Mineralization of 14C-Ring-Labeled Synthetic Lignin Correlates with the Production of Lignin Peroxidase, not of Manganese Peroxidase or Laccase

Recently, Mn(II) has been shown to induce manganese peroxidases (MnPs) and repress lignin peroxidases (LiPs) in defined liquid cultures of several white rot organisms. The present work shows that laccase is also regulated by Mn(II). We therefore used Mn(II) to regulate production of LiP, MnP, and laccase activities while determining the effects of Mn(II) on mineralization of ring-labeled synthetic lignin. At a low Mn(II) level, Phanerochaete chrysosporium and Phlebia brevispora produced relatively high titers of LiPs but only low titers of MnPs. At a high Mn(II) level, MnP titers increased 12- to 20-fold, but LiPs were not detected in crude broths. P. brevispora formed much less LiP than P. chrysosporium, but it also produced laccase activity that increased more than sevenfold at the high Mn(II) level. The rates of synthetic lignin mineralization by these organisms were similar and were almost seven times higher at low than at high Mn(II). Increased synthetic lignin mineralization therefore correlated with increased LiP, not with increased MnP or laccase activities.     

Mineralization of C-Ring-Labeled Synthetic Lignin Correlates with the Production of Lignin Peroxidase, not of Manganese Peroxidase or Laccase

Recently, Mn(II) has been shown to induce manganese peroxidases (MnPs) and repress lignin peroxidases (LiPs) in defined liquid cultures of several white rot organisms. The present work shows that laccase is also regulated by Mn(II). We therefore used Mn(II) to regulate production of LiP, MnP, and laccase activities while determining the effects of Mn(II) on mineralization of ring-labeled synthetic lignin. At a low Mn(II) level, Phanerochaete chrysosporium and Phlebia brevispora produced relatively high titers of LiPs but only low titers of MnPs. At a high Mn(II) level, MnP titers increased 12- to 20-fold, but LiPs were not detected in crude broths. P. brevispora formed much less LiP than P. chrysosporium, but it also produced laccase activity that increased more than sevenfold at the high Mn(II) level. The rates of synthetic lignin mineralization by these organisms were similar and were almost seven times higher at low than at high Mn(II). Increased synthetic lignin mineralization therefore correlated with increased LiP, not with increased MnP or laccase activities.     

Transformation of Industrial Dyes by Manganese Peroxidases from Bjerkandera adusta and Pleurotus eryngii in a Manganese-Independent Reaction

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn³⁺-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn²⁺. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn²⁺-independent manner. The reactions with the dyes were characterized by apparent K_m values ranging from 4 to 16 μM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn²⁺, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn³⁺-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1.     

Secretion of Ligninolytic Enzymes and Mineralization of 14C-Ring-Labelled Synthetic Lignin by Three Phlebia tremellosa Strains

Production of ligninolytic enzymes by three strains of the white rot fungus Phlebia tremellosa (syn. Merulius tremellosus) was studied in bioreactor cultivation under nitrogen-limiting conditions. The Mn(II) concentration of the growth medium strongly affected the secretion patterns of lignin peroxidase and laccase. Two major lignin peroxidase isoenzymes were expressed in all strains. In addition, laccase and glyoxal oxidase were purified and characterized in one strain of P. tremellosa. In contrast, manganese peroxidase was not found in fast protein liquid chromatography profiles of extracellular proteins under either low (2.4 μM) or elevated (24 and 120 μM) Mn(II) concentrations. However, H₂O₂- and Mn-dependent phenol red-oxidizing activity was detected in cultures supplemented with higher Mn(II) levels. Mineralization rates of ¹⁴C-ring-labelled synthetic lignin (i.e., dehydrogenation polymerizate) by all strains under a low basal Mn(II) level were similar to those obtained for Phanerochaete chrysosporium and Phlebia radiata. A high manganese concentration repressed the evolution of ¹⁴CO₂ even when a chelating agent, sodium malonate, was included in the medium.     

Lignin-Modifying Enzymes of the White Rot Basidiomycete Ganoderma lucidum

Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (M_r of 40,000 and 66,000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity (~100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.     

Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta

A peroxidase oxidizing Mn²⁺ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysosporium. However, the interest of the MnP described here is related to its ability to catalyze Mn²⁺-mediated as well as Mn²⁺-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.     

>Decolourisation and depolymerisation of solubilised low-rank coal by the white-rot basidiomycete Phanerochaete chrysosporium

Peroxidases secreted by the white-rot basidiomycete Phanerochaete chrysosporium can oxidise a wide range of recalcitrant compounds including lignin and aromatic xenobiotics. Since low-rank coals such as brown coal and lignite retain structural features of the parent lignin, we investigated the possibility that P. chrysosporium is capable of acting on a brown coal, with the production of useful low-molecular-mass compounds. In nitrogen-limiting liquid medium containing 0.03% solubilised Morwell brown coal, P. chrysosporium was found to convert about 85% of the coal after 16 days incubation to a form not recoverable by alkali-washing and acid-precipitation. The modal molecular mass of the residual coal macromolecules was reduced from the initial 65kDa to 32 kDa. Extensive bleaching of the coal coincided with the presence of extracellular lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP), although both LiP and MnP activity were lower in cultures containing coal. These reductions are accounted for by interference with the enzyme assays by solubilised coal and by binding of MnP to precipitated coal. LiP was about eight times more sensitive than MnP to inhibition by solubilised coal. In nitrogen-sufficient medium containing solubilised coal, neither coal modification nor LiP activity were observed, suggesting that LiP is an essential component of the bleaching process.     

