Highest Laccase Research Articles

Thermophilic laccase from xerophyte species Opuntia vulgaris

Two laccase temperature isoforms capable of oxidizing phenolic compounds to quinones were isolated and purified to homogeneity from the cladodes of the xerophyte species Opuntia vulgaris. These catalytically active proteins exhibit apparent molecular masses of 137 and 90 kDa. Under reducing conditions, both isoforms yielded a subunit molecular mass of 43 kDa, suggesting that the enzyme is a multimer of the 43 kDa subunit. The 137 kDa isoform when heated at 80°C for 3 min generated three polypeptide bands on activity stained polyacrylamide gels exhibiting 137, 90 and 43 kDa molecular forms. All isoforms of the enzyme exhibited an optimum pH of 10 when 2,6-dimethoxyphenol was used as a substrate. The optimum temperature of the 137 kDa enzyme form was noted to be 80°C and that of the 90 kDa enzyme form was 70°C. Denaturation kinetics of both the laccase isoforms carried out at their respective optimum temperatures for 30 min exhibited enzyme activity in excess of their t(1/2) values throughout the assay period. The K(m) for the 137 kDa form was determined to be 2.2 ± 0.3 mm and the V(max) was 2.8 ± 0.2 IU/mL. These high temperature stable laccase isoforms having alkaline pH optima can find significant industrial use.

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

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 and size exclusion chromatography. The molecular weight of purified laccase was estimated as 68 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme showed maximum substrate specificity toward 2,2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid than any other substrate. The optimum pH and temperature for laccase activity were 4.0 and 60°C, respectively. The purified enzyme was stable up to 50°C, and high laccase activity was maintained at pH 5.0 ∼ 7.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol, and L-cysteine. Purified laccase decolorized various textile dyes within 4 h in the absence of redox mediators. HPLC and FTIR analysis confirmed degradation of methyl orange. The metabolite formed after decolorization of methyl orange was characterized as p-N,N′-dimethylamine phenyldiazine using GCMS.

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

In this study, decolorization of dyestuffs, such as Reactive Red 198, Rem Blue RR, Dylon Navy 17, Rem Red RR, and Rem Yellow RR was studied using laccase and laccase-mediated system. The laccases are known to have an important potential for remediation of pollutants. Among these dyestuffs, decolorization of Rem Blue RR and Dylon Navy 17 was performed with crude laccase under optimized conditions. Vanillin was selected as laccase mediator after screening six different compounds with Rem Yellow RR, Reactive Red 198, and Rem Red RR as substrates. However, Rem Yellow RR was not decolorized by either laccase or laccase-mediated system. It is observed that the culture supernatant contained high laccase activity after treatment with catalase that was responsible for the decolorization. Besides, culture supernatant with high laccase activity as enzyme source was treated with catalase; in this way, the hypothesis that laccase was the enzyme responsible for decolorization was supported. The Rem Blue RR was decolorized with 64.84% under the optimum conditions and Dylon Navy 17 with 75.43% with crude laccase. However, using the laccase and vanillin, the decolorization of Reactive Red 198 and Rem Red RR was found to be 62% and 68%, respectively. Our study demonstrated that the decolorization abilities of laccase and/or laccase mediator systems were based on the types of mediator, the dye structure, and the standard experimental conditions. Also, the electrochemical behaviors of some samples were studied. The redox potentials of these samples were determined using cyclic voltammetry on glassy carbon electrode in phosphate buffer (pH6) solution.

Ecology of coarse wood decomposition by the saprotrophic fungus Fomes fomentarius

Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cellulose in dead wood and as such they attracted considerable attention. The aims of this work were to quantify the activity and spatial distribution of extracellular enzymes in coarse wood colonised by the white-rot basidiomycete Fomes fomentarius and in adjacent fruitbodies of the fungus and to analyse the diversity of the fungal and bacterial community in a fungus-colonised wood and its potential effect on enzyme production by F. fomentarius. Fungus-colonised wood and fruitbodies were collected in low management intensity forests in the Czech Republic. There were significant differences in enzyme production by F. fomentarius between Betula pendula and Fagus sylvatica wood, the activity of cellulose and xylan-degrading enzymes was significantly higher in beech wood than in birch wood. Spatial analysis of a sample B. pendula log segment proved that F. fomentarius was the single fungal representative found in the log. There was a high level of spatial variability in the amount of fungal biomass detected, but no effects on enzyme activities were observed. Samples from the fruiting body showed high β-glucosidase and chitinase activities compared to wood samples. Significantly higher levels of xylanase and cellobiohydrolase were found in samples located near the fruitbody (proximal), and higher laccase and Mn-peroxidase activities were found in the distal ones. The microbial community in wood was dominated by the fungus (fungal to bacterial DNA ratio of 62-111). Bacterial abundance composition was lower in proximal than distal parts of wood by a factor of 24. These results show a significant level of spatial heterogeneity in coarse wood. One of the explanations may be the successive colonization of wood by the fungus: due to differential enzyme production, the rates of biodegradation of coarse wood are also spatially inhomogeneous.

