spring. In autumn, this pattern was only found in the control plot, receiving current N input. Principal component analysis confirmed the depth profile and distinguished a response of the fungal community to reduced N deposition for most organic layers in spring and a roof effect for the Oe layer in autumn. These responses of the fungal community did not translate into changes in enzyme activity and lignin content and decomposition, suggesting that transformation processes in soils are well buffered despite the rapid response of the microbial community to environmental factors. PMID:20491921

Theuerl, Susanne; Dörr, Nicole; Guggenberger, Georg; Langer, Uwe; Kaiser, Klaus; Lamersdorf, Norbert; Buscot, François

2010-07-01

135

Cloning, expression and phylogenetic analysis of a divergent laccase multigene family in Auricularia auricula-judae.  

PubMed

Laccases (p-diphenol: oxygen oxidoreductase; EC 1.10.3.2) are multi-copper oxidases encoded by gene family in white rot fungi. Auricularia auricula-judae is one kind of white rot fungi with a soft, jelly-like texture and an ear-like shape. In the present study, seven laccase genes containing the signature sequences L1-L4 were isolated from A. auricula-judae strain Au916 on the basis of the mycelium-derived transcriptome. In the basidiomycetes, the predicted substrate binding loops of the A. auricula-judae laccases were found to be uncommon. Phylogenetic analysis showed that the laccases of the Auricularia were nested into the ascomycete laccases, indicating that the laccase genes from Auricularia are distinctly different in function from other basidiomycetes. Among the seven laccases, the intron positions and cluster distributions in the NJ tree varied from each other and the expression patterns of seven genes estimated by qRT-PCR were also discrepant. The lcc3 gene was highly expressed not only in the free-living mycelium but also in substrate mycelium, furthermore, the lcc5 gene was mostly expressed during the fruiting body formation and maturation indicating that lcc5 might play a major role during the sexual reproduction stage. PMID:24055313

Fan, XiuZhi; Zhou, Yan; Xiao, Yang; Xu, ZhangYi; Bian, YinBing

2014-01-01

136

Paper pulp delignification using laccase and natural mediators  

Microsoft Academic Search

Three plant phenols, namely acetosyringone, syringaldehyde and p-coumaric acid, were selected as laccase redox mediators to investigate the enzymatic delignification of paper pulp (obtained from kraft cooking of eucalypt wood) in combination with peroxide bleaching. The effects of these natural mediators were compared with those obtained using the synthetic mediator 1-hydroxybenzotriazole. p-Coumaric acid only caused minor increase of pulp brightness

Susana Camarero; David Ibarra; Ángel T. Martínez; Javier Romero; Ana Gutiérrez; José C. del Río

2007-01-01

137

The clone of laccase gene and its potential function in cuticular penetration resistance of Culex pipiens pallens to fenvalerate  

Microsoft Academic Search

Decreased insecticides cuticular penetration, as one of resistant mechanisms in insect, has been extensively documented. Laccases, are enzymes with p-diphenol oxidase activity, was related to the cuticular tanning in insect. In this study, one laccase 2 gene (CpLac2) was cloned from Culex pipiens pallens. The CpLac2 contains an open reading frame (ORF) of 2289bp and encodes a putative 762 amino

Chengyuan Pan; Yun Zhou; Jianchu Mo

2009-01-01

138

Mutagenicity screening of reaction products from the enzyme-catalyzed oxidation of phenolic pollutants  

SciTech Connect

Phenol-oxidizing enzymes such as peroxidases, laccases, and mushroom polyphenol oxidase are capable of catalyzing the oxidation of a wide range of phenolic pollutants. Although the use of these enzymes in waste-treatment applications has been proposed by a number of investigators, little information exists on the toxicological characteristics of the oxidation products. The enzymes chloroperoxidase, horseradish peroxidase, lignin peroxidase, and mushroom polyphenol oxidase were used in this study to catalyze the oxidation of phenol, several mono-substituted phenols, and pentachlorophenol. Seventeen reaction mixtures representing selected combinations of enzyme and parent phenol were subjected to mutagenicity screening using the Ames Salmonella typhimurium plate incorporation assay; five selected mixtures were also incubated with the S9 microsomal preparation to detect the possible presence of promutagens. The majority of reaction mixtures tested were not directly mutagenic, and none of those tested with S9 gave a positive response. Such lack of mutagenicity of enzymatic oxidation products provides encouragement for establishing the feasibility of enzyme-catalyzed oxidation as a waste-treatment process. The only positive responses were obtained with reaction products from the lignin peroxidase-catalyzed oxidation of 2-nitrophenol and 4-nitrophenol. Clear positive responses were observed when strain TA100 was incubated with 2-nitrophenol reaction-product mixtures, and when strain TA98 was incubated with the 4-nitrophenol reaction mixture. Additionally, 2,4-dinitrophenol was identified as a reaction product from 4-nitrophenol, and preliminary evidence indicates that both 2,4- and 2,6-dinitrophenol are produced from the oxidation of 2-nitrophenol. Possible mechanism by which these nitration reactions occur are discussed.

Massey, I.J.; Aitken, M.D.; Ball, L.M.; Heck, P.E. (Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Environmental Sciences and Engineering)

1994-11-01

139

Structural and Kinetic Characterization of Native Laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii  

Microsoft Academic Search

A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the

Anna Maria Garzillo; Maria Chiara Colao; Vincenzo Buonocore; Romina Oliva; Lucia Falcigno; Michele Saviano; Anna Maria Santoro; Riccardo Zappala; Raffaele Pietro Bonomo; Carmelina Bianco; Paola Giardina; Gianna Palmieri; Giovanni Sannia

2001-01-01

140

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

PubMed

The potential of brewer's spent grain (BSG), a common waste from the brewing industry, as a support-substrate for laccase production by the well-known laccase producer Trametes versicolor ATCC 20869 under solid-state fermentation conditions was assessed. An attempt was made to improve the laccase production by T. versicolor through supplementing the cultures with inducers, such as 2,2-azino bis(3-ethylbenzthiazoline-6-sulfonic acid), copper sulfate, ethanol, gallic acid, veratryl alcohol, and phenol. A higher laccase activity of 13506.2 ± 138.2 IU/gds (gram dry substrate) was obtained with a phenol concentration of 10 mg/kg substrate in a tray bioreactor after 12 days of incubation time. The flocculation properties of the laccase treated crude beer samples have been studied by using various parameters, such as viscosity, turbidity, ? potential, total polyphenols, and total protein content. The present results indicated that laccase (25 IU/L) showed promising results as a good flocculating agent. The laccase treatment showed better flocculation capacity compared to the industrial flocculation process using stabifix as a flocculant. The laccase treatments (25 IU/L) at 4 ± 1 °C and room temperature have shown almost similar flocculation properties without much variability. The study demonstrated the potential of in-house produced laccase using brewer's spent grain for the clarification and flocculation of crude beer as a sustainable alternative to traditional flocculants, such as stabifix and bentonite. PMID:22866699

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

2012-08-15

141

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

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.

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

2007-01-01

142

[Spectra analysis of lignin small molecular guaiacyl coniferyl alcohol biological modification treated by laccase].  

PubMed

The enzymatic modification mechanism of lignin small molecular lignin guaiacyl coniferyl alcohol existing in softwood and hardwood treated by laccase was studied. Gas chromatography-mass spectrometry (GC-MS), gel permeation chromatography (GPC), FTIR spectrum and PCD(particle charge detector) etc were used for the measurement. GC-MS could not detect the coniferyl alcohol monomer after treated by laccase, so it was speculated that coniferyl alcohol participated in the reaction totally, and the structure of coniferyl alcohol was changed. GPC demonstrated that the molecular weight of coniferyl alcohol increased when it was treated by laccase, and coniferyl alcohol was polymerized. FTIR spectrum determined the reaction point of coniferyl alcohol when treated by laccase, and it is mainly phenolic hydroxyl, aromatic ring side chain substituent such as methoxy, double bound in side chain, beta-carbonyl groups, and gamma-carbonyl groups. PCD gave a result that the cationic demandv(CD) decreased by 88.38%. PMID:20707131

Liu, Hai-Tang; Pei, Ji-Cheng; Hu, Hui-Ren; Pei, Ya

2010-06-01

143

Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus kraft pulp.  

PubMed

The natural phenolic compounds syringaldehyde and vanillin were compared to the synthetic mediators 1-hydroxybenzotriazole, violuric acid and promazine in terms of boosting efficiency in a laccase-assisted biobleaching of eucalyptus kraft pulp. Violuric acid and 1-hydroxybenzotriazole revealed to be the most effective mediators of the bioprocess. Nevertheless, laccase-syringaldehyde system also improved the final pulp properties (28% delignification and 63.5% ISO brightness) compared to the process without mediator (23% and 61.5% respectively), in addition to insignificant denaturation effect over laccase. The efficiency of the biobleaching process was further related to changes in non-conventionally used optical and chromatic parameters of pulp, such as (L*), chroma (C*) and dye removal index (DRI) showing good correlation. Adverse coupling reactions of the natural phenolic mediators on pulp lignin were predicted by electrochemical studies, demonstrating the complexity of the laccase-mediator reaction on pulp. PMID:18499450

Moldes, D; Díaz, M; Tzanov, T; Vidal, T

2008-11-01

144

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

PubMed

Fungal laccases from basidiomycetous fungi are thoroughly investigated in respect of catalytic mechanism and industrial applications, but the number of reported and well characterized ascomycetous laccases is much smaller although they exhibit interesting catalytic properties. We report on a highly chloride tolerant laccase produced by the plant pathogen ascomycete Botrytis aclada, which was recombinantly expressed in Pichia pastoris with an extremely high yield and purified to homogeneity. In a fed-batch fermentation, 495 mg L(-1) of laccase was measured in the medium, which is the highest concentration obtained for a laccase by a yeast expression system. The recombinant B. aclada laccase has a typical molecular mass of 61,565 Da for the amino acid chain. The pI is approximately 2.4, a very low value for a laccase. Glycosyl residues attached to the recombinant protein make up for approximately 27% of the total protein mass. B. aclada laccase exhibits very low K(M) values and high substrate turnover numbers for phenolic and non-phenolic substrates at acidic and near neutral pH. The enzyme's stability increases in the presence of chloride ions and, even more important, its substrate turnover is only weakly inhibited by chloride ions (I(50)=1.4M), which is in sharp contrast to most other described laccases. This high chloride tolerance is mandatory for some applications such as implantable biofuel cells and laccase catalyzed reactions, which suffer from the presence of chloride ions. The high expression yield permits fast and easy production for further basic and applied research. PMID:22178779

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

2012-01-20

145

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

NASA Astrophysics Data System (ADS)

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.

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

2012-05-01

146

[Purification and properties of laccase from Basidiomycete].  

PubMed

Laccase produced by Basidiomycete was purified to electrophoretic homogeneity by the steps of ammonium sulfate precipitation, DEAE-cellulose and hydrophobic interaction column chromatography. Purification of about 318.4 fold was achieved with an overall yield of 18.6%. Its molecular weight was estimated to be about 60.3 kD by SDS-PAGE, and that of it was 55.94 kD by mass spectrum. The optimum temperature and pH of the enzyme activity were 65 degrees C and 2.2 - 2.8 respectively. The isoelectric point was 4.02 (room temperature). Its N-terminal sequence was AIGPVTDL. The carbohydrate content was 49.2% by the phenol-sulfuric acid method. Michaelis constant of the enzyme for ABTS was 17.5 micromol/L. The enzyme activity was stable under 45 degrees C and in the pH range of 3.0 - 9.5. The activity was enhanced by Cu2+, and was strongly inhibited by Fe2+. While Mn2+ and Ag+ had no effect on laccase activity. Dithiothreitol and sodium azide inhibited completely the activity. Trp was possible essential residue for enzyme activity. PMID:16276874

Liu, Shuzhen; Qian, Shijun

2003-02-01

147

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

PubMed

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

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

2008-12-01

148

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

PubMed Central

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.

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

2000-01-01

149

Insect multicopper oxidases: Diversity, properties, and physiological roles  

Microsoft Academic Search

Multicopper oxidases (MCOs) are a group of related proteins that are ubiquitous in nature. They perform a wide variety of functions including pigmentation, lignin synthesis and degradation, iron homeostasis, and morphogenesis. The laccases of fungi are intensely studied for their biotechnological potential as a more environmentally friendly alternative to harsh or toxic chemicals used for certain industrial applications. Research into

Neal T. Dittmer; Michael R. Kanost

2010-01-01

150

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

PubMed Central

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.

Saparrat, Mario C. N.; Guillen, Francisco; Arambarri, Angelica M.; Martinez, Angel T.; Martinez, Maria Jesus

2002-01-01

151

Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis  

PubMed Central

Background Laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions they are able to catalyze. These include, for example, the formation and degradation of dimers, oligomers, polymers, and ring cleavage as well as oxidation of aromatic compounds. Potential applications of laccases include detoxification of industrial effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenolic acids. We have recently published a report on the cloning and characterization of a CotA Bacillus licheniformis laccase, an enzyme that catalyzes dimerization of phenolic acids. However, the broad application of this laccase is limited by its low expression level of 26 mg l-1 that was achieved in Escherichia coli. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of CotA. Results A CotA double mutant, K316N/D500G, was constructed by combining random and site-directed mutagenesis. It can be functionally expressed at an 11.4-fold higher level than the wild-type enzyme. In addition, it is able to convert ferulic acid much faster than the wild-type enzyme (21% vs. 14%) and is far more efficient in decolorizing a range of industrial dyes. The investigation of the effects of the mutations K316N and D500G showed that amino acid at position 316 had a major influence on enzyme activity and position 500 had a major influence on the expression of the laccase. Conclusion The constructed double mutant K316N/D500G of the Bacillus licheniformis CotA laccase is an appropriate candidate for biotechnological applications due to its high expression level and high activity in dimerization of phenolic acids and decolorization of industrial dyes.

Koschorreck, Katja; Schmid, Rolf D; Urlacher, Vlada B

2009-01-01

152

Evaluation of the potential of fungal and plant laccases for active-packaging applications.  

PubMed

Laccases from Trametes versicolor (TvL), Myceliophthora thermophila (MtL), and Rhus vernicifera (RvL) were investigated with regard to their potential utilization as oxygen scavengers in active packages containing food susceptible to oxidation reactions. The substrate selectivity of the laccases was investigated with a set of 17 reducing substrates, mainly phenolic compounds. The temperature dependence of reactions performed at low temperatures (4-31 °C) was studied. Furthermore, the laccases were subjected to immobilization in a latex/clay matrix and drying procedures performed at temperatures up to 105 °C. The results show that it is possible to immobilize the laccases with retained activity after dispersion coating, drying at 75-105 °C, and subsequent storage of the enzyme-containing films at 4 °C. TvL and, to some extent, MtL were promiscuous with regard to their reducing substrate, in the sense that the difference in activity with the 17 substrates tested was relatively small. RvL, on the other hand, showed high selectivity, primarily toward substrates resembling its natural substrate urushiol. When tested at 7 °C, all three laccases retained >20% of the activity they had at 25 °C, which suggests that it would be possible to utilize the laccases also in refrigerated food packages. Coating and drying resulted in a remaining enzymatic activity ranging from 18 to 53%, depending on the drying conditions used. The results indicate that laccases are useful for active-packaging applications and that the selectivity for reducing substrates is an important characteristic of laccases from different sources. PMID:21524087

Chatterjee, Robin; Johansson, Kristin; Järnström, Lars; Jönsson, Leif J

2011-05-25

153

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

PubMed Central

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.

Chefetz, Benny; Chen, Yona; Hadar, Yitzhak

1998-01-01

154

Grape seeds: the best lignocellulosic waste to produce laccase by solid state cultures of Trametes hirsuta.  

PubMed

Grape seeds were used by Trametes hirsuta as a substrate for laccase production giving 23 kU l(-1), which was 10-fold the value attained in the cultures with no lignocellulosic waste addition. The dyes, Indigo Carmine and Bromophenol Blue, were easily decolourised (100% in 24 h) by the extracellular liquid obtained in such cultures, whereas Methyl Orange (65% in 24 h) and Phenol Red (36% in 24 h) were more resistant to degradation. This shows the specificity of laccase towards different dye structures. PMID:12882277

Moldes, D; Gallego, P P; Rodríguez Couto, S; Sanromán, A

2003-03-01

155

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

PubMed Central

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.

Johannes, Christian; Majcherczyk, Andrzej

2000-01-01

156

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

SciTech Connect

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

Li Xu [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Wei Zhiyi [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Zhang Min [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Peng Xiaohui [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Yu Guangzhe [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Teng Maikun [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Gong Weimin [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); E-mail: wgong@ibp.ac.cn

2007-03-02

157

Thermostability, pH stability and dye degrading activity of a bacterial laccase are enhanced in the presence of Cu2O nanoparticles.  

PubMed

The present study relates to a nanotechnology enabled method in which purified laccase from Escherichia coli AKL2 was supplemented with 100 ?M copper oxide nanoparticles (Cu(2)O) (NP-laccase). The activity, half life and stability of NP-laccase were enhanced by 4, 42 and 36-fold respectively at high temperature (80 °C) and also over a wide range of pH (4-12) than laccase (in the presence of 0.18 mM CuSO(4)). Thermodynamic analysis of the nanoparticle-induced enzyme stability revealed an enhanced entropy-enthalpy compensation at 80 °C, which reflected the maintenance of its native structure. This was further supported by CD studies. The enhanced activity and thermostability of NP-laccase can be utilized for efficient decolorisation of dyes (both phenolic and azo). PMID:23131620

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

2013-01-01

158

Constitutive expression of Botrytis aclada laccase in Pichia pastoris.  

PubMed

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

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

2012-01-01

159

Oligonucleotide primers for specific detection of actinobacterial laccases from superfamilies I and K.  

PubMed

Although many putative laccase-like genes have been assigned to members of the phylum Actinobacteria, few of the related enzymes have been characterized so far. It is noteworthy, however, that this small number of enzymes has presented properties with industrial relevance. This observation, combined with the recognized biotechnological potential and the capability of this phylum to degrade recalcitrant soil polymers, has attracted attention for bioprospective approaches. In the present work, we have designed and tested primers that were specific for detection of sub-groups of laccase-like genes within actinomycetes, which corresponded to the superfamilies I and K from the classification presented by the laccase and multicopper oxidase engineering database. The designed primers have amplified laccase-like gene fragments from actinomycete isolates that were undetectable by primers available from the literature. Furthermore, phylogenetic alignments suggest that some of these fragments may belong to new laccases-like proteins, and thus emphasize the benefits of designing subgroup-specific primers. PMID:24846052

Fernandes, Tatiana Alves Rigamonte; da Silveira, Wendel Batista; Passos, Flávia Maria Lopes; Zucchi, Tiago Domingues

2014-08-01

160

A new Stenotrophomonas maltophilia strain producing laccase. Use in decolorization of synthetics dyes.  

PubMed

Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani (LB) medium added by 0.4 mM copper sulfate. The addition of CuSO(4) in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide gel electrophoresis. The enzyme showed syringaldazine (K (m) = 53 microM), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K (m) = 700 microM), and pyrocatechol (K (m) = 25 microM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange, and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions. PMID:18931956

Galai, Said; Limam, Ferid; Marzouki, M Nejib

2009-08-01

161

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

NASA Astrophysics Data System (ADS)

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.

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

2009-01-01

162

Mechanisms underlying dioxygen reduction in laccases. Structural and modelling studies focusing on proton transfer  

Microsoft Academic Search

BACKGROUND: Laccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water. They are the simplest members of the multi-copper oxidases and contain at least two types of copper centres; a mononuclear T1 and a trinuclear that includes two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear centre whereas reduction

Isabel Bento; Catarina S Silva; Zhenjia Chen; Lígia O Martins; Peter F Lindley; Cláudio M Soares

2010-01-01

163

An Optical Biosensor based on Immobilization of Laccase and MBTH in Stacked Films for the Detection of Catechol  

Microsoft Academic Search

The fabrication of an optical biosensor by using stacked films where 3-methyl- 2-benzothiazolinone hydrazone (MBTH) was immobilized in a hybrid nafion\\/sol-gel silicate film and laccase in a chitosan film for the detection of phenolic compounds was described. Quinone and\\/or phenoxy radical product from the enzymatic oxidation of phenolic compounds was allowed to couple with MBTH to form a colored azo-dye

Jaafar Abdullah; Musa Ahmad; Lee Yook Heng; Nadarajah Karuppiah; Hamidah Sidek

2007-01-01

164

A new approach to the biobleaching of flax pulp with laccase using natural mediators.  

PubMed

The phenols syringaldehyde (SA), acetosyringone (AS) and p-coumaric acid (PCA) were used as natural laccase mediators in combination with a laccase from Pycnoporus cinnabarinus to bleach flax fibres. Their performance was compared with 1-hydroxybenzotriazole (HBT) in terms of enzyme stability, and pulp and effluent properties. HBT and PCA were found to inactivate laccase in the absence of pulp. However, in the presence of unbleached flax pulp stability was increased; for example with PCA, laccase retained 77% of its initial activity, in contrast with complete inactivation in the absence of pulp. This suggests a protective effect of the pulp against denaturalization of the enzyme. All natural mediators resulted in a reduced kappa number after the subsequent alkaline treatment with hydrogen peroxide; the reduction being especially marked with SA (about 2 units - with respect to the control sample) and comparable to that obtained by HBT. Brightness was significantly increased by all natural mediators, but especially by AS and SA (23% with both), which performed very similarly to HBT in this respect. Natural mediators therefore might constitute an effective alternative to synthetic mediators for flax pulp biobleaching. This paper demonstrates for the first time the use of natural mediators in the laccase-assisted delignification of flax pulp and their effect on the properties of the resulting effluents. PMID:20138756

Fillat, Amanda; Colom, Josep F; Vidal, Teresa

2010-06-01

165

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

PubMed Central

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.

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

2009-01-01

166

Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase  

SciTech Connect

A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial wastewater samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.

Atlow, S.C.; Bonadonna-Aparo, L.; Klibanov, A.M.

1984-01-01

167

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

PubMed Central

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

Teixeira, Ricardo Sposina S.; Pereira, Patricia Maia; Ferreira-Leitao, Viridiana S.

2010-01-01

168

Laccase immobilization and insolubilization: from fundamentals to applications for the elimination of emerging contaminants in wastewater treatment.  

PubMed

Over the last few decades many attempts have been made to use biocatalysts for the biotransformation of emerging contaminants in environmental matrices. Laccase, a multicopper oxidoreductase enzyme, has shown great potential in oxidizing a large number of phenolic and non-phenolic emerging contaminants. However, laccases and more broadly enzymes in their free form are biocatalysts whose applications in solution have many drawbacks rendering them currently unsuitable for large scale use. To circumvent these limitations, the enzyme can be immobilized onto carriers or entrapped within capsules; these two immobilization techniques have the disadvantage of generating a large mass of non-catalytic product. Insolubilization of the free enzymes as cross-linked enzymes (CLEAs) is found to yield a greater volume ratio of biocatalyst while improving the characteristics of the biocatalyst. Ultimately, novel techniques of enzymes insolubilization and stabilization are feasible with the combination of cross-linked enzyme aggregates (combi-CLEAs) and enzyme polymer engineered structures (EPESs) for the elimination of emerging micropollutants in wastewater. In this review, fundamental features of laccases are provided in order to elucidate their catalytic mechanism, followed by different chemical aspects of the immobilization and insolubilization techniques applicable to laccases. Finally, kinetic and reactor design effects for enzymes in relation with the potential applications of laccases as combi-CLEAs and EPESs for the biotransformation of micropollutants in wastewater treatment are discussed. PMID:23051065

Ba, Sidy; Arsenault, Alexandre; Hassani, Thanina; Jones, J Peter; Cabana, Hubert

2013-12-01

169

Preparation of starch-sodium lignosulfonate graft copolymers via laccase catalysis and characterization of antioxidant activity.  

