In vitro depolymerization of lignin by manganese peroxidase of Phanerochaete chrysosporium

Homogeneous manganese peroxidase catalyzed the in vitro partial depolymerization of four different 14C-labeled synthetic lignin preparations. Gel permeation profiles demonstrated significant depolymerization of 14C-sidechain-labeled syringyl lignin, a 14C-sidechain-labeled syringyl-guaiacyl copolymer (angiosperm lignin), and depolymerization of 14C-sidechain- and 14C-ring-labeled guaiacyl lignins (gymnosperm lignin). 3,5-Dimethoxy-1,4-benzo-quinone, 3,5-dimethoxy-1,4-hydroquinone, and syringylaldehyde were identified as degradation products of the syringyl and syringyl-guaiacyl lignins. These results suggest that manganese peroxidase plays a significant role in the depolymerization of lignin by Phanerochaete chrysosporium.     

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.     

Activation of lignin and manganese peroxidase excretion in Phanerochaete chrysosporium by fungal extracts

Summary Lignin (LiP) and manganese peroxidase (MnP) excretion by Phanerochaete chrysosporium INA-12 was significantly increased in response to fungal extract supplementation. LiP and MnP production was increased 1.7- and 1.8-fold, respectively, with fungal extracts from agitated pellet cultures of strain INA-12, namely fungal extracts P₆ and P₄. In cultures supplemented with a fungal extract harvested from static cultures of strain INA-12 (fungal extract S₄), LiP and MnP production was increased 1.8- and 1.6-fold, respectively. Succinate dehydrogenase activity, a mitochondrial marker, was significantly enhanced (2.7-fold) in cultures with the addition of fungal extracts. Correspondence to: M. Asther     

Optimisation of lignin peroxidase production from Phanerochaete chrysosporium using response surface methodology

The ligninolytic white rot fungus Phanerochaete chrysosporium, holds good promise as a biological treatment tool due to its ability to produce the lignin peroxidase enzyme which has the potential to degrade a wide variety of hazardous compounds. The effective application of this technique requires optimisation of the process variables to maximise the enzyme production. Response surface methodology was applied to determine the effects of glucose, ammonium tartarate and ferrous sulphate and their mutual interactions on lignin peroxidase production. With a view to simultaneously reducing the number of experiments and obtaining more information on the mutual interactions between the variables, a 2³ full-factorial central composite experimental design was adopted. The experimental data were fitted to a second order polynomial equation using multiple regression analysis and also analysed by appropriate statistical methods. Solving the regression equation using the multi-stage monte-carlo optimisation techniques, the optimum process conditions for enhanced production of lignin peroxidase were obtained as: glucose 0.9728 kg/m³, ammonium tartarate 0.288 kg/m³, and ferrous sulphate 0.097 kg/m³.     

Solid-state fermentation of natural birch lignin by Phanerochaete chrysosporium

A strain of white rot fungus, Phanerochaete chrysosporium Burds. ME446, has been characterized with respect to the extent and rate of Betula nigrificans lignin and non-lignin conversion by solid-substrate fermentation for different culture conditions. Moisture content, inoculum density, nitrogen supplementation and autoclaving of birch solids significantly affected lignin conversion rates and yields in 20 day fermentations. Oxygen favoured lignin over non-lignin conversion at partial pressures of 1.0 atm. Oxygen pressures of 2.0 atm severely inhibited both lignin and non-lignin conversions. Carbon dioxide partial pressures of 0.25, 0.5 and 1.0 atm at oxygen pressures of 1.0 atm increasingly inhibited both lignin and non-lignin conversion rates and yields. The results of these studies demonstrate the effects of major process variables and suggest a need to control the gas environment for process optimization.     

The effect of agitation and nitrogen concentration on lignin peroxidase (LiP) isoform composition during fermentation of Phanerochaete chrysosporium

Convective Interaction Media (CIM) monolithic columns were applied for the HPLC monitoring of Phanerochaete chrysosporium lignin peroxidase (LiP) isoforms during cultivation. The influence of the agitation mode (circular, elliptic) and rate (130 and 200 rpm), as well as the initial nitrogen concentration (1.6-6 mM) in the growth medium was investigated. Identical rotation rate but different agitation modes resulted in different LiP activities and isoenzyme compositions. On the other hand, at different agitation types and rates, similar LiP activities were obtained at different isoenzyme compositions. Although LiP H2 and LiP H6/H7 were predominant isoenzymes obtained at various cultivation conditions, relative isoenzyme amounts differ considerably when initial nitrogen concentration was changed between 1.6 and 5 mM.     

