Ligninolytic Complex Research Articles

CAFFEIC ACID STIMULATES IN VIVO LUMINESCENCE OF THE MYCELIA OF THE HIGHER FUNGI NEONOTHOPANUS NAMBI AND ARMILLARIA BOREALIS

CAFFEIC ACID STIMULATES IN VIVO LUMINESCENCE OF THE MYCELIA OF THE HIGHER FUNGI NEONOTHOPANUS NAMBI AND ARMILLARIA BOREALIS

Ligninolytic basidiomycetes as promising organisms for the mycoremediation of PAH-contaminated Environments

Primary screening of ligninolytic fungi belonging to wood- and soil-inhabiting basidiomycetes revealed their ability to degrade three-ringed PAHs with formation of quinone metabolites at the first stage. The degradative activity was both species and strain specific, and some differences in the “chances” for the formed quinones were found. They were the main end metabolites in the degradation of PAHs by Stropharia rugosoannulata and Agaricus bisporus. During PAH degradation by strains of Trametes versicolor, Pleurotus ostreatus, Schizophyllum commune, and Bjerkandera adusta similar metabolites were detected during the cultivation, but they were utilized further. The results supported the hypothesis that the degree of PAH degradation may depend on the composition of the extracellular ligninolytic complex of the fungi: in the presence of a single ligninolytic enzyme, laccase, the accumulation of quinone metabolites takes place; their further utilization is possible with the participation of ligninolytic peroxidases. The data obtained showed the necessity not only to identify the metabolites formed, but also to study the activity of the basic ligninolytic enzymes. It is important for the correct selection of fungal strains for mycoremediation.

Relation between ligninolytic and phospholipase activities in the fungus Lentinus tigrinus

Effect of hydrocortisone, NaF, and FeSO4 on ligninolytic and phosphatase activity of the fungus Lentinus (Panus) tigrinus VKM F-3616D was investigated, Hydrocortisone and NaF were shown to inhibit the enzymes of the ligninolytic complex-laccase (EC 1.10.3.2), secretory peroxidase (EC 1.11.1.7), and Mn peroxidase (EC 1.11.1.13). FeSO4 exhibited no significant effect on the activity of these enzymes. Decreased activity of the enzymes of the ligninolytic complex was associatedwith inhibition of the activity and changes in the substrate specificity of phospholipase A2 (EC 3.1.1.4) in the presence of hydrocortisone of NaF. Cultivation of L. tigrinus in the presence of these compounds resulted in higher affinity of this enzyme to saturated fatty acids, while in the control and in the presence of FeSO4 affinity to unsaturated fatty acids was higher.

LIGNINOLYTIC ACTIVITY PATTERNS OF Pleurotus ostreatus OBTAINED BY SUBMERGED FERMENTATION IN PRESENCE OF 2,6-DIMETHOXYPHENOL AND REMAZOL BRILLIANT BLUE R DYE

The degradation of 2,6-dimethoxyphenol (DMP) and decolorization of Remazol brilliant blue R dye (RBB), added to culture media of Pleurotus ostreatus developed in submerged fermentation, and the laccase, manganese peroxidase and veratryl alcohol oxidase activities produced in these systems were evaluated. Both compounds were removed from the culture medium mainly by enzymatic action. These compounds decreased the specific growth rate and the effect on the maximal biomass values was not important. The enzymatic activities were increased by DMP and/or RBB; however, the DMP showed a higher inducer effect on all enzymes than RBB. On the other hand, the RBB showed a larger inducer effect on manganese peroxidase activity than on the laccases and veratryl alcohol oxidase activities. These results show that DMP was a better inducer of ligninolytic enzymes than dye, and the process of dye decolorization and degradation of DMP requires the action of all enzymes of the ligninolytic complex.

