Degradation of bisphenol A by the lignin-degrading enzyme, manganese peroxidase, produced by the white-rot basidiomycete, Pleurotus ostreatus

Degradation of 2,2-Bis(4-hydroxyphenyl)propane (bisphenol A, BPA), an endocrine-disturbing chemical, by the growing mycelia of the white-rot basidiomycete, Pleurotus ostreatus, was examined. About 80% of BPA initially present decreased in 12 days of culture with this fungus. By in vitro experiments using the lignin-degrading enzyme manganese peroxidase (MnP), BPA was metabolized to phenol, 4-isopropenylphenol, 4-isopropylphenol, and hexestrol. The degradation products of BPA were assumed to be formed by the one-electron oxidation of the substrate.     

Depolymerisation of lignosulfonate by peroxidase of the white-rot basidiomycete, Pleurotus ostreatus

In the presence of a H₂O₂-generating system, the peroxidase of Pleurotus ostreatus that decolorise Remazol Brilliant Blue R catalysed the partial depolymerisation of lignosulfonate. The UV spectrum of the lignosulfonate changed with time with the absorbance at 280 nm increasing gradually. Gel permeation profile showed that the pattern of molecular weight distribution was changed to a lower molecular weight region. Oxidation products were identified as 2,6-dimethoxy-1,4-benzoquinone (I), benzoic acid (II), butyl phthalate (III), and bis (2-ethylhexyl) phthalate (IV) by GC/MS procedures.     

Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus.

The gene (pox1) encoding a phenol oxidase from Pleurotus ostreatus, a lignin-degrading basidiomycete, was cloned and sequenced, and the corresponding pox1 cDNA was also synthesized and sequenced. The isolated gene consists of 2,592 bp, with the coding sequence being interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA consensus sequences could be identified at positions -174 and -84, respectively. The isolation of a second cDNA (pox2 cDNA), showing 84% similarity, and of the corresponding truncated genomic clones demonstrated the existence of a multigene family coding for isoforms of laccase in P. ostreatus. PCR amplifications of specific regions on the DNA of isolated monokaryons proved that the two genes are not allelic forms. The POX1 amino acid sequence deduced was compared with those of other known laccases from different fungi.     

Spectral characterization of manganese peroxidase, an extracellular heme enzyme from the lignin-degrading basidiomycete, Phanerochaete chrysosporium

Manganese peroxidase (MnP) is a component of the lignin degradation system of the basidiomycetous fungus, Phanerochaete chrysosporium. This novel MnII-dependent extracellular enzyme (Mr = 46,000) contains a single protoporphyrin IX prosthetic group and oxidizes phenolic lignin model compounds as well as a variety of other substrates. To elucidate the heme environment of this enzyme, we have studied its electron paramagnetic resonance and resonance Raman spectroscopic properties. These studies indicate that the native enzyme is predominantly in the high-spin ferric form and has a histidine as fifth ligand. The reduced enzyme has a high-spin, pentacoordinate ferrous heme. Fluoride and cyanide readily bind to the sixth coordination position of the heme iron in the native form, thereby changing MnP into a typical high-spin, hexacoordinate fluoro adduct or a low-spin, hexacoordinate cyano adduct, respectively. EPR spectra of 14NO- and 15NO-adducts of ferrous MnP were compared with those of horseradish peroxidase (HRP); the presence of a proximal histidine ligand was confirmed from the pattern of superhyperfine splittings of the NO signals centered at g approximately equal to 2.005. The appearance of the FeII-His stretch at approximately 240 cm-1 and its apparent lack of deuterium sensitivity suggest that the N delta proton of the proximal histidine of the enzyme is more strongly hydrogen bonded than that of oxygen carrier globins and that this imidazole ligand may be described as having a comparatively strong anionic character. Although resonance Raman frequencies for the spin- and coordination-state marker bands of native MnP, nu 3 (1487), nu 19 (1565), and nu 10 (1622 cm-1), do not fall into frequency regions expected for typical penta- or hexacoordinate high-spin ferric heme complexes, ligation of fluoride produces frequency shifts of these bands very similar to those observed for cytochrome c peroxidase and HRP. Hence, these data strongly suggest that the iron in native MnP is predominantly high-spin pentacoordinate. Analysis of the Raman frequencies indicates that the dx2-y2 orbital of the native enzyme is at higher energy than that of metmyoglobin. These features of the heme in MnP must be favorable for the peroxidase catalytic mechanism involving oxidation of the heme iron to FeIV. Consequently, it is most likely that the heme environment of MnP resembles those of HRP, cytochrome c peroxidase, and lignin peroxidase.     

