Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites

Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP.     

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.     

Effects of temperature on the production of manganese peroxidase and lignin peroxidase byPhanerochaete chrysosporium

Growth temperature played an important role in the appearance, maximum level and ratio of manganese peroxidase (MnP) and lignin peroxidase (LIP) activities in the cultures ofPhanerochaete chrysosporium. While at higher temperatures (39, 33, and 28°C) both enzymes were produced (with LIP being the major one) at 23°C MnP was dominant. At 18°C, of the two ligninolytic peroxidases only MnP activity was detected. Decrease of proteolytic activity at lower temperatures probably contributed to the retention of MnP and LIP activities.     

The relationship between lignin peroxidase and manganese peroxidase production capacities and cultivation periods of mushrooms

Mushrooms are able to secrete lignin peroxidase (LiP) and manganese peroxidase (MnP), and able to use the cellulose as sources of carbon. This article focuses on the relation between peroxidase-secreting capacity and cultivation period of mushrooms with non-laccase activity. Methylene blue and methyl catechol qualitative assay and spectrophotometry quantitative assay show LiP secreting unvaryingly accompanies the MnP secreting in mushroom strains. The growth rates of hyphae are detected by detecting the dry hyphal mass. We link the peroxidase activities to growth rate of mushrooms and then probe into the relationship between them. The results show that there are close relationships between LiP- and/or MnP-secretory capacities and the cultivation periods of mushrooms. The strains with high LiP and MnP activities have short cultivation periods. However, those strains have long cultivation periods because of the low levels of secreted LiP and/or MnP, even no detectable LiP and/or MnP activity. This study provides the first evidence on the imitate relation between the level of secreted LiP and MnP activities and cultivation periods of mushrooms with non-laccase activity. Our study has significantly increased the understanding of the role of LiP and MnP in the growth and development of mushrooms with non-laccase activity.     

Substrate specificity of peroxidase isoenzymes for hydrogen donors

The investigation of the substrate specificity of the anionic peroxidase isoenzymes, isolated from the zone of differentiation of the primary roots ofZea mays, for some representatives of phenolic compounds and aromatic amines, as hydrogen donors, is reported. The investigation was carried out electrophoretically with peroxidase isoenzymes partially purified by a combination of gel filtration by Sephadex G-25 and Sephadex G-100. A difference in the substrate specificity of the individual isoenzymes is observed. It was established that the anionic peroxidase isoenzymes showed a similarity in total number and relative activity on staining with bivalent phenols and difference on staining with trivalent phenols, as hydrogen donors. A greater number of isoenzymes was stained with benzidine ando-dianisidine and a lesser number witho- andp-phenylendiamine. The substrate specificity of the peroxidase isoenzymes was compared for guaiacol and benzidine. The substrate specificity of peroxidase soenzymes was discussed as regards their diverse role in the plant metabolism.     

Substrate specificity of african oil palm tree peroxidase

The optimal conditions for catalysis by the peroxidase isolated from leaves of African oil palm tree (AOPTP) have been determined. The pH optimum for oxidation of the majority of substrates studied in the presence of AOPTP is in the interval of 4.5-5.5. A feature of AOPTP is low pH value (3.0) at which the peroxidase shows its maximal activity toward 2,2"-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS). Increasing the buffer concentration changes the AOPTP activity, the degree of the effect depending upon the chemical structure of the substrate. Under optimal conditions of AOPTP catalysis, the values of second order rate constant characterizing efficiency of enzymatic oxidation of substrates have been calculated. It was shown that among 12 peroxidase substrates studied, ABTS and ferulic acid are the best substrates for AOPTP. The results show that substrate specificities of AOPTP and royal palm tree peroxidase are similar, but different from substrate specificity of other plant peroxidases.     

Degradation of synthetic lignin by the protoplasts of Phanerochaete chrysosporium in the presence of lignin peroxidase or manganese peroxidase

Lignin was mineralized in the experiments in which ¹⁴C-lignin was incubated with lignin peroxidase or manganese peroxidase in a tartrate buffer in the presence of cycloheximide-treated protoplasts obtained from the ligninolytic mycelia of Phanerochaete chrysosporium. The rate of lignin mineralization was dependent on the lignin peroxidase or manganese peroxidase concentration in the medium. In the experiments in which lignin was incubated with lignin peroxidase or manganese peroxidase, lignin was repolymerized irrespective of the presence of protoplasts mineralizing lignin, suggesting that an active degradation of lignin and repolymerization took place. Taking into account that lignin peroxidase and manganese peroxidase were the only extracellular enzymes in the experiments in which lignin was mineralized by the protoplasts, it is postulated that lignin peroxidase and/or manganese peroxidase can degrade lignin into small fragments which can then be further absorbed by the fungal cells and subsequently degraded to CO₂.     

In vitro depolymerization of lignin by manganese peroxidase of Phanerochaete chrysosporium

Homogeneous manganese peroxidase catalyzed the in vitro partial depolymerization of four different 14C-labeled synthetic lignin preparations. Gel permeation profiles demonstrated significant depolymerization of 14C-sidechain-labeled syringyl lignin, a 14C-sidechain-labeled syringyl-guaiacyl copolymer (angiosperm lignin), and depolymerization of 14C-sidechain- and 14C-ring-labeled guaiacyl lignins (gymnosperm lignin). 3,5-Dimethoxy-1,4-benzo-quinone, 3,5-dimethoxy-1,4-hydroquinone, and syringylaldehyde were identified as degradation products of the syringyl and syringyl-guaiacyl lignins. These results suggest that manganese peroxidase plays a significant role in the depolymerization of lignin by Phanerochaete chrysosporium.     