PubMed

Graft copolymers of waxy maize starch and sodium lignosulfonate (SLS) were prepared by Trametes versicolor laccase catalysis in aqueous solution. Amount of SLS grafted based on phenol analysis was 0.5% and 1.0% in the absence and presence of 1-hydroxybenzotriazole (HBT), respectively. Starch-SLS graft copolymers were effective antioxidants as judged by 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The presence of laccase caused a reduction in starch molecular weight although a cross-linked gel fraction was also observed when HBT was present. This new method for preparing starch chemically modified with phenolic compounds is simple and the resulting antioxidant polymers have potential in food, cosmetic and packaging applications. PMID:23121948

Shogren, Randal L; Biswas, Atanu

2013-01-16

170

Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor.  

PubMed Central

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).

Archibald, F; Roy, B

1992-01-01

171

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

NASA Astrophysics Data System (ADS)

“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.

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

2008-01-01

172

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

PubMed Central

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.

Rodriguez, Alexander; Osma, Johann F.; Almeciga-Diaz, Carlos J.; Sanchez, Oscar F.

2013-01-01

173

Crystal structure of the blue multicopper oxidase from the white-rot fungus Trametes trogii complexed with p-toluate  

Microsoft Academic Search

A multicopper oxidase, the fungal laccase glycoenzyme from the white-rot basidiomycete fungus Trametes (Funalia) trogii, was crystallized and its crystal structure was solved at 1.58Å using molecular replacement techniques.Model refinement resulted in R-factor and R-free values of 17.4% and 19.0%, respectively. The T. trogii laccase structural model reveals the presence of a ligand bound to the T1 active site which

Irene Matera; Antonella Gullotto; Silvia Tilli; Marta Ferraroni; Andrea Scozzafava; Fabrizio Briganti

2008-01-01

174

Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies.  

PubMed

A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58.0 kDa. The enzyme had an isoelectric point of around pH 6.9. The optimum pH for enzyme activity was around 3.0 against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 degrees C and stable up to 50 degrees C. The enzyme contained 8.6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. Beta-(3,4-dihydroxyphenyl)alanine (L-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of L-dopa was identified as L-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain. PMID:12949171

Nagai, Masaru; Kawata, Maki; Watanabe, Hisayuki; Ogawa, Machiko; Saito, Kumiko; Takesawa, Toshikazu; Kanda, Katsuhiro; Sato, Toshitsugu

2003-09-01

175

First evidence of laccase activity in the Pacific oyster Crassostrea gigas.  

PubMed

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defence mechanisms in various invertebrates. The aim of this study was to thoroughly identify the PO-like activity present in the hemolymph of the Pacific oyster Crassostrea gigas, by using different substrates (i.e. dopamine and p-phenylenediamine, PPD) and different PO inhibitors. In order to go deeper in this analysis, we considered separately plasma and hemocyte lysate supernatant (HLS). In crude plasma, oxygraphic assays confirmed the presence of true oxidase activities. Moreover, the involvement of peroxidase(s) was excluded. In contrast to other molluscs, no tyrosinase-like activity was detected. With dopamine as substrate, PO-like activity was inhibited by the PO inhibitors tropolone, phenylthiourea (PTU), salicylhydroxamic acid and diethyldithio-carbamic acid, by a specific inhibitor of tyrosinases and catecholases, i.e. 4-hexylresorcinol (4-HR), and by a specific inhibitor of laccases, i.e. cetyltrimethylammonium bromide (CTAB). With PPD as substrate, PO-like activity was inhibited by PTU and CTAB. In precipitated protein fractions from plasma, and with dopamine and PPD as substrates, PTU and 4-HR, and PTU and CTAB inhibited PO-like activity, respectively. In precipitated protein fractions from hemocyte lysate supernatant, PTU and CTAB inhibited PO-like activity, independently of the substrate. Taken together, these results suggest the presence of both catecholase- and laccase-like activities in plasma, and the presence of a laccase-like activity in HLS. To the best of our knowledge, this is the first time that a laccase-like activity is identified in a mollusc by using specific substrates and inhibitors for laccase, opening new perspectives for studying the implication of this enzyme in immune defence mechanisms of molluscs of high economic value such as C. gigas. PMID:20109560

Luna-Acosta, Andrea; Rosenfeld, Eric; Amari, Myriam; Fruitier-Arnaudin, Ingrid; Bustamante, Paco; Thomas-Guyon, Hélène

2010-04-01

176

Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature.  

PubMed

Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 degrees C instead of 28 degrees C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance. PMID:10531652

Cassland, P; Jönsson, L J

1999-09-01

177

Blood tolerant laccase by directed evolution.  

PubMed

High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnology. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific composition of human blood (above neutral pH, high chloride concentration) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chemical syntheses, and detoxification. PMID:23438751

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

2013-02-21

178

Features and applications of bilirubin oxidases.  

PubMed

Discovered in 1981 by Tanaka and Murao (Agric Biol Chem 45:2383-2384, 1981), bilirubin oxidase (BOD) is a sub-group of multicopper oxidases (MCOs) also utilizing four Cu(+/2+) ions. It catalyzes the oxidation of bilirubin to biliverdin, hence the classification of bilirubin oxidase, and has been primarily used in the determination of bilirubin in serum and thereby in the diagnostic of jaundice. Unlike laccases, the most studied MCOs, BODs display a high activity and stability at neutral pH, a high tolerance towards chloride anions and other chelators, and for some species, a high thermal tolerance. Therefore, BODs could potentially be an alternative to laccase which are so far mainly restricted to applications in acid media. Because of growing interest in BODs for numerous applications under mild pH conditions, based on the number of patents and publications published in the last 5 years, here I will summarize the available data on the biochemical properties of BODs, their occurrence, and their possible biotechnological use in (1) the field of Healthcare for the elaboration of biofuel cells or bilirubin sensors or (2) the field of environmentally desirable applications such as depollution, decolorization of dyes, and pulp bleaching. PMID:22878843

Mano, Nicolas

2012-10-01

179

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

PubMed Central

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.

2010-01-01

180

Modeling the 3-D structure of a recombinant laccase from Trametes trogii active at a pH close to neutrality.  

PubMed

A cDNA encoding a novel laccase from the white-rot fungus Trametes trogii was cloned and expressed in Pichia pastoris. The recombinant protein (Lcc2) exhibited kinetic parameters for both phenolic and non phenolic substrates that were different from the previously described Lcc1, the main laccase isoform expressed by T. trogii; in addition, the pH/activity profiles for phenolic substrates of Lcc2 were shifted upward by 1-1.5 pH units towards neutrality as compared to Lcc1. Comparative modeling of the two laccases (69.2% identity) showed that the overall fold of Lcc2 is very similar to Lcc1 and other laccases. The substrate cavity of Lcc2 contains the Asp residue which is thought to mediate the laccase activity at acidic pHs, whereas two hydrophobic residues (Phe, Ile) on the cavity orifice of Lcc2 replace the two polar residues (Thr, Ser) of Lcc1. These structural differences may be responsible for the unique kinetic performances of Lcc2. PMID:19806440

Colao, Maria Chiara; Caporale, Carlo; Silvestri, Federica; Ruzzi, Maurizio; Buonocore, Vincenzo

2009-12-01

181

Biochemical and Molecular Characterization of a Laccase from Marasmius quercophilus  

PubMed Central

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak (Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the “blue copper oxidase” family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75°C, and the Km was 7.1 ?M. The structural gene, lac1, was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.

Dedeyan, Boghos; Klonowska, Agnieszka; Tagger, Simone; Tron, Thierry; Iacazio, Gilles; Gil, Gerard; Le Petit, Jean

2000-01-01

182

[FTIR spectra analysis of the reactive activity of lignin when modified by laccase].  

PubMed

In company with the development of nonaqueous enzymology, the enzymatic modification of lignin has gained increasing interests, especially in the synthesis of high molecular material. In the present article, the enzymatic modification of spruce alkali lignin in cetyltrimethylammonium bromide (CTAB) reversed micelles (100 mmol x L(-1) 1, pH 6.0, W/O = 40), alcohol lignin in ethanol solution (50%), lignin sulphonate in sodium phosphate buffer (pH 5.8, 20 mmol x L(-1)) and steam-explosion wheat straw alkali lignin in alkaline solution (pH 10.0, 20 mmol x L(-1) NaOH) by mycelia sterilia YY-5 laccase was studied. Laccase was isolated from Mycelia Sterilia YY-5 (CGMCC-1462) which was an entophytic fungus of Rhus Chinensis Mill. FTIR spectrum was used to assay the structure of lignins and gel permeation chromatography (GPC) was used to determine the molecular weight and molecular weight polydispersity of lignins. Bands of lignin in FTIR spectra of all lignins changed obviously after treated with YY-5 laccase, which indicated that some bond breakage or rearrangement occurred to lignin. The shift of non-conjugated C=O and conjugated carbonyl groups (alpha-carbonyl groups) stretching vibration, the decrease of phenol hydroxyl stretching vibration and the increase of C--O--C stretching vibration of ester bond proved that phenolic hydroxyl, carbonyl group and side chain substituent all might participate in the laccase modification reactions of lignin. Meanwhile, the results of GPC indicated that the molecular of lignins all have certain increase and molecular polydispersity decreased. From the point of the molecular mass polydispersity, the modification effect of YY-5 laccase on steam-exploded wheat straw alkali lignin and spruce alkali lignin was more significantly than other two lignins. The molecular mass polydispersity for steam-exploded wheat straw alkali lignin and spruce alkali lignin was 1.211 and 1.375 respectively, which might contribute to the alkali-stable enzyme for YY-5 laccase. Correspondingly, alkali solution was chosen as the optimum medium for YY-5 laccase to modify lignins. PMID:18844148

Qiu, Wei-Hua; Chen, Hong-Zhang

2008-07-01

183

Enhanced the enzymatic hydrolysis efficiency of wheat straw after combined steam explosion and laccase pretreatment.  

PubMed

Laccase, capable of selectively degrading lignin while keeping cellulose intact, has been widely applied for the modification and bio-bleaching of pulp. In this study Sclerotium sp. laccase (MSLac) was employed in combination with steam explosion to evaluate the effect of this treatment on cellulose hydrolysis. Combined steam explosion with laccase pretreatment enhanced the cellulose conversion rate of wheat straw no matter in the case of successive (MSLac-Cel) and simultaneous (MSLac+Cel) MSLac and cellulase hydrolysis. The highest cellulose conversion rate of 84.23% was obtained when steam-exploded wheat straw (SEWS) (1.3 MPa, 5 min) was treated by MSLac+Cel at a laccase loading of 0.55 U g(-1) substrate. FT-IR and SEM analyses indicated that MSLac oxidized the phenol and changed electron configuration of the ring, which contributed to loosening the compact wrap of lignin-carbohydrate complex and consequently enhancing the enzymatic hydrolysis efficiency of cellulose. This article provided a promising method for lignocellulose bio-pretreatment. PMID:22695139

Qiu, Weihua; Chen, Hongzhang

2012-08-01

184

Laccase applications in biofuels production: current status and future prospects.  

PubMed

The desire to reduce dependence on the ever diminishing fossil fuel reserves coupled with the impetus towards green energy has seen increased research in biofuels as alternative sources of energy. Lignocellulose materials are one of the most promising feedstocks for advanced biofuels production. However, their utilisation is dependent on the efficient hydrolysis of polysaccharides, which in part is dependent on cost-effective and benign pretreatment of biomass to remove or modify lignin and release or expose sugars to hydrolytic enzymes. Laccase is one of the enzymes that are being investigated not only for potential use as pretreatment agents in biofuel production, mainly as a delignifying enzyme, but also as a biotechnological tool for removal of inhibitors (mainly phenolic) of subsequent enzymatic processes. The current review discusses the major advances in the application of laccase as a potential pretreatment strategy, the underlying principles as well as directions for future research in the search for better enzyme-based technologies for biofuel production. Future perspectives could include synergy between enzymes that may be required for optimal results and the adoption of the biorefinery concept in line with the move towards the global implementation of the bioeconomy strategy. PMID:24841120

Kudanga, Tukayi; Le Roes-Hill, Marilize

2014-08-01

185

Comparative Studies of Extracellular Fungal Laccases  

PubMed Central

Various basidiomycetes, ascomycetes, and deuteromycetes, grown in a sugar-rich liquid medium, were compared for laccase-producing ability and for the inducing effect of 2,5-xylidine on laccase production. Clear stimulation of the extracellular enzyme formation by xylidine was obtained in the cultures of Fomes annosus, Pholiota mutabilis, Pleurotus ostreatus, and Trametes versicolor, whereas Rhizoctonia praticola and Botrytis cinerea were not affected by the xylidine, and in the case of Podospora anserina a decrease in laccase activity was observed. The laccases were purified, and electrophoresis on polyacrylamide gels indicated a particular pattern for each laccase. The bands of the induced forms appeared only with basidiomycetes. The optimal pH of R. praticola laccase was in the neutral region, whereas the optima of all the other exolaccases were significantly lower (between pH 3.0 and 5.7). All laccases oxidized the methoxyphenolic acids under investigation, but there existed quantitative differences in oxidation efficiencies which depended on pH and on the nature (noninduced or induced) of the enzyme. The sensitivity of all enzymes to inhibitors did not differ considerably.

Bollag, Jean-Marc; Leonowicz, Andrzej

1984-01-01

186

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

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 H(2)O(2)-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H(2)O(2) 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 H(2)O(2). The stimulation of laccase gene expression in response to exogenous H(2)O(2) 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 H(2)O(2)-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage. PMID:22706050

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

2012-08-01

187

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

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.

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

2012-01-01

188

Oxidase Test Protocol  

NSDL National Science Digital Library

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).

American Society For Microbiology;

2010-11-11

189

Lignin changes after steam explosion and laccase-mediator treatment of eucalyptus wood chips.  

PubMed

Eucalyptus globulus chips were steam exploded followed by treatment with a laccase-mediator system (LMS) under different experimental conditions. Removal of hemicelluloses and, to a lesser extent, lignin was observed. Thermogravimetic analyses of whole meal obtained from chips before and after steam explosion indicated an increase in lignin degradation temperature due to lignin condensation. In contrast, application of LMS treatment caused a reduction in lignin and polysaccharide degradation temperatures. Lignins were isolated from wood samples before and after each treatment and analyzed by 2D NMR and (13)C NMR. An increase in carboxyl and phenolic hydroxyl groups and a significant decrease in ?-O-4 structures were found in steam-exploded samples. The most relevant changes observed after laccase treatment were increased secondary OH and degree of condensation. PMID:21749069

Martin-Sampedro, Raquel; Capanema, Ewellyn A; Hoeger, Ingrid; Villar, Juan C; Rojas, Orlando J

2011-08-24

190

Role of laccase and low molecular weight metabolites from Trametes versicolor in dye decolorization.  

PubMed

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

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

2012-01-01

191

Recent developments and applications of immobilized laccase.  

PubMed

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

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

2013-12-01

192

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

PubMed Central

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.

Camarero, Susana; Ibarra, David; Martinez, Maria Jesus; Martinez, Angel T.

2005-01-01

193

Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts.  

PubMed

To obtain laccase-gene-specific sequences from the white-rot fungus Trametes sanguinea M85-2, a PCR screening method was used. Degenerate primers were designed based on highly conserved copper-binding regions I and IV of known laccases and used to amplify laccase sequences from T. sanguinea M85-2 genomic DNA. A single 1.6-kbp DNA band was amplified and cloned into a vector. Partial sequences of 21 clones were classified into five groups (lcc1-5) and the deduced amino acid sequences were all homologous to known laccase sequences. Based on the partial sequence of lcc1, the 5'-end of its cDNA was obtained by a PCR termed 5' rapid amplification of cDNA ends (5'-RACE), and RT-PCR was then carried out using the 5'-primer and the poly-dT primer to obtain the full-length lcc1 cDNA. The obtained cDNA encoded a protein consisting of 518 amino acid residues and its first 21 amino acid residues were predicted to be the signal peptide for secretion. The conserved characteristic structures of laccase, such as copper-binding ligands, N-glycosylation sites, and cysteine residues for disulfide bridges, were observed. The genomic DNA sequence of the lcc1 gene was also cloned by PCR method and the sequence revealed 10 introns. The lcc1 cDNA was inserted into yeast vectors for heterologous expression by Saccharomyces cerevisiae and Pichia pastoris. Phenol-oxidizing activity was detected from transformants of the yeasts, indicating that the obtained cDNA encodes a laccase. Previously, two laccase isozymes were biochemically characterized and purified from T. sanguinea M85-2. Using the sequential PCR method presented here, we have obtained partial sequences of at least five laccase genes and one cDNA clone encoding a protein with laccase activity but without any enzymatic information, suggesting that expressed enzymes under restricted conditions may not represent all the isozymes in target microorganisms. PCR cloning and heterologous expression of the cloned genes can be an alternative method of screening enzymes if these enzymes have conserved sequences. PMID:16233113

Hoshida, H; Nakao, M; Kanazawa, H; Kubo, K; Hakukawa, T; Morimasa, K; Akada, R; Nishizawa, Y

2001-01-01

194

Phenolic removal in a model olive oil mill wastewater using Pleurotus ostreatus in bioreactor cultures and biological evaluation of the process  

Microsoft Academic Search

Pleurotus ostreatus grown in bioreactor batch cultures in a model phenolic wastewater (diluted and sterilized olive oil mill wastewater—OMW), caused significant phenolic removal. Laccase, the sole ligninolytic enzyme detected in the growth environment, was produced during primary metabolic growth. The bioprocess was simulated with the aid of a mathematical model and the parameters of growth were determined. When the fungal

G. Aggelis; D. Iconomou; M. Christou; D. Bokas; S. Kotzailias; G. Christou; V. Tsagou; S. Papanikolaou

2003-01-01

195

Laccase-mediated oxidation of natural glycosides  

Microsoft Academic Search

Regioselective oxidations of the primary OH's of natural glycosides (thiocolchicoside, colchicoside, amygdalin, asiaticoside, ginsenoside RE) have been performed on a preparative scale by exploiting the laccase–2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) methodology. The influence of water-miscible organic cosolvents on the stability and activity of a laccase from Trametes pubescens has been investigated. The enzyme has been covalently linked to Eupergit C250L and its performances

Lara Baratto; Andrea Candido; Mattia Marzorati; Francesca Sagui; Sergio Riva; Bruno Danieli

2006-01-01

196

Mesosilica-coated ultrafine fibers for highly efficient laccase encapsulation.  

PubMed

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. PMID:24821021

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

2014-05-29

197

Modification of old corrugated container pulp with laccase and laccase-mediator system.  

PubMed

Modification of the physical properties of old corrugated container (OCC) pulp with laccase or a laccase-mediator (ABTS, HBT, VA) system was investigated under select enzymatic concentrations and reaction times. The optimal conditions for laccase treatment shown to be using a laccase dose of 160U/g o.d. pulp, a treatment time of 20h at 25°C, pH 7 with a pulp consistency of 5%. Results showed that the Lac-HBT treated OCC pulp gave the best strength properties, improving tensile strength by 15.7%. The increase in the carboxyl group content of OCC laccase or Lac-HBT treated pulp led to the increase in the swelling ability and bonding between fibers. Microscope images showed the fiber surface became rougher and more collapsible after Lac-HBT treatment. FT-IR data showed that new carboxylic acid groups were formed during Lac-HBT treatment. PMID:22326330

Chen, Yangmei; Wan, Jinquan; Ma, Yongwen; Tang, Bing; Han, Wenjia; Ragauskas, Arthur J

2012-04-01

198

In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.  

PubMed

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds. PMID:21877154

Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

2012-05-01

199

Mesosilica-coated ultrafine fibers for highly efficient laccase encapsulation  

NASA Astrophysics Data System (ADS)

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

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

2014-05-01

200

Natural and recombinant fungal laccases for paper pulp bleaching  

Microsoft Academic Search

Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus

C. Sigoillot; E. Record; V. Belle; J. L. Robert; A. Levasseur; P. J. Punt; C. A. M. J. J. van den Hondel; A. Fournel; J. C. Sigoillot; M. Asther

2004-01-01

201

Laccase expression in murine pulmonary Cryptococcus neoformans infection.  

PubMed

Cryptococcus neoformans laccase expression during murine infection was investigated in lung tissue by immunohistochemistry and immunogold electron microscopy. Laccase was detected in the fungal cell cytoplasm, cell wall, and capsule in vivo. The amount of laccase found in different sites varied as a function of the time of infection. PMID:15845520

Garcia-Rivera, Javier; Tucker, Stephanie C; Feldmesser, Marta; Williamson, Peter R; Casadevall, Arturo

2005-05-01

202

Laccase Expression in Murine Pulmonary Cryptococcus neoformans Infection  

PubMed Central

Cryptococcus neoformans laccase expression during murine infection was investigated in lung tissue by immunohistochemistry and immunogold electron microscopy. Laccase was detected in the fungal cell cytoplasm, cell wall, and capsule in vivo. The amount of laccase found in different sites varied as a function of the time of infection.

Garcia-Rivera, Javier; Tucker, Stephanie C.; Feldmesser, Marta; Williamson, Peter R.; Casadevall, Arturo

2005-01-01

203

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

PubMed

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

Chhaya, Urvish; Gupte, Akshaya

2013-06-15

204

Electrochemical characterization of a unique, "neutral" laccase from Flammulina velutipes.  

PubMed

The flac1 gene consisted of 1488 bases encodes a novel laccase (Flac1) from Flammulina velutipes. The deduced amino acid sequence of Flac1 with 496 amino acids shows 58-64% homologies with other fungal laccases. The recombinant Flac1 (rFlac1) was heterologously expressed in Pichia pastoris, with sugars of approximately 4 kDa attached on the protein molecule, which has the calculated molecular mass of 53,532 Da. rFlac1 was shown to be a multi-copper oxidase from spectroscopies. The optimum pHs of rFlac1 for oxidations of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and o-aminophenol, were 5.0, 5.0, and 6.0-6.5, respectively, showing higher pH values than those from many other fungal laccases. The slightly acidic or neutral optimum pH that is not strongly dependent on substrates is a unique property of rFlac1. Effective O(2) reduction was realized by the direct electron transfer of rFlac1 at a highly oriented pyrolytic graphite electrode modified with fine carbon particles (Ketjen Black) in O(2)-saturated solution. The pHs showing the maximum ?E°' [=E°'(enzyme) - E°'(substrate)] coincided well with the optimum pHs shown by rFlac1 under steady-state conditions. The present electrochemical results of rFlac1 indicate that ?E°' is one of the primary factors to determine the activity of multi-copper oxidases. PMID:23063242

Otsuka Saito, Kaori; Kurose, Shinji; Tsujino, Yoshio; Osakai, Toshiyuki; Kataoka, Kunishige; Sakurai, Takeshi; Tamiya, Eiichi

2013-02-01

205

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

PubMed

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

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

2009-12-01

206

Induction of a laccase Lcc9 from Coprinopsis cinerea by fungal coculture and its application on indigo dye decolorization.  