Genetic linkage map of the edible basidiomycete Pleurotus ostreatus

We have constructed a genetic linkage map of the edible basidiomycete Pleurotus ostreatus (var. Florida). The map is based on the segregation of 178 random amplified polymorphic DNA and 23 restriction fragment length polymorphism markers; four hydrophobin, two laccase, and two manganese peroxidase genes; both mating type loci; one isozyme locus (est1); the rRNA gene sequence; and a repetitive DNA sequence in a population of 80 sibling monokaryons. The map identifies 11 linkage groups corresponding to the chromosomes of P. ostreatus, and it has a total length of 1,000.7 centimorgans (cM) with an average of 35.1 kbp/cM. The map shows a high correlation (0.76) between physical and genetic chromosome sizes. The number of crossovers observed per chromosome per individual cell is 0.89. This map covers nearly the whole genome of P. ostreatus.     

Aryl alcohol oxidases from the white-rot basidiomycete Pleurotus ostreatus

 Three aryl alcohol oxidases (AAOs; EC 1.1.3.7) I, II, and III from the culture filtrate of a strain of white-rot fungus Pleurotus ostreatus were purified by multistep chromatography. Each of the purified AAOs I, II, and III had the same molecular masses of 70 kDa and 72 kDa on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Their optimum temperature was 40°C, but their optimum pHs differed slightly. The N-terminal amino acid sequence of AAOs I, II, and III was determined to be Ala-Asp-Lys-Asp-Tyr-Ile-Val-Val-Gly-Ala, which showed significant similarity to those of Pleurotus eryngii (80% identity) and Pleurotus ostreatus Florida (60% identity). Received: May 30, 2002 / Accepted: July 10, 2002 Acknowledgment This work was supported in part by a Grant-in-Aid for the Encouragement of Young Scientists (no. 12760117) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Correspondence to:K. Okamoto     

Effect of culture medium composition on peroxidase biosynthesis by the fungus Pleurotus ostreatus UzBI-I105

Production of extracellular peroxidase during submerged cultivation of the xylotrophic bazidiomycete Pleurotus ostreatus UZBI-I105 in nutrient media with lignocellulosic wastes, exhausted cottonseed oil cake, cotton stalks, rice husks, or ambary hemp was studied. The enzyme production increased threefold to fivefold in the presence of exhausted cottonseed oil cake extract in the nutrient medium. The dynamics of peroxidase production in various media was investigated.     

Variability of laccase activity in the white-rot basidiomycete Pleurotus ostreatus

The production of laccase in liquid cultures of the white-rot fungusPleurotus ostreatus was highly variable. During the first days of cultivation, the relative variability was as high as 80–100% and it decreased to 30% in the course of cultivation. The main source of variability was assumed to be the independent development of enzyme activity in individual cultures. Cultures with high laccase production showed also high production of the other ligninolytic enzyme—Mn-dependent peroxidase. The variability was probably due to the source of inoculum, deactivation of the enzyme in culture liquid and genetic variations among the cultures. Variability of laccase activities was lower during solid-state fermentation on wheat straw and during the growth in nonsterile soil.     

Production of biomass and ligninolytic enzymes by Pleurotus ostreatus in submerged culture.

