Production of lignin peroxidase and laccase by Phlebia radiata

Summary A cultivation method using carrierbound mycelium was developed for the production of lignin-modifying enzymes by Phlebia radiata. Laccase and lignin peroxidase were produced in batch and semi-continuous cultivations. Laccase activity was clearly enhanced by veratryl alcohol. The presence of both veratryl alcohol and Tween 80 was required for lignin peroxidase production in submerged cultivations. During the course of the semi-continuous cultivations production of lignin peroxidase activity increased fourfold compared with static cultivations.     

Cellulolytic enzymes of the ligninolytic white-rot fungus Phlebia radiata

The ligninolytic fungus Phlebia radiata growing in a low-nitrogen medium with Avicel cellulose as the sole carbon source produced a full spectrum of celluloytic enzymes. Some properties of these enzymes were investigated during the growth of the fungal culture.     

Biodegradation of chestnut shell and lignin-modifying enzymes production by the white-rot fungi Dichomitus squalens,Phlebia radiata

As a discarded lignocellulosic biomass, chestnut shell is of great potential economic value, thus a sustainable strategy is needed and valuable for utilization of this resource. Herein, the feasibility of biological processes of chestnut shell with Dichomitus squalens, Phlebia radiata and their co-cultivation for lignin-modifying enzymes (LMEs) production and biodegradation of this lignocellulosic biomass was investigated under submerged cultivation. The treatment with D. squalens alone at 12 days gained the highest laccase activity (9.42 ± 0.73 U mg^?1). Combined with the data of laccase and manganese peroxidase, oxalate and H₂O₂ were found to participate in chestnut shell degradation, accompanied by a rapid consumption of reducing sugar. Furthermore, specific surface area of chestnut shell was increased by 77.6–114.1 % with the selected fungi, and total pore volume was improved by 90.2 % with D. squalens. Meanwhile, the surface morphology was observably modified by this fungus. Overall, D. squalens was considered as a suitable fungus for degradation of chestnut shell and laccase production. The presence of LMEs, H₂O₂ and oxalate provided more understanding for decomposition of chestnut shell by the white-rot fungi.     

Hemicellulolytic enzymes of the ligninolytic white-rot fungus Phlebia radiata. Influence of phenolic compounds on the synthesis of hemicellulolytic enzymes

The ligninolytic fungus Phlebia radiata growing in a low-nitrogen medium with wheat bran as the sole carbon source was induced by some lignin monomers, e.g. vanillic, veratric and ferulic acids. In the medium these substances showed a mainly stimulating influence on the hemicellulolytic enzymes activity except for arabinofuranosidase and ferulic acid esterase.     

Conversion of Milled Pine Wood by Manganese Peroxidase from Phlebia radiata

Purified manganese peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tween 20, Mn²⁺, Mn-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid redsidues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from P. radiata (MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37°C with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to >6.5. An aromatic nonphenolic lignin structure (β-O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols.     

Purification and properties of alpha-galactosidase isosymes from Phlebia radiata

Phlebia radiata formed extracellular alpha-galactosidase when it was grown in a culture containing wheat bran or locus bean gum as a carbon source. Their activities were optimal at pH 5.0, and demonstrated the highest level of activity at 60 degrees C. Highly purified isoforms of alpha-galactosidase (AGaS-m1, AGaS-m2, AGaS-m3) isolated from the media with galactomannan and (AGaS-b1, AGaS-b2, AGaS-b3) from the media with wheat bran were obtained by means of the column chromatography on Q-Sepharose and chromatofosussing on Polybuffer Exchanger PBE-94.     

Conversion of milled pine wood by manganese peroxidase from Phlebia radiata

Purified manganese peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tween 20, Mn(2+), Mn-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid residues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from P. radiata (MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37 degrees C with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to >6.5. An aromatic nonphenolic lignin structure (beta-O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols.     

