Lignin and veratryl alcohol are not inducers of the ligninolytic system of Phanerochaete chrysosporium.

Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. In this work, we investigated the roles of veratryl alcohol and lignin in the ligninolytic system of P. chrysosporium BKM-F-1767 cultures grown under nitrogen-limited conditions. Cultures supplemented with 0.4 to 2 mM veratryl alcohol showed increased lignin peroxidase activity. Addition of veratryl alcohol had no effect on Mn-dependent peroxidase activity and inhibited glyoxal oxidase activity. Azure-casein analysis of acidic proteases in the extracellular fluid showed that protease activity decreased during the early stages of secondary metabolism while lignin peroxidase activity was at its peak, suggesting that proteolysis was not involved in the regulation of lignin peroxidase activity during early secondary metabolism. In cultures supplemented with lignin or veratryl alcohol, no induction of mRNA coding for lignin peroxidase H2 or H8 was observed. Veratryl alcohol protected lignin peroxidase isozymes H2 and H8 from inactivation by H2O2. We conclude that veratryl alcohol acts as a stabilizer of lignin peroxidase activity and not as an inducer of lignin peroxidase synthesis.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.     

Production of lignin peroxidase by Phanerochaete chrysosporium immobilized on porous poly(styrene-divinylbenzene) carrier and its application to the degrading of 2-chlorophenol

Porous poly(styrene-divinylbenzene) carriers, for the immobilization of white rot fungus Phanerochaete chrysosporium have been prepared by the concentrated emulsion polymerization method. The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase, and water as the dispersed phase. The polymerization of the monomers of the continuous phase generated the polymer carrier with a porcus structure. The white rot fungus Phanerochaete chrysosporium has been immobilized on porous poly(styrene-divinylbenzene) carriers and used for the batch production and the repeated batch production of lignin peroxidase in shake cultures based on a carbon-limited medium containing veratryl alcohol. The best results were achieved when a spore inoculum was used for immobilization instead of 1-day-old mycelial pellets, for both the batch production and the repeated batch production. The porous poly(styrene-divinylbenzene) immobilized Phanerochaete chrysosporium and freely suspended mycelial pellets were used as biocatalysts for the degradation of 2-chilorophenol in a 2-L bioreactor. The porous poly(styrene-divinylbenzene) particle (diameter congruent with 0.2 cm) immobilized spores exhibited a much higher activity in the degradation of 2-chlorophenol than the freely suspended mycelial pellets. (c) 1994 John Wiley & Sons, Inc.     

Manganese Regulation of Veratryl Alcohol in White Rot Fungi and Its Indirect Effect on Lignin Peroxidase

Many white rot fungi are able to produce de novo veratryl alcohol, which is known to be a cofactor involved in the degradation of lignin, lignin model compounds, and xenobiotic pollutants by lignin peroxidase (LiP). In this study, Mn nutrition was shown to strongly influence the endogenous veratryl alcohol levels in the culture fluids of N-deregulated and N-regulated white rot fungi Bjerkandera sp. strain BOS55 and Phanerochaete chrysosporium BKM-F-1767, respectively. Endogenous veratryl alcohol levels as high as 0.75 mM in Bjerkandera sp. strain BOS55 and 2.5 mM in P. chrysosporium were observed under Mn-deficient conditions. In contrast, veratryl alcohol production was dramatically decreased in cultures supplemented with 33 or 264 (mu)M Mn. The LiP titers, which were highest in Mn-deficient media, were shown to parallel the endogenous veratryl alcohol levels, indicating that these two parameters are related. When exogenous veratryl alcohol was added to Mn-sufficient media, high LiP titers were obtained. Consequently, we concluded that Mn does not regulate LiP expression directly. Instead, LiP titers are enhanced by the increased production of veratryl alcohol. The well-known role of veratryl alcohol in protecting LiP from inactivation by physiological levels of H(inf2)O(inf2) is postulated to be the major reason why LiP is apparently regulated by Mn. Provided that Mn was absent, LiP titers in Bjerkandera sp. strain BOS55 increased with enhanced fungal growth obtained by increasing the nutrient N concentration while veratryl alcohol levels were similar in both N-limited and N-sufficient conditions.     