PubMed

A fungal coculture system comprised of Coprinopsis cinerea Okayama 7 (#130) and Gongronella sp. w5 produced 900 times higher laccase activity than that in pure culture. A fungal laccase named Lcc9 was induced from C. cinerea for the first time by coculture. Lcc9 was purified, characterized, and found to have high activity toward phenolic substrates at the optimum pH of 6.5 and temperature of 60°C. The laccase was stable at alkaline pH values, and its activity was not significantly affected by cations and organic solvents. Lcc9 showed decolorization capability toward indigo dye in the presence of 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate), with 75% of indigo was decolorized by 50U/L enzyme after 1h of incubation under optimal catalytic conditions. These results showed that fungal coculture could active silent laccase gene, and the unusual properties make Lcc9 a candidate for specific industrial and environmental applications. PMID:24736211

Pan, Kai; Zhao, Nannan; Yin, Qiang; Zhang, Tianwei; Xu, Xiaolan; Fang, Wei; Hong, Yuzhi; Fang, Zemin; Xiao, Yazhong

2014-06-01

207

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus.  

PubMed

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae ?-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 ?kat/?g, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics. PMID:22526780

Hildén, Kristiina; Mäkelä, Miia R; Lundell, Taina; Kuuskeri, Jaana; Chernykh, Alexey; Golovleva, Ludmila; Archer, David B; Hatakka, Annele

2013-02-01

208

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

NASA Astrophysics Data System (ADS)

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.

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

2003-12-01

209

Simple laccase-based biosensor for formetanate hydrochloride quantification in fruits.  

PubMed

This work describes the development of an electrochemical enzymatic biosensor for quantification of the pesticide formetanate hydrochloride (FMT). It is based on a gold electrode modified with electrodeposited gold nanoparticles and laccase. The principle behind its development relies on FMT's capacity to inhibit the laccase catalytic reaction that occurs in the presence of phenolic substrates. The optimum values for the relevant experimental variables such as gold nanoparticles electrochemical deposition (at -0.2V for 100s), laccase immobilization (via glutaraldehyde cross-linking), laccase concentration (12.4mg/mL), substrate selection and concentration (5.83×10(-5)M of aminophenol), pH (5.0), buffer (Britton-Robinson), and square-wave voltammetric parameters were determined. The developed biosensor was successfully applied to FMT determination in mango and grapes. The attained limit of detection was 9.5×10(-8)±9.5×10(-10)M (0.02±2.6×10(-4)mg/kg on a fresh fruit weight basis). Recoveries for the five tested spiking levels ranged from 95.5±2.9 (grapes) to 108.6±2.5% (mango). The results indicated that the proposed device presents suitable characteristics in terms of sensitivity (20.58±0.49A/?M), linearity (9.43×10(-7) to 1.13×10(-5)M), accuracy, repeatability (RSD of 1.4%), reproducibility (RSD of 1.8%) and stability (19days) for testing of compliance with established maximum residue limits of FMT in fruits and vegetables. PMID:24161938

Ribeiro, Francisco Wirley Paulino; Barroso, Maria Fátima; Morais, Simone; Viswanathan, Subramanian; de Lima-Neto, Pedro; Correia, Adriana N; Oliveira, Maria Beatriz Prior Pinto; Delerue-Matos, Cristina

2014-02-01

210

Purification and biochemical characterization of a new alkali-stable laccase from Trametes sp. isolated in Tunisia: role of the enzyme in olive mill waste water treatment.  

PubMed

A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes sp, was selected in a broad plate screening because of its ability to decolorize and dephenolize olive oil mill wastewater (OMW) efficiently. The major laccase was purified and characterized as a monomeric protein with apparent molecular mass of 61 kDa (SDS-PAGE). It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 4.0 and a temperature of 60 °C. The purified laccase is stable at alkaline pH values. The enzyme retained 50 % of its activity after 90 min of incubation at 55 °C. Using ABTS, this laccase presented K m and V max values of 0.05 mM and 212.73 ?moL min(-1) mg(-1), respectively. It has shown a degrading activity towards a variety of phenolic compounds. The purified laccase was partially inhibited by Fe(2+), Zn(2+), Cd(2+) and Mn(2+), while Cu(2+) acted as inducer. EDTA (10 mM) and NaN3 (10 mM) were found to completely inhibit its activity. 73 % OMW was dephenolized after 315 min incubation at 30 °C with 2 U mL(-1) of laccase and 2 mM HBT. PMID:23712478

Daâssi, Dalel; Zouari-Mechichi, Héla; Prieto, Alicia; Martínez, María Jesús; Nasri, Moncef; Mechichi, Tahar

2013-11-01

211

Construction and direct electrochemistry of orientation controlled laccase electrode.  

PubMed

A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, using genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O2 reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells. PMID:24583131

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

2014-03-28

212

Biodegradation of tetrabromobisphenol A by oxidases in basidiomycetous fungi and estrogenic activity of the biotransformation products.  

PubMed

Tetrabromobisphenol A (TBBPA) degradation was investigated using white rot fungi and their oxidative enzymes. Strains of the Trametes, Pleurotus, Bjerkandera and Dichomitus genera eliminated almost 1 mM TBBPA within 4 days. Laccase, whose role in TBBPA degradation was demonstrated in fungal cultures, was applied to TBBPA degradation alone and in combination with cellobiose dehydrogenase from Sclerotium rolfsii. Purified laccase from Trametes versicolor degraded approximately 2 mM TBBPA within 5 h, while the addition of cellobiose dehydrogenase increased the degradation rate to almost 2.5 mM within 3 h. Laccase was used to prepare TBBPA metabolites 2,6-dibromo-4-(2-hydroxypropane-2-yl) phenol (1), 2,6-dibromo-4-(2-methoxypropane-2-yl) phenol (2) and 1-(3,5-dibromo-4-hydroxyphen-1-yl)-2,2',6,6'-tetrabromo-4,4'-isopropylidene diphenol (3). As compounds 1 and 3 were identical to the TBBPA metabolites prepared by using rat and human liver fractions (Zalko et al., 2006), laccase can provide a simple means of preparing these metabolites for toxicity studies. Products 1 and 2 exhibited estrogenic effects, unlike TBBPA, but lower cell toxicity. PMID:21865031

Uhnáková, Bronislava; Ludwig, Roland; P?knicová, Jana; Homolka, Ladislav; Lisá, Ludmila; Šulc, Miroslav; Pet?í?ková, Alena; Elzeinová, Fatima; Pelantová, Helena; Monti, Daniela; K?en, Vladimír; Haltrich, Dietmar; Martínková, Ludmila

2011-10-01

213

Reactions of pentachlorophenol with laccase from Coriolus versicolor  

Microsoft Academic Search

Laccase, purified from Coriolus versicolor, removed pentachlorophenol (PCP) from solution at pH 5, depending on initial PCP concentration and amount of laccase. With\\u000a 100 units of laccase, 100% of 25??g?ml?1 PCP and 60% of 200??g?ml?1 PCP were removed respectively over 72?h. No free chloride was released in the reaction. In reaction with 100??g PCP, products\\u000a were primarily polymers (about 80,000?MW)

M. A. Ullah; C. T. Bedford; C. S. Evans

2000-01-01

214

Bilirubin oxidases in bioelectrochemistry: features and recent findings.  

PubMed

Bilirubin oxidases, a sub class of the Multicopper oxidases family, were discovered in 1981 by Tanaka and Murao (Murao and Tanaka, 1981) and first used for the detection of bilirubin. Since 2001 and the pioneering work of Tsujimura, these BODs have attracted a lot of attention for the reduction of O2. Unlike laccases, these BODs are stable in physiological conditions (20mM phosphate buffer, pH 7.4, 0.14 M NaCl, 37 °C) and more than 120 papers have been published in the last 7 years. Here, we will first briefly describe some general features of BODs and then review the use of BODs for bilirubin biosensors and the recent achievements and progress toward the elaboration of efficient O2 reducing cathodes. PMID:23911663

Mano, Nicolas; Edembe, Lise

2013-12-15

215

Comparing the efficiency of the laccase–NHA and laccase–HBT systems in eucalyptus pulp bleaching  

Microsoft Academic Search

Laccase–mediator systems have the disadvantage that the mediator is expensive and potentially toxic. In this work, we used N-hydroxyacetanilide (NHA) in combination with laccase for the first time to bleach eucalypt pulp and found it to be a very promising, advantageous alternative to 1-hydroxybenzotriazole (HBT) as mediator. Thus, NHA is efficiently oxidized by laccase to a radical that absorbs light

Cristina Valls; José F. Colom; Carole Baffert; Isabelle Gimbert; M. Blanca Roncero; Jean-Claude Sigoillot

2010-01-01

216

Molecular modeling and docking of novel laccase from multiple serotype of Yersinia enterocolitica suggests differential and multiple substrate binding.  

PubMed

Multi-copper oxidases (MCOs) are widely distributed in bacteria, where they are responsible for metal homeostasis, acquisition and oxidation. Using specific primers, yacK coding for MCO was amplified from different serotypes of Yersinia enterocolitica biovar 1A. Homology modeling of the protein followed by docking with five well-known substrates for different MCO's (viz., 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid [ABTS], syringaldazine, l-tyrosine, ammonium ferrous sulfate and guaiacol), lignin monomers (Coniferyl alcohol, p-coumaryl alcohol and sinapyl alcohol) and two inhibitors i.e., kojic acid and N-hydroxyglycine was done. The docking gave maximum GoldScore i.e., 91.93 and 72.64 with ammonium ferrous sulfate and ABTS, respectively. Similarly, docking with ICM gave -82.10 and -83.61 docking score, confirming the protein to be true laccase with ferroxidase activity. Further, validation with ammonium ferrous sulfate as substrate gave laccase activity of 0.36Units/L/min. Guaiacol, l-tyrosine, and lignin monomers showed good binding affinity with protein models with GoldScores of 35.89, 41.82, 40.41, 41.12 and 43.10, respectively. The sequence study of all the cloned Yack genes showed serotype specific clade in dendrogram. There was distinct discrimination in the ligand binding affinity of Y. enterocolitica laccase, among strains of same clonal groups, suggesting it as a tool for phylogenetic studies. PMID:24832734

Singh, Deepti; Sharma, Krishna Kant; Dhar, Mahesh Shanker; Virdi, Jugsharan Singh

2014-06-20

217

Removal of chlorophenolic derivatives by soil isolated ascomycete of Paraconiothyrium variabile and studying the role of its extracellular laccase.  

PubMed

The ability of Paraconiothyrium variabile, a laccase producing ascomycete recently isolated from soil, was studied to eliminate chlorophenol derivatives in submerged culture medium. Among the tested compounds, ?-chlorophenol (?-CP) and pentachlorophenol (PCP) were found to have minimum and maximum toxic effects, respectively, on the growth of the microorganism and at the same time high and low bioelimination percentages. The fungal strain was able to remove 86% of ?-CP (with initial concentration of 40 mg l(-1)) and 56% of 2,4-dichlorophenol (2,4-DCP; with same concentration as ?-CP) after 9 days of incubation while no elimination was observed in the presence of 2,4,6-trichlorophenol (2,4,6-TCP) and PCP. Monitoring of laccase production level in the fermentation broth together with pollutant removal confirmed the key role of this copper-containing oxidase in chlorophenol derivatives elimination. The type of laccase inducer (guaiacol) and its final concentration (250 ?M) and also initial pH of the fermentation broth (pH=5.5) in the elimination of ?-CP increased the final removal yield from 86% to 94.3%. PMID:22277342

Forootanfar, Hamid; Movahednia, Mohammad Mehdi; Yaghmaei, Soheila; Tabatabaei-Sameni, Minoosadat; Rastegar, Hossein; Sadighi, Armin; Faramarzi, Mohammad Ali

2012-03-30

218

Pervaporation of phenols  

DOEpatents

Aqueous phenolic solutions are separated by pervaporation to yield a phenol-depleted retentate and a phenol-enriched permeate. The separation effect is enhanced by phase segregation into two immiscible phases, phenol in water'' (approximately 10% phenol), and water in phenol'' (approximately 70% phenol). Membranes capable of enriching phenols by pervaporation include elastomeric polymers and anion exchange membranes, membrane selection and process design being guided by pervaporation performance and chemical stability towards phenolic solutions. Single- and multiple-stage processes are disclosed, both for the enrichment of phenols and for purification of water from phenolic contamination. 8 figs.

Boddeker, K.W.

1989-02-21

219

Crystallization and preliminary X-ray crystallographic analysis of the small subunit of the heterodimeric laccase POXA3b from Pleurotus ostreatus.  

PubMed

Laccases are multicopper oxidases of great biotechnological potential. While laccases are generally monomeric glycoproteins, the white-rot fungus Pleurotus ostreatus produces two closely related heterodimeric isoenzymes composed of a large subunit, homologous to the other fungal laccases, and a small subunit. The sequence of the small subunit does not show significant homology to any other protein or domain of known function and consequently its function is unknown. The highest similarity to proteins of known structure is to a putative enoyl-CoA hydratase/isomerase from Acinetobacter baumannii, which shows an identity of 27.8%. Diffraction-quality crystals of the small subunit of the heterodimeric laccase POXA3b (sPOXA3b) from P. ostreatus were obtained using the sitting-drop vapour-diffusion method at 294?K from a solution consisting of 1.8?M sodium formate, 0.1?M Tris-HCl pH 8.5. The crystals belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 126.6, c = 53.9?Å. The asymmetric unit contains two molecules related by a noncrystallographic twofold axis. A complete data set extending to a maximum resolution of 2.5?Å was collected at 100?K using a wavelength of 1.140?Å. PMID:24419623

Ferraroni, Marta; Scozzafava, Andrea; Ullah, Sana; Tron, Thierry; Piscitelli, Alessandra; Sannia, Giovanni

2014-01-01

220

Purification and characterization of a new laccase from the filamentous fungus Podospora anserina.  

PubMed

A new laccase from the filamentous fungus Podospora anserina has been isolated and identified. The 73 kDa protein containing 4 coppers, truncated from its first 31 amino acids, was successfully overexpressed in Pichia pastoris and purified in one step with a yield of 48% and a specific activity of 644Umg(-1). The kinetic parameters, k(cat) and K(M), determined at 37 °C and optimal pH are 1372 s(-1) and 307 ?M for ABTS and, 1.29 s(-1) and 10.9 ?M, for syringaldazine (SGZ). Unlike other laccases, the new protein displays a better thermostability, with a half life>400 min at 37 °C, is less sensitive to chloride and more stable at pH 7. Even though, the new 566 amino-acid enzyme displays a large homology with Bilirubin oxidase (BOD) from Myrothecium verrucaria (58%) and exhibits the four histidine rich domains consensus sequences of BODs, the new enzyme is not able to oxidize neither conjugated nor unconjugated bilirubin. PMID:23220637

Durand, Fabien; Gounel, Sébastien; Mano, Nicolas

2013-03-01

221

Decolourization and detoxification of textile industry wastewater by the laccase-mediator system  

Microsoft Academic Search

Decolourization and detoxification of a textile industry effluent by laccase from Trametes trogii in the presence and the absence of laccase mediators was investigated. Laccase alone was not able to decolourize the effluent efficiently even at the highest enzyme concentration tested: less than 10% decolourization was obtained with 9U\\/mL reaction mixture. To enhance effluent decolourization, several potential laccase mediators were

Rim Khlifi; Lassad Belbahri; Steve Woodward; Mariem Ellouz; Abdelhafidh Dhouib; Sami Sayadi; Tahar Mechichi

2010-01-01

222

Fungal laccases degradation of endocrine disrupting compounds.  

PubMed

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

Macellaro, Gemma; Pezzella, Cinzia; Cicatiello, Paola; Sannia, Giovanni; Piscitelli, Alessandra

2014-01-01

223

Fungal Laccases Degradation of Endocrine Disrupting Compounds  

PubMed Central

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.

Macellaro, Gemma; Cicatiello, Paola; Sannia, Giovanni

2014-01-01

224

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

PubMed

The method of combining white rot fungal strains was used to enhance laccase activity, and the interaction mechanism between strains was also studied. The laccase activity of combined fungi of strain 55 (Trametes trogii) and strain m-6 (Trametes versicolor) were 24.13 and 4.07-fold higher than that of strain 55 and strain m-6, respectively. No inhibitory effect was observed when the two strains were co-cultivated. On plate cultivation, there was hyphal interference in the contact area, where laccase activity was the highest followed by brown pigmentation. In liquid cultivation, strain m-6 played much more important role on enhancement of laccase activity, and the laccase activity of strain 55 by adding strain m-6 was 7.03-fold higher than that of strain m-6 by adding strain 55, furthermore, filter sterilized- and high temperature autoclaved-extracellular substances of strain m-6 could also stimulate strain 55 to excrete more laccase, which led to 6.79-fold and 4. 60-fold increase in laccase activity by adding 20 mL, respectively. The native staining results of Native-PAGE showed that the types of laccase isozymes were not changed when strains were co-cultured, but the concentration of three types increased. PMID:20391719

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

2010-02-01

225

Degradation of Azo Dyes by Laccase and Ultrasound Treatment  

Microsoft Academic Search

The goal of this work was to investigate the decomposition of azo dyes by oxidative methods, such as laccase and ultrasound treatments. Each of these methods has strong and feeble sides. The laccase treatment showed high decolorization rates but cannot degrade all investigated dyes (reactive dyes), and high anionic strength led to enzyme deactivation. Ultrasound treatment can decolorize all tested

Michael M. Tauber; Georg M. Guebitz; Astrid Rehorek

2005-01-01

226

Marinomonas mediterranea MMB-1 Transposon Mutagenesis: Isolation of a Multipotent Polyphenol Oxidase Mutant  

PubMed Central

Marinomonas mediterranea is a melanogenic marine bacterium expressing a multifunctional polyphenol oxidase (PPO) able to oxidize substrates characteristic for laccases and tyrosinases, as well as produce a classical tyrosinase. A new and quick method has been developed for screening laccase activity in culture plates to detect mutants differentially affected in this PPO activity. Transposon mutagenesis has been applied for the first time to M. mediterranea by using different minitransposons loaded in R6K-based suicide delivery vectors mobilizable by conjugation. Higher frequencies of insertions were obtained by using mini-Tn10 derivatives encoding kanamycin or gentamycin resistance. After applying this protocol, a multifunctional PPO-negative mutant was obtained. By using the antibiotic resistance cassette as a marker, flanking regions were cloned. Then the wild-type gene was amplified by PCR and was cloned and sequenced. This is the first report on cloning and sequencing of a gene encoding a prokaryotic enzyme with laccase activity. The deduced amino acid sequence shows the characteristic copper-binding sites of other blue copper proteins, including fungal laccases. In addition, it shows some extra copper-binding sites that might be related to its multipotent enzymatic capability.

Solano, Francisco; Lucas-Elio, Patricia; Fernandez, Eva; Sanchez-Amat, Antonio

2000-01-01

227

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

PubMed

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

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

2013-06-28

228

Improving the fermentation performance of Saccharomyces cerevisiae by laccase during ethanol production from steam-exploded wheat straw at high-substrate loadings.  

PubMed

Operating the saccharification and fermentation processes at high-substrate loadings is a key factor for making ethanol production from lignocellulosic biomass economically viable. However, increasing the substrate loading presents some disadvantages, including a higher concentration of inhibitors (furan derivatives, weak acids, and phenolic compounds) in the media, which negatively affect the fermentation performance. One strategy to eliminate soluble inhibitors is filtering and washing the pretreated material. In this study, it was observed that even if the material was previously washed, inhibitory compounds were released during the enzymatic hydrolysis step. Laccase enzymatic treatment was evaluated as a method to reduce these inhibitory effects. The laccase efficiency was analyzed in a presaccharification and simultaneous saccharification and fermentation process at high-substrate loadings. Water-insoluble solids fraction from steam-exploded wheat straw was used as substrate and Saccharomyces cerevisiae as fermenting microorganism. Laccase supplementation reduced strongly the phenolic content in the media, without affecting weak acids and furan derivatives. This strategy resulted in an improved yeast performance during simultaneous saccharification and fermentation process, increasing significantly ethanol productivity. PMID:23143932

Alvira, Pablo; Moreno, Antonio D; Ibarra, David; Sáez, Felicia; Ballesteros, Mercedes

2013-01-01

229

Laccase2 is required for cuticular pigmentation in stinkbugs.  

PubMed

During the maturation of insect cuticle, protein-protein and protein-chitin crosslinkages are formed by the action of diphenoloxidases. Two types of diphenoloxidases, laccases and tyrosinases, are present in the insect cuticle. In coleopteran and hymenopteran insects, laccase2 gene has been identified as encoding an enzyme principally responsible for cuticular pigmentation and hardening, whereas biological roles of laccase genes in hemimetabolous insects remain to be established. Here we identified laccase2 genes from three hemipteran stinkbugs, Riptortus pedestris (Alydidae), Nysius plebeius (Lygaeidae) and Megacopta punctatissima (Plataspidae). In R. pedestris, laccase2 gene was highly expressed in epidermal tissues prior to molting. When the gene expression was suppressed by an RNA interference technique, cuticular pigmentation after molting were blocked depending on the dose of injected double-stranded RNA targeting the laccase2 gene. Similar results were obtained for N. plebeius and M. punctatissima. In all the stinkbug species, injecting 20 ng of double-stranded RNA was sufficient to prevent the cuticular maturation. These results indicate that laccase2 gene is generally required for cuticular pigmentation in different stinkbug families, highlighting its conserved biological function across diverse insect taxa. PMID:21167282

Futahashi, Ryo; Tanaka, Kohjiro; Matsuura, Yu; Tanahashi, Masahiko; Kikuchi, Yoshitomo; Fukatsu, Takema

2011-03-01

230

Induction of laccases in Trametes versicolor by aqueous wood extracts.  

PubMed

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

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

2014-01-01

231

Oxidases and related redox systems  

SciTech Connect

This book contains the proceedings of a symposium on oxidases and related redoxsystems. Topics covered include: Oxidases and related redoxsystems, Flavoprotein oxidases and oxygenases, Peroxidases, and Cytochrome P-450 and related proteins.

King, T.E. (Inst. for Structural and Functional Studies, Univ. City Science Center, Philadelphia, PA (US)); Mason, H.S. (Dept. of Biochemistry, Oregon Health Sciences Univ., Portland, OR (US)); Morrison, M. (Saint Jude Children's Hospital, Memphis, TN (USA). Dept. of Biochemical and Chemical Pharmacology)

1988-01-01

232

Laccase immobilized on magnetic carriers for biotechnology applications  

NASA Astrophysics Data System (ADS)

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.

