Laccase Formation Research Articles

Laccase mediator system obtained from a marine spore exhibits decolorization potential in harsh environmental conditions.

Laccases play a significant role in remedying dye pollutants. Most of these enzymes are originated from terrestrial fungi and bacteria, thus they are not proper to be used in the environments with neutral/alkaline pH, or they may require laborious extraction/purification steps. These limitations can be solved using marine spore laccases through high stability and easy to use application. In the current study, laccase activity of the marine spore -forming Bacillus sp. KC2 was measured according to the guaiacol and syringaldazine oxidation. Abiotic stresses like pH of 6, temperature of 37°C and 0.3mM CuSO4 (in comparison with optimal sporulation conditions: pH of 8, temperature of 20°C and 0.0mM CuSO4) enhanced laccase formation in sporal coat. Maximum activity of enzyme was observed at 50°C and pH 7, which did not change in the alkaline pH and temperature range of 20-70°C. Results indicated ions, inhibitors and solvent stability of the enzyme and its activity were stimulated by Co2+, Mn2+, PMSF, acetone, acetonitrile, ethanol, and methanol. The spore laccase could decolorize synthetic dyes from various chemical groups including azo (acid orange, amaranth, trypan blue, congo red, and amido black), indigo (indigo carmine), thiazine (methylene blue, and toluidine blue), and triarylmethane (malachite green) with ABTS/syringaldazine mediators after 5h. Degradation products were not toxic against Sorghum vulgare and Artemia salina model organisms. The enzyme mediator system showed high potentials for dye bioremediation over a wide range of harsh conditions.

Bioelectrocatalytic Oxygen Reduction by Laccase Immobilized on Various Carbon Carriers

Laccase is an enzyme that is used for fabricating cathodes of biofuel cells. Many studies have been aimed at searching the ways for enhancing specific electrochemical characteristics of cathode with the laccase- based catalyst. The electroreduction of oxygen on the electrode with immobilized laccase proceeds under the conditions of direct electron transfer between the electrode and active enzyme center. In this work, the effect of oxygen partial pressure on the electrocatalytic activity of laccase is studied. It is shown that, at the concentrations of oxygen dissolved in the electrolyte higher than 0.28 mM, the process is controlled by the kinetics of the formation of laccase–oxygen complex, whereas at lower concentrations and a polarization higher than 0.3 V, the process is limited by the oxygen diffusion. A wide range of carbon materials are studied as the carriers for laccase immobilization: carbon black and nanotubes with various BET specific surface areas. The conditions, which provide the highest surface coverage of carbon material with enzyme in the course of spontaneous adsorptive immobilization and the highest specific characteristics when using a “floating” electrode simulating a gas-diffusion electrode, are determined: 0.2 M phosphate-acetate buffer solution; oxygen atmosphere; the carrier material (nanotubes with a BET surface area of 210 m2/g and a mesopore volume of 3.8 cm3/g); and the composition of active mass on the electrode (50 wt % of carbon material + 50 wt % of hydrophobized carbon black).

Production of Laccase and Optimization of Its Production by Bacillus sp. Using Distillery Spent Wash as Inducer

ABSTRACT A bacillus sp. isolated from the sediments of a distillery mill was used for laccase production under optimized culture conditions. The distillery effluent was used as an inducer for overproduction of laccase by employing the Taguchi approach. Screening of different medium components and their effect on laccase production was studied using an M-16 orthogonal array. The formation of laccase was considerably increased by addition of 1 mM copper sulfate (51.95 U/ml), which was further enhanced by the use of different inducers. The usefulness of the Taguchi method for optimization of culture conditions was investigated with five selected factors at four levels, and it was observed that the optimized medium resulted in a 9-fold increase in extracellular laccase production compared with the control. The optimized medium composition for laccase production was dextrose (1%), tryptone (0.1%), CuSO4 (1 mM), and an inducer (distillery effluent 10% [v/v]) at pH 7, which altogether resulted in 107.32 U/ml extracellular laccase activity. Hence, the Taguchi approach proved to be a reliable tool in optimizing culture conditions and achieving the best possible combination for enhanced laccase production.