Continuous production of lignin peroxidase by Phanerochaete chrysosporium

Phanerochaete chrysosporium spores were immobilized both in agarose and agar gel beads, and used for the production of lignin peroxidase in repeated batch cultures on carbon-limited medium both with 0.5 g l⁻¹ glucose and without glucose. Veratryl alcohol was used as an activator of enzyme production. The biocatalyst was more stable in agarose gel with the maximum activity of 245 U l⁻¹ obtained in a 70 h batch. The biocatalyst could be used for at least 12 batches on the glucose medium with a gradual decrease in lignin peroxidase activity after the sixth batch. Further, mycelium pellets grown on carbon-limited medium were employed both in vertical and horizontal column reactors for the continuous production of lignin peroxidase. The bioreactor produced lignin peroxidase for at least 20 days in the horizontal system at 49 h residence time, with a maximum activity of 95 U l⁻¹.     

Continuous production of lignin peroxidase by immobilized Phanerochaete chrysosporium in a pilot scale bioreactor

Lignin peroxidase was continuously produced by nylon-web or polyurethane immobilized Phanerochaete chrysosporium ATCC 24725 in a modified Biostate E® bioreactor, agitated either with the air and oxygen flow alone or in combination with a mechanical stirrer. Lignin peroxidase production started rapidly, and activities as high as ∼600 U l⁻¹ were reached as early as in 3 days. At best a total activity yield of ∼10 000 U was obtained during one week's continuous production with the maximum activity of ∼750 U l⁻¹ with nylon-web immobilized fungus, veratryl alcohol as an activator, and 2,2-dimethylsuccinate as buffer, although the relatively inexpensive benzyl alcohol and sodium tartrate peformed satisfactorily as the activator and buffer, respectively.     

Production of Phanerochaete chrysosporium lignin peroxidase

Liginin peroxidase (ligninase) of the white rot fungus Phanerochaete chrysosporium Burdsall was discovered in 1982 as a secondary metabolite. Today multiple isoenzymes are known, which are often collectively called as lignin peroxidase. Lignin peroxidase has been characterized as a veratryl alcohol oxidizing enzyme, but it is a relatively unspecific enzyme catalyzing a variety of reactions with hydrogen peroxide as the electron acceptor. P. chrysosporium ligninases are heme glycoproteins. At least a number of isoenzymes are also phosphorylated. Two of the major isoenzymes have been crystallized. Until recently lignin peroxidase could only be produced in low yields in very small scale stationary cultures owing to shear sensitivity. Most strains produce the enzyme only after grown under nitrogen or carbon limitation, although strains producing lignin peroxidase under nutrient sufficiency have also been isolated. Activities over 2000 U dm(-3) (as determined at 30 degrees to 37 degrees C) have been reported in small scale Erlenmeyer cultures with the strain INA-12 grown on glycerol in the presence of soybean phospholipids under nitrogen sufficiency. In about 8 dm(3) liquid volume pilot scale higher than 100 U dm(-3) (as determined at 23 degrees C) have been obtained under agitation with immobilized P. chrysosporium strains ATCC 24725 or TKK 20512. Good results have been obtained for example with nylon web, polyurethane foam, sintered glass or silicon tubing as the carrier. The immobilized biocatalyst systems have also made large scale repeated batch and semicontinuous production possible. With nylon web as the carrier, lignin peroxidase production has recently been scaled up to 800 dm(3) liquid volume semicontinuous industrial production process.     

Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium.

The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme H2 (LiPH2), H2O2, veratryl alcohol, oxalate, ferric chloride, and 1,10-phenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPH2-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants.     