Color removal ability of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in relation to lignin peroxidase and manganese peroxidase produced in molasses wastewater

Decolorization of molasses wastewater (MWW) from an ethanolic fermentation plant by Phanerochaete chrysosporium was studied. By diluting MWW properly (10%v/v) and incubating it with an appropriate concentration of the spores (2.5 × 10⁶/ml), extensive decolorization occurred (75%) on day 5 of the incubation. The colour removal ability was found to be correlated to the activity of ligninolytic enzyme system: lignin peroxidase (LiP) activity was 185 U/l while manganese peroxidase (MnP) activity equaled 25 U/l. Effects of some selected operating variables were studied: manganese(II), veratryl alcohol (VA), glucose as a carbon source and urea and ammonium nitrate, each as a source of nitrogen. Results showed that the colour reduction and LiP activity were highest (76% and 186 U/l, respectively) either when no Mn(II) was added or added at the lowest level tested (0.16 mg/l to provide 0.3 mg/l). Activity of MnP was highest (25 U/l) when Mn(II) added to the diluted MWW at the highest level (100 ppm) while activity of LiP was lowest (7.1 U/l) at this level of added Mn(II). The colour reduction in the presence of the added VA was shown to be little less than in its absence (70 vs. 75%). When urea as an organic source of nitrogen for the fungus, was added to the MWW, the decolorizing activity of P. chrysosporium decreased significantly (15 vs. 75%) and no activities were detected for LiP and MnP. Use of ammonium nitrate as an inorganic source of nitrogen did not show such a decelerating effects, although no improvements in the metabolic behavior of the fungus (i.e., LiP and MnP activities) deaccelerating was observed. Effects of addition of glucose was also discussed.     

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l^–1 and 356 U l^–1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l^–1 min^–1 using the extracellular culture filtrate.     

Degradation of the herbicide bentazon as related to enzyme production by Phanerochaete chrysosporium in two solid substrate fermentation systems

Enzyme production and degradation of the herbicide bentazon by Phanerochaete chrysosporium growing on straw (solid substrate fermentation, SSF) and the effect of nitrogen and the hydraulic retention time (HRT) were studied using a small bioreactor and batch cultures. The best degradation of bentazon was obtained in the low nitrogen treatments, indicating participation of the ligninolytic system of the fungus. The treatments that degraded bentazon also had manganese peroxidase (MnP) activity, which seemed to be necessary for degradation. Pure MnP (with Mn(II) and H₂O₂) did not oxidize bentazon. However, in the presence of MnP, Mn(II) and Tween 80, bentazon was slowly oxidized in a H₂O₂-independent reaction. Bentazon was a substrate of pure lignin peroxidase (LiP) and was oxidized significantly faster (22,000–29,000 times) as compared to the MnP-Tween 80 system. Although LiP was a better enzyme for bentazon oxidation in vitro, its role in the SSF systems remains unclear since it was detected only in treatments with high nitrogen and high HRT where no degradation of bentazon occurred. Inhibition of LiP activity may be due to phenols and extractives present in the straw.     

Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw

The white rot fungus Phlebia radiata 79 (ATCC 64658) produces lignin peroxidase (LiP), manganese peroxidase (MnP), glyoxal oxidase (GLOX), and laccase in the commonly used glucose low-nitrogen liquid medium. However, the enzymes which this fungus utilizes for selective removal of lignin during degradation of different lignocellulosic substrates have not been studied before. Multiple forms of LiP, MnP, GLOX, and laccase were purified from P. radiata culture extracts obtained after solid-state fermentation of wheat straw. However, the patterns of extracellular lignin-modifying enzymes studied were different from those of the enzymes usually found in liquid cultures of P. radiata. Three LiP isoforms were purified. The major LiP isoform from solid-state cultivation was LiP2. LiP3, which has usually been described as the major isoenzyme in liquid cultures, was not expressed during straw fermentation. New MnP isoforms have been detected in addition to the previously reported MnPs. GLOX was secreted in rather high amounts simultaneously with LiP during the first 2 weeks of growth. GLOX purified from P. radiata showed multiple forms, with pIs ranging from 4.0 to 4.6 and with a molecular mass of ca. 68 kDa.     

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     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.     