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

2009-05-01

233

Screening Diverse Fungi for Laccases of Varying Properties  

Microsoft Academic Search

Qualitative screening of 295 fungi for laccases yielded 125 laccase positive ones, mostly basidiomycetes. Fifty of these were\\u000a tested for laccase activity at pH 3.0, 4.5 and 6.0. Most showed maximum activity at pH 4.5, a few showed a broad activity\\u000a range, two were optimal at pH 3.0 and only the mitosporic fungus Beltraniella sp. was best at pH 6.

Pranali M. Bodke; Gunasekaran Senthilarasu; Seshagiri Raghukumar

234

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

PubMed

Pycnoporus cinnabarinus laccase and a chimeric laccase-CBM were applied in softwood kraft pulp biobleaching in the presence of 1-hydroxybenzotriazole (HBT). The presence of CBM could enhance the laccase biobleaching potential as a decrease in the enzymatic charge and chlorine dioxide consumption, as well as an increase in pulp brightness were observed. Laccase/HBT treatment could be improved by increasing oxygen pressure from 1 to 3bar and pulp consistency from 5% to 10%. Conversely, under the same conditions, no improvement of laccase-CBM/HBT treatment was observed, indicating a different behavior of both systems. However, laccase-CBM/HBT treatment led to a better preservation of pulp properties. This effect was probably due to fiber surface modifications involving the action of the CBM. Transmission electron microscopy examination of pulp fibers indicated a retention of laccase-CBM inside the pulp fibers due to CBM binding and an increased external microfibrillation of the fibers due to enzymatic treatments. PMID:22854132

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

2012-10-01

235

Stimulation of indoleacetic acid production in a Rhizobium isolate of Vigna mungo by root nodule phenolic acids  

Microsoft Academic Search

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA\\u000a metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots.\\u000a Three most abundant endogenous root nodule

Santi Mandal; Mahitosh Mandal; Amit Das; Bikas Pati; Ananta Ghosh

2009-01-01

236

Textile dye degrading laccase from Pseudomonas desmolyticum NCIM 2112  

Microsoft Academic Search

A laccase requiring optimum temperature 60°C, pH 4.0 for the activity and having apparent molecular weight 43,000Da was purified from Pseudomonas desmolyticum NCIM 2112 by three steps, including heating, anion exchange, and molecular sieve chromatography. The purification fold and yield of laccase obtained through Biogel P100 were 45.75 and 19%, respectively. Staining of native gel with L-dopa showed dark brown

Satish Kalme; Sheetal Jadhav; Mital Jadhav; Sanjay Govindwar

2009-01-01

237

Degradation of Bisphenol A by Purified Laccase from Trametes villosa  

Microsoft Academic Search

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

Tetsuya Fukuda; Hiroyuki Uchida; Yoshiko Takashima; Takayuki Uwajima; Takahiro Kawabata; Motoshi Suzuki

2001-01-01

238

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

PubMed Central

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.

Brander, S?ren; Mikkelsen, J?rn D.; Kepp, Kasper P.

2014-01-01

239

CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity  

PubMed Central

Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10?6±0.21 M·min?1 and 0.32±0.02 s?1, respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal.

Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

2013-01-01

240

Investigation of hydroxamic acids as laccase-mediators for pulp bleaching.  

PubMed

A number of hydroxamic acids have been synthesized and investigated as laccase-mediators for pulp bleaching. As compared with N-hydroxyacetanilide (NHA), one of the most effective laccase-mediators reported so far, N-(4-cyanophenyl)acetohydroxamic acid (NCPA), resulted in the highest brightness and lowest kappa number of hardwood kraft pulp of all the laccase-mediators studied. The bleaching efficacy of a laccase/7-cyano-4-hydroxy-2H-1,4-benzoxazin-3-one system was also comparable with that of a laccase/NHA system. A laccase/NCPA system was further studied for the bleaching of unbleached softwood kraft pulp. The effects of pulp consistency, laccase dosage, NCPA dosage, incubation time, and oxygen pressure on the bleaching efficacy of a laccase/NCPA system were studied. PMID:14605773

Geng, X; Li, K; Xu, F

2004-05-01

241

Effect of three trifluoromethanesulfonate ionic liquids on the activity, stability and conformation of laccase.  

PubMed

The activity, stability and conformation of laccase were first investigated in an aqueous solution of ionic liquids 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim]TfO), 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate ([Bmpyr]TfO) or tetramethylammonium trifluoromethanesulfonate ([TMA]TfO). Compared with control system, high level of [Bmim]TfO or [Bmpyr]TfO destabilizes laccase while [TMA]TfO stabilizes laccase. These effects are more pronounced with the extension of the incubation time. The activity variations are well correlated with the changes of the conformation of laccase evidenced by fluorescence and circular dichroism spectra under specified conditions. The effects of the three ionic liquids on laccase are associated with the chaotropicity of the cations in Hofmeister series. For laccase, [TMA]TfO is not a good activating agent but it greatly enhances the stability of laccase in addition to maintaining the catalytic efficiency of laccase, showing its great potential in real application. PMID:23403026

Yu, Xinxin; Zou, Feixue; Li, Ying; Lu, Lu; Huang, Xirong; Qu, Yinbo

2013-05-01

242

In situ encapsulation of laccase in microfibers by emulsion electrospinning: preparation, characterization, and application.  

PubMed

Laccase from Trametes versicolor was successfully in situ encapsulated into the poly(D,L-lactide) (PDLLA)/PEO-PPO-PEO (F108) electrospun microfibers by emulsion electrospinning. The porous morphology of electrospun microfibers was observed with scanning electron microscope, and the core-shell structure of microfibers and existence of laccase in microfibers were proved by laser confocal scanning microscopy micrograph. In this study, fibrous porosity and core-shell structure are advantageous to the activity and stability preservation of immobilized laccase. The activity of immobilized laccase could retain over 67% of that of the free enzyme. After 10 successive runs in the enzyme reactor, the immobilized laccase could also maintain 50% of its initial activity. Crystal violet dye was successfully degraded by the PDLLA/F108-laccase electrospun microfiber membranes. It was observed that the immobilized laccase possessed a broadening pH range of catalysis activity compared to free laccase. PMID:20673716

Dai, Yunrong; Niu, Junfeng; Liu, Jia; Yin, Lifeng; Xu, Jiangjie

2010-12-01

243

Antioxidant activity and enzyme inhibition of phenolic acids from fermented rice bran with fungus Rizhopus oryzae.  

PubMed

The solid-state fermentation (SSF) has been employed as a form making available a higher content of functional compounds from agroindustrial wastes. In this work, the effect of SSF with the Rhizopus oryzae fungus on the phenolic acid content of rice bran was studied. Phenolic extracts derived from rice bran and fermented rice bran were evaluated for their ability to reduce free radical 1,1-diphenyl-2-picrihidrazil (DPPH) and for the ability to inhibit the enzymes peroxidase and polyphenol oxidase. The phenolic compound content increased by more than two times with fermentation. A change in the content of phenolic acids was observed, with ferulic acid presenting the greatest increase with the fermentation, starting from 33mg/g in rice bran and reaching 765mg/g in the fermented bran. The phenolic extracts showed an inhibition potential for DPPH and for the peroxidase enzyme, however did not inhibit the polyphenol oxidase enzyme. PMID:24176356

Schmidt, Cristiano G; Gonçalves, Letícia M; Prietto, Luciana; Hackbart, Helen S; Furlong, Eliana B

2014-03-01

244

Cholesterol oxidase: physiological functions  

PubMed Central

An important aspect of catalysis by cholesterol oxidase (3?-hydroxysteroid oxidase) is the nature of its association with the lipid bilayer that contains the sterol substrate. Efficient catalytic turnover is affected by the association of the protein with the membrane as well as the solubility of the substrate in the lipid bilayer. In this review, the binding of cholesterol oxidase to the lipid bilayer, its turnover of substrates presented in different physical environments, and how these conditions affect substrate specificity are discussed. The physiological functions of the enzyme in bacterial metabolism, pathogenesis, and macrolide biosynthesis are reviewed in this context.

Kreit, Joseph; Sampson, Nicole S.

2009-01-01

245

Anthraquinone dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase  

Microsoft Academic Search

A new Trametes trogii laccase was purified and its biochemical properties were subsequently characterized. After a survey of other T. trogii laccases, this laccase showed a lower isoelectric point, different N-terminal sequence and kinetic parameters. Recently most laccase-catalyzed decolorizations of synthetic dyes are single-solute studies with commercially available dyes as model pollutants and need the employment of redox mediators. In

Xiangkang Zeng; Yujie Cai; Xiangru Liao; Xianglong Zeng; Shoupeng Luo; Dabing Zhang

246

Remazol Brilliant Blue R decolourization by the laccase from Trametes trogii  

Microsoft Academic Search

The decolourization of the recalcitrant dye RBBR by the culture filtrate of Trametes trogii and its isolated laccase was investigated. Both filtrates from Cu-induced cultures as well as purified laccase decolourized the dye RBBR. The purified laccase decolourized the dye down to 97% of 100mgl?1 initial concentration of RBBR when only 0.2Uml?1 of laccase was used in the reaction mixture.

Tahar Mechichi; Nejla Mhiri; Sami Sayadi

2006-01-01

247

Modification of high-lignin kraft pulps with laccase. Part 2. Xylanase-enhanced strength benefits.  

PubMed

The effects of xylanase pretreatment of high lignin content softwood (SW) kraft pulp on subsequent pulp treatment with laccase in combination with gallic acid were investigated. Although xylanase pretreatment was ineffective in enhancing the laccase-facilitated biografting of gallic acid to kraft fibers, it was beneficial for subsequent treatment with laccase exclusively. Treating pulp fibers with xylanase followed by laccase provided a collective 25% and 46% increase in dry and wet tensile strength properties, respectively. PMID:16080715

Chandra, Richard P; Ragauskas, Arthur J

2005-01-01

248

Partial characterization of lettuce ( Lactuca sativa L.) polyphenol oxidase  

Microsoft Academic Search

Polyphenol oxidase (PPO) from garden lettuce (Lactuca sativa L.) was partially purified by ammonium sulphate ((NH4)2SO4) precipitation and dialysis, and then some of its kinetic properties such as optimum pH and temperature, substrate specificity,\\u000a thermal inactivation and inhibition were investigated. The total phenolic and protein contents of Lactuca sativa L. extracts were determined according to the Folin-Ciocalteu and Bradford methods,

Serap Do?an; Ümran Salman

2007-01-01

249

A NOVEL BIOSENSOR BASED ON Lactobacillus acidophilus FOR DETERMINATION OF PHENOLIC COMPOUNDS IN MILK PRODUCTS AND WASTEWATER  

Microsoft Academic Search

Different branches of industry need to use phenolic compounds (PCs) in their production, so determination of PCs sensitively, accurately, rapidly, and economically is very important. For the sensitive determination of PCs, some biosensors based on pure polyphenol oxidase, plant tissu,e and microorganisms were developed before. But there has been no study to develop a microbial phenolic compounds biosensor based on

Ayten Sagiroglu; Hatice Paluzar; Hakki Mevlut Ozcan; Suzan Okten; Burhan Sen

2011-01-01

250

Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase  

Microsoft Academic Search

The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two

Alexander Schouten; Lia Wagemakers; Francesca L. Stefanato; Kaaij van der R. M; Kan van J. A. L

2002-01-01

251

Effect of cucurbitacins on mRNA coding for laccase in Botrytis cinerea  

Microsoft Academic Search

The effect of cucurbitacin and of Ecballium extract on the formation of mRNA coding for laccase was examined in cultures of Botrytis cinerea grown with inducers of laccase formation, in the presence or absence of the inhibitory compounds. RNA was isolated from the cultures and probed with specific DNA probes for laccase. As an internal control, the RNA was probed

Lyat Gonen; Ada Viterbo; Frank Cantone; Richard C. Staples; Alfred M. Mayer

1996-01-01

252

Decolorization of textile dyes by laccases from a newly isolated strain of Trametes modesta  

Microsoft Academic Search

Four ligninolytic fungi, Trametes modesta, Trametes hirsuta, Trametes versicolor and Sclerotium rolfsii, were compared for their ability to produce laccases. The fungal laccases were screened for their ability to decolorize eight synthetic dyes (anthraquinone, azo, indigo and triarylmethane). The decolorization rate depended both on the source of the enzyme preparation and on the structure of the dye. Based on laccase

G. S Nyanhongo; J Gomes; G. M Gübitz; R Zvauya; J Read; W Steiner

2002-01-01

253

Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies  

Microsoft Academic Search

The present investigation addresses the problem of the binding mode of phenolic inhibitors and the substrate ascorbate to the active site of ascorbate oxidase. The results from both types of compounds indicate that the binding site is located in a pocket near the type 1 copper center. This information is of general interst for blue multicopper oxidases. Docking calculations performed

Laura Santagostini; Michele Gullotti; Luca De Gioia; Piercarlo Fantucci; Elena Franzini; Augusto Marchesini; Enrico Monzani; Luigi Casella

2004-01-01

254

Antioxidant, ?-glucosidase and xanthine oxidase inhibitory activity of bioactive compounds from maize (Zea mays L.).  

PubMed

Chemical investigations into maize (Zea mays L.) kernels yielded phenolic compounds, which were structurally established using chromatographic and spectroscopic methods. The isolated phenolic compounds from maize kernel were examined in vitro for their antioxidant abilities by DPPH (2,2-diphenyl-1-picryl hydrazine) radical, OH radical scavenging activity, and reducing ability, along with ?-glucosidase and xanthine oxidase (XO) inhibition. The isolated maize phenolics revealed significant xanthine oxidase and ?-glucosidase inhibitory activity to that of allopurinol and acarbose in vitro and in vivo, respectively. The kinetics study with xanthine oxidase revealed competitive type of inhibition by isolated maize vanillic acid (M2), ferulic acid (M5), 3'-methoxyhirsutrin (M7), and peonidin-3-glucoside (M10) as compared to control allopurinol. Overall, with few exceptions, all the phenolic compounds from maize kernel revealed significant biological activities with all parameters examined. Also, the phenolic compounds from maize were found to be more reactive toward DPPH radical and had considerable reducing ability and OH radical scavenging activity. These findings suggest that maize kernel phenolic compounds can be considered as potential antioxidant, ?-glucosidase, and XO inhibitory agents those might be further explored for the design of lead antioxidant, antidiabetic and antigout drug candidates using in vivo trials. PMID:23957301

Nile, Shivraj H; Park, Se W

2014-01-01

255

Degradation of Azo Dyes by Laccase and Ultrasound Treatment  

PubMed Central

The goal of this work was to investigate the decomposition of azo dyes by oxidative methods, such as laccase and ultrasound treatments. Each of these methods has strong and feeble sides. The laccase treatment showed high decolorization rates but cannot degrade all investigated dyes (reactive dyes), and high anionic strength led to enzyme deactivation. Ultrasound treatment can decolorize all tested dyes after 3 h at a high energy input, and prolonged sonication leads to nontoxic ionic species, which was demonstrated by ion chromatography and toxicity assays. For the first time, it was shown that a combination of laccase and ultrasound treatments can have synergistic effects, which was shown by higher degradation rates. Bulk light absorption and ion-pairing high-performance liquid chromatography (IP-HPLC) were used for process monitoring, while with reversed-phase HPLC, a lower number of intermediates than expected by IP-HPLC was found. Liquid chromatography-mass spectrometry indicated that both acid orange dyes lead to a common end product due to laccase treatment. Acid Orange 52 is demethylated by laccase and ultrasound treatment. Further results confirmed that the main effect of ultrasound is based on ?OH attack on the dye molecules.

Tauber, Michael M.; Guebitz, Georg M.; Rehorek, Astrid

2005-01-01

256

Electrochemical studies of a truncated laccase produced in Pichia pastoris.  

PubMed

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 (k(het) for LCCIa = 1.3 x 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

Gelo-Pujic, M; Kim, H H; Butlin, N G; Palmore, G T

1999-12-01

257

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

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.

2013-01-01

258

Phenolic Wastewater Treatment Alternatives.  

National Technical Information Service (NTIS)

The Air Force uses phenol compounds primarily in its paint stripping and carbon removal operations, the major portions of which are performed at the Air Logistics Centers (ALCs). Depending on the type operation, the spent phenol either enters the industri...

R. G. Blum

1980-01-01

259

Phenol removal pretreatment process  

DOEpatents

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.

Hames, Bonnie R. (Westminster, CO) [Westminster, CO

2004-04-13

260

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode.  

PubMed

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35°C when operated at a scan rate of 50 mV s(-1). Linear range, response time, and detection limit were 0.1-500 ?M, 6 s, and 0.1 ?M, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4°C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment. PMID:21855525

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

2011-12-15

261

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

PubMed Central

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.

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

2011-01-01

262

Extra- and Intracellular Laccases of the Chestnut Blight Fungus, Cryphonectria parasitica  

PubMed Central

A double-stranded RNA virus of the chestnut blight pathogen, Cryphonectria parasitica, has been shown previously to reduce accumulation of mRNAs of extracellular laccase (laccase A) produced by this fungus. Both extra- and intracellular laccases have been detected after growth of the fungus in liquid culture. In addition to cellular localization, the two laccases are distinguishable by time of appearance during growth and electrophoretic mobility. Laccase A was purified from the culture filtrate by standard protein purification procedures. The enzyme was characterized as a glycoprotein with a molecular mass of approximately 77 kDa. Both laccase A and laccase B activities were significantly reduced in the hypovirulent (double-stranded RNA-infected) strain UEP1 compared with the isogenic virulent (double-stranded RNA-free) strain EP155/2. Images

Rigling, Daniel; Van Alfen, Neal K.

1993-01-01

263

Mesoporous silicas with tunable morphology for the immobilization of laccase.  

PubMed

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

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

2014-01-01

264

Phenol oxidase activity in secondary transformed peat-moorsh soils  

Microsoft Academic Search

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

K. Styla; L. Szajdak

2009-01-01

265

Laccase-catalysed functionalisation of chitosan by ferulic acid and ethyl ferulate: evaluation of physicochemical and biofunctional properties.  

PubMed

Chitosan and its derivatives functionalized by laccase-catalyzed oxidation of ferulic acid (FA) and ethyl ferulate (EF) were characterised for their physico-chemical, antioxidant and antibacterial properties. The enzymatic grafting of oxidised phenols led to FA-coloured and EF-colourless chitosan derivatives with good stability of colour and grafted phenols towards the chemical treatment by organic solvents. The efficiency of FA-products grafting onto chitosan was higher than that of EF-products. Moreover, the enzymatic grafting of phenols onto chitosan changed its morphological surface, increased its molecular weight and its viscosity. Furthermore, the chitosan derivatives presented improved antioxidant properties especially for FA-chitosan derivative when compared with chitosan with good antioxidant stability towards thermal treatment (100°C/1h). Chitosan and its derivatives showed also similar antibacterial activities and more precisely bactericidal activities. This enzymatic procedure provided chitosan derivatives with improved properties such as antioxidant activity, thermal antioxidant stability as well as the preservation of initial antibacterial activity of chitosan. PMID:24837951

Aljawish, Abdulhadi; Chevalot, Isabelle; Jasniewski, Jordane; Revol-Junelles, Anne-Marie; Scher, Joël; Muniglia, Lionel

2014-10-15

266

Phenolic metabolism of Matricaria chamomilla plants exposed to nickel.  

PubMed

We examined accumulation of phenolic acids, total soluble phenolics and flavonoids, and activities of phenolic metabolism-related enzymes (shikimate dehydrogenase (SKDH), phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), polyphenol oxidase (PPO)) in Matricaria chamomilla plants exposed to 3, 60 and 120 microM of nickel (Ni) for 10 days. Ni showed low toxicity as indicated by unaltered content of total soluble phenolics in the leaf rosettes. In the roots, the effects of Ni were more visible, including increased total phenolics and PAL activity, but a decrease in PPO activity was observed. CAD activity was not affected by any of the Ni concentrations. Cinnamic acid derivatives were affected more than benzoic acid derivatives. Accumulation of chlorogenic acid, an important antioxidant compound, was enhanced by Ni treatment (ca. 4-fold in 120 microM Ni). Accumulation of protocatechuic acid, a phenol with high chelating strength, even decreased in the leaf rosettes. These observations are discussed in connection to antioxidative properties of phenolic metabolites and previously tested metals (cadmium and copper). PMID:19380176

Kovácik, Jozef; Klejdus, Borivoj; Backor, Martin

2009-09-01

267

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

PubMed Central

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.

2014-01-01

268

Oxidation of phenolic compounds from Aloe barbadensis by peroxidase activity: Possible involvement in defence reactions  

Microsoft Academic Search

Sephadex LH-20 chromatography and reverse phase-high performance liquid chromatography (RP-HPLC) have been combined to analyse different phenolics in Aloe barbadensis Mill. Among them, a new chromone peak was found. Whole phenolics, and anthrone and chromone fractions were assayed as substrates of endogenous peroxidases (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7) and polyphenol oxidases (1,2-benzenediol:oxygen oxidoreductase; EC 1.10.3.1) by following the disappearance of specific

Alberto Esteban-Carrasco; Matías López-Serrano; José Miguel Zapata; Bartolomé Sabater; Mercedes Martín

2001-01-01

269

Synthetic dye decolorization by three sources of fungal laccase.  

PubMed

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

Forootanfar, Hamid; Moezzi, Atefeh; Aghaie-Khozani, Marzieh; Mahmoudjanlou, Yasaman; Ameri, Alieh; Niknejad, Farhad; Faramarzi, Mohammad Ali

2012-01-01

270

Novel laccase redox mediators: spectral, electrochemical, and kinetic properties.  

PubMed

The screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from Coriolus hirsutus and Coriolus zonatus. It was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. The characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4- methylamino-pyrazolone-5-N(4)-methanesulfonate and 1-(3'-sulfophenyl)-3- methylpyrazolone-5, in the reaction catalyzed by laccase was carried out using spectral, electrochemical, and enzyme kinetics methods. The kinetic parameters for the oxidation of the newly discovered substrates were comparable with those for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by laccase. Electrochemical experiments demonstrated that oxidation of these compounds yielded two high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, to the corresponding aldehyde and acid. 1-(3'-Sulfophenyl)-3- methylpyrazolone-5 was about 30-40% as effective in degrading veratryl alcohol compared to ABTS as judged from high-performance liquid chromatography kinetic studies. 1-Phenyl-3-methyl-pyrazolones may be of commercial interest for oxidoreductase-catalyzed biodegradation of organic compounds. PMID:14665736

Shleev, S V; Khan, I Gvon; Gazaryan, I G; Morozova, O V; Yaropolov, A I

2003-12-01

271

Synthetic dye decolorization by three sources of fungal laccase  

PubMed Central

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.

