Stability and activity of a phenol oxidase from the ligninolytic fungus Pleurotus ostreatus

Three different phenol oxidases produced by the basidiomycete fungus Pleurotus ostreatus have been isolated and their main structural, enzymatic and physico-chemical properties characterized. Studies have forcaused on the most abundantly secreated of these proteins, a copper-e nzyme specific towards ortho-diphenol substrates. This protein was purified to homogeneity and part of its primary structure determined by direct protein sequencing. The ingluence of pH, temperature and presence of water-soluible or water-insoluble organic solvents on the activity and stability of the enzyme were also investigated. These data can be used for applying bioarectors to problems of environmental concern such as waste-water treatmentCorrespondens to: G. Sannia     

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.     

Identification of naphthalene metabolism by white rot fungus Pleurotus eryngii

The use of biomaterials or microorganisms in PAHs degradation had presented an eye-catching performance. Pleurotus eryngii is a white rot fungus, which is easily isolated from the decayed woods in the tropical rain forest, used to determine the capability to utilize naphthalene, a two-ring polycyclic aromatic hydrocarbon as source of carbon and energy. In the meantime, biotransformation of naphthalene to intermediates and other by-products during degradation was investigated in this study. Pleurotus eryngii had been incubated in liquid medium formulated with naphthalene for 14 days. The presence of metabolites of naphthalene suggests that Pleurotus eryngii begin the ring cleavage by dioxygenation on C1 and C4 position to give 1,4-naphthaquinone. 1,4-Naphthaquinone was further degraded to benzoic acid, where the proposed terepthalic acid is absent in the cultured extract. Further degradation of benzoic acid by Pleurotus eryngii shows the existence of catechol as a result of the combination of decarboxylation and hydroxylation process. Unfortunately, phthalic acid was not detected in this study. Several enzymes, including manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase and 2,3-dioxygenase are enzymes responsible for naphthalene degradation. Reduction of naphthalene and the presence of metabolites in liquid medium showed the ability of Pleurotus eryngii to utilize naphthalene as carbon source instead of a limited glucose amount.     

Purification and characterization of two laccase isoenzymes from a ligninolytic fungus Trametes gallica

Constant laccase activities were detected in culture supernatant of newly isolated basidiomycete Trametes gallica. Tryptone and glucose have great effects on the production of laccase. Two laccase isoenzymes (Lac I and Lac II) produced by T. gallica were purified to homogeneity (51- and 50-fold, respectively) by gel filtration chromatography, anion exchange chromatography, and improved native PAGE, with an overall yield of 24.8%. Lac I and Lac II from this fungus are glycoproteins with 3.6% and 4% carbohydrate content, the same molecular masses (by SDS-PAGE) of 60 kDa, and the pI of 3.1 and 3.0, respectively. Native gel electrophoresis indicates that the two laccases have different migration ratios. Lac I and Lac II have the same optimal pH of 3.0 on 2,6-dimethoxyphenol (DMP), pH 2.2 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and of pH 4.0 on guaiacol. The highest rate of ABTS oxidation for both laccases was reached at 70 degrees C. Both laccases are stable from pH 6 to 9, retaining 88-90% activity after 24 hr incubation, and show good stability when incubated at temperatures lower than 40 degrees C. The Km values of Lac I for ABTS, DMP, and guaiacol are 0.118 x 10(-2), 0.420, and 0.405 mM, respectively; the Km values of Lac II for ABTS, DMP, and guaiacol are 0.086 x 10(-2), 0.41, and 0.40 mM, respectively. Their N-terminal sequences are determined and show strong similarity with those from other basidiomycetes. Graphite-furnace atomic absorption analysis revealed that both laccases have four copper atoms per protein molecule, but they have no type I copper signal at around 600 nm and a type III copper signal near 330 nm. Cyanide, azide, and halides completely inhibit the enzyme activity, whereas EDTA has less inhibition.     

Eryngin, a novel antifungal peptide from fruiting bodies of the edible mushroom Pleurotus eryngii

An antifungal peptide with a molecular mass of 10k Da was isolated from fruiting bodies of the mushroom Pleurotus eryngii. The peptide, designated as eryngin, inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola. It was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and S-Sepharose. Its N-terminal sequence demonstrated some similarity to the antifungal protein from the mushroom Lyophyllum shimeiji and little resemblance to thaumatin and thaumatin-like proteins.     

Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase

Lignin degradation by fungal peroxidases is initiated by one-electron transfer to an exposed tryptophan radical, a reaction mediated by veratryl alcohol (VA) in lignin peroxidase (LiP). Versatile peroxidase (VP) differs not only in its oxidation of Mn2+ at a second catalytic site but also in its ability to directly oxidize different aromatic compounds. The catalytic tryptophan environment was compared in LiP and VP crystal structures, and six residues near VP Trp164 were modified by site-directed mutagenesis. Oxidation of Mn2+ was practically unaffected. However, several mutations modified the oxidation kinetics of the high-redox-potential substrates VA and Reactive Black 5 (RB5), demonstrating that other residues contribute to substrate oxidation by the Trp164 radical. Introducing acidic residues at the tryptophan environment did not increase the efficiency of VP oxidizing VA. On the contrary, all variants harboring the R257D mutation lost their activity on RB5. Interestingly, this activity was restored when VA was added as a mediator, revealing the LiP-type behavior of this variant. Moreover, combination of the A260F and R257A mutations strongly increased (20-50-fold) the apparent second-order rate constants for reduction of VP compounds I and II by VA to values similar to those found in LiP. Dissociation of the enzyme-product complex seemed to be the limiting step in the turnover of this improved variant. Nonexposed residues in the vicinity of Trp164 can also affect VP activity, as found with the M247F mutation. This was a direct effect since no modification of the surrounding residues was found in the crystal structure of this variant.     