Delignification of Pinus radiata kraft pulp by treatment with a yeast genetically modified to produce laccases

Cellulose pulp bleaching is one of the main biotechnological applications of fungal laccases due to their capacity to degrade lignin from the unbleached pulp. For this application, not only a low cost enzyme production, but also higher enzyme concentrations than those obtained by its natural producers are needed. Heterologous expression of laccase in yeasts is an option for producing these enzymes at industrial scale. In this work the heterologous expression of the cglcc1 gene, responsible for laccase production in the basidiomicetous fungus Coriolopsis gallica, in the yeast Kluyveromyces lactis has been tested. In order to know if the transformed yeast obtains delignificant capability, a Pinus radiata kraft pulp has been incubated with it. After the treatment, an important decrease in kappa number (13%) and in lignin content (22%) has been observed. These results showed the delignificant capacity of this transformed yeast. It can be concluded that the employment of genetically modified microorganisms, without cellulolitic activity, to produce high laccase levels could be a feasible option to delignify cellulose pulps with a potential application in cellulose pulp bleaching.

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

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 this study, laccase production in the repeated-batch cultures of F. trogii and T. versicolor pre-grown pellets were investigated under various conditions. Retention time, temperature, agitation, pH and the amount of pellets were found to be important for laccase production during repeated-batch studies. The culture filtrate showed one protein band. Zymogram gel also showed only one band of activity. The enzyme remained fully active when frozen for 300 days. The immobilized fungi were also able to produce high amounts of laccase during reuse. It is possible to obtain high laccase amounts with free and immobilized repeated-batch cultures under the most appropriate culture conditions determined. Considering the various biotechnological applications of laccase, an enhancement in laccase production through the selection of appropriate culture conditions could facilitate the development of more economical and environmentally friendly processes.

Dephenolization and decolorization of olive mill wastewater through sequential batch and co-culture applications

In a previous work it was reported adapted Trametes versicolor FPRL 28A INI culture was used to treat undiluted olive mill wastewater (OMW) without addition of any nutrients with significant amount of total phenolics were removed. However, decolorization was not so pronounced. Therefore, the aim of this study is to enhance the efficiency of dephenolization and decolorization of the primary treatment with adapted Trametes versicolor, incorporating a secondary biological treatment step using different microorganisms with sequential batch and co-culture applications. Through sequential batch applications Funalia trogii ATCC 20080 was found to have a higher potential in terms of total phenolics removal and decolorization amongst the tested organisms and better results were obtained from sequential batch applications as compared to co-culture experiments. In sequential batch applications, up to 91% total phenolics were removed and 64% decolorization was achieved after 24 days with 20% (v/v) inoculation rate of F. trogii when malt extract broth was used in inoculum preparation. In addition, significant accumulation of laccase (2019 ± 121.13Ul−1) and manganese peroxidase (463 ± 33.89 Ul−1) activities were attained. In co-culture applications highest total phenolics removal and decolorization were 78 and 39%, respectively, with non-adapted T. versicolor, whereas highest laccase and manganese peroxidase acitivities were obtained with F. trogii as 2219 Ul−1 ± 176.14. and 513 ± 4.12 Ul−1, respectively.

High Current Density Air-Breathing Laccase Biocathode

Although biofuel cell research has progressed over the past decade, there were still problems with employing enzymes at air-breathing cathodes, because enzymes need to remain hydrated and most enzyme reactions occur in solution and not in gas phase. This research details an approach to the development of an air-breathing biocathode employing direct electron transfer. This laccase biocathode is studied in two different fuel cell configurations: a proton exchange membrane hydrogen/air fuel cell and a direct methanol fuel cell (DMFC) with an anion exchange membrane. The laccase from the Rhus vernificera biocathode with an enzyme loading of provides fuel crossover tolerance and provides a high operational current density of and a maximum power density of in a 40% methanol DMFC. The laccase biocathode shows a lifetime of 290 h in a DMFC. The hydrogen/air fuel cell provides a stable current for a total of 350 discontinuous hours when operated for 8 h daily.