Biodegradation of 2,4,6‐TCA by the white‐rot fungus Phlebia radiata is initiated by a phase I (O‐demethylation)–phase II (O‐conjugation) reactions system: implications for the chlorine cycle

Thirteen species of white-rot fungi tested have been shown to efficiently biodegrade 1 mM 2,4,6-trichloroanisole (2,4,6-TCA) in liquid cultures. The maximum biodegradation rate (94.5% in 10-day incubations) was exhibited by a Phlebia radiata strain. The enzymes of the ligninolytic complex, laccase, lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) were not able to transform 2,4,6-TCA in in vitro reactions, indicating that the ligninolytic complex was not involved in the initial attack to 2,4,6-TCA. Instead, the first biodegradative steps were carried out by a phase I and phase II reactions system. Phase I reaction consisted on a O-demethylation catalysed by a microsomal cytochrome P-450 monooxygenase to produce 2,4,6-trichlorophenol (2,4,6-TCP). Later, in a phase II reaction catalysed by a microsomal UDP-glucosyltransferase, 2,4,6-TCP was detoxified by O-conjugation with D-glucose to produce 2,4,6-TCP-1-O-d-glucoside (TCPG). This compound accumulated in culture supernatants, reaching its maximum concentration between 48 and 72 h of growth. TCPG levels decreased constantly by the end of fermentation, indicating that it was subsequently metabolized. A catalase activity was able to break in vitro the glycosidic link to produce 2,4,6-TCP, whereas ligninolytic enzymes did not have a significant effect on the biotransformation of that compound. Once formed, 2,4,6-TCP was further degraded as detected by a concomitant release of 2.6 mol of chloride ions by 1 mol of initial 2,4,6-TCA, indicating that this compound underwent almost a complete dehalogenation and biodegradation. It was concluded that P. radiata combines two different degradative mechanisms in order to biodegrade 2,4,6-TCA. The significance of the capability of white-rot fungi to O-demethylate chloroanisoles for the global chlorine cycle is discussed.

Biodegradation of remazol brilliant blue R by ligninolytic enzymatic complex produced by Pleurotus ostreatus

Pleurotus ostreatus (shimeji) is produced in Brazil on a commercial scale using various lignocellulosic residues. Efforts have been made to reuse the culture residue to obtain products of greater aggregate value such as enzymes or in processes of bioremediation. We evaluated the Remazol brilliant blue R (RBBR) degradation potential of extracts from solid substrate colonized by P. ostreatus and extracts from residue of the shimeji mushroom yield. Colonized substrates and residue were provided by Toyobo do Brasil Ltda. Extraction was performed with sodium acetate buffer (50 mM, pH 4.6). RBBR decolorization was monitored at 592 nm and peroxidase and laccase activities were measured by monitoring the oxidation of ABTS. Horseradish peroxidase was used as reference. The time of growth of P. ostreatus influenced RBBR degradation and peroxidase and laccase activities. Concentration of 1 mM H2O2 and pH 4.0 were the best for RBBR decolorization. Complete RBBR decolorization was obtained with the addition of only one aliquot of 50 µL of 1 mM H2O2. The stability of the extracts was higher when they were kept under refrigeration than when stored frozen. The potential application of the ligninolytic complex derived from P. ostreatus and mushroom residue for xenobiotic degradation was demonstrated.

Screening of supports and inducers for laccase production by Trametes versicolor in semi-solid-state conditions

The production of laccase by Trametes versicolor under semi-solid-state conditions has been studied. Several supports (polyurethane foam, wheat straw, barley straw, wood shavings and barley bran) have been tested in order to determine the most suitable for laccase production by the above-mentioned microorganism. Barley bran led to the highest activity levels, reaching maximum values of about 1200 U/l. In this stage of research, several factors affecting laccase production (veratryl alcohol, xylidine, fresh support and the initial C/N ratio) were investigated. Xylidine was shown to be the best inducer of laccase activity, attaining values of about 1700 U/l. Moreover, the addition of fresh support not only prolonged culture lifetime but also enhanced activity levels, reaching in all the cases values higher than 2000 U/l. In addition, the decolourisation of three structurally different dyes by cultures grown on the best operating conditions determined in the present study, was monitored in order to assess the degrading capability of the ligninolytic complex secreted by such cultures. The decolourisation of all the dyes tested was almost total (85–96%) after 6 days of incubation.