Effect of veratryl alcohol and manganese (IV) oxide on ligninolytic activity in semi solid cultures of Phanerochaete chrysosporium

An inert carrier (nylon sponge), a non-inert carrier (barley straw) and the addition of veratryl alcohol or manganese (IV) oxide to the cultures were used to study the production of ligninolytic enzymes by Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) during semi solid state fermentation conditions. By supplementing the medium with these compounds we could stimulate the ligninolytic system of this fungus. The different carriers employed and the effect of adding veratryl alcohol or manganese (IV) oxide to the cultures were compared in order to determine the best system to produce high activities of ligninolytic enzymes. Lignin peroxidase (LiP) activities higher than 500 U/L and manganese-dependent peroxidase (MnP) activities about 1100 U/L were achieved.     

Comparison of different methods of immobilization for lignin peroxidase production by Phanerochaete chrysosporium

Summary Phanerochaete chrysosporium was immobilized in agar, agarose and -carrageenan gel beads, nylon web, and polyurethane foam, and used for the production of lignin peroxidase in shake cultures on a carbon-limited medium. Nylon was found to be the best carrier, with the maximum lignin peroxidase activity (340 U/l) reached on the 7th day. The enzyme production rate was significantly lower with freely suspended mycelial pellets. Both nylon and polyurethane based biocatalysts were active for at least 38 days after the addition of veratryl alcohol. Best results were obtained when a spore inoculum was used instead of day-old pellets. -Carrageenan was found unsuitable as a carrier for lignin peroxidase production.     

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

A proposed role of oxalic acid in wood decay systems of wood-rotting basidiomycetes

Abstract: The possible roles of oxalic acid, veratryl alcohol, and manganese were investigated in relation to lignin biodegradation by white-rot basidiomycetes. Oxalate inhibited both lignin peroxidase (LiP) and manganese-peroxidase (MnP). and was decarboxylated by the mediation of veratryl alcohol and Mn. Oxalate was shown to regulate the mineralization of lignin in the in vivo system of Phanerochaete chrysosporium . In the brown-rot wood decay process, oxalic acid may serve as an acid catalyst as well as an electron donor for the Fenton reaction, to breakdown cellulose and hemicellulose. Oxaloacetase and glyoxylate oxidase may play a key role in production of oxalic acid by white-rot and brown-rot basidiomycetes such as Phanerochaete chrysosporium, Coriolus versicolor and Tyromyces palustris . A possible role of oxalate metabolism is discussed in relation to the physiology of wood-rotting fungi.     

Engineering of a manganese-binding site in lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

A Mn(2+)-binding site was created in the recombinant lignin peroxidase isozyme H8 from Phanerochaete chrysosporium. In fungal Mn peroxidase, the Mn-binding site is composed of Glu35, Glu39, and Asp179. We generated a similar site in lignin peroxidase by generating an anionic binding site. We generated three mutations: Asn182Asp, Asp183Lys, and Ala36Glu. Its activity, veratryl alcohol, and Mn(2+) oxidation were compared to those of native recombinant enzyme and to fungal Mn peroxidase isozyme H4, respectively. The mutated enzyme was able to oxidize Mn(2+) and still retain its ability to oxidize veratryl alcohol. Steady-state results indicate that the enzyme's ability to oxidize veratryl alcohol was lowered slightly. The K(m) for Mn(2+) was determined to be 1.57 mM and the k(cat) = 5.45 s(-1). These results indicate that the mutated lignin peroxidase is less effective in Mn(2+) oxidation that the wild type fungal enzyme. The pH optima of veratryl alcohol and Mn oxidation were altered by the mutation. They are one unit of pH value higher than those of recombinant H8 and wild type fungal Mn peroxidase isozyme H4.     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

Kinetics of Veratryl Alcohol Oxidation by Lignin Peroxidase and in situ Generated Hydrogen Peroxide in an Electrochemical Reactor