Effect of nitrogen sources and vitamins on ligninolytic enzyme production by some white-rot fungi. Dye decolorization by selected culture filtrates

The effect of amino acids, complex nitrogen sources and vitamin addition on Trametes trogii, Trametes villosa and Coriolus versicolor var. antarcticus ligninolytic enzyme production, was evaluated. Dye decolorization by their culture filtrates was compared. Glutamic acid followed by peptone, were the best N sources for laccase and manganese peroxidase production. The three fungi produced two laccase isoenzymes (molecular weights from 38 up to 150kDa); their pattern of production was not affected by medium composition. Although the response was not uniform, vitamin addition sometimes stimulated ligninolytic enzyme production, but never inhibited it. Thiamine induced manganese peroxidase production. T. trogii grown in glutamic acid produced culture filtrates with the highest laccase (188.3 U/ml) and manganese peroxidase activities (4.5 U/ml), rendering the best results in decolorization. These crude filtrates were able to decolorize in half hour (at pH 4.5, 30°C): 13%, 23%, 40%, 46%, 82%, 94% and 95% of Gentian Violet, Xylidine, Congo Red, Malachite Green, Remazol Brilliant Blue R, Indigo Carmine and Anthraquinone Blue, respectively.

A Combination of Evolutionary Trace Method, Molecular Surface Accessibility and Hydrophobicity Analysis to Design a High Hydrophobicity Laccase

Laccases are industrially attractive enzymes and their applications have expanded to the field of bioremediation. The challenge of today's biotechnology in enzymatic studies is to design enzymes that not only have a higher activity but are also more stable and could fit well with the condition requirements. Laccases are known to oxidize non-natural substrates like polycyclic aromatic hydrocarbons (PAHs). We suppose by increasing the hydrophobicity of laccase, it would increase the chance of the enzyme to meet the hydrophobic substrates in a contamination site, therefore increasing the bioremediation efficacy of PAHs from environment. In this attempt, the applications of evolutionary trace (ET), molecular surface accessibility and hydrophobicity analysis on laccase sequences and laccase's crystal structure (1KYA) are described for optimal design of an enzyme with higher hydrophobicity. Our analysis revealed that Q23A, Q45I, N141A, Q237V, N262L, N301V, N331A, Q360L and Q482A could be promising exchanges to be tested in mutagenesis experiments.

Cloning, expression in Escherichia coli, and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology (more than 60%) with bacterial laccases in the genome and protein databases and the highest degree of similarity (61% identity) was observed with the multicopper oxidase of Klebsiella sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid) and DMP (2,6-dimethoxyphenol), respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Biodegradation of Vanillate Derivatives by White-Rot Fungus, Phlebia Radiata

14 C either at carboxyl, methoxyl, or aromatic ring was studied during the 10-day cultivation of Phlebia radiata. Seventy percents of carbon dioxide evolved from carboxyl-labeled vanillate and about 40% from ring carbon-labeled vanillate and car- boxyl-labeled benzylvanillate. The degradation of methoxyl group from vanillic and benzylvanillic acids reached a similar level, yielding about 25% after one week growth of P. radiata in these conditions. The radioactivity inside mycelium reached two maximas, the first on the 2 nd -3 rd day after the induction and the second on the 7 th day after the induction. The first peak could be derived from high polymerization which can accompany high laccase activity. These polymers of decarboxylated quinones probably attach them- selves to the mycelium and can be subsequently degraded by the P. radiata extracellular enzymatic sys- tem. These enzymes can demethylate monomeric and polymeric structures as well as cleave the aromatic ring. The late second peak inside the mycelium can be attributed to the cleavage of monomers originating from the degradation of polyquinones or from the secondary metabolites.

Electrochemical and AFM characterization on gold and carbon electrodes of a high redox potential laccase from Fusarium proliferatum

The redox potential of the T1 copper site of laccase from Fusarium proliferatum was determined by titration to be about 510 mV vs. SCE (750 mV vs. NHE), which makes it a high redox potential enzyme. Anaerobic electron transfer reactions between laccase and carbon and gold electrodes were detected, both in solution and when the enzyme was adsorbed on these surfaces. In solution, a single high-potential signal (660 mV vs. SCE) was recorded at the carbon surfaces, attributable to the T1 copper site of the enzyme. However, a well-defined oxidative process at about 660 mV and an anodic wave at 350 mV vs. SCE were recorded at the gold electrode, respectively associated with the T1 and T2 copper sites. Laccase-modified carbon electrodes behaved analogously when the enzyme was in solution, unlike laccase adsorbed on gold, which showed only a low-potential signal. Laccase molecules were successfully imaged by AFM; obtaining a thick compact stable film on Au(111), and large aggregates forming a complex network of small branches leaving voids on the HOPG surface. Laccase-modified carbon electrodes retained significant enzymatic activity, efficiently oxidising violuric acid and reducing molecular oxygen. Explanations are proposed for how protein-film organisation affects the electrode function.