Optimization of Laccase Production from <i>Penicillium martensii </i>NRC 345

Laccase production by <i>Penicillium martensii </i>NRC 345 was optimized. Eight media with different components were screened. The enzyme formed by <i>P. martensii</i> NRC 345 was detected mainly exocellularly under shake culture condition. Laccase formation reaches its maximum value with specific activities of about 7.18 U/mg protein at the twenty-sixth day of incubation at pH 5.5 and 30°C. Galactose (5 g/l) and sodium nitrate (0.2 g/l) were found to be the best carbon and nitrogen sources for laccase formation, respectively. Replacement of galactose instead of glucose at the same concentration increased laccase production by about more than ten-fold. Among the various wastes used wheat bran induces the highest laccase production with specific activity of 39.52 U/mg protein. Tween-80 and CuSO<SUB>4</SUB>.5H<SUB>2</SUB>O, were also tested for laccase induction.

Optimization of Cultural and Nutritional Parameters for the Production of Laccase by Pleurotus ostreatus ARC280

Aims: To optimize laccase production by submerged fermentation using an edible mushroom Pleurotus ostreatus ARC280. Study Design: Laccase activity was assayed by monitoring the product formation rate of enzymatic oxidation of syringaldazine spectrophotometrically at 525 nm. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Cairo, Egypt, between May 2009 and October 2010. Methodology: Pleurotus ostreatus ARC280 was maintained on potato dextrose agar medium. The liquid medium used for the laccase production by the fungal culture during its growth in submerged fermentation was selected from eight liquid media for inducing laccase production. Parameters such as incubation period, temperature, pH of the production medium, carbon and nitrogen sources and other nutritional parameters were studied using syringaldazine as a model substrate for laccase activity determination. Results: In the present work, Eight media with different components were screened. The enzyme formed by Pl. ostreatus ARC280 was localized mainly in the extra-cellular fraction. Laccase formation reaches its maximum value with specific activity of about 140 U/mg protein at the twenty-sixth day of incubation, pH 5.0 and 28oC. Among the various wastes used, corn stover induces the highest laccase production with specific activity of Research Article British Biotechnology Journal, 2(3): 115-132, 2012 116 75.48 U/mg protein. Soluble starch at 1.5% (w/v) and ammonium sulfate was found to be the best carbon and nitrogen sources for laccase formation, respectively. The optimal concentrations of Tween-80 and CuSO4. 5H2O, were found to be 0.1% (v/v) and 100μM and cause enzyme induction by about 44% and 19% than control, respectively. Conclusion: Laccase production by Pl. ostreatus ARC280 has been shown to depend markedly on the composition of the culture medium, carbon, nitrogen content and inducer compounds and governed by parameters such as pH of the production medium and other nutrition parameters.

Increased production of extracellular laccase by the white rot fungus Coriolus versicolor MTCC 138

The white rot fungus Coriolus versicolor MTCC 138 has been identified as an excellent producer of the industrially important enzyme laccase. The basal medium containing glucose gave laccase activity of 155 U/ml. Screening of different media components and their effects on laccase production by Coriolus versicolor was studied using one factor at a time and L9 (34) orthogonal array method. A two-fold increase (305 U/ml) in laccase production was observed using a combination of glucose and starch as carbon source and yeast extract as nitrogen source. This activity is very high as compared to most of the reported strains. Hence this strain was explored for enhancement in laccase. The formation of extracellular laccase could be considerably stimulated by the addition of copper in the optimized medium. Addition of 1 mM copper enhanced laccase activity to 460 U/ml. Laccase production was further enhanced using different aromatic inducers. Among different inducers used 2,5-xylidine was found to be the best, and gave maximum laccase activity of 820 U/ml with 1 mM concentration. Thus, this strain could be an efficient and attractive source for laccase production.

Influence of culture conditions on laccase production and isozyme patterns in the white‐rot fungus Trametes gallica

Laccase production by the white-rot fungus Trametes gallica was studied, using twelve different media under static or shaking condition. The results indicated that organic nitrogen sources such as tryptone and peptone strongly improved laccase production. The application of an amino acid mixture and a lignin preparation also increased the formation of laccase, which was not observed in the presence of potato extract. Native polyacryl amide gel electrophoresis (PAGE) followed by laccase activity staining using guaiacol as the substrate was performed to analyze the laccase isozyme patterns under the different culture conditions employed. Zymograms revealed a total of twenty different laccase activity bands that appeared in individual patterns, dependent on the respective culture condition applied. This indicates that both the medium composition and the mode of incubation (static or shaking) influenced the laccase isozyme gene expression. This was the first time to report so many laccase isozymes in a fungus. Native PAGE with silver staining showed that laccases were the main protein productions in several media providing a potentially convenient way in purifying laccases from T. gallica.

Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens

The white-rot fungus Trametes pubescens MB 89 is an excellent producer of the industrially important enzyme laccase. Extracellular laccase formation can be considerably stimulated by the addition of Cu(II) in the millimolar range to a simple, glucose-based culture medium. When using glucose, a typically repressing substrate, as the main carbon source, significant laccase formation by T. pubescens only started when glucose was completely consumed from the culture medium. In addition, the nitrogen source employed had an important effect on laccase synthesis. When using an optimized medium containing glucose (40 g l−1), peptone from meat (10 g l−1), and MgSO4·7H2O and stimulating enzyme formation by the addition of 2.0 mM Cu, maximal laccase activities obtained in a batch cultivation were approximately 330 U ml−1. Under these conditions it was not necessary to add aromatic compounds that are routinely used as inducers of laccase in fungi. By applying a fed-batch strategy, in which a glucose solution was fed continuously to a cultivation of T. pubescens so that the glucose concentration in the medium never exceeded a certain low, critical value, glucose repression could be avoided and production of laccase was almost doubled as compared to the batch cultivation (740 U ml−1).

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

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 laccase could be considerably stimulated by the addition of Cu(II) to a simple, glycerol-based culture medium. In this study, optimal concentrations of copper were found to be 0.5-1 mM, which were added during the growth phase of the fungus. Other medium components important for laccase production are the carbon and nitrogen sources employed. When using an optimized medium containing glycerol (40 g/L), peptone from meat (15 g/L), and MgSO4 x 7H2O and stimulating enzyme formation by the addition of 1.0 mM Cu, maximal laccase activities obtained in shake-flask cultures were approx 85 U/mL. These results, however, could not be scaled up to a laboratory fermentor cultivation. Laccase production by T. multicolor decreased considerably when the fungus was grown in a stirred-tank reactor, presumably because of damage of the mycelia caused by shear stress and/or changes in the morphology of the fungus.

Enhanced formation of laccase activity by the white-rot fungus Trametes pubescens in the presence of copper.

The white-rot fungus Trametes pubescens MB 89 has been identified as an outstanding, although not-yet-described, producer of the industrially important enzyme laccase. Extracellular laccase formation could be greatly stimulated by the addition of Cu(II) to a simple, glucose-based culture medium. Using optimum Cu concentrations (1.5-2.0 mM), maximum values for laccase activity of approximately 65 U/ml were obtained. The synthesis of the laccase protein depended on the presence of Cu in the medium as shown by Western blot analysis. Copper had to be supplemented during the exponential phase of growth for its maximal effect; addition during the stationary phase, during which laccase activity is predominantly formed, resulted in markedly reduced laccase productivity. As was shown by X-ray microanalysis of T pubescens mycelia obtained from a laboratory fermentation, Cu was rapidly taken up by the fungal biomass. A possible explanation for this stimulatory effect of Cu on laccase biosynthesis could be a role for this enzyme activity in melanin synthesis. The stimulatory effect of Cu on laccase synthesis was also effective for several other basidiomycetes and hence could be used as a simple method to boost the production of this enzyme.

Evidence for rapid breakdown of hydrogen peroxide by Botrytis cinerea

Spores of Botrytis cinerea can germinate in the presence of up to 180 mM hydrogen peroxide and its mycelium can develop in the presence of even higher concentrations. The mycelium can breakdown hydrogen peroxide present in its culture medium rapidly in a time-dependent manner. This breakdown depends on the composition of the culture medium, is not inhibited by inhibitors of catalase activity, but is considerably reduced by inhibitors of peroxidase, such as benzhydroxamic acid, and by caffeic acid. In addition, growth in the presence of inducers of laccase formation appears to promote subsequent breakdown of hydrogen peroxide.

Evidence for rapid breakdown of hydrogen peroxide byBotrytis cinerea

Spores of Botrytis cinerea can germinate in the presence of up to 180 mM hydrogen peroxide and its mycelium can develop in the presence of even higher concentrations. The mycelium can breakdown hydrogen peroxide present in its culture medium rapidly in a time-dependent manner. This breakdown depends on the composition of the culture medium, is not inhibited by inhibitors of catalase activity, but is considerably reduced by inhibitors of peroxidase, such as benzhydroxamic acid, and by caffeic acid. In addition, growth in the presence of inducers of laccase formation appears to promote subsequent breakdown of hydrogen peroxide.