2012-01-01

272

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

PubMed

Newly large-pore magnetic mesoporous silica nanoparticles (MMSNPs) with wormhole framework structures were synthesized for the first time by using tetraethyl orthosilicate as the silica source and amine-terminated Jeffamine surfactants as template. Iminodiacerate was attached on these MMSNPs through a silane-coupling agent and chelated with Cu(2+). The Cu(2+)-chelated MMSNPs (MMSNPs-CPTS-IDA-Cu(2+)) showed higher adsorption capacity of 98.1 mg g(-1)-particles and activity recovery of 92.5% for laccase via metal affinity adsorption in comparison with MMSNPs via physical adsorption. The Michaelis constant (K(m)) and catalytic constant (k(cat)) of laccase immobilized on the MMSNPs-CPTS-IDA-Cu(2+) were 3.28 mM and 155.4 min(-1), respectively. Storage stability and temperature endurance of the immobilized laccase on MMSNPs-CPTS-IDA-Cu(2+) increased significantly, and the immobilized laccase retained 86.6% of its initial activity after 10 successive batch reactions operated with magnetic separation. PMID:20655206

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

2010-12-01

273

Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: the role of laccase and versatile peroxidase  

Microsoft Academic Search

The ability of Pleurotus eryngii, Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus sajor-caju to degrade the aromatic pollutants 2,4-dichorophenol (2,4-DCP) and benzo(a)pyrene [B(a)P] in liquid culture and microcosm (using wheat straw as growth substrate and sea sand as a xenobiotic carrier) was investigated by HPLC and 14CO2 release from labeled pollutants. We found that 100 ?M 2,4-DCP was very quickly transformed

E Rodr??guez; O Nuero; F Guillén; A. T Mart??nez; M. J Mart??nez

2004-01-01

274

Enhancing the Laccase Production and Laccase Gene Expression in the White-Rot Fungus Trametes velutina 5930 with Great Potential for Biotechnological Applications by Different Metal Ions and Aromatic Compounds  

PubMed Central

Laccase is useful for various biotechnological and industrial applications. The white-rot fungus Trametes velutina 5930 and its laccase, isolated from the Shennongjia Nature Reserve in China by our laboratory, has great potential for practical application in environmental biotechnology. However, the original level of laccase produced by Trametes velutina 5930 was relatively low in the absence of any inducer. Therefore, in order to enhance the laccase production by Trametes velutina 5930 and make better use of this fungus in the field of environmental biotechnology, the regulation of laccase production and laccase gene expression in Trametes velutina 5930 were investigated in this study. Different metal ions such as Cu2+ and Fe2+ could stimulate the laccase synthesis and laccase gene transcription in Trametes velutina 5930. Some aromatic compounds structurally related to lignin, such as tannic acid, syringic acid, cinnamic acid, gallic acid and guaiacol, could also enhance the level of laccase activity and laccase gene transcription. We also found that there existed a positive synergistic effect of aromatic compound and metal ion on the laccase production and laccase gene transcription in Trametes velutina 5930. Taken together, our study may contribute to the improvement of laccase productivity by Trametes velutina 5930.

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

2013-01-01

275

Molecular Structure of Phenol  

NSDL National Science Digital Library

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.

2003-05-08

276

Decolorization of Alizarin Red and other synthetic dyes by a recombinant laccase from Pichia pastoris.  

PubMed

A cDNA encoding for a laccase was isolated from the white-rot fungus Lenzites gibbosa by RT-PCR and expressed in the Pichia pastoris. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as pH, cultivation temperature, copper concentration and methanol concentration, were optimized. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a MW of ~61.5 kDa. The purified enzyme behaved similarly to the native laccase produced by L. gibbosa and efficiently decolorized Alizarin Red, Neutral Red, Congo Red and Crystal Violet, without the addition of redox mediators. The decolorization capacity of this recombinant enzyme suggests that it could be a useful biocatalyst for the treatment of dye-containing effluents. This study is the first report on the synthetic dye decolorization by a recombinant L. gibbosa laccase. PMID:24078122

Zheng, Miaomiao; Chi, Yujie; Yi, Hongwei; Shao, Shuli

2014-01-01

277

Flow-cell fibre-optic enzyme sensor for phenols  

SciTech Connect

A solid-state fibre-optic luminescent oxygen sensor was used for flow-through measurements. It acts as a transducer in a new flow-cell enzyme sensor arrangement. This arrangement comprises a flow path, sample injector, microcolumn with the immobilized enzyme, oxygen membrane and fibre-optic connector joined together to form an integral unit. Laccase enzyme was used as a recognition system which provided specific oxidation of the substrates with the dissolved oxygen being monitored. The assay procedure was optimized and performance of the new system studied. The sensor was applied to the determination polyphenol content in tea, brandy, etc. (quality control test). The sensitivity to some important phenolic compounds was tested with the view of industrial wastewater control applications. 5 refs., 6 figs., 1 tab.

Papkovsky, D.B.; Ghindilis, A.L.; Kurochkin, I.N. (Research Center of Molecular Diagnostics and Therapy, Moscow (Russian Federation))

1993-07-01

278

Enhanced production of laccase in repeated-batch cultures of Funalia trogii and Trametes versicolor  

Microsoft Academic Search

The biotechnologically important enzyme laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is secreted by white rot fungi. However, these organisms produce insufficient amount of laccase for use in various biotechnological areas. The main aim of this study is to enhance the laccase production in the repeated-batch cultures of Funalia trogii ATCC 200800 and Trametes versicolor ATCC 200801 isolated in Turkey. In

Emre Birhanli; Ozfer Yesilada

2010-01-01

279

Purification, characterization and decolourization ability of Fomes fomentarius laccase produced in solid medium  

Microsoft Academic Search

Laccase produced by Fomes fomentarius grown on wheat bran in solid cultures was purified to electrophoretic homogeneity by ammonium sulfate precipitation, size-exclusion chromatography and anion-exchange chromatography. A single laccase was found having apparent molecular mass of 51kDa. The N-terminal amino acid sequence was IGPKTDLTIATGDVSPDG and the highest similarity value was found to the laccase from Trametes sp. 420 (94%). The

Mohamed Neifar; Atef Jaouani; Raoudha Ellouze-Ghorbel; Semia Ellouze-Chaabouni

2010-01-01

280

Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system  

Microsoft Academic Search

High-quality flax pulp was bleached in a totally-chlorine-free (TCF) sequence using a laccase-mediator system. Three fungal laccases (from Pycnoporus cinnabarinus, Trametes versicolor and Pleurotus eryngii) and two mediators, 2,2?-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and 1-hydroxybenzotriazole (HBT), were compared. P. cinnabarinus and T. versicolor laccases in the presence of HBT gave the best results in terms of high brightness and low lignin content (kappa

Susana Camarero; Olga Garc??a; Teresa Vidal; José Colom; José C del R??o; Ana Gutiérrez; José M Gras; Rebeca Monje; Mar??a J Mart??nez; Ángel T Mart??nez

2004-01-01

281

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

Microsoft Academic Search

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

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

2008-01-01

282

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

Microsoft Academic Search

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

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

1997-01-01

283

Multicopper oxidases and oxygenases  

Microsoft Academic Search

Copper is an essential trace element in living systems, present in the parts per million concentration range. It is a key cofactor in a diverse array of biological oxidation-reduction reactions. These involve either outer-sphere electron transfer, as in the blue copper proteins and the Cu{sub A} site of cytochrome oxidase and nitrous oxide redutase, or inner-sphere electron transfer in the

Edward I. Solomon; Uma M. Sundaram; Timothy E. Machonkin

1996-01-01

284

Increasing Pleurotus ostreatus laccase production by culture medium optimization and copper/lignin synergistic induction.  

PubMed

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 2??² where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively. PMID:20694851

Tinoco, Raunel; Acevedo, Abisaí; Galindo, Enrique; Serrano-Carreón, Leobardo

2011-04-01

285

Controlling the simultaneous production of laccase and lignin peroxidase from Streptomyces cinnamomensis by medium formulation  

PubMed Central

Background Use of crude ligninase of bacterial origin is one of the most promising ways to improve the practical biodegradation of lignocellulosic biomass. However, lignin is composed of diverse monolignols with different abundance levels in different plant biomass and requires different proportions of ligninase to realize efficient degradation. To improve activity and reduce cost, the simultaneous submerged fermentation of laccase and lignin peroxidase (LiP) from a new bacterial strain, Streptomyces cinnamomensis, was studied by adopting formulation design, principal component analysis, regression analysis and unconstrained mathematical programming. Results The activities of laccase and LiP from S. cinnamomensis cultured with the optimal medium formulations were improved to be five to eight folders of their initial activities, and the measured laccase:LiP activity ratios reached 0.1, 0.4 and 1.7 when cultured on medium with formulations designed to produce laccase:LiP complexes with theoretical laccase:LiP activity ratios of 0.05 to 0.1, 0.5 to 1 and 1.1 to 2. Conclusion Both the laccase and LiP activities and also the activity ratio of laccase to LiP could be controlled by the medium formulation as designed. Using a crude laccase-LiP complex with a specially designed laccase:LiP activity ratio has the potential to improve the degradation of various plant lignins composed of diverse monolignols with different abundance levels.

2012-01-01

286

Enzymatic removal of phenols from aqueous solution by artichoke ( Cynara scolymus L.) extracts  

Microsoft Academic Search

The effects of extracts from artichoke (Cynara scolymus L.) flower bracts on model wastewaters containing a range of phenolic contaminants have been studied. The extracts contained various isoenzymes of both peroxidase (POD) and polyphenol oxidase (PPO). HPLC measurements showed that the monophenol, 4-chlorophenol, was most effectively oxidized in the presence of both extract and hydrogen peroxide (H2O2), suggesting that this

Dorotea López-Molina; Alexander N. P Hiner; José Tudela; Francisco Garc??a-Cánovas; José Neptuno Rodr??guez-López

2003-01-01

287

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

Microsoft Academic Search

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

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

288

Bilirubin Oxidase from Bacillus pumilus: A promising enzyme for the elaboration of efficient cathodes in Biofuel cells  

PubMed Central

A CotA Multicopper Oxidase (MCO) from Bacillus pumilus, previously identified as a laccase, has been studied and characterized as a new bacterial Bilirubin Oxidase (BOD). The 59kDa protein containing four coppers, was successfully over-expressed in Escherichia coli and purified to homogeneity in one step. This 509 amino-acid enzyme, having 67% and 26% sequence identity with CotA from Bacillus subtilis and BOD from Myrothecium verrucaria, respectively, shows higher turnover activity towards bilirubin compared to other bacterial MCOs. The current density for O2 reduction, when immobilized in a redox hydrogel, is only 12% smaller than the current obtained with Trachyderma tsunodae BOD. Under continuous electrocatalysis, an electrode modified with the new BOD is more stable, and has a higher tolerance towards NaCl, than a T. tsunodae BOD modified electrode. This makes BOD from B. pumilus an attractive new candidate for application in biofuel cells and biosensors.

Durand, Fabien; Kjaergaard, Christian Hauge; Suraniti, Emmanuel; Gounel, Sebastien; Hadt, Ryan G.; Solomon, Edward I; Mano, Nicolas

2013-01-01

289

Cloning and functional analysis of a new laccase gene from Trametes sp. 48424 which had the high yield of laccase and strong ability for decolorizing different dyes.  

PubMed

The laccase gene lac48424-1 and its corresponding full-length cDNA were cloned and characterized from a novel white-rot fungi Trametes sp. 48424 which had the high yield of laccase and strong ability for decolorizing different dyes. The 1563 bp full-length cDNA of lac48424-1 encoded a mature laccase protein containing 499 amino acids preceded by a signal peptide of 21 amino acids. The deduced protein sequence of LAC48424-1 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. The functionality of lac48424-1 gene encoding active laccase was verified by expressing the gene in the yeast Pichia pastoris successfully. It was found that the recombinant laccase produced by the yeast transformant could decolorize different dyes. The 5'-flanking sequence upstream of start codon was obtained by Self-Formed Adaptor PCR. Many putative cis-acting responsive elements involved in the transcriptional regulation were identified in the promoter region of lac48424-1. PMID:21094600

Fan, Fangfang; Zhuo, Rui; Sun, Su; Wan, Xia; Jiang, Mulan; Zhang, Xiaoyu; Yang, Yang

2011-02-01

290

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

PubMed

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

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

2012-05-15

291

Hybrid biobattery based on arylated carbon nanotubes and laccase.  

PubMed

Single-walled carbon nanotubes (SWCNT) were covalently modified with anthracene and anthraquinone and used for the construction of cathodes for biocatalytic reduction of dioxygen. The nanotubes with aromatic groups casted onto the electrode increased the working surface of the electrode and enabled efficient direct electron transfer (DET) between the enzyme and the electrode. The aryl groups enter the hydrophobic pocket of the T1 center of laccase responsible for exchanging electrons with the substrate. Glassy carbon electrode covered with arylated SWCNT and coated with a layer of neutralized Nafion containing laccase was found to be a very efficient cathode in the hybrid battery. Zn wire covered with a Nafion film served as the anode. The cell parameters were determined: power density was 2 mW/cm(2) and the open circuit potential was 1.5 V. PMID:22078125

Stolarczyk, Krzysztof; Sepelowska, Ma?gorzata; Lyp, Dominika; Zelechowska, Kamila; Biernat, Jan F; Rogalski, Jerzy; Farmer, Kevin D; Roberts, Ken N; Bilewicz, Renata

2012-10-01

292

Modeling of growth and laccase production by Pycnoporus sanguineus.  

PubMed

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L(-1 )days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L(-1). PMID:24005762

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

2014-05-01

293

Decolourization and detoxification of textile industry wastewater by the laccase-mediator system.  

PubMed

Decolourization and detoxification of a textile industry effluent by laccase from Trametes trogii in the presence and the absence of laccase mediators was investigated. Laccase alone was not able to decolourize the effluent efficiently even at the highest enzyme concentration tested: less than 10% decolourization was obtained with 9 U/mL reaction mixture. To enhance effluent decolourization, several potential laccase mediators were tested at concentrations ranging from 0 to 1mM. Most potential mediators enhanced decolourization of the effluent, with 1-hydroxybenzotriazol (HBT) being the most effective. The effect of several physico-chemical parameters that could influence enzyme activity, such as pH, temperature and dye concentration was tested. Optimal decolourization occurred with 20% effluent at pH 5, a temperature of 50 degrees C, and in the presence of 1mM HBT. The toxicities of crude, laccase-HBT treated and laccase-acetosyringone treated effluent were evaluated using the Microtox assay. Only laccase-acetosyringone treated effluent was not toxic; crude and laccase-HBT treated effluent retained toxicity. PMID:19945786

Khlifi, Rim; Belbahri, Lassad; Woodward, Steve; Ellouz, Mariem; Dhouib, Abdelhafidh; Sayadi, Sami; Mechichi, Tahar

2010-03-15

294

Degradation of azo dyes by oxidative processes – Laccase and ultrasound treatment  

Microsoft Academic Search

Azo dyes are of synthetic origin and their environmental fate is not well understood. They are resistant to direct aerobic bacterial degradation and form potentially carcinogenic aromatic amines by reduction of the azo group. This study shows that applying the oxidative processes of enzymatic treatment with laccase and ultrasound treatment, both alone and in combination, leads to dye degradation. Laccase

Michael M. Tauber; Georg M. Gübitz; Astrid Rehorek

2008-01-01

295

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

PubMed

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

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

2014-03-01

296

Influence of different magnetic composites carriers on the immobilization of laccase  

NASA Astrophysics Data System (ADS)

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.

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

2006-01-01

297

Evidence of an active laccase-like enzyme in deepwater pink shrimp ( Parapenaeus longirostris)  

Microsoft Academic Search

This paper demonstrates the presence of an active laccase-like enzyme from deepwater pink shrimp (Parapenaeus longirostris) using polyacrylamide gel electrophoresis. This enzyme was found in all anatomical parts of the deepwater pink shrimp, but particularly in the cephalothorax, and became active during the course of storage. Gel staining with laccase-specific substrates such as ADA, DMP and DAB was used to

Oscar Martínez-Alvarez; Pilar Montero; Carmen Gómez-Guillén

2008-01-01

298

Effect of onion-type multilamellar liposomes on Trametes versicolor laccase activity and stability.  

PubMed

Trametes versicolor laccase was encapsulated into onion-type, lipid-based multilamellar vesicles (MLVs). When encapsulated, laccase was isolated from the assay medium but was still active once freed from its capsule. The encapsulation efficiency was larger than 65% at 25 °C and 37 °C and decreased to 55% by introducing 140 mM NaCl into the buffered medium (pH = 4.5). MLVs were shown to drastically improve both laccase stability and activity. At 25 °C, laccase activity was doubled in the presence of MLVs. At 37 °C in the salt-free medium, the half-life time of laccase was increased from 2 hr 30-65 h without and with MLVs, respectively. This effect was even more pronounced in the salted medium where laccase activity was unchanged for 6 days in the presence of MLVs. These beneficial effects were attributed to the immobilization of laccase onto MLV surface. Laccase activity as well as stability was notably shown to be directly correlated to MLV stability. PMID:22051377

Prévoteau, Antonin; Faure, Chrystel

2012-01-01

299

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

SciTech Connect

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

Collins, P.J.; Dobson, A.D.W. [Univ. College, Cork (Ireland); Kotterman, M.J.J.; Field, J.A. [Wageningen Agricultural Univ. (Netherlands)

1996-12-01

300

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

PubMed

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

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

2014-01-15

301

Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple  

Microsoft Academic Search

Polyphenol oxidase (PPO), a copper-containing metalloprotein, catalyzes the oxidation of phenolics to quinones which make brown pigments in wounded tissues. Because the phenomena decrease fruit quality, PPO has been regarded to be a critical enzyme in food technology. In the course of expressed sequence tags (ESTs) analysis of the Fuji apple (Malus domesticus Borkh.), we identified two partial PPO cDNA

Joo Young Kim; Young Sam Seo; Jee Eun Kim; Soon-Kee Sung; Kwan Jeong Song; Gynheung An; Woo Taek Kim

2001-01-01

302

Bacillus amyloliquefaciens laccase--from soil bacteria to recombinant enzyme for wastewater decolorization.  

PubMed

One hundred wild type strains of Bacillus sp. were isolated from industrial and agricultural soil across Serbia and screened for laccase activity. Three strains showed high laccase activity temperature optimum of 65 and 80 °C towards ABTS. A new laccase gene from the strain with highest temperature optimum, namely Bacillus amyloliquefaciens 12B was cloned and expressed in Escherichia coli. Recombinant laccase degraded dye Reactive blue 52 at pH 7.0 and pH 4.0 and at elevated temperature, while fungal laccases was unable to act on this substrate at pH higher than 4.0 and was quickly inactivated at temperatures higher than 45 °C. Degradation of dye was monitored by HPLC-DAD and resulting precipitate was analyzed by FTIR spectroscopy. Single product peak without chromophore was detected in solution, while water insoluble aggregate, presumably dye polymer is formed retaining blue color. PMID:23994699

Lon?ar, Nikola; Boži?, Nataša; Lopez-Santin, Josep; Vuj?i?, Zoran

2013-11-01

303

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

PubMed

A cDNA encoding for laccase (Lcc1) was isolated from the ligninolytic fungus Trametes versicolor by reverse transcriptase polymerase chain reaction. The Lcc1 gene was subcloned into the Pichia methanolica expression vector pMETalphaA and transformed into the P. methanolica strains PMAD11 and PMAD16. The extracellular laccase activity of the PMAD11 recombinants was found to be 1.3-fold higher than that of the PMAD16 recombinants. The identity of the recombinant protein was further confirmed by immunodetection using the Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form. The effects of copper concentration, cultivation temperature, pH and methanol concentration in the BMMY on laccase expression were investigated. The laccase activity in the PMAD11 recombinant was up to 12.6 U ml(-1) by optimization. PMID:16292528

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

2006-08-01

304

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

PubMed

Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k1', value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ? 22 kJ mol(-1), while energy for laccase inactivation was 18 kJ mol(-1). Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (?T = 40 K) as opposed to fungal biomass (?T = 15 K). Comparison of entropy change (?S) values indicated metabolism of the dye by the biomass. PMID:24464534

Razak, N N A; Annuar, M S M

2014-03-01

305

Iodination of phenol.  

National Technical Information Service (NTIS)

Phenol is iodinated in aqueous solution at pH 5 (acetate buffer) by elemental iodine or, if the iodine is present as iodide, enzymatically controlled by peroxidases. Generally mono-, di- and triiodophenols are obtained, the overall product composition bei...

J. V. Christiansen A. Feldthus L. Carlsen

1990-01-01

306

Transitions in Phenolic Polymer.  

National Technical Information Service (NTIS)

A phenolic polymer has been polymerized under conditions which lead to a crack free solid casting and its compressibility, electrical resistivity and specific heat have been determined. A secondary transition was found by compressibility measurements at a...

R. W. Warfield

1974-01-01

307

Microbial Production of Phenols.  

National Technical Information Service (NTIS)

Most processes for the oxidation, particularly hydroxylation of aromatic hydrocarbons depend on the organic chemical reactions which sometimes require complicated steps in commercial applications as they are not without by-products generation. If phenols ...

A. Yoshikawa

1985-01-01

308

Phenol burns and intoxications.  

PubMed

Phenol burns and intoxications are life-threatening injuries. Roughly 50 per cent of all reported cases have a fatal outcome. Only a small number of cases have been reported with high serum concentrations after phenol burns who survived. In our own experience a patient with 20.5 per cent total body surface area deep partial skin thickness phenol burns and serum concentrations of 17,400 micrograms/litre survived after immediate and repeated treatment of the scalds with polyethylene glycol (PEG) and silver sulphadiazine. A literature review of experiences with phenol intoxications reveals the advantages of PEG application. Questions on the need for enforced diuresis and haemodialysis as well as the initial treatment procedures are discussed. Advantages of different solutions for local therapy are reported. PMID:8148075

Horch, R; Spilker, G; Stark, G B

1994-02-01

309

Electrochemical sensor for predicting transformer overload by phenol measurement.  

PubMed

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

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

2003-03-10

310

Characterization of the multicopper oxidase gene family in Anopheles gambiae  

PubMed Central

The multicopper oxidase (MCO) family of enzymes includes laccases, which oxidize a broad range of substrates including diphenols, and several oxidases with specific substrates such as iron, copper or ascorbic acid. We have identified five putative MCO genes in the genome of Anopheles gambiae and have cloned cDNAs encompassing the full coding region for each gene. MCO1 mRNA was detected in all developmental stages and in all of the larval and adult tissues tested. We observed an increase in MCO1 transcript abundance in the midguts and Malphighian tubules of adult females following a blood meal and in adult abdominal carcasses in response to an immune challenge. Two alternatively spliced isoforms of MCO2 mRNA were identified. The A isoform of MCO2 was previously detected in larval and pupal cuticle where it probably catalyzes sclerotization reactions (He et al., 2007). The B isoform was transcriptionally upregulated in ovaries in response to a blood meal. MCO3 mRNA was detected in the adult midgut, Malpighian tubules, and male reproductive tissues; like MCO1, it was upregulated in response to an immune challenge or a blood meal. MCO4 and MCO5 were observed primarily in eggs and in the abdominal carcass of larvae. A phylogenetic analysis of insect MCO genes identified putative orthologs of MCO1 and MCO2 in all of the insect genomes tested, whereas MCO3, MCO4 and MCO5 were found only in the two mosquito species analyzed. MCO2 orthologs have especially high sequence similarity, suggesting that they are under strong purifying selection; the A isoforms are more conserved than the B isoforms. The mosquito specific group shares a common ancestor with MCO2. This initial study of mosquito MCOs suggests that MCO2 may be required for egg development or eggshell tanning in addition to cuticle tanning, while MCO1 and MCO3 may be involved in metal metabolism or immunity.