Extracellular ligninolytic enzymes by Lentinus polychrous Lév. under solid-state fermentation of potential agro-industrial wastes and their effectiveness in decolorization of synthetic dyes

Six agro-industrial wastes were evaluated as a support for ligninolytic enzyme production by the white-rot fungus Lentinus polychrous Lev. under solid-state fermentation. Enzyme production was markedly different according to the substrate used. Rice bran (RB) yielded the highest laccase activity of 1,449 U/L (after 21 days of culture) with specific activity of 4.4 U/g substrate. Rice bran supplemented with rice husk (RH) (2:1 by wt) showed high laccase activity of 1,425 U/L with specific activity of 10.0 U/g substrate (after 17 days of culture). The crude enzyme of the RH-RB culture also contained manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities in relative proportions of 1.9:1.4:1 of laccase:MnP:MIP, respectively. Zymogram studies showed the same isoenzyme pattern with these ligninolytic enzymes. The high enzyme production level and low substrate cost of SSF-L. polychrous Lev. suggest that it has potential for industrial applications. Our studies showed that the crude enzyme from this culture exhibited in vitro decolorization of Indigo Carmine. The highest efficiency of dye decolorization was observed under alkaline conditions (pH 9.0) at an initial dye concentration of 10 mg/L. The rather high pH conditions and high efficiency in Indigo Carmine decolorization make the enzyme further interest for the applications in treatment of waste water from the textile industry, which contains synthetic dyes.

Degradation of aflatoxin B 1 by fungal laccase enzymes

The enzymatic degradation of aflatoxin B 1 (AFB 1) by white rot fungi through laccase production was investigated in different liquid media. A significant ( P < 0.0001) correlation was observed between laccase activity and AFB 1 degradation exhibited by representatives of Peniophora and Pleurotus ostreatus cultivated in minimal salts (MSM) ( r = 0.93) and mineral salts — malt extract (MSB–MEB) ( r = 0.77) liquid media. Peniophora sp. SCC0152 cultured in MSB–MEB liquid medium supplemented with veratryl alcohol and sugarcane bagasse showed high laccase activity (496 U/L), as well as 40.45% AFB 1 degradation as monitored using high performance liquid chromatography. P. ostreatus St2-3 cultivated in MSM liquid medium supplemented with veratryl alcohol resulted in laccase activity of 416.39 U/L and 35.90% degradation of AFB 1. Aflatoxin B 1 was significantly ( P < 0.0001) degraded when treated with pure laccase enzyme from Trametes versicolor (1 U/ml, 87.34%) and recombinant laccase produced by Aspergillus niger D15-Lcc2#3 (118 U/L, 55%). Aflatoxin B 1 degradation by laccase enzyme from T. versicolor and recombinant laccase enzyme produced by A. niger D15-Lcc2#3 coincided with significant ( P < 0.001) loss of mutagenicity of AFB 1, as evaluated in the Salmonella typhimurium mutagenicity assay. The degradation of AFB 1 by white rot fungi could be an important bio-control measure to reduce the level of this mycotoxin in food commodities.

Effect of culturing processes and copper addition on laccase production by the white-rot fungusFomes fomentariusMUCL 35117

To produce high laccase activities from the white-rot fungus Fomes fomentarius. Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius. The SSF study expresses the highest laccase activities, nearly to 6400 U l(-1) after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l(-1) copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l(-1). With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1.5 l) leading to a laccase activity of about 6230 U l(-1) on day 13. The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l(-1) copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor. The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius- SSF for industrial purposes.