Design of different bioreactor configurations: application to ligninolytic enzyme production in semi-solid-state cultivation

The production of ligninolytic enzymes by Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) in laboratory-scale bioreactors was studied. The cultivations were carried out in semi-solid-state conditions, employing corncob as carrier, which functioned both as a place of attachment and as a source of nutrients. Several bioreactor configurations were investigated in order to determine the most suitable one for ligninolytic enzyme production: a 1-dm3-static-bed bioreactor, a 1-dm3-static-bed bioreactor with air diffusers into the bed, a 0.5-dm3-static-bed bioreactor with air diffusers into the bed and a tray bioreactor. Although the static-bed configurations produced maximum individual lignin peroxidase (LiP) activities about 400 U dm−3 (1.0-dm3 bioreactor) and about 700 U dm−3 (0.5-dm3 bioreactor), manganese-dependent peroxidase (MnP) was not detected throughout the cultures. Nevertheless, the tray configuration led to maximum individual MnP and LiP activities of about 200 U dm−3 and 300 U dm−3, respectively. Therefore, this configuration is the most adequate of the different bioreactor configurations tested in the present work, since the ligninolytic complex formed by MnP and LiP is more efficient for its application to bio-processing systems. In addition, the results indicated the influence of the oxygen in ligninolytic enzyme production. © 2001 Society of Chemical Industry

In vivo decolourization of the polymeric dye poly R‐478 by corncob cultures of Phanerochaete chrysosporium

AbstractIn this paper, the in vivo decolourization of the polymeric dye Poly R‐478 by semi‐solid‐state cultures of Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725) was investigated, employing corncob as a support. In order to stimulate the ligninolytic system of the fungus, the cultures were supplemented with veratryl alcohol (2 mM) or manganese (IV) oxide (1 g/l).Maximum manganese‐dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of around 2,000 U/l and 400 U/l were attained by the former, whereas the activities reached by the latter were of about 1,500 U/l and 200 U/l, respectively. Furthermore, laccase activity (around 150 U/l) was only detected in manganese (IV) oxide supplemented cultures.The polymeric dye Poly R‐478 (0.02 w/v) was added to three‐day‐old cultures. A percentage of biological decolourization of about 85% was achieved using cultures supplemented with veratryl alcohol, whereas MnO2 cultures showed a rather lower percentage of around 58% after nine days of dye incubation. Moreover, a correlation between MnP activity and Poly R‐478 decolourization could be observed, indicating that this enzyme is mainly responsible for dye degradation.In the present work, the in vivo decolourizing capability of the ligninolytic complex secreted by P. chrysosporium was investigated under the above‐mentioned cultivation conditions, employing a model compound, such as the polymeric dye Poly R‐478.

Substrate specificity of laccase from Lentinus edodes

AbstractIn previous studies, the white‐rot basidiomycete Lentinus edodes, strain SC‐495, was proved to be a “selective” lignin degrader and its extracellular crude preparations arising from solid‐state cultures were successfully employed in biopulping experiments on annual plants. This fungus produced extracellular laccase as the predominant phenoloxidase when growing in solid‐state fermentation on corn stalks. Laccase from this strain was purified and partially characterized, as an initial approach towards the study of its ligninolytic complex. Laccase was purified 69.6‐fold by anion‐exchange chromatography and two affinity‐chromatography steps with an overall yield of 7.45%. The native enzyme exhibited a molecular mass of 74 kDa, an isoelectric point of 3.42 and a carbohydrate content of 7.5%. The absorption spectrum of laccase showed a maximum at 605 nm, typical of blue‐copper oxidases. The optimum pH and temperature for the activity of laccase were 4.0–4.2 and 50°C, respectively. Kinetic experiments, performed with a wide range of phenolic compounds, showed that the reaction rate and the substrate affinity greatly varied depending on the nature of substituents and their reciprocal positions on the aromatic ring. In particular, the enzyme showed high affinity to phenolic compounds bearing methoxyl or methyl groups, but no affinity to those bearing the nitro group directly attached to the benzene ring, nor to non‐phenolic lignin‐related compounds, such as trans‐cinnamic acid or 3,4‐dimethoxycinnamic acid. The huge differences in terms of reactivity of the enzyme towards phenolic compounds suggests that a preliminary systematic screening should be advisable when using laccase in effluent treatment applications.