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.     

Differential regulation of manganese peroxidases and characterization of two variable MnP encoding genes in the white-rot fungus Physisporinus rivulosus

Manganese peroxidase (MnP) production in the white-rot basidiomycete Physisporinus rivulosus T241i was studied. Separate MnP isoforms were produced in carbon-limited liquid media supplemented with Mn²⁺, veratryl alcohol, or sawdust. The isoforms had different pH ranges for the oxidation of Mn²⁺ and 2,6-dimethoxyphenol. Although lignin degradation by white-rot fungi is often triggered by nitrogen depletion, MnPs of P. rivulosus were efficiently produced also in the presence of high-nutrient nitrogen, especially in cultures supplemented with veratryl alcohol. Two MnP encoding genes, mnpA and mnpB, were identified, and their corresponding cDNAs were characterized. Structurally, the genes showed marked dissimilarity, and the expression of the two genes implicated quantitative variation and differential regulation in response to manganese, veratryl alcohol, or sawdust. The variability in regulation and properties of the isoforms may widen the operating range for efficient lignin degradation by P. rivulosus.     

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.     

Effects of Mn2+ and NH+ 4 concentrations on laccase and manganese peroxidase production and Amaranth decoloration by Trametes versicolor

Extracellular lignin peroxidase (LiP) was not detected during decoloration of the azo dye, Amaranth, by Trametes versicolor. Approximately twice as much laccase and manganese peroxidase (MnP) was produced by decolorizing cultures compared to when no dye was added. At a low Mn²⁺ concentration (3 M), N-limited (1.2 mM NH₄ ⁺) cultures decolorized eight successive additions of Amaranth with no visible sorption to the mycelial biomass. At higher Mn²⁺ concentrations (200 M), production of MnP increased and that of laccase decreased, but the rate or number of successive Amaranth decolorations was unaffected. There was always a 6-h to 8-h lag prior to decoloration of the first aliquot of Amaranth, regardless of MnP and laccase concentrations. Although nitrogen-rich (12 mM NH₄ ⁺) cultures at an initial concentration of 200 M Mn²⁺ produced high laccase and MnP levels, only three additions of Amaranth were decolorized, and substantial mycelial sorption of the dye occurred. While the results did not preclude roles for MnP and laccase, extracellular MnP and laccase alone were insufficient for decoloration. The cell-free supernatant did not decolorize Amaranth, but the mycelial biomass separated from the whole broth and resuspended in fresh medium did. This indicates the involvement of a mycelial-bound, lignolytic enzyme or a H₂O₂-generating mechanism in the cell wall. Nitrogen limitation was required for the expression of this activity. Received: 19 May 1998 / Received revision: 22 October 1998 / Accepted: 7 November 1998     

Lignin and manganese peroxidase activity in extracts from straw solid substrate fermentations

Manganese and lignin peroxidase (MnP, LiP) activities were measured in straw extracts from cultures of Phanerochaete chrysosporium. Out of six MnP substrates, the MBTH/DMAB (3-methyl-2-benzothiazolinone hydrazone/3-(dimethylamino)benzoic acid), gave the highest MnP activity. Detection of LiP activity as veratryl alcohol oxidation was inhibited by phenols in the straw culture extracts. Appropriate levels of veratryl alcohol and peroxide (4 mM and 0.4 mM, respectively), and a restricted sample volume (not larger than 10%) were necessary to detect activity.     

Optimization of manganese peroxidase and laccase production in the South American fungus<Emphasis Type="Italic"> Fomes sclerodermeus</Emphasis> (Lév.) Cke

Fomes sclerodermeus produces manganese peroxidase (MnP) and laccase as part of its ligninolytic system. A Doehlert experimental design was applied in order to find the optimum conditions for MnP and laccase production. The factors studied were Cu²⁺, Mn²⁺ and asparagine. The present model and data analysis allowed us not only to define optimal media for production of both laccase and MnP, but also to show the combined effects between the factors. MnP was strongly influenced by Mn²⁺, which acts as an inducer. Under these conditions Cu²⁺ negatively affected MnP activity. At 13 days of growth 0.75 U ml^–1 were produced in the optimized culture medium supplemented with 1 mM MnSO₄ and 4 g l^–1 asparagine. The laccase titer under optimized conditions reached maximum values at 16 days of growth: 13.5 U ml^–1 in the presence of 0.2 mM CuSO₄, 0.4 mM MnSO₄ and 6 g l^–1 asparagine. Mn²⁺ promoted production of both enzymes. There were important interactions among the nutrients evaluated, the most significant being those between Cu²⁺ and asparagine.     

Response surface analysis for enzymatic decolorization of Congo red by manganese peroxidase

The enzymatic decolorization process of manganese peroxidase (MnP) is a complex system, which is greatly affected by the concentrations of H₂O₂, Mn²⁺, dye and enzyme. This work aimed to study these factors and investigate the combined interactions between them by applying response surface methodology (RSM) for decolorization of Congo red with MnP from Schizophyllum sp. F17, meanwhile conventional one-factor-at-a-time analysis was carried out. Through the one-factor-at-a-time analysis the optimized H₂O₂, Mn²⁺, Congo red and MnP extract was 0.2 mM, 0.5 mM, 50 mg/l and 0.8 ml, respectively, and the maximum decolorization attained under such conditions was 24.2%. Response surface analysis was conducted through Box–Behnken design and a second-order polynomial model (R² = 0.8565) was generated to describe the combined effect and the interactions quantificationally. ANOVA analysis indicated that the interactions between H₂O₂ and MnP, between dye and MnP were significant; the optimum condition through RSM was found to be 0.35 mM H₂O₂, 0.5 mM Mn²⁺, 75 mg/l Congo red and 1.4 ml MnP extract, for maximum decolorization of 30.8%.     