The effect of manganese on the production of Phanerochaete flavido-alba ligninolytic peroxidases in nitrogen limited cultures

Manganese supplementation of culture medium affected Phanerochaete flavido-alba FPL 106507 growth, glucose consumption and extracellular protein accumulation. Both the titre and time of detection of lignin peroxidase (LiP) were affected by manganese concentration in the medium, whereas with manganese peroxidase (MnP) only the titre was affected. In high Mn(II) containing cultures highest manganese peroxidase levels and a decrease in extracellular veratryl alcohol accumulation were observed. After FPLC a number of haemprotein peaks showing manganese peroxidase activity were detected in Mn(II) supplemented cultures. On the contrary, only haemprotein peaks of lignin peroxidase were detected in culture medium not supplemented with Mn(II).     

Amelioration of ligninolytic enzyme production by Phanerochaete chrysosporium in airlift bioreactors

Maximum activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) in free cultures of Phanerochaete chrysosporium (ATCC 24725) were 258 U l^–1 and 103 U l^–1, respectively, in an airlift bioreactor. Immobilisation of the fungus on an inert carrier as well as several design modifications of the bioreactor employed gave MnP activities around 500–600 U l^–1 during 9 days' operation. The continuous operation of the latter led to MnP and LiP activities about 140 U l^–1 and 100 U l^–1, respectively, for two months, without operational problems. Furthermore, the extracellular liquid secreted decolourised the polymeric dye Poly R-478 about 56%.     

Purification and biochemical characterization of two extracellular peroxidases from Phanerochaete chrysosporium responsible for lignin biodegradation

Two extracellular peroxidases from Phanerochaete chrysosporium, namely a lignin peroxidase (LiP) and manganese peroxidase (MnP), were purified simultaneously by applying successively, ultrafiltration, ion-exchange and gel filtration chromatography. LiP and MnP have a molecular mass of 36 and 45 kDa, respectively. The optimal pHs for LiP and MnP activities were 3.0 and 4.5, respectively. Both peroxidases showed maximal activity at 30 °C and moderate thermostability. MnP activity was strongly inhibited by Fe²⁺, Zn²⁺, Mg²⁺ and Hg²⁺, and enhanced by Mn²⁺, Ca²⁺ and Cu²⁺. LiP activity was enhanced by Ca²⁺, Na⁺ and Co²⁺ and it was inhibited in the presence of K⁺, Hg⁺, Fe²⁺, Mg²⁺ and high concentrations of Cu²⁺ and Zn²⁺. The K_m and V_max for LiP toward veratryl alcohol as a substrate were 0.10 mM and 15.2 U mg⁻¹, respectively and for MnP toward Mn²⁺, they were respectively 0.03 mM and 25.5 U mg⁻¹. The two peroxidases were also able to break down rice lignin in a small-scale solid state treatment system. Data suggest these two peroxidases may be considered as potential candidates for the development of enzyme-based technologies for lignin degradation.     

Removal of Aroclor 1254 by the white rot fungus Coriolus versicolor in the presence of different concentrations of Mn(IV)oxide

Lignin peroxidase (LiP) plays an active role in the biodegradation of lignin and phenolic structures resembling lignin. The role of other enzymes in the biodegradation of recalcitrant compounds, e.g. manganese(II)-peroxidase, is uncertain. Solid manganese(IV)oxide addition improved the production of manganese(II)-dependant peroxidase (MnP) and H₂O₂ and increased the rate of biodegradation of Aroclor 1254 in a nitrogen-limited medium by the white rot fungus Coriolus versicolor. MnP activity was detected 48 h after the addition of MnO₂ to the cultures and was absent in cultures that did not receive MnO₂. The rate of Aroclor 1254 removal by C. versicolor was influenced by the concentration of MnO₂. 34.5 mM concentrations only increased the H₂O₂ production. Removal of Aroclor 1254 in the absence of MnO₂ still took place which implied the presence of (LiP) or nonspecific absorption. The cultures containing 57.5 mM MnO₂ removed ca. 84% of the initial 750 mg l⁻¹ Aroclor in 6 days of incubation. Cultures with no MnO₂ and 34.5 mM removed 79 and 76%, respectively. Cultures with MnP or LiP as the dominant enzyme species removed penta- and hexachlorobiphenyls at a slower rate than tri- and tetrachlorobiphenyl.     

Lipid Peroxidation by the Manganese Peroxidase of Phanerochaete chrysosporium Is the Basis for Phenanthrene Oxidation by the Intact Fungus

The manganese peroxidase (MnP) of Phanerochaete chrysosporium supported Mn(II)-dependent, H₂O₂-independent lipid peroxidation, as shown by two findings: linolenic acid was peroxidized to give products that reacted with thiobarbituric acid, and linoleic acid was peroxidized to give hexanal. MnP also supported the slow oxidation of phenanthrene to 2,2′-diphenic acid in a reaction that required Mn(II), oxygen, and unsaturated lipids. Phenanthrene oxidation to diphenic acid by intact cultures of P. chrysosporium occurred to the same extent that oxidation in vitro did and was stimulated by Mn. These results support a role for MnP-mediated lipid peroxidation in phenanthrene oxidation by P. chrysosporium.