Gorman, Maureen J.; Dittmer, Neal T.; Marshall, Jeremy L.; Kanost, Michael R.

2008-01-01

311

Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase.  

PubMed

Laccase (Lcc) is a lignin-degrading enzyme produced by white-rot fungi and has been the subject of much interest in the field of bioremediation due to its ability to oxidize phenolic compounds. In this report, we describe the isolation and characterization of lcc1, a novel gene of Lentinula edodes that encodes Lcc1, and demonstrate that recombinant Lcc1 is expressed in an active, secreted form in tobacco BY-2 cells in culture. The open reading frame of lcc1 was 1,557 base pairs in length and encoded a putative protein of 518 amino acids. We introduced a chimeric form of lcc1 (CaMV35Sp:clcc1) into tobacco BY-2 cells and obtained several stable clcc1 transformants that expressed active Lcc1. Lcc1 activity in BY-2 culture media was higher than in cellular extracts, which indicated that recombinant Lcc1 was produced in a secreted form. Recombinant Lcc1 had a smaller apparent molecular weight and exhibited a different pattern of posttranslational modification than Lcc1 purified from L. edodes. The substrate specificity of purified recombinant Lcc1 was similar to L. edodes Lcc1, and both enzymes were able to decolorize the same set of dyes. These results suggest that heterologous expression of fungal Lcc1 in BY-2 cells will be a valuable tool for the production of sufficient quantities of active laccase for bioremediation. PMID:18488166

Sakamoto, Yuichi; Nakade, Keiko; Yano, Akira; Nakagawa, Yuko; Hirano, Tatsuya; Irie, Toshikazu; Watanabe, Hisayuki; Nagai, Masaru; Sato, Toshitsugu

2008-07-01

312

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

PubMed

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

Sathishkumar, P; Murugesan, K; Palvannan, T

2010-08-01

313

Conversion of tyrosine to phenolic derivatives by Taiwan cobra venom.  

PubMed

We have examined the ability of Taiwan cobra (Naja naja atra) venom to transform in vitro the amino acid tyrosine to phenolic oxidation products via 4-hydroxyphenylpyruvate. This amino acid can be released from neuropeptide substrates by oligopeptidases present in the venom. Using a variety of analytical techniques to probe a complicated series of reactions, we confirm that the L-amino acid oxidase present in the venom initially releases the keto form of 4-hydroxyphenylpyruvic acid and hydrogen peroxide after reacting with the tyrosine. Thereafter, there is evidence that a tautomerase in the venom promotes a partial conversion of the keto-form 4-hydroxyphenylpyruvic acid into an enol form. The enol is oxidised primarily to 4-hydroxybenzaldehyde and 4-hydroxyphenol (hydroquinone). The keto form is oxidised through to 4-hydroxyphenylacetic acid by the hydrogen peroxide co-released by the L-amino acid oxidase. The venom promotes both these spontaneous oxidation routes and also generates traces of other phenolics, some of which are as yet unidentified. We propose that reactions between the precursors of the major oxidation products may be responsible for generating unusual short-lived phenolics, possibly giving rise to special bioactivities that are relevant to venom action. PMID:9690784

Nucaro, E; Jodra, M; Russell, E; Anderson, L; Dennison, P; Dufton, M

1998-08-01

314

NADPH Oxidase and Neurodegeneration  

PubMed Central

NADPH oxidase (Nox) is a unique, multi-protein, electron transport system that produces large amounts of superoxide via the reduction of molecular oxygen. Nox-derived reactive oxygen species (ROS) are known to be involved in a variety of physiological processes, including host defense and signal transduction. However, over the past decade, the involvement of (Nox)-dependent oxidative stress in the pathophysiology of several neurodegenerative diseases has been increasingly recognized. ROS produced by Nox proteins contribute to neurodegenerative diseases through distinct mechanisms, such as oxidation of DNA, proteins, lipids, amino acids and metals, in addition to activation of redox-sensitive signaling pathways. In this review, we discuss the recent literature on Nox involvement in neurodegeneration, focusing on Parkinson and Alzheimer diseases.

Hernandes, Marina S; Britto, Luiz R G

2012-01-01

315

In vitro antioxidant, xanthine oxidase and acetylcholinesterase inhibitory activities of Balanites aegyptiaca (L.) Del. (Balanitaceae).  

PubMed

The present study aimed to test the validity of Balanites aegyptiaca remedies used for the treatment of rheumatisms and mental disorders by examining the antioxidant, xanthine oxidase and acetylcholinesterase inhibitory activities of galls and leaves extracts and fractions. The total phenolics and flavonoids were measured using Folin-Ciocalteu and AlCl3 reagents, respectively. Two methods i.e., FRAP and ABTS were used to estimate the total antioxidant capacity of the plant materials. The FRAP and ABTS antioxidant activities showed that among all extracts and fractions tested, the best antioxidant activities were found with the galls dichloromethane and the leaves ethyl acetate fractions. The antioxidant activities did correlated significantly with the total phenolic and flavonoid contents. The study also showed that B. aegyptiaca galls and leaves fractions exhibited a moderate xanthine oxidase inhibitory activity comparatively to the acetylcholinesterase which was weakly inhibited by the tested extracts and fractions. PMID:20836295

Meda, N T R; Lamien-Meda, A; Kiendrebeogo, M; Lamien, C E; Coulibaly, A Y; Millogo-Rasolodimby, J; Nacoulma, O G

2010-04-15

316

Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance  

Microsoft Academic Search

Polyphenol oxidases (PPOs; EC 1.10.3.2 or EC 1.14.18.1) catalyzing the oxygen-dependent oxidation of phenols to quinones are ubiquitous among angiosperms and assumed to be involved in plant defense against pests and pathogens. In order to investigate the role of PPO in plant disease resistance, we made transgenic tomato (Lycopersicon esculentum Mill. cv. Money Maker) plants that overexpressed a potato (Solanum

Li Li; John C. Steffens

2002-01-01

317

Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris.  

PubMed

Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol. PMID:12115407

Hong, Feng; Meinander, Nina Q; Jönsson, Leif J

2002-08-20

318

Molecular characteristics of two laccase from the basidiomycete fungus Polyporus brumalis.  

PubMed

Two laccase cDNAs, pblac1 and pblac2, were cloned from a white-rot fungus strain, Polyporus brumalis (KFRI 20912). The cloned cDNAs consisted of 1,829 bp and 1,804 bp, and their open reading frames encoded proteins of 520 and 524 amino acids, with calculated molecular masses of approximately 55.9 kDa and 56 kDa, respectively. The deduced amino acid sequences of each protein showed 70% similarity. The copper binding regions were conserved in both proteins, as in other fungal laccases. RT-PCR analysis revealed that the transcript levels of the two laccases increased progressively in shallow stationary culture liquid medium. The transcript level of each laccase was induced when the fungus was exposed to di-butyl phthalate (DBP), suggesting that the two laccases are involved in DBP degradation. The overexpression of the pblac1 gene was derived by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase, using a homologous system. The activity of laccase in the transformants was significantly higher than that of the wild type. The identification of these laccase cDNAs was a first step to characterize the molecular events related to the lignin degradation ability of this basidiomycetous fungus, as well as the degradation of many recalcitrant xenobiotics. PMID:18337695

Ryu, Sun-Hwa; Lee, A-Young; Kim, Myungkil

2008-02-01

319

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

PubMed Central

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.

Zhao, J.; Kwan, H. S.

1999-01-01

320

Extended Heating Ablation of Carbon Phenolic and Silica Phenolic.  

National Technical Information Service (NTIS)

An analysis was made of experimental and analytical investigations of the ablation of carbon phenolic and silica phenolic composites under extended heating conditions. Specimens of up to 8.75 sq. in. in area and instrumented with indepth thermocouples wer...

R. W. Farmer

1974-01-01

321

Phenolic Acids and Phenolic Glycosides of Gaultheria Species.  

National Technical Information Service (NTIS)

Twenty-two species of Gaultheria were examined for phenols and phenolic acids obtained by hydrolysis of ethanolic extracts. Most species yielded p-hydroxybenzoic, o-pyrocatechuic, protocatechuic, gentisic, vanillic, p-coumaric, caffeic and ferulic acids. ...

G. H. N. Towers A. Tse W. S. G. Maass

1965-01-01

322

Xenobiotics enhance laccase activity in alkali-tolerant ?-proteobacterium JB  

PubMed Central

Various genotoxic textile dyes, xenobiotics, substrates (10 µM) and agrochemicals (100 µg/ml) were tested for enhancement of alkalophilic laccase activity in ?-proteobacterium JB. Neutral Red, Indigo Carmine, Naphthol Base Bordears and Sulphast Ruby dyes increased the activity by 3.7, 2.7, 2.6 and 2.3 fold respectively. Xenobiotics/substrates like p-toluidine, 8-hydroxyquinoline and anthracine increased it by 3.4, 2.8 and 2.3 fold respectively. Atrazine and trycyclozole pesticides enhanced the activity by 1.95 and 1.5 fold respectively.

Singh, Gursharan; Batish, Mona; Sharma, Prince; Capalash, Neena

2009-01-01

323

Myocardial xanthine oxidase/dehydrogenase.  

PubMed

High-energy phosphates in heart muscle deprived of oxygen are rapidly broken down to purine nucleosides and oxypurines. We studied the role of xanthine oxidase/dehydrogenase (EC 1.2.3.2/EC 1.2.1.37) in this process with novel high-pressure liquid chromatographic techniques. Under various conditions, including ischemia and anoxia, the isolated perfused rat heart released adenosine, inosine and hypoxanthine, and also substantial amounts of xanthine and urate. Allopurinol, an inhibitor of xanthine oxidase, greatly enhanced the release of hypoxanthine. From the purine release we calculated that the rat heart contained about 18 mU xanthine oxidase per g wet weight. Subsequently, we measured a xanthine oxidase activity of 9 mU/g wet wt. in rat-heart homogenate. When endogenous low molecular weight inhibitors were removed by gel-filtration, the activity increased to 31 mU/g wet wt. Rat myocardial xanthine oxidase seems to be present mainly in the dehydrogenase form, which upon storage at -20 degrees C is converted to the oxidase form. PMID:6575831

Schoutsen, B; De Jong, J W; Harmsen, E; De Tombe, P P; Achterberg, P W

1983-07-14

324

Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA.  

PubMed Central

Transmissible hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, is associated with cytoplasmic double-stranded-RNA (dsRNA) viruses. The fungal laccase has attracted interest because its activity is reduced in hypovirulent dsRNA-containing strains. A laccase cDNA clone was isolated by screening a cDNA expression library with antibodies against the purified extracellular laccase. The amino acid sequence deduced from part of the cDNA clone revealed high homology to other fungal laccases, especially to the Neurospora crassa laccase. A major laccase transcript 2.3 kb in length was detected in Northern (RNA) blots. In liquid culture, extracellular laccase activity was reduced by about 75% in the hypovirulent (dsRNA-free) strain EP155/2. In contrast, production of biomass was not affected by the dsRNA. Northern blot analysis indicated that dsRNA down regulates laccase biosynthesis by reducing laccase mRNA accumulation. The laccase gene is one of several developmentally regulated genes affected by the presence of dsRNA. Images FIG. 1 FIG. 4

Rigling, D; Van Alfen, N K

1991-01-01

325

Transcriptional, biochemical and histochemical investigation on laccase expression during Tuber melanosporum Vittad. development.  

PubMed

The cDNAs of Tuber melanosporum laccases (Tmellcc1 and Tmellcc2) have been cloned. From the cloned cDNAs probes were prepared to investigate the expression levels of the Tmellcc1 and Tmellcc2 genes in the free living mycelium (FLM), ectomycorrhizae (ECM) and different developmental stages of fruit body (FB) by quantitative PCR (qPCR). The mRNA expression levels agree with the changes of laccase activities. The histochemical data agree with the qPCR and biochemical results. The highest laccase expression occurs in the ECM, when the host plant roots are invaded by the fungal mycelium. PMID:23276677

Zarivi, Osvaldo; Bonfigli, Antonella; Colafarina, Sabrina; Aimola, Pierpaolo; Ragnelli, Anna Maria; Miranda, Michele; Pacioni, Giovanni

2013-03-01

326

Effect of Wash Water Temperature and Chlorination on Phenolic Metabolism and Browning of Stored Iceberg Lettuce Photosynthetic and Vascular Tissues  

Microsoft Academic Search

Cut tissues from distinct anatomical locations in iceberg lettuce (Lactuca sativa L.) were subjected to washing in cold (4 °C) and warm (47 °C) water with or without chlorine to assess their propensity to discoloration during storage. Total protein (Bradford method) and phenolic (TPH; Folin-Ciocalteu method) contents and polyphenol oxidase (PPO; spectrophotometric method using catechol as a substrate), peroxidase (POD;

Lana R. Fukumoto; Peter M. A. Toivonen; Pascal J. Delaquis

2002-01-01

327

THERMOPHILIC ANAEROBIC BIODEGRADATION OF PHENOLICS  

EPA Science Inventory

The report gives results of a series of anaerobic microbial acclimation and treatment performance tests with synthetic phenolic substrates. The research is a feasibility level assessment of substituting anaerobic biodegradation of phenolics for solvent extraction. The tests showe...

328

NADPH Oxidases in Vascular Pathology  

PubMed Central

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.

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

2014-01-01

329

NADPH oxidases in vascular pathology.  

PubMed

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

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

2014-06-10

330

Phenol metabolism in the leaves of the olive tree (Olea europaea L.) cv. Picual, Verdial, Arbequina, and Frantoio during ripening.  

PubMed

The kinetic behavior and protein-expression level of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) have been determined in the leaves of the olive tree (Olea europaea L.) of cv. Picual, Verdial, Arbequina, and Frantoio during fruit ripening. Moreover, the concentration of total phenolic compounds, oleuropein, hydroxytyrosol, and tyrosol has been also determined. This study was carried out in 20-year-old olive trees grown in Jaén (Spain). The concentration of total and specific phenols showed a specific pattern in each cultivar. Frantoio showed the highest phenol concentration followed by Arbequina, Picual, and Verdial. A coordinated response between PAL, PPO, and the concentration of total phenols in the four cultivars was found. Also, specific changes were shown over the course of ripening, indicating a regulation of PAL, PPO, and phenol concentration in the olive-tree leaves during fruit ripening. PMID:21047129

Ortega-García, Francisca; Peragón, Juan

2010-12-01

331

Electrochemical oxidation of chlorinated phenols  

Microsoft Academic Search

Electrochemical oxidation has been proposed as a remediation method for chlorinated phenols but is hampered by anode fouling. In this work the authors explore the mechanism of anode fouling by chlorinated phenols, compare structure vs reactivity for phenols differing in the extent of chlorination, and relate the efficiency of oxidation to the mechanism of oxidation at different electrode types. Linear

James D. Rodgers; Wojciech Jedral; Nigel J. Bunce

1999-01-01

332

Oxidation of anthracene by immobilized laccase from Trametes versicolor.  

PubMed

The laccase of Trametes versicolor was immobilized on the functionalized nanoparticles SBA-15 with the average diameter less than 10 nm. Laccase mediated oxidations of anthracene (ANT) were investigated in the presence of two mediators, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and 1-hydroxybenzotriazole (HBT). Oxidation of ANT was more efficiently enhanced by adding 1 mM of HBT than that by adding ABTS. After 48 h oxidation HBT group significantly oxidized ANT with residue 58% relative to 88% in the ABTS group. HPLC and GC/MS analyses indicated the main product of ANT oxidation was anthraquinone (ANQ). The fluorescein diacetate (FDA) uptake of two human cell lines was used to assess the cytotoxicity and genotoxicity of ANT and ANQ. Treatments with ANT and ANQ at 5 and 10 microM exhibited significant cytotoxicity to the HaCaT cells and the A3 lymphocytes and no significant genotoxicity was observed. The results illustrated that ANQ is less toxic than ANT as well. PMID:19564104

Hu, Xiaoke; Wang, Peng; Hwang, Huey-min

2009-11-01

333

Laccase-induced grafting on plasma-pretreated polypropylene.  

PubMed

A new environmentally friendly strategy for the sustainable functionalization of inert man-made polymer surfaces is mapped out for the first time using a combination of plasma pretreatment and enzymatic postgrafting. The efficiency of enzymatic covalent binding is investigated by grafting methacrylate monomers possessing different amino groups, primary, tertiary, and quaternary, onto a polypropylene surface using plasma pretreatment. Subsequent enzymatic grafting, using laccase and guaiacol sulfonic acid (GSA), is determined by surface analytical techniques, such as attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The grafting of GSA in the presence of a laccase is proven by a 10-fold increase in sulfur compared to the control. The covalent coupling between GSA and primary amine groups is determined by HPLC-MS using hexylamine as a model substrate. The advantage of technology is in the strong covalent binding of functional groups onto the synthetic polymer's surface, which could then be suitably tailored by enzymes possessing substrate specificity and regional selectivity. PMID:18771316

Schroeder, M; Fatarella, E; Kovac, J; Guebitz, G M; Kokol, V

2008-10-01

334

Phenol and phenolics from lignocellulosic biomass by catalytic microwave pyrolysis  

SciTech Connect

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.

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

2011-07-01

335

Phenol and phenolics from lignocellulosic biomass by catalytic microwave pyrolysis.  

PubMed

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 bio-based phenols for chemical industry. PMID:21531545

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

2011-07-01

336

Cloning of a laccase gene from a novel basidiomycete Trametes sp. 420 and its heterologous expression in Pichia pastoris.  

PubMed

The laccase gene lacD, cloned from a novel laccase-producing basidiomycete Trametes sp. 420, contained 2,052 base pairs (bp) interrupted by 8 introns. lacD displayed a relatively high homology with laccase genes from other white rot fungi, whereas the homology between lacD and laccase genes from plants, insects, or bacteria was less than 25%. A 498-amino acid peptide encoded by the lacD cDNA was heterologously expressed in the Pichia pastoris strain GS115, resulting in the highest yield of laccase (8.3 x 10(4) U/l) as determined with ABTS (2,2'-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) as the substrate. Additionally, the enzyme activity of recombinant laccase on decolorization of some industrial dyes was assessed. PMID:17334840

Hong, Yu-zhi; Zhou, Hong-min; Tu, Xiao-ming; Li, Jian-feng; Xiao, Ya-zhong

2007-04-01

337

Natural laccase mediators separated from water-washed solution of steam exploded corn straw by nanofiltration and organic solvent fractionation.  

PubMed

Artificially synthetic mediators of laccase had the limitation of high cost and possible toxicity. The separation of natural laccase mediators from water-washed solution (WWS) of steam exploded corn straw (SECS) was studied using nano-filtration and successive organic solvents extraction. Results indicated that the UV absorption intensity of nano-filtrated WWS was significantly enhanced. The UV absorption intensity of each extractive from WWS could be ranked as ether extractive (EE)>ethyl acetate extractive (EAE)>chloroform extractive (CE). Decoloration of crystal violet catalyzed by laccase/EE was higher than that by laccase/ABTS, which was 66.95% and 61.9% at 8h, respectively. All the decoloration rates of malachite green at 60min using EE, EAE and ABTS as mediator were both more than 80%. This research would benefit for broaden the source of laccase mediator and reduce the using cost of laccase/mediator system. PMID:24513027

Qiu, Weihua; Zhang, Wenyan; Chen, Hongzhang

2014-03-01

338

Elimination of carbamazepine by repeated treatment with laccase in the presence of 1-hydroxybenzotriazole.  

PubMed

Carbamazepine (CBZP) is used as an antiepileptic drug and is highly persistent. In this study, CBZP was treated with laccase from white rot fungus Trametes versicolor in the presence of a redox mediator 1-hydroxybenzotriazole (HBT). A single treatment with laccase and HBT eliminated CBZP by about 22% after 24h, and repeated treatments with laccase and HBT, which were added to the reaction mixture every 8h, helped eliminate about 60% of CBZP after 48h. This suggests that repeated treatment is effective in eliminating CBZP. Mass spectrometric analyses demonstrated that two degradation products of CBZP, 10,11-dihydro-10,11-epoxycarbamazepine and 9(10H)-acridone, were formed via repeated treatment with laccase and HBT. PMID:20619797

Hata, Takayuki; Shintate, Hideaki; Kawai, Shingo; Okamura, Hideo; Nishida, Tomoaki

2010-09-15

339

[Relationship between mycelium morphology and laccase production of Pleurotus ferulae in submerged cultivation].  

PubMed

In this study, the relationship between mycelium morphology and laccase production was studied. The results indicated that the morphology of P. ferulae pellets was changed when glass beads were added. Laccase production showed higher with spherical mycelium than with filamentous or flocculent mycelium. In addition, the spherical mycelium with a diameter of 0.2-0.4 mm highly affected laccase production. Effect of the composition of culture medium on pellets was investigated and results indicated that various concentrations of glucose, corn meal and wheat bran were important to the formation of pellets in diameter of 0.2-0.4 mm. Besides nutrients, the addition of non-nutritional substrates influenced the distribution of P. ferulae pellets. However, the production of laccase was not promoted by non-nutritional substrates. PMID:24701838

Chen, Youzhi; Wang, Lu; Peng, Lin; Ding, Zhongyang; Zhang, Liang; Gu, Zhenghua; Shi, Guiyang; Zhang, Kechang

2013-11-01

340

Halotolerant laccases from Chaetomium sp., Xylogone sphaerospora, and Coprinopsis sp. isolated from a Mediterranean coastal area.  

PubMed

Laccases (EC 1.10.3.2) are phenoloxidases involved in the transformation of the recalcitrant fraction of organic matter in soil. These enzymes are also able to transform certain aromatic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and are known to be inhibited by chloride ions. This study aims to test the potential of some fungal strains newly isolated from natural environments subjected to high osmotic pressure such as coastal ecosystems, to produce chloride tolerant laccases. Three strains were identified as Chaetomium sp., Xylogone sphaerospora (two Ascomycota), and Coprinopsis sp. (a Basidiomycota) and the laccases produced by these fungi were weakly inhibited by chloride ions compared with previous data from literature. Moreover, we tested their reactivity towards various PAHs which are widespread anthropic pollutants. They were able to transform anthracene to 9,10-anthraquinone and we determine 7.5 eV as the threshold of ionization potential for PAH oxidation by these laccases. PMID:23063188

Qasemian, Leila; Billette, Christophe; Guiral, Daniel; Alazard, Emilie; Moinard, Magalie; Farnet, Anne-Marie

2012-10-01

341

Studies of laccase from Trametes versicolor in aqueous solutions of several methylimidazolium ionic liquids.  