The production of biomass and ligninolytic enzymes by Pleurotus ostreatus was analysed in synthetic medium with yeast extract and different glucose concentrations (0.5 - 20 g/l), at different pH (3.5-6.5) and incubation temperatures (23-32 degrees C). The best culture condition were: initial glucose concentration of 5 g/l, initial pH between 5.5-6.5 and incubation temperature between 26-29 degrees C. The saturation constant for glucose (Ks) was 1.75 g/l. The biomass concentration reached 8.6 g/l with a glucose addition of 20.0 g/l to the culture medium. The control of pH allowed an increment of 0.5 g/l of biomass concentration. The birreactor produced pellets with a homogeneous distribution of diameter size of 3.4 -/+ 0.2 mm. Approximately, 307 U/l of laccase and 0.41 U/l of manganese peroxidase were obtained in extracellular liquid medium and 0.015 U/g of laccase and 0.809 U/g of manganese peroxidase were obtained in solid substrate. Lignin peroxidase activity was not detected at any condition.     

Decolorisation of artificial dyes by peroxidase from the white-rot fungus, Pleurotus ostreatus

The Remazol Brilliant Blue R (RBBR) decolorising peroxidase of Pleurotus ostreatus decolorised several recalcitrant dyes. Eight different types of dyes, including triphenyl methane, heterocyclic, azo, and polymeric dyes, were decolorised to some extent. The best decolorisation was obtained for Bromophenol blue (98%). The enzyme oxidised triphenyl methane and azo dyes effectively. However, heterocyclic dyes, Methylene Blue and Toluidine Blue O were decolorised only by 10%. © Rapid Science Ltd. 1998     

Oxidation of syringic acid by extracellular peroxidase of white-rot fungus,Pleurotus ostreatus

The oxidative degradation of syringic acid by the extracellular peroxidase ofPleurotus ostreatus was studied. Three products formed in the oxidation of syringic acid by the peroxidase in the presence of O₂ and H₂O₂ were identified as 2,6-dimethoxyphenol, 2,6-dimethoxy-1,4-dihydroxybenzene, and 2,6-dimethoxy-1,4-benzoquinone. A free radical was detected as the reaction intermediate of the extracellular peroxidase-catalyzed oxidation of acetosyringone. These results can be explained by mechanisms involving the production of a phenoxy radical and subsequent decarboxylation. This is the first time that 2,6-dimethoxyphenol has been identified in extracellular peroxidase-catalyzed reactions.     

Production of 6-aminopenicillanic acid by immobilized Pleurotus ostreatus

Summary 6-Aminopenicillanic acid from penicillin V is produced by Pleurotus ostreatus immobilized by entrapment in a chitosan matrix. In these carriers the cell concentration increases after network formation by irreversible shrinking of the biocatalyst. Specific activity of the biocatalyst for the hydrolysis reaction is 1,31 mol.min^-1. (g wet weight of catalyst)^-1 corresponding to a relative activity of 38%. Catalytic half-life of immobilized Pl. ostreatus is 25 days compared to 2.5 days for free suspended cells.This paper is part of the Dissertation of Michael Kluge, Technical University Braunschweig 1981.     

Manganese peroxidase isoenzymes produced by Pleurotus ostreatus grown on wood sawdust

The white rot basidiomycete Pleurotus ostreatus produces two manganese peroxidase (MnP) isoenzymes when grown in solid stationary conditions on poplar sawdust, whereas a lower production of these same enzymes is observed on fir sawdust. Addition of Mn(2+) to poplar culture resulted in a threefold increase of MnP activity; the same addition to fir culture was able to increase tenfold the MnP production. The two MnP isoenzymes (MnP2 and MnP3) were purified from P. ostreatus poplar culture. The isoenzymes differ in their pI values, molecular masses, and N-terminal sequences. MnP3 has the same N-terminal sequence as that of a P. ostreatus MnP previously reported. Both isoenzymes exhibit Mn(2+)-dependent and Mn(2+)-independent peroxidase activities when tested on phenolic substrates. The gene coding for the new isoenzyme MnP2 was cloned and sequenced and the promoter region analyzed. Furthermore, the chromosomal localization of all known P. ostreatus genes was determined.     