Purification and characterization of an extracellular alpha-D-glucuronidase from Phlebia radiata

Alpha-D-glucuronidase was isolated from the culture filtrate of Phlebia radiata grown on wheat bran and purified to homogeneity by chromatographic methods. The final enzymic preparation was purified 65-fold with an activity yield of 58%; it showed a high level of specific activity (over 23,000 nkat/mg protein). The molecular and hydrolytic properties of the purified enzyme were studied. The secreted alpha-glucuronidase had a molecular weight of 110 kDa, as established by gel permeation chromatography (GP HPLC), had a determined pI just below 4.4, and was stable at pH 5.5 for prolonged times. The carbohydrate content in protein molecules was found to be 15%. The activity of alpha-D-glucuronidase peaked at pH 3,8 and 60 degrees C with aldouronic acids preparation as the substrate. The Michaelis-Menten constant (K(m)), the maximum reaction velocity (V(max)), and the activation energy (E(a)) were 0.18 mM, 0.13 microM/min and 5.91 kJ/mol, respectively. The alpha-glucuronidase was active mainly on small substituted xylooligomers. When this enzyme was used with endoxylanase for the degradation of oat xylan, synergistic effects were observed.     

Production of Ligninolytic Enzymes by Phlebia Floridensis

Summary The present work reports the production of laccase, lignin peroxidase and manganese peroxidase by the little studied white-rot fungus Phlebia floridensis under a variety of nutritional and physicochemical conditions. Among the different media and supplements the highest yields of laccase, lignin peroxidase and manganese peroxidase were recorded in the presence of sugarcane bagasse, wheat straw and rice straw, respectively. Laccase and manganese peroxidase activities were best expressed at a pH of 4.5 while lignin peroxidase was optimally active at a lower pH. Laccase proved to be much more thermostable as compared to the other two enzymes.     

Structure of extracellular polysaccharide produced by lignin-degrading fungus Phlebia radiata in liquid culture

The extracellular material (EM) produced by the white rot fungus Phlebia radiata cultured in an N-limited liquid medium was studied. Carbohydrate analysis showed maximum concentration of glucose as the major monosaccharide component of EM was found on postinoculation day 9. Beyond day 9 of cultivation the proportion of glucose decreased suggesting that the glucan component of EM had been further metabolized. The analysis of EM at day 9 revealed the presence of the following monosaccharides (in relative %): glucose (62); galactose (16); mannose (13); xylose (4); and fucose (5). The carbohydrate analysis together with the presence of protein in EM corresponds to a mixture of glucan and glycoprotein. Purification by trypsin treatment yielded an enriched glucose-containing extracellular polysaccharide (EPS). Methylation analysis identified EPS as (1-3)-beta-D-glucan highly branched at C-6. The structure of the glucan was confirmed by 13C-NMR spectroscopy. The results suggest that P. radiata's EPS is entangled with a glycoprotein in a complex that makes the extracellular sheath surrounding the hyphae.     

Biodegradation of 2,4,6-trinitrotoluene (TNT) by the white-rot basidiomycete Phlebia radiata

Phlebia radiatatransformed 2,4,6-trinitrotoluene (TNT), as well as its first reduction products, the aminodinitrotoluenes, into 4-hydroxylamino-2,6-dinitrotoluene (4-OHA-2,6-DNT) and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT). No extracellular peroxidases were involved in this step. The ligninolytic extracellular fluid, assumed to contain peroxidases, did not reduce TNT. However, ligninolytic peroxidases are implicated in the transformation of the first reduction products of TNT.     

Mineralization and solubilization of synthetic lignin by manganese peroxidases from Nematoloma frowardii and Phlebia radiata

Crude and purified manganese peroxidase from the white-rot fungi Nematoloma frowardii and Phlebia radiata catalyzed the partial depolymerization of a [¹⁴C-ring]labelled synthetic lignin into water-soluble fragments (30–50%). The in vitro depolymerization of the ¹⁴C-labelled lignin was accompanied by a release of ¹⁴CO₂ ranging from 4 to 6%. Small quantities of the thiol mediator glutathione stimulated the depolymerization of lignin resulting in a mineralization and solubilization of up to 10 and 64%, respectively. Most of the water-soluble substances formed had molecular masses around 0.7 kDa, although a higher-molecular mass fraction was also detectable (>2 kDa). Photometric assays using 2,2′-azinobis(3-ethylbenzothiazolinesulphonate) as an indicator demonstrated that high levels of Mn(III), which were very probably responsible for the depolymerization and mineralization of the ¹⁴C-labelled lignin, were adjusted within the first 24 h of incubation. The manganese peroxidase catalyzed depolymerization process was not necessarily dependent on H₂O₂; also in the absence of the H₂O₂-generating system glucose/glucose oxidase, effective solubilization and mineralization of lignin dehydrogenation polymerizate occurred, due to the in part superoxide dismutase sensitive, ‘oxidase-like’ activity of MnP which probably produces radical species and peroxides from malonate.     