Effect of cucurbitacins on mRNA coding for laccase in Botrytis cinerea

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 for Botrytis β-tubulin mRNA. From an analysis of the results it is clear that cucurbitacin I and Ecballium extract specifically repress the amount of mRNA coding for laccase. This could account for the previously observed repression of laccase formation by cucurbitacins.

Induction of laccase production inCyathus bulleri under shaking and static culture conditions

Laccase production byCyathus bulleri was lower in lignins and phenolic compounds as compared to malt extract medium (8 U/mL) which increased significantly on supplementing these compounds with malt extract. Of the different lignins and phenolic compounds, Reax, lignin and orcinol exhibited maximum laccase formation (12 and 68 U/mL, respectively) under static culture conditions, while sugars repressed it. Laccase activity inC. bulleri was higher under static than under shaking cultivation conditions. Moreover, agitation repressed laccase formation even in the presence of inducers.

Selective mode of action of cucurbitacin in the inhibition of laccase formation in Botrytis cinerea

Polyclonal antibodies were raised against LAC1, one of two isoelectric forms of laccase, and the antibodies, which recognized both of the laccases, LAC1 and LAC2, were then used to demonstrate that both forms have the same molecular range, 72 0000 M r . A procedure was developed for labelling laccase at the stage of its secretion, into the culture medium, using radioactive leucine. Immuno-precipitation of the [ 14C]-labelled laccase showed that synthesis of the protein of laccase under inducing conditions was inhibited by cucurbitacins. The degree of inhibition of synthesis of the enzyme protein corresponded to the amount of reduction of laccase activity in the culture medium. Tritiated 2,16,25-cucurbitacin D triacetate was taken up rapidly by young mycelium and appears to be effective as an inhibitor of formation of laccase at micromolar concentrations at the site of action.

Induction of laccase formation in Botrytis and its inhibition by cucurbitacin: Is gallic acid the true inducer?

We demonstrate that it is possible to reduce the induction time for formation of extracellular laccase by Botrytis cinerea. This was achieved by preincubating inductive medium with mycelium, followed by inoculation with fresh mycelium. We suggest that the active inducer is a product of gallic acid metabolism. Its formation can be prevented by trapping the quinone formed from gallic acid, using benzenesulphinic acid. We also show that induction of laccase formation can be inhibited by pre-treatment of the mycelium with 2,16,25-cucurbitacin D triacetate, prior to transfer to inductive medium. The implications for control of laccase formation are discussed.

Dependence of activity of cucurbitacin in repression of Botrytis laccase on its structure

The activity of cucurbitacin D and I in repressing laccase formation by Botrytis cinerea, under inducing conditions was compared with some of their derivatives. The activity of cucurbitacin D and its 25-monoacetate were similar. The 2,16-cucurbitacin D diacetate and 2,16,25-cucurbitacin D triacetate were four and 16 times more effective than the parent compound, while the activity of the 2,16-valproate derivative of cucurbitacin D did not differ significantly from cucurbitacin D itself. The 2- O-β- d-glucopyranosyl-cucurbitacin I was inactive, but its hexaacetate had the same activity as cucurbitacin I. The difference in activity of the compounds may be related to their relative solubilities in cell membranes, which determine to what extent they penetrate to the site of their activity.

Botrytis cinereaによるラッカービの生成

Botrytis cinereaによるラッカービの生成

Cucurbitacins protect cucumber tissue against infection by Botrytis cinerea

Extracts of Ecballium elaterium or cucurbitacin I were applied to cucumber fruits or plants or to cabbage leaves, prior to inoculation with Botrytis cinerea. This treatment prevented infection of the tissue, the infecting fungus being restricted to the site of infection. Infection was always accompanied by localized lignification. The protective effect was not due to the induction of lignification. It is suggested that the ability of cucurbitacin I to inhibit induction of laccase formation by Botrytis is responsible for its protective effect.

Cucurbitacins-repressors of induction of laccase formation

It is demonstrated that some members of the Cucurbitacea contain compounds which inhibit the induction of laccase formation by Botrytis cinerea. The general properties of the inhibitory substances correspond to those of cucurbitacins. Cucurbitacin I and D inhibit induction of laccase formation as do the extracts of plant tissue. In extracts of Ecballium the inhibiting activity was tentatively identified as being due to the presence of cucurbitacins. The possible role of the cucurbitacins in plant tissue in relation to infection is discussed.