PubMed

Stability and kinetic behavior of laccase from Trametes versicolor in the presence of several ionic liquids from the methylimidazolium family have been investigated. In general laccase stability diminished as the size of the alkylic substitute in the methylimidazolium ring increased. Higher concentrations of ionic liquids caused more destabilization than lower ones. Thus, low concentrations of [C(2)mim(+)][EtSO(4)(-)] allowed maintaining enzymatic stability. [C(4)mim(+)][Cl(-)] appeared to have a stabilizing effect on laccase, as little activity decay was observed within three weeks. Kinetic studies indicated that both [C(2)mim(+)][EtSO(4)(-)] and [C(4)mim(+)][Cl(-)] inhibited laccase activity, although 10-fold more [C(2)mim(+)][EtSO(4)(-)] than [C(4)mim(+)][Cl(-)] was required to cause the same degree of inhibition. A kinetic model was developed to represent the experimental data. PMID:21669518

Domínguez, Alberto; Rodríguez, Oscar; Tavares, Ana Paula M; Macedo, Eugenia A; Longo, María Asunción; Sanromán, María Angeles

2011-08-01

342

Coupling in cytochrome c oxidase  

PubMed Central

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K+ when cytochrome oxidase is incorporated into liposomes containing acidic phospholipids or by the combination of lipid cytochrome c and bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid. Images

Kessler, R. J.; Blondin, G. A.; Zande, H. Vande; Haworth, R. A.; Green, D. E.

1977-01-01

343

Optimizing Production of Extracellular Laccase from Grammothele Subargentea CLPS No. 436 Strain  

Microsoft Academic Search

The production of extracellular laccase by the Grammothele subargentea CLPS no. 436 strain in liquid cultures grown on a carbon-limited basal medium was significantly enhanced when culture conditions,\\u000a including the addition of CuSO45H2O or veratryl alcohol, were consecutively optimized. A laccase activity as high as 1954.5 mU ml?1 of liquid medium was obtained under optimum conditions, which corresponded to non-agitated

Mario Carlos Nazareno Saparrat; Nazareno Saparrat

2004-01-01

344

Primary structure and transcription analysis of a laccase-encoding gene from the basidiomycete Trametes trogii  

Microsoft Academic Search

A cDNA coding for laccase was isolated from the white-rot fungus Trametes trogii 201. This cDNA corresponded to the lcc1 gene, which coded for a precursor protein of 517 amino acids with a 21 amino acid signal peptide. Comparison of the deduced sequence with known laccases showed that this enzyme was most closely related to Lac1 from basidiomycete PM1 and

M. Ch. Colao; A. M. Garzillo; V. Buonocore; A. Schiesser; M. Ruzzi

2003-01-01

345

Purification and characterization of an extracellular laccase of a fungus (family Chaetomiaceae) isolated from soil  

Microsoft Academic Search

A laccase-producing fungus was newly isolated from soil and shown to belong to family Chaetomiaceae. The extracellular laccase was purified to electrophoretic homogeneity from coffeic acid-induced culture medium by ammonium sulfate precipitation and anion exchange column chromatography. The enzyme was determined to be a monomeric protein with an apparent molecular mass of approximately 73–80kDa and an isoelectric point (pI) of

Takao Saito; Peng Hong; Katsuya Kato; Masaharu Okazaki; Hidetoshi Inagaki; Sumio Maeda; Yoshiyuki Yokogawa

2003-01-01

346

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

Microsoft Academic Search

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

TREVOR M. D'SOUZA; K. Boominathan; C. A. Reddy

1996-01-01

347

Affinity chromatography as a rapid and convenient method for purification of fungal laccases.  

PubMed

A rapid and convenient method for graduation, isolation, and purification of laccase from Trametes versicolor and Fomes fomentarius culture fluids was developed. For purification affinity chromatography on syringyl- and vanillyl-controlled porosity glass (CPG) columns was applied. The purified laccase of F. fomentarius was immobilized on porous glass. Some properties of the immobilized enzyme in comparison to the free one are discussed. PMID:18600674

Rogalski, J; Wojtas-Wasilewska, M; Apalovic, R; Leonowicz, A

1991-04-01

348

Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa  

Microsoft Academic Search

Summary  Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early\\/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at

Z. T. Xing; J. H. Cheng; Q. Tan; Y. J. Pan

2006-01-01

349

Sorption-assisted surface conjugation: a way to stabilize laccase enzyme  

Microsoft Academic Search

Enyzme immobilization on solid surfaces is one of the most relevant methods to improve enzyme activity and stability under\\u000a harsh conditions over extended periods. A typically interesting application is the immobilization of laccases, multicopper\\u000a enzymes oxidizing aromatic compounds, to solid surfaces in order to develop valuable tools for the elimination of micropollutants\\u000a in wastewater. Laccase of the white-rot fungus Coriolopsis

Yannick-Serge Zimmermann; Patrick Shahgaldian; Philippe F. X. Corvini; Gregor Hommes

350

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

Microsoft Academic Search

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

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

2005-01-01

351

Expression, characterization and 2,4,6-trichlorophenol degradation of laccase from Monilinia fructigena  

Microsoft Academic Search

A novel laccase gene from Monilinia fructigena was synthesized chemically according to the yeast bias codon and integrated into the genome of Pichia pastoris GS115 by electroporation. The expressed enzyme was recovered from the culture supernatant and purified. The result of enzyme\\u000a activity assay and SDS-PAGE demonstrated that the recombinant laccase was induced and extracellularly expressed in P. pastoris. Main

Wenhua BaoRihe; Rihe Peng; Zhen Zhang; Yongsheng Tian; Wei Zhao; Yong Xue; Jianjie Gao; Quanhong Yao

352

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

PubMed

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

Knutson, Kristina; Ragauskas, Arthur

2004-01-01

353

Enhanced formation of extracellular laccase activity by the white-rot fungus Trametes multicolor  

Microsoft Academic Search

The white-rot fungus Trametes multicolor MB 49 has been identified as an excellent producer of the industrially important enzyme laccase. The formation of extracellular\\u000a laccase could be considerably stimulated by the addition of Cu(II) to a simple, glycerol-based culture medium. In this study,\\u000a optimal concentrations of copper were found to be 0.5–1 mM, which were added during the growth phase

Johann Hess; Christian Leitner; Christiane Galhaup; Klaus D. Kulbe; Barbara Hinterstoisser; Martin Steinwender; Dietmar Haltrich

2002-01-01

354

Biochemical characterization and potential for textile dye degradation of blue laccase from Aspergillus ochraceus NCIM-1146  

Microsoft Academic Search

In our study, we produced intracellular blue laccase by growing the filamentous fungus Aspergillus ochraceus NCIM-1146 in potato dextrose broth. The enzyme was then purified 22-fold to a specific activity of 4.81 U\\/mg using anion-exchange\\u000a and size exclusion chromatography. The molecular weight of purified laccase was estimated as 68 kDa using sodium dodecyl sulfate\\u000a polyacrylamide gel electrophoresis. The enzyme showed

Amar A. Telke; Avinash A. Kadam; Sujit S. Jagtap; Jyoti P. Jadhav; Sanjay P. Govindwar

2010-01-01

355

Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum  

Microsoft Academic Search

Ganoderma lucidum, a medicinal white-rot basidiomycete, produces many laccase isozymes in liquid culture. Three laccase isozymes (GaLc 1, 2, 3) have been purified 32.4-fold from the crude enzyme protein through anion exchange chromatography, preparative gel electrophoresis, and electroelution. Their estimated molecular weights are 65-68 kDa, and they contain 7-10% N-linked carbohydrates. The three isozymes have identical N-terminal amino acid sequences:

E.-M. Ko; Y.-E. Leem; H. Choi

2001-01-01

356

Oxygen Activation during Oxidation of Methoxyhydroquinones by Laccase from Pleurotus eryngii  

PubMed Central

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%.

Guillen, Francisco; Munoz, Carmen; Gomez-Toribio, Victor; Martinez, Angel T.; Jesus Martinez, Maria

2000-01-01

357

Redox-mediated decolorization of recalcitrant textile dyes by Trichoderma harzianum WL1 laccase  

Microsoft Academic Search

The efficiency of crude and partially purified Trichoderma harzianum WL1 laccase for the decolorization of synthetic dyes (Rhodamine 6G, Erioglaucine and Trypan blue) with complex aromatic structures\\u000a were evaluated. Selection of dyes was based on their extensive usage in local dyeing and textile industries around the study\\u000a area. Studies on the role of redox potential of laccases on dye decolorization

S. Sadhasivam; S. Savitha; K. Swaminathan

2009-01-01

358

Laccase of Cryptococcus neoformans Is a Cell Wall-Associated Virulence Factor  

PubMed Central

Virulence is the outcome of an interaction between the host and a microbe and is characterized by a large array of opposing reactions operating at the host-pathogen interface. Cryptococcus neoformans is an important opportunistic pathogen in immunocompromised patients, including those with human immunodeficiency virus, and expresses a virulence-associated laccase which is believed to oxidize brain catecholamines and iron as a defense against host immune cells. In the present report, we investigated the cellular location of laccase to understand more fully how it contributes to cryptococcal virulence. A monoclonal antibody to the C. neoformans laccase was generated and used to show localization in the cell walls of representative serotype A (H99) and serotype D (B-3501) strains by immunoelectron microscopy. In addition, confocal microscopy was used to show a peripheral location of green fluorescent protein-tagged laccase expressed in live H99 cells. Biochemical studies showed that laccase could be released from intact cells or cell wall fractions with glucanase enzymes but was retained in the cell wall after sequential extraction with 1 M NaCl, 6 M urea, and 1% sodium dodecyl sulfate. The presence of a hydrolyzable bond linking laccase to the cell wall was suggested by removal of laccase from cell wall preparations after they were boiled in 1% sodium dodecyl sulfate, as was the presence of a disulfide or thioester bond by removal with dithiothreitol or ?-mercaptoethanol. These data show that laccase is present as a tightly associated cell wall enzyme that is readily accessible for interactions with host immune cells.

Zhu, Xudong; Gibbons, Jack; Garcia-Rivera, Javier; Casadevall, Arturo; Williamson, Peter R.

2001-01-01

359

Laccase of Cryptococcus neoformans is a cell wall-associated virulence factor.  

PubMed

Virulence is the outcome of an interaction between the host and a microbe and is characterized by a large array of opposing reactions operating at the host-pathogen interface. Cryptococcus neoformans is an important opportunistic pathogen in immunocompromised patients, including those with human immunodeficiency virus, and expresses a virulence-associated laccase which is believed to oxidize brain catecholamines and iron as a defense against host immune cells. In the present report, we investigated the cellular location of laccase to understand more fully how it contributes to cryptococcal virulence. A monoclonal antibody to the C. neoformans laccase was generated and used to show localization in the cell walls of representative serotype A (H99) and serotype D (B-3501) strains by immunoelectron microscopy. In addition, confocal microscopy was used to show a peripheral location of green fluorescent protein-tagged laccase expressed in live H99 cells. Biochemical studies showed that laccase could be released from intact cells or cell wall fractions with glucanase enzymes but was retained in the cell wall after sequential extraction with 1 M NaCl, 6 M urea, and 1% sodium dodecyl sulfate. The presence of a hydrolyzable bond linking laccase to the cell wall was suggested by removal of laccase from cell wall preparations after they were boiled in 1% sodium dodecyl sulfate, as was the presence of a disulfide or thioester bond by removal with dithiothreitol or beta-mercaptoethanol. These data show that laccase is present as a tightly associated cell wall enzyme that is readily accessible for interactions with host immune cells. PMID:11500433

Zhu, X; Gibbons, J; Garcia-Rivera, J; Casadevall, A; Williamson, P R

2001-09-01

360

Detoxification of malachite green by Pleurotus florida laccase produced under solid-state fermentation using agricultural residues  

Microsoft Academic Search

Laccase was produced from Pleurotus florida under solid-state fermentation, and the production was optimized by response surface methodology. The predicted maximum laccase production of 8.81 U g was obtained by the optimum concentration of malt extract, banana peel, wheat bran and CuSO 4, which was found to be 0.69 g, 10.61 g, 10.68 g and 77.15 ppm, respectively. The validation results suggested that the laccase production

Palanivel Sathishkumar; Thayumanavan Palvannan; Kumarasamy Murugesan; Seralathan Kamala-Kannan

2012-01-01

361

Oxygen cathode based on a layer-by-layer self-assembled laccase and osmium redox mediator  

Microsoft Academic Search

Trametes trogii laccase has been studied as biocatalyst for the oxygen electro-reduction in three different systems: (i) soluble laccase was studied in solution; (ii) an enzyme monolayer was tethered to a gold surface by dithiobis N-succinimidyl propionate (DTSP), with a soluble osmium pyridine-bipyridine redox mediator in both cases. The third case (iii) consisted in the sequential immobilization of laccase and

R. Szamocki; V. Flexer; L. Levin; F. Forchiasin; E. J. Calvo

2009-01-01

362

Expression of alternative oxidase in tomato  

SciTech Connect

Tomato fruit ripening is characterized by an increase in ethylene biosynthesis, a burst in respiration (i.e. the climacteric), fruit softening and pigmentation. As whole tomatoes ripened from mature green to red, there was an increase in the alternative oxidase capacity. Aging pink tomato slices for 24 and 48 hrs also showed an increase of alternative oxidase and cytochrome oxidase capacities. Monoclonal antibodies prepared to the Sauromatum guttatum alternative oxidase were used to follow the appearance of alternative oxidase in tomato fruits. There is a corresponding increase in a 36kDa protein with an increase in alternative oxidase capacity. Effects of ethylene and norbornadiene on alternative oxidase capacity were also studied. We are using an alternative oxidase cDNA clone from potato to study the expression of mRNA in ripening and wounded tomatoes to determine if the gene is transcriptionally regulated.

Kakefuda, M.; McIntosh, L. (Michigan State Univ., East Lansing (USA))

1990-05-01

363

Biochemical and kinetic study of laccase from Ganoderma cupreum AG-1 in hydrogels.  

PubMed

In the present study, three different types of hydrogels i.e., (poly (-acrylamide)/alginate (P (AAm)/Alg), poly (acrylamide-N-isopropylacrylamide) (P (AAm-NIPA)), and poly (acrylamide-N-isopropylacrylamide)/alginate (P (AAm-NIPA)/Alg)) were synthesized by acrylamide, alginate, and N-isopropylacrylamide for the entrapment of laccase. The hydrogel-entrapped and free laccase showed optimum temperature of 50 °C for the oxidation of ABTS, but the entrapped laccase showed high temperature, pH, and storage stability as compared to the free enzyme. The K m values of free laccase, (P (AAm)/Alg)-L, (P (AAm-NIPA))-L, and (P (AAm-NIPA)/Alg)-L were found to be 0.13, 0.28, 0.33, and 0.50 mM, respectively. The V max values of free laccase, (P (AAm)/Alg)-L, (P (AAm-NIPA))-L, and (P (AAm-NIPA)/Alg)-L were found to be 22.22?×?10(2), 5.55?×?10(2), 5.0?×?10(2), and 4.54?×?10(2) mM/min, respectively. The entrapped laccase hydrogels were used for the decolorization of Reactive Violet 1 dye, with 39 to 45 % decolorization efficiency till the 10th cycle. PMID:24740356

Gahlout, Mayur; Gupte, Shilpa; Gupte, Akshaya

2014-05-01

364

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

SciTech Connect

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.

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

1996-10-01

365

Improved Laccase Production by Trametes pubescens MB89 in Distillery Wastewaters  

PubMed Central

Various culture parameters were optimised for laccase synthesis by Trametes pubescens MB89, including pH, carbon source, nitrogen source, lignocellulosic supplements, and reported inducers. Glucose, in conjunction with a complex nitrogen source at pH 5.0, resulted in the highest laccase yield. Adding ethanol, copper, or 2,5-xylidine prior to inoculation further improved laccase concentrations. The addition of 2,5-xylidine was further investigated with multiple additions applied at varying times. This novel application substantially improved laccase production when applied regularly from inoculation and during the growth phase, and also countered glucose repression of laccase synthesis. Single and multiple factor changes were studied in three distillery wastewaters and a wine lees. A synergistic increase in laccase synthesis was observed with the addition of glucose, copper, and 2,5-xylidine. Single addition of 2,5-xylidine proved most beneficial with distillery wastewaters, while copper addition was most beneficial when using the wine lees as a culture medium.

Strong, P. J.

2011-01-01

366

Primary structure and transcription analysis of a laccase-encoding gene from the basidiomycete Trametes trogii.  

PubMed

A cDNA coding for laccase was isolated from the white-rot fungus Trametes trogii 201. This cDNA corresponded to the lcc1 gene, which coded for a precursor protein of 517 amino acids with a 21 amino acid signal peptide. Comparison of the deduced sequence with known laccases showed that this enzyme was most closely related to Lac1 from basidiomycete PM1 and Trametes C30 (98% similarity). The expression of lcc1 was analysed under different growth conditions; transcription of this gene was enhanced by the addition of organic nitrogen to the medium. The level of lcc1 transcription was higher when T. trogii was grown on synthetic medium supplemented with yeast extract rather than mycological peptone or tryptone. The transcription data were in agreement with total laccase activity measured in the supernatant and suggested that laccase production and lcc1 transcription are coordinately regulated in this organism. The lcc1 cDNA was expressed in the methylotrophic yeast Pichia pastoris and the detection of laccase activity indicated that this cDNA encodes a laccase. PMID:13680201

Colao, M Ch; Garzillo, A M; Buonocore, V; Schiesser, A; Ruzzi, M

2003-12-01

367

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

PubMed Central

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.

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

1996-01-01

368

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

PubMed Central

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

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

1997-01-01

369

Immobilization of laccase on SiO? nanocarriers improves its stability and reusability.  

PubMed

Laccases have a broad range of industrial applications. In this study, we immobilized laccase on SiO2 nanoparticles to overcome problems associated with stability and reusability of the free enzyme. Among different reagents used to functionally activate the nanoparticles, glutaraldehyde was found to be the most effective for immobilization. Optimization of the immobilization pH, temperature, enzyme loading, and incubation period led to a maximum immobilization yield of 75.8% and an immobilization efficiency of 92.9%. The optimum pH and temperature for immobilized laccase were 3.5 and 45°C, respectively, which differed from the values of pH 3.0 and 40°C obtained for the free enzyme. Immobilized laccase retained high residual activities over a broad range of pH and temperature. The kinetic parameter Vmax was slightly reduced from 1,890 to 1,630 ?mol/min/mg protein, and Km was increased from 29.3 to 45.6. The thermal stability of immobilized laccase was significantly higher than that of the free enzyme, with a half-life 11- and 18-fold higher at temperatures of 50°C and 60°C, respectively. In addition, residual activity was 82.6% after 10 cycles of use. Thus, laccase immobilized on SiO2 nanoparticles functionally activated with glutaraldehyde has broad pH and temperature ranges, thermostability, and high reusability compared with the free enzyme. It constitutes a notably efficient system for biotechnological applications. PMID:24509251

Patel, Sanjay K S; Kalia, Vipin C; Choi, Joon-Ho; Haw, Jung-Rim; Kim, In-Won; Lee, Jung Kul

2014-05-01

370

Purification and characterization of a laccase from Coprinopsis cinerea in Pichia pastoris.  

PubMed

A modified laccase gene, CcLCC6, from Coprinopsis cinerea was chemically synthesized according to the yeast codon bias and expressed in Pichia pastoris. The main properties of laccase, effects of ions and inhibitors, and optimal condition for decolouring malachite green (MG) were investigated in this study. The optimal pH level and temperature of laccase are 3.0 and 40 °C, respectively. The metal ions Mn²?, Zn²?, Fe³? and Al³? could inhibit laccase activity, as well as 1 mM of sodium dodecyl sulphate and sodium thiosulphate. 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), as a mediator, was necessary in decolorizing MG. The optimal pH and temperature for MG decolorization were 3.0 and 50 °C, respectively. Approximately 0.02 ?M recombinant laccase could effectively decolour 0.05 mM of MG in 1 h. CcLCC6I could inhibit the toxicity of MG to P. pastoris. This is the first report on the successful expression in P. pastoris of CcLCC6I and its enzymatic property. Laccase can also be considered as a candidate for treating industrial effluent containing MG. PMID:24178808

Wang, Bo; Wang, Lijuan; Lin, Yaqiu; Han, Qing; Han, Jing; Gao, Jianjie; Tian, Yongsheng; Zhao, Wei; Peng, Rihe; Yao, Quanhong

2014-04-01

371

Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger.  

PubMed

Convenient expression systems for efficient heterologous production of different laccases are needed for their characterization and application. The laccase cDNAs lcc1 and lcc2 from Trametes versicolor were expressed in Pichia pastoris and Aspergillus niger under control of their respective glyceraldehyde-3-phosphate dehydrogenase promoters and with the native secretion signal directing catalytically active laccase to the medium. P. pastoris batch cultures in shake-flasks gave higher volumetric activity (1.3 U/L) and a better activity to biomass ratio with glucose than with glycerol or maltose as carbon source. Preliminary experiments with fed-batch cultures of P. pastoris in bioreactors yielded higher activity (2.8 U/L) than the shake-flask experiments, although the levels remained moderate and useful primarily for screening purposes. With A. niger, high levels of laccase (2700 U/L) were produced using a minimal medium containing sucrose and yeast extract. Recombinant laccase from A. niger harboring the lcc2 cDNA was purified to homogeneity and it was found to be a 70-kDa homogeneous enzyme with biochemical and catalytic properties similar to those of native T. versicolor laccase A. PMID:16915640

Bohlin, Christina; Jönsson, Leif J; Roth, Robyn; van Zyl, Willem H

2006-01-01

372

Demonstration of Laccase in the White Rot Basidiomycete Phanerochaete chrysosporium BKM-F1767  

PubMed Central

It has been widely reported that the white rot basidiomycete Phanerochaete chrysosporium, unlike most other white rot fungi, does not produce laccase, an enzyme implicated in lignin biodegradation. Our results showed that P. chrysosporium BKM-F1767 produces extracellular laccase in a defined culture medium containing cellulose (10 g/liter) and either 2.4 or 24 mM ammonium tartrate. Laccase activity was demonstrated in the concentrated extracellular culture fluids of this organism as determined by a laccase plate assay as well as a spectrophotometric assay with ABTS [2,2(prm1)-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] as the substrate. Laccase activity was observed even after addition of excess catalase to the extracellular culture fluid to destroy the endogenously produced hydrogen peroxide, indicating that the observed activity is not due to a peroxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by activity staining with ABTS revealed the presence of a laccase band with an estimated M(infr) of 46,500.

Srinivasan, C.; Dsouza, T. M.; Boominathan, K.; Reddy, C. A.

1995-01-01

373

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

PubMed

Laccase production by solid state fermentation (SSF) using an indigenously isolated litter dwelling fungus Fusarium incarnatum LD-3 was optimized. Fourteen medium components were screened by the initial screening method of Plackett-Burman. Each of the components was screened on the basis of 'p' (probability value) which was above 95% confidence level. Ortho-dianisidine, thiamine HCl and CuSO(4) . 5 H(2)O were identified as significant components for laccase production. The Central Composite Design response surface methodology was then applied to further optimize the laccase production. The optimal concentration of these three medium components for higher laccase production were (g/l): CuSO(4) . 5 H(2)O, 0.01; thiamine HCl, 0.0136 and ortho-dianisidine, 0.388 mM served as an inducer. Wheat straw, 5.0 g was used as a solid substrate. Using this statistical optimization method the laccase production was found to increase from 40 U/g to 650 U/g of wheat straw, which was sixteen times higher than non optimized medium. This is the first report on statistical optimization of laccase production from Fusarium incarnatum LD-3. PMID:20082375

Chhaya, Urvish; Gupte, Akshaya

2010-02-01

374

Solid-state fermentation for enhanced production of laccase using indigenously isolated Ganoderma sp.  