Free-radical polymerization of acrylamide by manganese peroxidase produced by the white-rot basidiomycete Bjerkandera adusta

Acrylamide was polymerized to give polyacrylamide using manganese peroxidase (MnP) produced by the basidiomycete Bjerkandera adusta. The molecular weight of the polymer synthesized by MnP was 155000, higher than those obtained with other reaction systems using horseradish peroxidase and a redox initiator. The ¹³C-NMR spectrum showed that polyacrylamide was atactic. Electron spin resonance analysis revealed that 2,4-pentanedione added as an initiator was first oxidized to generate a carbon-centered radical, which initiated radical additive polymerization of acrylamide.     

Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus

Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.     

Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism

Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.12-8.29 with a second-order rate constant of (2.0 +/- 0.1) x 10(6) M-1 s-1. The activation energy for MnPI formation is 20 kJ mol-1. MnPI formation also occurs with organic peroxides such as peracetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid with second-order rate constants of 9.7 x 10(5), 9.5 x 10(4), and 5.9 x 10(4) M-1 s-1, respectively. The reactions of MnPI and MnPII with p-cresol strictly obeyed second-order kinetics. The second-order rate constant for the reaction of MnPII with p-cresol is extremely low, (9.5 +/- 0.5) M-1 s-1. Kinetic analysis of the reaction of MnII with MnPI and MnPII showed a binding interaction with the oxidized enzymes which led to saturation kinetics. The first-order dissociation rate constants for the reaction of MnII with MnPI and MnPII are (0.7 +/- 0.1) and (0.14 +/- 0.01) s-1, respectively, when the reaction is conducted in lactate buffer. Rate constants are considerably lower when the reactions are conducted in succinate buffer. Single turnover experiments confirmed that MnII serves as an obligatory substrate for MnPII and that both oxidized forms of the enzyme form productive complexes with MnII. Finally, these results suggest the alpha-hydroxy acids such as lactate facilitate the dissociation of MnIII from the enzyme.     

Polymerization of guaiacol by lignin-degrading manganese peroxidase from Bjerkandera adusta in aqueous organic solvents

 Lignin-degrading manganese (II) peroxidase (MnP) purified from the culture of a wood-rotting basidiomycete, Bjerkandera adusta, was used in the polymerization of guaiacol. MnP was found to catalyze polymerization of guaiacol in 50% aqueous acetone, dimethyl formamide, methanol, ethanol, dioxane, acetonitrile, ethylene glycol and methylcellosolve. Maximum yield of polyguaiacol was achieved in 50% aqueous acetone. The weight average molecular weight (M _w) of the polymer was estimated to be 30 300 by gel permeation chromatography. However, matrix-assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF-MS) analysis gave a more reliable M _w of 1690. IR, ¹³C-NMR, MALDI-TOF-MS and pyrolysis GC-MS analyses showed the presence of C–C and C–O linkages and quinone structure in polyguaiacol. It was also indicated that polyguaiacol has a methoxy-phenyl group as the terminal moiety. This suggests that polyguaiacol is a branched polymer in which guaiacol units are cross-linked at the phenolic group. Thermal gravimetric and differential scanning calorimetric analyses were also carried out. MnP also catalyzed the polymerization of o-cresol, 2,6-dimethoxyphenol and other phenolic compounds and aromatic amines. M _w of these polymers ranged from around 1000 to 1500. Received: 2 August 1999 / Received revision: 10 December 1999 / Accepted: 4 January 2000     

Relationship between monokaryotic growth rate and mating type in the edible basidiomycete Pleurotus ostreatus

The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBalpha and matBbeta) and to correlate the presence of specific matBalpha and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.