Decolorization of textile reactive dyes by the crude laccase produced from solid-state fermentation of agro-byproducts

Reactive dyes are one of the major sources of waste-water pollution. Efficient degradation of these dyes with enzymes produced from agricultural waste has attracted tremendous recent interests in both the scientific community and the general public. In this study, we took advantage of solid-state fermentation of four agro-byproducts (rape stem, wheat bran, peanut shell and rice hull) for producing laccase from the fungus Trametes sp. AH28-2. Higher laccase activities were obtained in multiple-substrates media than in single substrate media. The maximum yield of laccase (2.10 × 106 U/kg) was obtained in the medium containing 60% rape stem, 20% peanut shell and 20% wheat bran, without the supplement of any toxic inducers. Our results further demonstrated that the textile reactive dyes Levafix Blue CA and Cibacron Blue FN-R (1.0 g/l) were completely decolorized by the crude laccase (5.0 U/l) obtained within 15 h in the absence of any mediator. Therefore, the agro-byproducts could be re-utilized to produce laccase for the decolorization of textile reactive dyes.

Effect of different culture conditions and inducers on production of laccase by a basidiomycete fungal isolate Pleurotus ostreatus HP-1 under solid state fermentation

The production of laccase by an indigenous strain of Pleurotus ostreatus HP-1 was studied on solid state fermentation. Culture parameters such as type and concentration of substrate, inoculum size, moisture content, pH, surfactant presence, temperature, and nitrogen source were optimized by conventional “one factor at a time” methodology. A maximum laccase yield of 3952 U g-1 of dry substrate optimized was obtained with wheat straw as substrate with five agar plugs as the inoculum, 60% moisture content, pH 5.0, surfactant concentration 0.015 gl-1, and nitrogen source (combination of L-asparagine and NH4NO3 at 10 mM concentration each) at incubation temperature 28oC. Enhancement in laccase activity was achieved with the use of various aromatic inducers and copper sulphate. Highest laccase activity of 14189 U g-1 of dry substrate was achieved using 0.28 mM copper sulphate under optimized conditions. Thus, the indigenous isolate seems to be a potential producer of laccase using SSF and can be exploited for further biotechnological applications. The process also promises economic utilization and value addition of agro-residues.

Laccase isoforms with unusual properties from the basidiomyceteSteccherinum ochraceumstrain 1833

To isolate and characterize the laccase isoforms from S. ochraceum 1833 - a new active producer of high extracellular laccase activity. Three laccase isoforms (laccases I, II and III) with 57.5, 59.5 and 63 kDa molecular masses respectively were purified from S. ochraceum 1833 and in contrast to the known laccases had strongly pronounced absorption at 611 nm with molar extinction coefficients ranging from 7170 to 7830 mol(-1) l cm(-1). All isoforms showed maximal activity with ABTS at low pH (<or=2) and temperatures in the range 70-80 degrees C, were stable for long time of incubation at high temperature (60-80 degrees C) and at pH values ranging from 2 to 6. Laccase II showed a higher activity and wider substrate specificity. N-terminal amino acid sequence analysis of the purified laccase II (VQIGPVTDLH) showed 80% identity with the N-terminal amino acid sequence of laccase from Lentinula edodes [Appl Microbiol Biotechnol 60 (2002) 327]. Elevated temperature optima, high thermo- and pH-stabilities, the broad substrate specificity of the isoforms make the laccases from S. ochraceum 1833 a suitable model for biotechnological processes proceeding at high temperatures. For the first time, new basidiomycete strain S. ochraceum was reported as a producer of novel thermostable, pH stable, acidophilic laccases with unusual spectral properties.

Study of laccase production byPleurotus ostreatusin a 5 l bioreactor and application of the enzyme to determine the antioxidant concentration of human plasma

To achieve high laccase production from Pleurotus ostreatus in a bench top bioreactor and to utilize the enzyme for determination of the total antioxidant concentration (TAC) of human plasma. Laccase production by P. ostreatus studied in a benchtop bioreactor was as high as, 8740 U ml(-1) in presence of copper sulfate. The enzyme was used to replace metmyoglobin and hydrogen peroxide for the estimation of TAC in human plasma. The trolox equivalent antioxidant concentrations determined by the laccase-based method and metmyoglobin method ranged from 1.63 +/- 0.011 to 1.80 +/- 0.006 mmol l(-1) and from 1.41 +/- 0.004 to 1.51 +/- 0.008 mmol l(-1) plasma, respectively. Pleurotus ostreatus produced high amount of extracellular laccase in a benchtop bioreactor. The enzyme can be used to assay TAC of blood plasma without the interference encountered with the hydrogen peroxide and metmyoglobin mediated assay method. Laccase production by P. ostreatus obtained in this study was the highest among all reported laccase producing white-rot fungi. Moreover, an accurate laccase-based assay method was developed for detection of TAC in human plasma.