The cathodic reduction of oxygen to hydrogen peroxide, the current efficiency for the production of H₂O₂ and the oxidation of veratryl alcohol with an in situ generated hydrogen peroxide‐lignin peroxidase complex were studied in this paper. The complex was prepared by utilizing a novel preparation technique in an electrochemical reactor. The oxidation of veratryl alcohol (VA; 3,4‐dimethoxybenzyl alcohol) was carried out with or without lignin peroxidase under an electric field. The redox properties of veratryl alcohol on a carbon electrode in the presence of lignin peroxidase have been investigated using cyclic voltammetry. The kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to the oxidation when hydrogen peroxide was supplied externally. Further, the oxidation of veratryl alcohol by lignin peroxidase was optimized in terms of enzyme dosage, pH, and electrical potential. The novel electroenzymatic method was found to be effective using in situ generated hydrogen peroxide for the oxidation of veratryl alcohol by lignin peroxidase.     

Lignin and manganese peroxidase activity in extracts from straw solid substrate fermentations

Manganese and lignin peroxidase (MnP, LiP) activities were measured in straw extracts from cultures of Phanerochaete chrysosporium. Out of six MnP substrates, the MBTH/DMAB (3-methyl-2-benzothiazolinone hydrazone/3-(dimethylamino)benzoic acid), gave the highest MnP activity. Detection of LiP activity as veratryl alcohol oxidation was inhibited by phenols in the straw culture extracts. Appropriate levels of veratryl alcohol and peroxide (4 mM and 0.4 mM, respectively), and a restricted sample volume (not larger than 10%) were necessary to detect activity.     

Inhibition of lignin peroxidase-mediated oxidation activity by ethylenediamine tetraacetic acid and N-N-N'-N'-tetramethylenediamine

The mineralization rate of LC-[1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] (DDT) was reduced by 90% on the 18th day in fungal cultures of Phanerochaete chrysosporium in the presence of 8 mM ethylenediamine tetraacetic acid (EDTA). In the presence of 8 mM N-N-N'-N'-tetramethylenediamine (TEMED), the mineralization rate of 14C-DDT was reduced by 80%. In the presence of 2 mM or 10 mM EDTA, 95% inhibition of lignin peroxidase (LiP) mediated veratryl alcohol oxidase activity and 97% inhibition of LiP mediated iodide oxidase activity occurred. TEMED caused 79% inhibition of veratryl alcohol oxidase activity and 92% inhibition of iodide oxidase activity when the amount used was 2 mM and 10 mM, respectively. In the presence of Zn(II) with slight molar excess of the EDTA concentration, reversed the EDTA mediated non-competitive inhibition of LiP catalyzed veratryl alcohol or iodide oxidation, Zn(II) also reversed the inhibition of LiP catalyzed veratryl alcohol oxidase activity caused by chelators other than EDTA and TEMED. In addition to Zn(II), several other metal ions also relieved EDTA mediated inhibition of veratryl alcohol and iodide oxidase activity catalyzed by LiP. The ability of veratryl alcohol to inhibit iodide oxidation catalyzed by LiP showed that veratryl alcohol could inhibit LiP mediated iodide oxidase activity. Increasing the concentration of iodide was also shown to inhibit veratryl alcohol oxidation. Kinetic analysis showed that the reaction was competitive inhibition.     

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.     

Polymerisation of lignins by ligninases from Phanerochaete chrysosporium

Abstract Four major hemoproteins were purified by isoelectric focusing from an extracellular crude enzyme preparation, produced by the white rot fungus Phanerochaete chrysosporium under carbon-limited conditions. Both the crude enzyme and the purified proteins oxidised milled wood lignin, HCl-dioxane-extracted straw lignin and alkali straw lignin in the presence of hydrogen peroxide. The oxidation resulted mainly in further polymerisation of the lignins and was enhanced by addition of veratryl alcohol to the reaction mixture. Alkali straw lignin was also polymerised by horseradish peroxidase, although veratryl alcohol had no influence on this reaction.     

Metabolism of cyanide by Phanerochaete chrysosporium

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.