Phlebia radiata laccase forms induced by veratric acid and xylidine in relation to lignin peroxidase and manganese-dependent peroxidase

White-rot basidiomycete Phlebia radiata grown in the nitrogen-poor liquid medium was tested for the inducing effect of 2,5-xylidine and veratric acid on laccase production and properties of its preparations. Also lignin peroxidase and manganese-dependent peroxidase were assayed under the same conditions. The maximum of laccase activity for veratric acid as a stimulator was done much earlier than that of xylidine. It was very sharp and disappeared quickly. At that time only weak lignin peroxidase and manganese-dependent peroxidase activities were noticed. The maximum of laccase induced by xylidine was observed much later and kept longer. Both laccase preparations showed the same properties. For biotechnological reasons, the production of laccase induced by the nontoxic veratric acid is much more economic and better acceptable than that induced by xylidine.     

Production of laccase, manganese peroxidase and lignin peroxidase by Brazilian marine-derived fungi

Marine-derived fungi are a potential for the search of new compounds with relevant features. Among these, the ligninolytic enzymes have potential applications in a large number of fields, including the environmental and industrial sectors. This is the work aimed to evaluate the enzymatic activities of three marine-derived fungi (Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857 and Mucor racemosus CBMAI 847) under different carbon sources and salinity conditions by using statistical experimental design. MnP, LiP and laccase were detected when these fungi were cultured in malt extract, however when grown on basal medium containing glucose and wheat bran LiP was not detected and yet an increase in MnP and laccase was observed. Statistical analysis through surface responses was performed and results showed high values of MnP and laccase activities under 12.5% and 23% (w/v) salinity, highlighting the potential use of these fungi for industrial applications and in bioremediation of contaminated sites having high salt concentrations. The highest values for LiP (75376.34 UI L⁻¹), MnP (4484.30 IU L⁻¹) and laccase (898.15 UI L⁻¹) were obtained with the fungus M. racemosus CBMAI 847 and it is the first report concerning ligninolytic enzymes production by a zygomycete from this genus.     

Manganese and malonate are individual regulators for the production of lignin and manganese peroxidase isozymes and in the degradation of lignin by Phlebia radiata

 The effects of high manganese [180 μM Mn(II)] concentration and addition of malonate (10 mM) were studied in nitrogen-limited cultures of the white-rot fungus, Phlebia radiata. High levels of manganese alone showed no systematic influence on the production of lignin peroxidase (LiP), manganese peroxidase (MnP) or laccase. In contrast, high-manganese containing cultures of P. radiata showed lower efficiency in the mineralization of ¹⁴C-ring-labelled synthetic lignin ([¹⁴C]DHP). The highest rates of mineralization, up to 30% in 18 days, were reached in low- manganese(2 μM)-containing cultures when malonate was omitted. Degradation of [¹⁴C]DHP was substantially restricted by the addition of malonate. The combination of high manganese and malonate resulted in increased levels of MnP and laccase production, whereas LiP production was repressed. Also, the profiles of expression of the MnP and LiP isozymes were affected. A new P. radiata MnP isozyme of pI 3.6 (MnP3) was found in the high-manganese cultures. Addition of malonate alone caused some repression but also stimulating effects on distinctive MnP and LiP isozymes. The results indicate that manganese and malonate are individual regulators of MnP and LiP expression and have different roles in the degradation of lignin by P. radiata. Received: 30 August 1995/Received revision: 10 January 1996/Accepted: 12 February 1996     

Conversion of milled pine wood by manganese peroxidase from Phlebia radiata

Purified manganese peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tween 20, Mn(2+), Mn-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid residues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from P. radiata (MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37 degrees C with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to >6.5. An aromatic nonphenolic lignin structure (beta-O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols.     

Oxidation of 1-hydroxybenzotriazole by laccase and lignin peroxidase

A method to measure laccase and lignin peroxidase (LiP) activity at 408 nm (402–410 nm) using 1-hydroxybenzotriazole (HBT) was developed. The assay can be performed either as a kinetic measurement or as a stopped reaction using 5 mM Na-azide which improves the spectrum. Only white-rot fungal laccases and LiP were found to oxidize HBT to give shoulders or peaks at 402-410 nm. Phanerochaete and Phlebia manganese peroxidases did not give absorbance increase at 402–410 nm. © Rapid Science Ltd. 1998     

Mn-dependent peroxidase from the lignin-degrading white rot fungus Phlebia radiata

A homogeneous Mn-dependent peroxidase (MnP) was purified from the extracellular culture fluid of the lignin-degrading white rot fungus Phlebia radiata by anion exchange chromatography. The enzyme had a molecular weight of 49,000 and pI 3.8. It was a glycoprotein, containing carbohydrate moieties accounting for 10% of the molecular weight. Mn-peroxidase was capable of oxidizing phenolic compounds in the presence of H2O2, whereas the effect on nonphenolic lignin model compounds was insignificant. MnP contained protoporphyrin IX as a prosthetic group. During enzymatic reactions H2O2 converted the native MnP to compound II. Mn2+ was essential in completing the catalytic cycle by returning the enzyme to its native state. The oxidation of ultimate substrates was dependent on superoxide radicals, O2- and probably on Mn3+ generated during the catalytic cycle. MnP exhibited high activity of NADH oxidation without exogenously added H2O2. It was shown to produce H2O2 at the expense of NADH.     