Degradation of synthetic lignin by the protoplasts of Phanerochaete chrysosporium in the presence of lignin peroxidase or manganese peroxidase

Lignin was mineralized in the experiments in which ¹⁴C-lignin was incubated with lignin peroxidase or manganese peroxidase in a tartrate buffer in the presence of cycloheximide-treated protoplasts obtained from the ligninolytic mycelia of Phanerochaete chrysosporium. The rate of lignin mineralization was dependent on the lignin peroxidase or manganese peroxidase concentration in the medium. In the experiments in which lignin was incubated with lignin peroxidase or manganese peroxidase, lignin was repolymerized irrespective of the presence of protoplasts mineralizing lignin, suggesting that an active degradation of lignin and repolymerization took place. Taking into account that lignin peroxidase and manganese peroxidase were the only extracellular enzymes in the experiments in which lignin was mineralized by the protoplasts, it is postulated that lignin peroxidase and/or manganese peroxidase can degrade lignin into small fragments which can then be further absorbed by the fungal cells and subsequently degraded to CO₂.     

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.     

Removal of hazardous compounds by lignin peroxidase from Phanerochaete chrysosporium

Phenolic compounds, which are present in many industrial wastewaters, have become a cause for worldwide concern due to their persistence, toxicity and health risks. Enzymatic approaches to remove phenol have been tried for some years as they have several advantages compared with the conventional methods. This paper reports some studies on the use of the white rot fungus Phanerochaete chrysosporium which produces the enzyme lignin peroxidases for the removal of phenol, chlorophenol, and dyes. Batch studies in Erylenmeyer flasks showed complete removal of phenol (500 2 10^х kg/m³) in 30 h. It was also seen that phenol has a significant inhibitory effect on the biomass growth and the enzyme synthesis if added in the early stages of the growth. However, phenol was effectively removed when added after attaining the maximum enzyme activity. 90% of the dyes were removed in about three days, whereas only 62% of the added 4-chlorophenol was removed in about ten days.     

Influence of lignin peroxidase concentration and localisation in lignin biodegradation by Phanerochaete chrysosporium

Summary The lignin mineralization rate in cultures of Phanerochaete chrysosporium increases with lignin peroxidase concentration up to 20 nkat ml^–1. At higher concentrations the rate of lignin mineralization decreases with increasing lignin peroxidase concentration. The amount of mycelium is not a limiting factor for lignin mineralization at high exocellular lignin peroxidase in association with the mycelium as pellets and no free exocellular enzyme induce a lignin mineralization rate equivalent to cultures reconstituted with washed pellets supplemented with 15 nkat ml^–1 of exogenous free enzyme. These results show that although lignin degradation by lignin peroxidase seems to be facilitated when lignin peroxidase is localised on the surface of the mycelium, free exocellular lignin peroxidase can also efficiently enhance mineralization of lignin by P. chrysosporium.     

Utilisation of lignocellulosic wastes for lignin peroxidase production by semi-solid-state cultures of Phanerochaete chrysosporium

In the present work, the production of ligninolytic enzymes by semi-solid-statecultures of Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725),employing different lignocellulosic wastes as support, was investigated. Thewaste materials employed were grape seeds, wheat straw and wood shavings.Maximum lignin peroxidase activities of 1620 ± 123 U/l, 364 ± 35 U/l and 571 ± 42 U/l were attained, respectively. Nevertheless, lowmanganese-dependent peroxidase activities were found, being insignificantin the grape seed cultures. Moreover, the in vivo decolourisation of a model dye compound, the polymeric dye Poly R-478 (polyvinylamine sulfonateanthrapyridone), by the above-mentioned cultures was monitored to assessthe degrading capability of the extracellular liquid secreted by such cultures.The percentage of biological decolourisation attained by grape seed and woodshaving cultures was around 74% and 63%, respectively, whereas it was ratherlow (40%) in the wheat straw ones.     

Cleavage of nonphenolic beta-1 diarylpropane lignin model dimers by manganese peroxidase from Phanerochaete chrysosporium.

Purified manganese peroxidase (MnP) from Phanerochaete chrysosporium oxidizes nonphenolic beta-1 diarylpropane lignin model compounds in the presence of Tween 80, and in three- to fourfold lower yield in its absence. In the presence of Tween 80, 1-(3',4'-diethoxyphenyl)-1-hydroxy-2-(4'-methoxyphenyl)propane (I) was oxidized to 3,4-diethoxybenzaldehyde (II), 4-methoxyacetophenone (III) and 1-(3',4'-diethoxyphenyl)-1-oxo-2-(4'-methoxyphenyl)propane (IV), while only 3,4-diethoxybenzaldehyde (II) and 4-methoxyacetophenone (III) were detected when the reaction was conducted in the absence of Tween 80. In contrast to the oxidation of this substrate by lignin peroxidase (LiP), oxidation of substrates by MnP did not proceed under anaerobic conditions. When the dimer (I) was deuterated at the alpha position and subsequently oxidized by MnP in the presence of Tween 80, yields of 3,4-diethoxybenzaldehyde, 4-methoxyacetophenone remained constant, while the yield of the alpha-keto dimeric product (IV) decreased by approximately sixfold, suggesting the involvement of a hydrogen abstraction mechanism. MnP also oxidized the alpha-keto dimeric product (IV) to yield 3,4-diethoxybenzoic acid (V) and 4-methoxyacetophenone (III), in the presence and, in lower yield, in the absence of Tween 80. When the reaction was performed in the presence of 18O2, both products, 3,4-diethoxybenzoic acid and 4-methoxyacetophenone, contained one atom of 18O. Finally, MnP oxidized the substrate 1-(3',5'-dimethoxyphenyl)-1-hydroxy-2-(4'-methoxyphenyl)propane (IX) to yield 3,5-dimethoxybenzaldehyde (XI), 4-methoxyacetophenone (III) and 1-(3',5'-dimethoxyphenyl)-1-oxo-2-(4'-methoxyphenyl)propane (X). In sharp contrast, LiP was not able to oxidize IX. Based on these results, we propose a mechanism for the MnP-catalyzed oxidation of these dimers, involving hydrogen abstraction at a benzylic carbon, rather than electron abstraction from an aromatic ring.     