Comparison of two lignin-degrading fungi:Phlebia radiata andPhanerochaete chrysosporium

Summary The ligninolytic enzymes ofPhlebia radiata were produced in static conditions earlier developed forPhanerochaete chrysosporium. The production pattern of lignin peroxidases resembled that ofP. chrysosporium. The extracellular proteins ofPhlebia radiata were separated by isoelectric focusing. Four proteins with acidic isoelectric points (4.15) were detected by peroxidase staining. The peroxidases ofP. radiata reacted with antibodies produced against a peroxidase ofPhanerochaete chrysosporium and vice versa. Thus the lignin peroxidases of the two fungi have major similarities despite slight differences in their isoelectric points and molecular weights. Veratryl alcohol was produced by both fungi and degraded to veratraldehyde, two lactones and a quinone by the ligninolytic cultures.     

The synergistic effect on production of lignin-modifying enzymes through submerged co-cultivation of Phlebia radiata, Dichomitus squalens and Ceriporiopsis subvermispora using agricultural residues

The lignin-modifying enzymes (LMEs) play an important role in decomposition of agricultural residues, which contain a certain amount of lignin. In this study, the production of LMEs by three co-cultivated combinations of Phlebia radiata, Dichomitus squalens and Ceriporiopsis subvermispora and the respective monocultures was comparatively investigated. Laccase and manganese peroxidases (MnP) were significantly promoted in the co-culture of P. radiata and D. squalens, and corncob was verified to be beneficial for laccase and MnP production. Moreover, laccase production by co-culture of P. radiata and D. squalens with high ratio of glucose to nitrogen was higher than low ratio under carbon- and nitrogen-meager conditions. New laccase isoenzymes measured by Native-PAGE were stimulated by co-cultured P. radiata with D. squalens or C. subvermispora, respectively, growing in the defined medium containing corncob, but the expression of laccase was greatly restrained by the co-culturing of D. squalens with C. subvermispora. This study showed that the synergistic and depressing effects of co-cultivation of P. radiata, D. squalens and C. subvermispora on LMEs were species specific.     

Production of p-cresol by Clostridium difficile on different basal media

The production of p -cresol by Clostridium difficile on a variety of agar basal media was investigated using gas-liquid chromatography. None of the basal media studied supported the production of large amounts of p -cresol. The addition of 0–1% p -hydroxyphenylacetic acid to a basal medium stimulated the production of p -cresol. If detection of p -cresol is to be used for the presumptive identification of C1. difficile then the basal medium should be supplemented with p -hydroxyphenylacetic acid.     

Semi-continuous production of laccase byPhlebia radiata in different culture media

The white-rot fungusPhlebia radiata, immobilized on a polypropylene carrier, was cultivated in a laboratory fermentor under semi-continuous conditions on culture media varying in the content of nitrogen, glucose, vitamins and microelements. Moreover, two laccase inducers were used: veratryl alcohol and veratraldehyde. Throughout the cultivation except the growth phase in the first cycle of fermentation, the observed rate of laccase expression reached up to about 2.0 nkat/mL per 1 h of cultivation, as determined by ABTS oxidation. In most experiments, phenol oxidase activity was determined also in the reaction with syringaldazine, giving reaction rates almost two times lower than in the case of ABTS.     

Effect of wood modification on lignin consumption and synthesis of lignolytic enzymes by the fungus Panus (Lentinus) tigrinus

Lignin consumption and synthesis of lignolytic enzymes by the fungus Panus (Lentinus) tigrinus cultivated on solid phase (modified and unmodified birch and pine sawdusts) were studied. The fungus grew better and consumed more readily the birch lignin than the pine wood. Peroxidase activity was higher in the case of pine sawdust; laccase and lignolytic activities, in the case of birth sawdust. Treatment with ammonia or sulfuric acid decreased lignin consumption by the fungus cultivated on either medium. Modification of sawdust by ultrasound increased lignin consumption and may be recommended for accelerating biodegradation of lignocellulose substrates.