Mineralization and solubilization of synthetic lignin by manganese peroxidases from Nematoloma frowardii and Phlebia radiata

Crude and purified manganese peroxidase from the white-rot fungi Nematoloma frowardii and Phlebia radiata catalyzed the partial depolymerization of a [¹⁴C-ring]labelled synthetic lignin into water-soluble fragments (30–50%). The in vitro depolymerization of the ¹⁴C-labelled lignin was accompanied by a release of ¹⁴CO₂ ranging from 4 to 6%. Small quantities of the thiol mediator glutathione stimulated the depolymerization of lignin resulting in a mineralization and solubilization of up to 10 and 64%, respectively. Most of the water-soluble substances formed had molecular masses around 0.7 kDa, although a higher-molecular mass fraction was also detectable (>2 kDa). Photometric assays using 2,2′-azinobis(3-ethylbenzothiazolinesulphonate) as an indicator demonstrated that high levels of Mn(III), which were very probably responsible for the depolymerization and mineralization of the ¹⁴C-labelled lignin, were adjusted within the first 24 h of incubation. The manganese peroxidase catalyzed depolymerization process was not necessarily dependent on H₂O₂; also in the absence of the H₂O₂-generating system glucose/glucose oxidase, effective solubilization and mineralization of lignin dehydrogenation polymerizate occurred, due to the in part superoxide dismutase sensitive, ‘oxidase-like’ activity of MnP which probably produces radical species and peroxides from malonate.     

Production of lignolytic and feed-back type enzymes by Phlebia radiata on different media

Low molecular-weight compounds, structurally related to lignin, increase the production of laccase, lignin peroxidase, manganese dependent peroxidase, and feed-back type enzymes such as glucose oxidase, cellobioso-quinone oxidoreductase, and glyoxal oxidase in the culture of the white rot fungus Phlebia radiata growing on different carbon sources.     

Transformation and mineralization of 2,4,6-trinitrotoluene (TNT) by manganese peroxidase from the white-rot basidiomycete Phlebia radiata

The degradation of the nitroaromatic pollutant 2,4,6-trinitrotoluene (TNT) by the manganese-dependent peroxidase (MnP) of the white-rot fungus Phlebia radiata and the main reduction products formed were investigated. In the presence of small amounts of reduced glutathione (10 mM), a concentrated cell-free preparation of MnP from P. radiata exhibiting an activity of 36 nkat/ml (36 nmol Mn(II) oxidized per sec and per ml) transformed 10 mg/l of TNT within three days. The same preparation was capable of completely transforming the reduced derivatives of TNT. When present at 10 mg/l, the aminodinitrotoluenes were transformed in less than two days and the diaminonitrotoluenes in less than three hours. Experiments with ¹⁴C-U-ring labeled TNT and 2-amino-4,6-dinitrotoluene showed that these compounds were mineralized by 22% and 76%, respectively, within 5 days. Higher concentrations of reduced glutathione (50 mM) led to a severe inhibition of the degradation process. It is concluded that Phlebia radiata is a good candidate for the biodegradation of TNT as well as its reduction metabolites.     

Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes.

Lignin peroxidase oxidizes non-phenolic substrates by one electron to give aryl-cation-radical intermediates, which react further to give a variety of products. The present study investigated the possibility that other peroxidative and oxidative enzymes known to catalyse one-electron oxidations may also oxidize non-phenolics to cation-radical intermediates and that this ability is related to the redox potential of the substrate. Lignin peroxidase from the fungus Phanerochaete chrysosporium, horseradish peroxidase (HRP) and laccase from the fungus Trametes versicolor were chosen for investigation with methoxybenzenes as a homologous series of substrates. The twelve methoxybenzene congeners have known half-wave potentials that differ by as much as approximately 1 V. Lignin peroxidase oxidized the ten with the lowest half-wave potentials, whereas HRP oxidized the four lowest and laccase oxidized only 1,2,4,5-tetramethoxybenzene, the lowest. E.s.r. spectroscopy showed that this congener is oxidized to its cation radical by all three enzymes. Oxidation in each case gave the same products: 2,5-dimethoxy-p-benzoquinone and 4,5-dimethoxy-o-benzoquinone, in a 4:1 ratio, plus 2 mol of methanol for each 1 mol of substrate. Using HRP-catalysed oxidation, we showed that the quinone oxygen atoms are derived from water. We conclude that the three enzymes affect their substrates similarly, and that whether an aromatic compound is a substrate depends in large part on its redox potential. Furthermore, oxidized lignin peroxidase is clearly a stronger oxidant than oxidized HRP or laccase. Determination of the enzyme kinetic parameters for the methoxybenzene oxidations demonstrated further differences among the enzymes.     