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     

Mechanism of peroxidase inactivation in liquid cultures of the ligninolytic fungus pleurotus pulmonarius

It has recently been reported that Pleurotus pulmonarius secretes a versatile peroxidase that oxidizes Mn2+, as well as different phenolic and nonphenolic aromatic compounds; this enzyme has also been detected in other Pleurotus species and in Bjerkandera species. During culture production of the enzyme, the activity of the main peak was as high as 1,000 U/liter (measured on the basis of the Mn3+-tartrate formation) but this peak was very ephemeral due to enzyme instability (up to 80% of the activity was lost within 15 h). In culture filtrates inactivation was even faster; all peroxidase activity was lost within a few hours. Using different inhibitor compounds, we found that proteases were not responsible for the decrease in peroxidase activity. Peroxidase instability coincided with an increase in the H2O2 concentration, which reached 200 μM when filtrates were incubated for several hours. It also coincided with the onset of biosynthesis of anisylic compounds and a decrease in the pH of the culture. Anisyl alcohol is the natural substrate of the enzyme aryl-alcohol oxidase, the main source of extracellular H2O2 in Pleurotus cultures, and addition of anisyl alcohol to filtrates containing stable peroxidase activity resulted in rapid inactivation. A decrease in the culture pH could also dramatically affect the stability of the P. pulmonarius peroxidase, as shown by using pH values ranging from 6 to 3.25, which resulted in an increase in the level of inactivation by 10 μM H2O2 from 5 to 80% after 1 h. Moreover, stabilization of the enzyme was observed after addition of catalase, Mn2+, or some phenols or after dialysis of the culture filtrate. We concluded that extracellular H2O2 produced by the fungus during oxidation of aromatic metabolites is responsible for inactivation of the peroxidase and that the enzyme can protect itself in the presence of different reducing substrates.     

Pantoea beijingensis sp. nov., isolated from the fruiting body of Pleurotus eryngii

Four Gram-negative-staining, facultatively anaerobic bacterial isolates were obtained from a fruiting body of the edible mushroom Pleurotus eryngii showing symptoms of soft rot disease in Beijing, China. Partial 16S rRNA gene sequencing, together with partial rpoB sequencing, placed these isolates in the genus Pantoea. Multilocus sequence analysis based on the partial sequences of gyrB, rpoB, infB and atpD revealed Pantoea dispersa and Pantoea gaviniae as their closest phylogenetic relatives and indicated that these isolates constituted a possible novel species. DNA–DNA hybridization studies confirmed the classification of the new isolates as a novel species and phenotypic tests allowed for differentiation from the closest phylogenetic neighbours. The name Pantoea beijingensis sp. nov. [type strain LMG 27579^T = KCTC 32406^T = JZB2120001^T (deposited at Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences)] is proposed.     

Medium optimization, molecular characterization, and bioactivity of exopolysaccharides from Pleurotus eryngii

An optimal medium for exopolysaccharides (EPS) production was obtained through one-factor-at-a-time method and response surface methodology. Under optimal culture medium, the maximum EPS concentration in shake flask was 5.16 g/l. Two groups of EPSs (designated as Fr-I and Fr-II) were obtained from the culture filtrates by size exclusion chromatography/multiangle laser light scattering, and the weight average molar masses (M _w) of Fr-I and Fr-II were determined to be 4.098 × 10⁴ and 1.114 × 10⁴ g/mol, respectively. The molecular confirmation of Fr-I was revealed to be a rigid rod form in aqueous solution. Moreover, monosaccharide composition and characteristic groups were investigated by GC and Fourier transform infrared, respectively. Finally, pharmacology experiment in vitro indicated EPS Fr-II of Pleurotus eryngii exhibited higher antioxidant and antitumor abilities than Fr-I, which might be attributed to the different molecular weights and chemical compositions in the EPS fraction.     

Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungusPleurotus eryngii

Summary Production of extracellular hydrogen peroxide by fungal oxidases is been investigated as a requirement for lignin degradation. Aryl-alcohol oxidase activity is described in extracellular liquid and mycelium ofPleurotus eryngii and studied under non-limiting nitrogen conditions. This aryl-alcohol oxidase catalyses conversion of primary aromatic alcohols to the corresponding aldehydes and H₂O₂, showing no activity with aliphatic and secondary aromatic alcohols. The enzyme is stable at pH 4.0–9.0, has maximal activity at 45°–50°C and pH 6.0–6.5, is inhibited by Ag⁺, Pb²⁺ and NaN₃, and has aK _m of 1.2 mM using veratryl alcohol as substrate. A single protein band with aryl-alcohol oxidase activity was found in zymograms of extracellular and intracellular crude enzyme preparations fromP. eryngii.     

Effect of aromatic compounds and Mn2+ on the ligninolytic enzyme complex from the fungus Pleurotus floridae (FRIES) Kummer--a white-rot wood fungus

The influence of aromatic compounds and Mn ions on activities of ligninolityc enzymes from white-rot fungus Pleurotus floridae has been studied. The specific inducers: vanillic acid and vanillyl alcohol--for activity of manganese-dependent peroxidase; vanillyl alcohol--for activity of cellobiose: quinone oxidoreductase during submerged, fermentation of Pleurotus floridae in Kirk's medium have been revealed. The inducers of laccase activity among studied aromatic compounds have not been revealed. The influence of Mn2+ in concentration range 0.4-68.4 mM on activities of ligninolytic enzymes of submerged culture of fungus P. floridae has been studied. Concentration of Mn ions 32.4 mM was optimal for manganese-dependent peroxidase activity.