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.     

Gene cloning,heterologous expression,in vitro reconstitution and catalytic properties of a versatile peroxidase

The gene of a peroxidase described as being involved in carotenoid degradation was cloned from a strain that was conserved as Lepista irina (CBS 458.79). Gene sequencing revealed high nucleotide and amino-acid identity with Pleurotus eryngii gene vpl, which encodes a versatile peroxidase with unique catalytic properties, and only reported in Pleurotus and Bjerkandera species. Re-identification of the supposed L. irina strain revealed that, in fact, it is a P. eryngii strain. The new P. eryngii peroxidase was expressed in Escherichia coli, and the recombinant protein folded in the presence of cofactor to obtain the active form. The purified enzyme was able to oxidize Mn²⁺, veratryl alcohol, substituted phenols, and both low and high redox-potential dyes, demonstrating that it belongs to the versatile peroxidase family (named VPL3). These catalytic properties agreed with the presence of both Mn²⁺ and aromatic-substrate oxidation sites in its molecular structure.     

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.     

DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes

The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l⁻¹). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l⁻¹. Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as “dye-decolorizing peroxidases”. Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn²⁺) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases.     

Dynamics of enzymes generating hydrogen peroxide in solid-state fermentation ofPanus tigrinus on wheat straw

The relationship between the production of extracellular H₂O₂, hydrogen peroxide-producing enzymes and ligninolytic peroxidase was examined during solid-state cultivation ofPanus tigrinus on wheat straw. Glyoxal oxidase, Mn²⁺-dependent peroxidase and glucose oxidase, capable of H₂O₂ generation, were found in the extracellular enzyme preparation. The production of H₂O₂ has two maxima: the maximal production correlates well with the maximal activities of glyoxal oxidase and Mn²⁺-dependent peroxidase, while another, lower peak of H₂O₂ generation is related to the second peak of Mn²⁺-dependent peroxidase activity. The contribution of glucose oxidase to the production of hydrogen peroxide is probably only marginal. Comparison of the dynamics of these extracellular activities and the ligninolytic peroxidase showed good temporal correlation indicating an interrelation of the two processes.     

Purification and characterization of novel manganese peroxidase from <Emphasis Type="Italic">Rhizoctonia</Emphasis> sp. SYBC-M3

A novel manganese peroxidase of Rhizoctonia sp. SYBC-M3 (R-MnP) was purified by (NH₄)₂SO₄ fractionation, DEAE-cellulose-32 column chromatography, and Sephadex G100 column chromatography. The molecular mass of R-MnP was determined to be approximately 40.4 kDa by SDS-PAGE. The optimum temperature and pH for R-MnP were 55°C and 4.5, respectively. R-MnP was highly stability when the temperature was below 50°C. R-MnP could retain about 60% of its activity when the pH was between 4 and 6.5. However, R-MnP activity was inhibited by Fe³⁺, Cu²⁺, and Co³⁺ as well as increased by Zn²⁺ and Ca²⁺. R-MnP demonstrated oxidation of DMP, ABTS, veratryl alcohol, and guaiacol. The K _m values of RMnP for H₂O₂ and Mn²⁺ were 25.3 and 53.9 μmol/L, respectively.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

Glutathione-mediated mineralization of 14C-labeled 2-amino-4,6-dinitrotoluene by manganese-dependent peroxidase H5 from the white-rot fungus Phanerochaete chrysosporium

Manganese-dependent peroxidase (MnP) H5 from the white-rot fungus Phanerochaete chrysosporium, in the presence of either Mn(II) (10 mM) or GSH (10 mM), was able to mineralize ¹⁴C-U-ring-labeled 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) up to 29% in 12 days. When both Mn(II) and GSH were present, the mineralization extent reached 82%. On the other hand, no significant mineralization was observed in the absence of both Mn(II) and GSH, suggesting the requirement of a mediator [either Mn(II) or GSH] for the degradation of 2-A-4,6-DNT by MnP. Using electron spin resonance (ESR) techniques, it was found that the glutathionyl free radical (GS^•) was produced through the oxidation of GSH by MnP in the presence as well as in the absence of Mn(II). GS^• was also generated through the direct oxidation of GSH by Mn(III). Our results strongly suggest the involvement of GS^• in the GSH-mediated mineralization of 2-A-4,6-DNT by MnP. Received: 18 February 2000 / Received revision: 24 May 2000 / Accepted: 26 May 2000     

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.     

Hybrid Mn-peroxidases from basidiomycetes

The Mn-peroxidase from the fungus Panus tigrinus 8/18 is a hybrid enzyme. It catalyzes both Mn²⁺-dependent and Mn²⁺-independent oxidation of organic substrates. The spectral properties of intermediates and the pathway of the catalytic cycle are typical of hybrid Mn-peroxidases. The enzyme catalyzes the “oxidase” reaction (NADH oxidation) without peroxide and with the presence of Mn²⁺, which takes part in hydrogen peroxide production via Mn³⁺ and preserves the enzyme from inactivation. With the presence of organic mediators, the hybrid Mn-peroxidase oxidizes nonphenolic compounds: aromatic alcohols and a nonphenolic lignin model compound. The degree of conversion of 2,4,6-trichlorophenol is higher with the presence of 1-hydroxybenzotriazole.     

Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase

Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a Mn^II-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215–222, 1997; Gengenbach et al. in Biochemistry 38:11425–11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k _cat/K _M) than the variant in the original design, mostly due to a stronger K _M of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with Co^II at the designed Mn^II site were also obtained. The metal ion in the engineered metal-binding site overlays well with Mn^II bound in MnP, suggesting that this variant is the closest structural model of the Mn^II-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal–ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (Co^II rather than Mn^II) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.