Production of Phanerochaete chrysosporium lignin peroxidase

Liginin peroxidase (ligninase) of the white rot fungus Phanerochaete chrysosporium Burdsall was discovered in 1982 as a secondary metabolite. Today multiple isoenzymes are known, which are often collectively called as lignin peroxidase. Lignin peroxidase has been characterized as a veratryl alcohol oxidizing enzyme, but it is a relatively unspecific enzyme catalyzing a variety of reactions with hydrogen peroxide as the electron acceptor. P. chrysosporium ligninases are heme glycoproteins. At least a number of isoenzymes are also phosphorylated. Two of the major isoenzymes have been crystallized. Until recently lignin peroxidase could only be produced in low yields in very small scale stationary cultures owing to shear sensitivity. Most strains produce the enzyme only after grown under nitrogen or carbon limitation, although strains producing lignin peroxidase under nutrient sufficiency have also been isolated. Activities over 2000 U dm(-3) (as determined at 30 degrees to 37 degrees C) have been reported in small scale Erlenmeyer cultures with the strain INA-12 grown on glycerol in the presence of soybean phospholipids under nitrogen sufficiency. In about 8 dm(3) liquid volume pilot scale higher than 100 U dm(-3) (as determined at 23 degrees C) have been obtained under agitation with immobilized P. chrysosporium strains ATCC 24725 or TKK 20512. Good results have been obtained for example with nylon web, polyurethane foam, sintered glass or silicon tubing as the carrier. The immobilized biocatalyst systems have also made large scale repeated batch and semicontinuous production possible. With nylon web as the carrier, lignin peroxidase production has recently been scaled up to 800 dm(3) liquid volume semicontinuous industrial production process.     

Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase

In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O₂ during laccase and tyrosinase treatment and of H₂O₂ during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20–120 min after the addition of enzyme).     

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

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.     

A lignin peroxidase-encoding cDNA from the white-rot fungus Phlebia radiata: characterization and expression in Trichoderma reesei

The nucleotide sequence of a cDNA coding for a lignin peroxidase (Lgp) of the white-rot fungus, Phlebia radiata, has been determined. By amino acid (aa) sequencing, it has been shown that the protein product of this gene is the LIII Lgp of Pb. radiata. The isolated gene and the putative aa sequence are about 60% homologous to published Lgp sequences from the fungus, Phanerochaete chrysosporium. The aa thought to be involved in the catalysis of LIII are revealed by comparison with the yeast cytochrome c peroxidase. The P. radiata Lgp-encoding gene (lgp3) was expressed in the fungus, Trichoderma reesei, under the cellobiohydrolase-encoding cbh1 gene promoter. Lgp3 mRNA was produced by the T. reesei transformants. No Lgp protein, however, could be detected.     

Co-immobilization of manganese peroxidase from Phlebia radiata and glucose oxidase from Aspergillus niger on porous silica beads

Manganese peroxidase (MnP) from Phlebia radiata and glucose oxidase from Aspergillus niger were co-immobilized on porous silica beads. Immobilization of both enzymes on the same carrier provided an integrated system in which H₂O₂ required by MnP was produced by glucose oxidase. The immobilization process resulted in a decrease of both enzymatic activities and substrate affinities. However, immobilization improved the stability of MnP against H₂O₂ or high pH, as well as the storage stability of this enzyme.     

Production of manganese peroxidase and laccase in laboratory-scale bioreactors by Phanerochaete chrysosporium

The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) in laboratory-scale bioreactors was studied. One bioreactor was filled with cubes of polyurethane foam and the other with cubes of nylon sponge, in order to determine the more suitable carrier to produce high ligninolytic enzyme activities by this fungus. Both cultivations were carried out in batch. Manganese-dependent peroxidase activities about 600 U l^у were achieved in the bioreactor filled with cubes of nylon sponge, while up to 500 U l^у were detected in that filled with cubes of polyurethane foam. Furthermore, quite high levels of laccase appeared in both cultures: maximum activities of 114 U l^у and 62 U l^у were obtained on nylon and polyurethane supports, respectively.     

The selective visualization of lignin peroxidase,manganese peroxidase and laccase,produced by white rot fungi on solid media

A visual method for the selective screening of lignin degrading enzymes, produced by white rot fungi (WRF), was investigated by the addition of coloring additives to solid media. Of the additives used in the enzyme production media, guaiacol and RBBR could be used for the detection of lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. Syringaldazine and Acid Red 264 were able for the detection of both the MnP and laccase, and the LiP and laccase, respectively, and a combination of these two additives was able to detect each of the ligninases produced by the WRF on solid media.     

Purification and properties of alpha-galactosidase isosymes from Phlebia radiata

Phlebia radiata formed extracellular alpha-galactosidase when it was grown in a culture containing wheat bran or locus bean gum as a carbon source. Their activities were optimal at pH 5.0, and demonstrated the highest level of activity at 60 degrees C. Highly purified isoforms of alpha-galactosidase (AGaS-m1, AGaS-m2, AGaS-m3) isolated from the media with galactomannan and (AGaS-b1, AGaS-b2, AGaS-b3) from the media with wheat bran were obtained by means of the column chromatography on Q-Sepharose and chromatofosussing on Polybuffer Exchanger PBE-94.