PubMed

Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using wheat bran as substrate with 70% initial moisture content at 25 degrees C and the seven agar plugs as the inoculum. Further enhancement in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using SSF. The process also promises economic utilization and value addition of agro-residues. PMID:18025593

Revankar, Madhavi S; Desai, Kiran M; Lele, S S

2007-10-01

375

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

PubMed

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

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

2014-07-01

376

Laccase localized in hulle cells and cleistothecial primordia of Aspergillus nidulans.  

PubMed Central

Several species of the genus Aspergillus form sexual spores within minute (approximately 0.2 mm) spherical shells (cleisthothecia) which are woven from specialized hyphae. Aspergillus nidulans cleistothecia are uniquely characterized by their dark red coloration and an envelope of thick-walled globose cells (hulle cells). By use of a new chromogenic substrate, we have shown that the constitutent hyphae of young cleistothecia and the hulle cells which surround the cleistothecia of A. nidulans exhibit a strong phenoloxidase activity which has the substrate specificity of a laccase. This enzyme (laccase II) is distinct from the previously described phenoloxidase (laccase I) that participates in the synthesis of the conidial pigment of A. nidulans: the two enzymes differ electrophoretically, do not cross-react immunologically, appear at different times during colonial development, and are under different genetic control. Examination of seven additional species of Aspergillus showed that the hulle cells of three acleistothecial species were also laccase positive, whereas the pale or unpigmented cleistothecia of four species (which lack hulle cells) were laccase negative. The relevance of these findings to the role of hulle cells in cleistothecial development is discussed. The presence of histologically detectable laccase in cleistothecial primordia provides a valuable tool, previously unavailable, for quantitating the early stages of sexual development in A. nidulans. Images

Hermann, T E; Kurtz, M B; Champe, S P

1983-01-01

377

A homodimeric laccase with unique characteristics from the yellow mushroom Cantharellus cibarius.  

PubMed

The aim of the present study was to isolate a laccase from fruiting bodies of the yellow mushroom Cantharellus cibarius. The fruiting body extract was subjected to a purification protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and Con A-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on Con A-Sepharose. The laccase was composed of two identical subunits each with a molecular mass of 46 kDa. The laccase exhibited a temperature-dependent rise in activity over the temperature range 20-50 degrees C. When the temperature was raised above 60 degrees C there was a fall in enzyme activity. The enzyme manifested maximal activity at pH 4. At and above pH 6 there was a dramatic reduction in activity. The unique features of this fruiting body laccase compared with previously reported mycelial laccases include homodimeric nature, a distinctive N-terminal sequence, a higher optimal pH, and adsorption on only ConA-Sepharose among the various chromatographic media tested. PMID:14672694

Ng, T B; Wang, H X

2004-01-01

378

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

NASA Astrophysics Data System (ADS)

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.

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

2008-05-01

379

Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme  

Microsoft Academic Search

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration,\\u000a anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa\\u000a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including\\u000a 2,2?-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH

Lei Lu; Min Zhao; Bei-Bei Zhang; Shu-Yu Yu; Xi-Jun Bian; Wei Wang; Yan Wang

2007-01-01

380

CYP98A3 from Arabidopsis thaliana Is a 3'Hydroxylase of Phenolic Esters, a Missing Link in the Phenylpropanoid Pathway  

Microsoft Academic Search

The 4- and 5-hydroxylations of phenolic compounds in plants are catalyzed by cytochrome P450 enzymes. The 3-hydroxylation step leading to the formation of caffeic acid from p-coumaric acid remained elusive, however, alternatively described as a phenol oxidase, a dioxygen- ase, or a P450 enzyme, with no decisive evidence for the involvement of any in the reaction in planta. In this

Guillaume Schoch; Simon Goepfert; Marc Morant; Alain Hehn; Denise Meyer; Pascaline Ullmann; Daniele Werck-Reichhart

2001-01-01

381

Barlerisides A and B, new potent superoxide scavenging phenolic glycosides from Barleria acanthoides.  

PubMed

Barlerisides A (1) and B (2), new phenolic glycosides, have been isolated from the n-butanol soluble sub-fraction of Barleria acanthoides along with two known compounds acteoside (3) and p-hydroxycinnamic acid (4). Their structures have been assigned on the basis of spectral studies. Both 1 and 2 showed significant activity in the superoxide scavenging assay while weak inhibitory activity was observed against the enzyme xanthine oxidase. PMID:19912065

Karim, Aman; Noor, Atia Tun; Malik, Abdul; Qadir, Muhammad I; Choudhary, Muhammad I

2009-12-01

382

X-Ray absorption edge determination of the oxidation state and coordination number of copper: application to the type 3 site in rhus vernicifera laccase and its reaction with oxygen  

SciTech Connect

Cu X-ray absorption edge features of 19 Cu(I) and 40 Cu(II) model complexes have been systematically studied and correlated with oxidation state and geometry. Studies of Cu(I) model complexes with different coordination number reveal that an 8983-8984-eV peak (assigned as the Cu 1s ..-->.. 4p transition) can be correlated in energy, shape, and intensity with ligation and site geometry of the cuprous ion. These Cu(I) edge features have been qualitatively interpreted with ligand field concepts. Alternatively, no Cu(II) complex exhibits a peak below 8985.0 eV. The limited intensity observed in the 8983-8985-eV region for some Cu(II) complexes is associated with the tail of an absorption peak at approx. 8986 eV which is affected by the covalency of the equatorial ligands. These models studies allow accurate calibration of a normalized difference edge procedure which is used for the quantitative determination of Cu(I) content in copper complexes of mixed oxidation state composition. This normalized difference edge analysis is then used to quantitatively determine the oxidation states of the copper sites in type 2 copper-depleted (T2D) and native forms of the multicopper oxidase, Rhus vernicifera laccase. The type 3 site of the T2D laccase is found to be fully reduced and stable to oxidation by O/sub 2/ or by 25-fold protein equivalents of ferricyanide, but it can be oxidized by reaction with peroxide. The increase in intensity of the 330-nm absorption feature which results from peroxide titration of T2D laccase is found to correlate linearly with the percent of oxidation of the binuclear copper site.

Kau, L.S.; Spira-Solomon, D.J.; Penner-Hahn, J.E.; Hodgson, K.O.; Solomon, E.I.

1987-10-14

383

Relationship between production of 3-indoleacetic acid and peroxidase-laccase activities depending on the culture periods in Funalia trogii (Trametes trogii)  

Microsoft Academic Search

The relationship between production of 3-indoleacetic acid (IAA) and peroxidase and laccase activity was investigated in white-rot\\u000a fungusFunalia trogii (Trametes trogii). F. trogii produced IAA and peroxidase and laccase as both primary metabolite and secondary metabolite; the levels of IAA may be influenced\\u000a by peroxidase and laccase. A correlation exists between the levels of IAA and peroxidase-laccase activity.

S. Ünyayar; E. Ünal; A. Ünyayar

2001-01-01

384

Relationship between production of 3-indoleacetic acid and peroxidase-laccase activities depending on the culture periods in Funalia trogii (Trametes trogii).  

PubMed

The relationship between production of 3-indoleacetic acid (IAA) and peroxidase and laccase activity was investigated in white-rot fungus Funalia trogii (Trametes trogii). F. trogii produced IAA and peroxidase and laccase as both primary metabolite and secondary metabolite; the levels of IAA may be influenced by peroxidase and laccase. A correlation exists between the levels of IAA and peroxidase-laccase activity. PMID:11501398

Unyayar, S; Unal, E; Unyayar, A

2001-01-01

385

Laccase/AuAg Hybrid Glucose Microfludic Fuel Cell  

NASA Astrophysics Data System (ADS)

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 .

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

2013-12-01

386

Preparation and application of polyclonal antibody against a recombinant laccase.  

PubMed

A laccase gene from Trametes sp. 420 was recombinantly expressed in Pichia pastoris, producing the enzyme rLacD. Six mutant enzymes were produced by site-directed mutation at six potential glycosylation sites in the enzyme rLacD respectively. To probe the mutants with lower activities sensitively and specifically, the antiserum containing specific polyclonal antibodies were prepared by immunizing healthy male rabbits, about 4-month-old and 2 kilogram weight, using pure rLacD as an immunogen. Antibodies were collected after the fifth immunization injection. The antiserum had titres of 1:32 in double immunodiffusion test and of 1:128,000 in enzyme-linked immunosorbent assay (ELISA). The results obtained by Western blot analysis showed that the antiserum could react with rLacD and its mutants with highly specific and sensitive affinities. PMID:17941175

Xu, Yinghai; Hong, Yuzhi; Xiao, Yazhong; Fang, Wei

2007-08-01

387

Gold nanoparticles as electronic bridges for laccase-based biocathodes.  

PubMed

Direct electron transfer (DET) reactions between redox enzymes and electrodes can be maximized by oriented immobilization of the enzyme molecules onto an electroactive surface modified with functionalized gold nanoparticles (AuNPs). Here, we present such strategy for obtaining a DET-based laccase (Lc) cathode for O(2) electroreduction at low overpotentials. The stable nanostructured enzymatic electrode is based on the step-by-step covalent attachment of AuNPs and Lc molecules to porous graphite electrodes using the diazonium salt reduction strategy. Oriented immobilization of the enzyme molecules on adequately functionalized AuNPs allows establishing very fast DET with the electrode via their Cu T1 site. The measured electrocatalytic waves of O(2) reduction can be deconvoluted into two contributions. The one at lower overpotentials corresponds to immobilized Lc molecules that are efficiently wired by the AuNPs with a heterogeneous electron transfer rate constant k(0) ? 400 s(-1). PMID:23004683

Gutiérrez-Sánchez, Cristina; Pita, Marcos; Vaz-Domínguez, Cristina; Shleev, Sergey; De Lacey, Antonio L

2012-10-17

388

The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase.  

PubMed Central

The white rot fungus Pycnoporus cinnabarinus was characterized with respect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with low amounts of an unusual peroxidase. Neither lignin peroxidase nor manganese peroxidase was detected. Laccase was produced constitutively during primary metabolism. Addition of the most effective inducer, 2,5-xylidine, enhanced laccase production ninefold without altering the isoenzyme pattern of the enzyme. Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,000 Da, as determined by calibrated gel filtration chromatography, and a carbohydrate content of 9%. The enzyme displayed an unusual behavior on isoelectric focusing gels; the activity was split into one major band (pI, 3.7) and several minor bands of decreasing intensity which appeared at regular, closely spaced intervals toward the alkaline end of the gel. Repeated electrophoresis of the major band under identical conditions produced the same pattern, suggesting that the laccase was secreted as a single acidic isoform with a pI of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This appeared to be confirmed by N-terminal amino acid sequencing of the purified enzyme, which yielded a single sequence for the first 21 residues. Spectroscopic analysis indicated a typical laccase active site in the P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indicated the enzyme to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus laccase showed close homology to the N-terminal sequences determined for laccases from Trametes versicolor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The principal features of the P. cinnabarinus enzyme system, a single predominant laccase and a lack of lignin- or manganese-type peroxidase, make this organism an interesting model for further studies of possible alternative pathways of lignin degradation by white rot fungi.

Eggert, C; Temp, U; Eriksson, K E

1996-01-01

389

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

PubMed

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

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

2014-04-01

390

Degradation of phenol and phenolic compounds by Pseudomonas putida EKII  

Microsoft Academic Search

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

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

1992-01-01

391

Stability Mechanisms of a Thermophilic Laccase Probed by Molecular Dynamics  

PubMed Central

Laccases are highly stable, industrially important enzymes capable of oxidizing a large range of substrates. Causes for their stability are, as for other proteins, poorly understood. In this work, multiple-seed molecular dynamics (MD) was applied to a Trametes versicolor laccase in response to variable ionic strengths, temperatures, and glycosylation status. Near-physiological conditions provided excellent agreement with the crystal structure (average RMSD ?0.92 Å) and residual agreement with experimental B-factors. The persistence of backbone hydrogen bonds was identified as a key descriptor of structural response to environment, whereas solvent-accessibility, radius of gyration, and fluctuations were only locally relevant. Backbone hydrogen bonds decreased systematically with temperature in all simulations (?9 per 50 K), probing structural changes associated with enthalpy-entropy compensation. Approaching Topt (?350 K) from 300 K, this change correlated with a beginning “unzipping” of critical ?-sheets. 0 M ionic strength triggered partial denucleation of the C-terminal (known experimentally to be sensitive) at 400 K, suggesting a general salt stabilization effect. In contrast, F? (but not Cl?) specifically impaired secondary structure by formation of strong hydrogen bonds with backbone NH, providing a mechanism for experimentally observed small anion destabilization, potentially remedied by site-directed mutagenesis at critical intrusion sites. N-glycosylation was found to support structural integrity by increasing persistent backbone hydrogen bonds by ?4 across simulations, mainly via prevention of F? intrusion. Hydrogen-bond loss in distinct loop regions and ends of critical ?-sheets suggest potential strategies for laboratory optimization of these industrially important enzymes.

Christensen, Niels J.; Kepp, Kasper P.

2013-01-01

392

NADPH oxidases: progress and opportunities.  

PubMed

Abstract From the initial discovery in 1999 that NADPH oxidases comprise a family of enzymes to our current focus on drug development to treat multiple pathologies related to this enzyme family, progress has been swift and impressive. We have expanded our understanding of the extent of the family, the basic enzymatic biochemistry, the multiple cellular functions controlled by NADPH oxidases, and their varied roles in physiology and diseases. We have developed numerous cell culture tools, animal models, and human databases that have allowed us to delve deeply into the various roles of these enzymes. However, it is clear that much remains to be learned and that there are many opportunities for new tools and new research directions to more fully understand these critical enzymes. With this Antioxidants and Redox Signaling Forum, we explore in detail the progress, challenges, and opportunities in Nox biology. Progress so far has clearly shown that NADPH oxidases are integral to fully functioning organisms and that the dysregulation of Nox enzymes contributes to a wide variety of pathologies. We have the opportunity to develop new tools and small molecules that will not only help us to better understand the molecular underpinnings of NADPH oxidases but also to develop treatments for diverse human diseases. Antioxid. Redox Signal. 20, 2692-2694. PMID:24730700

San Martin, Alejandra; Griendling, Kathy K

2014-06-10

393

Characterization of catecholase and cresolase activities of eggplant polyphenol oxidase.  

PubMed

In the present paper the catecholase and cresolase activities of eggplant polyphenol oxidase (PPO) are described. To preserve the latter activity, a partially purified enzyme was used. Peroxidase was removed from the preparation to avoid its interference with PPO during phenol oxidation. The partially purified eggplant PPO was fully active. The catecholase/cresolase ratio of 41.1 indicated that, in a pH close to the physiological, diphenol oxidation predominates over monophenol oxidation. The characteristic lag phase of the cresolase activity is modulated by the pH, the monophenol and diphenol concentrations, and the enzyme's concentration. The effect of several inhibitors was also tested, and the K(i) values of the two most effective (tropolone and 4-hexylresorcinol) were determined. PMID:10725136

Pérez-Gilabert, M; García Carmona, F

2000-03-01

394

Distinct Stress Responses of Two Functional Laccases in Cryptococcus neoformans Are Revealed in the Absence of the Thiol-Specific Antioxidant Tsa1  

Microsoft Academic Search

Laccases are thought to be important to the virulence of many fungal pathogens by producing melanin, a presumed oxygen radical scavenger. A laccase in Cryptococcus neoformans has been shown to synthesize melanin and contributes to the virulence and the survival in macrophages of this fungal pathogen. One C. neoformans laccase gene, LAC1, previously called CNLAC1, has been extensively studied, and

Tricia A. Missall; Jason M. Moran; John A. Corbett; Jennifer K. Lodge

2005-01-01

395

Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa  

PubMed Central

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ?40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome–based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.

Lu, Shanfa; Li, Quanzi; Wei, Hairong; Chang, Mao-Ju; Tunlaya-Anukit, Sermsawat; Kim, Hoon; Liu, Jie; Song, Jingyuan; Sun, Ying-Hsuan; Yuan, Lichai; Yeh, Ting-Feng; Peszlen, Ilona; Ralph, John; Sederoff, Ronald R.; Chiang, Vincent L.

2013-01-01

396

Effect of metal ions on reactive dye decolorization by laccase from Ganoderma lucidum.  

PubMed

In this work, the influence of different metal ions on laccase activity and laccase-catalyzed dye decolorization was investigated under in vitro conditions using crude laccase obtained from a white rot fungus Ganoderma lucidum. Laccase activity was enhanced by metal ions such as Ca(2+), Co(2+), Cu(2+) and Zn(2+) at low concentrations (1mM). Increasing the concentration of metal ions except that of Cu(2+) and Zn(2+) up to 5mM and above decreased the enzyme activity. Among several heavy metals, Fe(2+) highly inhibited the enzyme activity. Effect of metal ions was tested on decolorization of two reactive dyes, namely Remazol black-B (RB-5) and Remazol brilliant blue R (RBBR) at a concentration of 50 mg l(-1). The presence of heavy metals generally did not exert much influence on the decolorization except Fe(2+). Cu(2+) and Cr(6+) enhanced the decolorization of both dyes. In the presence of 1mM Cu(2+), 94% of RB-5 and 35.5% of RBBR were decolorized during 1h incubation. G. lucidum laccase was able to tolerate mixture of several metal ions. Treatment of simulated reactive dye effluent by laccase showed that the redox mediator system is necessary for effluent decolorization. Syringaldehyde, a natural redox mediator, was very effective than the synthetic mediator 1-hydroxybenzotriazole (HBT). The initial rate of effluent decolorization in presence of syringaldehyde (0.0831 h(-1)) was 5.6 times higher than HBT (0.0152 h(-1)). Although the rate of decolorization was markedly decreased in the effluent containing mixed metal ions, presence of syringaldehyde showed effective decolorization. This study indicates that G. lucidum laccase and natural redox mediator system could be a potential candidate for color removal from reactive dye effluent. PMID:19356850

Murugesan, Kumarasamy; Kim, Young-Mo; Jeon, Jong-Rok; Chang, Yoon-Seok

2009-08-30

397

Plasma functionalized carbon electrode for laccase-catalyzed oxygen reduction by direct electron transfer.  

PubMed

For the first time, a fast and versatile technique, an atmospheric pressure plasma jet (APPJ), has been used to functionalise graphite carbon electrodes for biofuel cell applications. The bioelectrode was functionalized by an atmospheric pressure plasma jet (APPJ) system using air, oxygen (O2) and nitrogen (N2) plasmas applied for only a few seconds. XPS analysis showed that carboxylic groups were created on the carbon substrates using both air and O2 plasmas, while mainly carbonyl and amine/amide functionalities were generated using N2 plasmas. A purified laccase from Trametes versicolor was both adsorbed and covalently bound (NHS/EDC method) to the plasma modified carbon. Higher laccase activity was obtained for the covalently grafted laccase compared to the physically adsorbed one: 13.2 (±2) 10(-3)U of laccase on air treated graphite and two-fold less (5.3 (±1.1) 10(-3)U) were obtained on N2 plasma treated surfaces (1mM ABTS as a substrate, 30°C, pH=3.0), one unit (U) being the quantity of ABTS (?mole) oxidized by laccase per minute. Dioxygen reduction was performed by direct electron transfer (DET). The highest current density, 108?A/cm(2) (at 0.2V (vs. SCE), pH 4.2, room temperature), was recorded for covalently immobilized laccase on N2 plasma treated surfaces (geometric surface=0.38cm(2)). This could be explained by the fact that the highly conductive graphite structure was retained in the case of this surface treatment and could also suggest a preferential orientation of the T1 Cu center of the laccase toward the surface of the N2 plasma treated electrode. PMID:23416361

Ardhaoui, Malika; Zheng, Meihui; Pulpytel, Jerome; Dowling, Denis; Jolivalt, Claude; Khonsari, Farzaneh Arefi

2013-06-01

398

Phenoloxidase-mediated interactions of phenols and anilines with humic materials  

SciTech Connect

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.

Dec, J.; Bollag, J.M.

2000-06-01

399

Phenol cauterization for ingrown toenails.  

PubMed Central

A new clinic was set up to deal with all toenail problems referred to a large general hospital. Ingrowing toenails were the commonest problem dealt with and phenol cauterization, either partial or complete, was the standard form of treatment. Over a 2-year period 1013 phenol cauterizations were carried out on 631 patients. After a minimum of 12 months follow-up, the recurrence rate was 2.96%. All recurrences were successfully treated by repeat phenol cauterization. We conclude that phenol cauterization should replace surgical ablation in the treatment of ingrown toenails.

Ramsay, G; Caldwell, D

1986-01-01

400

Olive oil phenols and neuroprotection.  

PubMed

Olive oil is a rich source of phenolic components which have a wide variety of beneficial health effects in vitro, in vivo, and clinically. The beneficial effects of olive oil phenols attributed to a variety of biological activities including free radical scavenging/antioxidant actions, anti-inflammatory effects, anti-carcinogenic properties, and anti-microbial activities. On the other hand, olive oil phenols have been shown to be some of neuroprotective effects against cerebral ischemia, spinal cord injury, Huntington's disease, Alzheimer's diseases, multiple sclerosis, Parkinson's disease, aging, and peripheral neuropathy. This paper summarizes current knowledge on the mechanisms of neuroprotective effects of olive oil phenols. PMID:23406576

Khalatbary, Ali Reza

2013-11-01

401

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

PubMed Central

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

Dhakar, Kusum; Pandey, Anita

2013-01-01

402

Induction of Laccase Activity in Rhizoctonia solani by Antagonistic Pseudomonas fluorescens Strains and a Range of Chemical Treatments  

PubMed Central

Fungi often produce the phenoloxidase enzyme laccase during interactions with other organisms, an observation relevant to the development of biocontrols. By incorporating the laccase substrate 2,2?-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) into agar, we analyzed laccase induction in the plant-pathogenic fungus Rhizoctonia solani when paired against isolates of the soil bacterium Pseudomonas fluorescens. Substantial induction of R. solani laccase was seen only in pairings with strains of P. fluorescens known to produce antifungal metabolites. To study laccase induction further, a range of chemical treatments was applied to R. solani liquid cultures. p-Anisidine, copper(II), manganese(II), calcium ionophore A23187, lithium chloride, calcium chloride, cyclic AMP (cAMP), caffeine, amphotericin B, paraquat, ethanol, and isopropanol were all found to induce laccase; however, the P. fluorescens metabolite viscosinamide did not do so at the concentrations tested. The stress caused by these treatments was assessed by measuring changes in lipid peroxidation levels and dry weight. The results indicated that the laccase induction seen in pairing plate experiments was most likel