Activation of lignin and manganese peroxidase excretion in Phanerochaete chrysosporium by fungal extracts

Summary Lignin (LiP) and manganese peroxidase (MnP) excretion by Phanerochaete chrysosporium INA-12 was significantly increased in response to fungal extract supplementation. LiP and MnP production was increased 1.7- and 1.8-fold, respectively, with fungal extracts from agitated pellet cultures of strain INA-12, namely fungal extracts P₆ and P₄. In cultures supplemented with a fungal extract harvested from static cultures of strain INA-12 (fungal extract S₄), LiP and MnP production was increased 1.8- and 1.6-fold, respectively. Succinate dehydrogenase activity, a mitochondrial marker, was significantly enhanced (2.7-fold) in cultures with the addition of fungal extracts. Correspondence to: M. Asther     

Nitrogen regulation of lignin peroxidase and manganese-dependent peroxidase production is independent of carbon and manganese regulation in Phanerochaete chrysosporium

In this study, a N-deregulated mutant (der8-5) of Phanerochaete chrysosporium was used as a tool to investigate the interrelationships between N, C, and Mn(II) regulation of LIP and MNP production in this organism. The results showed that LIP and MNP production by der8-5 was blocked in excess C medium but not in excess N medium. Furthermore, LIP and MNP production in this organism was subject to Mn(II) regulation regardless of the fact whether it is grown in low N medium or in high N medium. These and other results indicate that N regulation of LIP and MNP production in P. chrysosporium is independent of C and Mn(II) regulation.Abbreviations LIP lignin peroxidase - MNP manganese-dependent peroxidase - WT wild-type - der8-5 nitrogen-deregulated mutant     

Substrate specificity of rat sera IgG antibodies with peroxidase and oxidoreductase activities

We have recently shown that intact IgGs from the sera of healthy Wistar rats oxidize 3,3'-diaminobenzidine (DAB) in the presence and in the absence of H(2)O(2) similar to horseradish peroxidase (HRP). Here we demonstrate for the first time that the peroxidase and oxidoreductase activities of IgGs can efficiently oxidize not only DAB but also o-phenylendiamine, phenol, p-dihydroquinone, alpha-naphthol, and NADH but, in contrast to HRP, cannot oxidize adrenalin. In contrast to IgGs, HRP cannot oxidize phenol, p-dihydroquinone, or alpha-naphthol in the absence of H(2)O(2). In contrast to plant and mammalian peroxidases, IgGs were more universal in their metal dependence. The specific wide repertoire of polyclonal peroxidase and oxidoreductase IgGs oxidizing various substances could play an important role in protecting the organism from oxidative stress and serve as an additional natural system destroying different toxic, carcinogenic, and mutagenic compounds.     

Effect of carbon and nitrogen limitation on lignin peroxidase and manganese peroxidaseproduction by Phanerochaete flavido-alba

The ligninolytic enzymes lignin peroxidase (LiP) and manganese dependent peroxidase(MnP), were detected in extracellular fluids of Phanerochaete flavido-alba FPL 106507cultures under carbon or nitrogen limitation. MnP activities were found to be higher than LiPactivities under all growth conditions tested. Higher titres of both peroxidases were obtainedunder carbon limitation in excess nitrogen. Isoelectric points (pIs) observed after FPLC and IEFof concentrated extracellular fluids revealed more acidic pIs for LiP enzymes obtained innitrogen-limited cultures than those in carbon-limited cultures. However, the change in thelimiting growth factor does not significantly affect MnP pIs.     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.     

Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase

The present study maps the active site of lignin peroxidase in respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant lignin peroxidases. A nonphenolic tetrameric lignin model was synthesized that contains beta-O-4 linkages. The fungal and recombinant wild type lignin peroxidase both oxidized the tetrameric model forming four products. The four products were identified by mass spectral analyses and compared with synthetic standards. They were identified as tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. All four of these products were also formed from single turnover experiments. This indicates that lignin peroxidase is able to attack any of the C(alpha)-C(beta) linkages in the tetrameric compound and that the substrate-binding site is well exposed. Mutation of the recombinant lignin peroxidase (isozyme H8) in the heme access channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidation of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidation of the tetrameric model. These results are consistent with lignin peroxidase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators.     

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.     

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     

木材白腐菌分解木质素的酶系统-锰过氧化物酶、漆酶和木质素过氧化物酶催化分解木质素的机制

<正>近年来许多研究者进行了木材白腐菌分解木质素的酶系统对木质素的催化分解机制的研究。木材白腐菌在分解木质素的过程中会产生分解木质素的酶系统,氧化与分解木质素,这些酶系统主要包括细胞外过氧化物酶(锰过氧化物酶-MnP、木质素过氧化物酶-LiP)和细胞外酚氧化酶-漆酶(laccase)。在降解