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.     

Effects of temperature on the production of manganese peroxidase and lignin peroxidase byPhanerochaete chrysosporium

Growth temperature played an important role in the appearance, maximum level and ratio of manganese peroxidase (MnP) and lignin peroxidase (LIP) activities in the cultures ofPhanerochaete chrysosporium. While at higher temperatures (39, 33, and 28°C) both enzymes were produced (with LIP being the major one) at 23°C MnP was dominant. At 18°C, of the two ligninolytic peroxidases only MnP activity was detected. Decrease of proteolytic activity at lower temperatures probably contributed to the retention of MnP and LIP activities.     

Decolorization of synthetic textile dyes by lignin peroxidase of Phanerochaete chrysosporium

Neem hull waste (containing a high amount of lignin and other phenolic compounds) was used for lignin peroxidase production byPhanerochaete chrysosporum under solid-state fermentation conditions. Maximum decolorization achieved by partially purified lignin peroxidase was 80% for Porocion Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for Navidol Fast Black MSRL. The effects of different concentrations of veratryl alcohol, hydrogen peroxide, enzyme and dye on the efficiency of decolorization have been investigated. Maximum decolorization efficiency was observed at 0.2 and 0.4 mmol/L hydrogen peroxide, 2.5 mmol/L veratryl alcohol and pH 5.0 after a 1-h reaction, using 50 ppm of dyes and 9.96 mkat/L of enzyme.     

Characterization of two lignin peroxidase clones from Phanerochaete chrysosporium

Two cDNA clones encoding lignin peroxidase isozymes from Phanerochaete chrysosporium have been isolated and characterized. One of the clones, lambda ML-4, encodes isozyme H8 as does the previously reported clone lambda ML-1 [Tien, M. and Tu, C.-P.D. Nature 326 (1987) 520-523; 328, 742]. Our data are consistent with lambda ML-1 and lambda ML-4 being allelic variants. The other clone, lambda ML-5, encodes a homologous isozyme. We have also isolated the genomic clone corresponding to lambda ML-4 cDNA. Conserved residues thought to be essential for peroxidase function were identified in the predicted amino acid sequences of both cDNA clones. Northern blot analyses indicate that these isozymes are expressed during secondary metabolism, appearing on day 4 of growth and increasing on days 5 and 6.     

Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium

The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter was used to drive expression of lip2, the gene encoding lignin peroxidase (LiP) isozyme H8, in primary metabolic cultures of Phanerochaete chrysosporium. The expression vector, pUGL, also contained the Schizophyllum commune ura1 gene as a selectable marker. pUGL was used to transform a P. chrysosporium Ura11 auxotroph to prototrophy. Ura+ transformants were screened for peroxidase activity in liquid cultures containing high-carbon and high-nitrogen medium. Recombinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous lip genes were not expressed under these conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained after purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPh8 (wt LiPH8), indicating that heme insertion, folding, and secretion functioned normally in the transformant. Steady-state and transient-state kinetic properties for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were also identical.     

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.     

Oxidation of aromatic sulfides by lignin peroxidase from Phanerochaete chrysosporium.

The reaction of H2O2 with 4-substituted aryl alkyl sulfides (4-XC6H4SR), catalysed by lignin peroxidase (LiP) from Phanerochaete chrysosporium, leads to the formation of sulfoxides, accompanied by diaryl disulfides. The yields of sulfoxide are greater than 95% when X = OMe, but decrease significantly as the electron donating power of the substituent decreases. No reaction is observed for X = CN. The bulkiness of the R group has very little influence on the efficiency of the reaction, except for R = tBu. The reaction exhibits enantioselectivity (up to 62% enantiomeric excess with X = Br, with preferential formation of the sulfoxide with S configuration). Enantioselectivity decreases with increasing electron density of the sulfide. Experiments in H218O show partial or no incorporation of the labelled oxygen into the sulfoxide, with the extent of incorporation decreasing as the ring substituents become more electron-withdrawing. On the basis of these results, it is suggested that LiP compound I (formed by reaction between the native enzyme and H2O2), reacts with the sulfide to form a sulfide radical cation and LiP compound II. The radical cation is then converted to sulfoxide either by reaction with the medium or by a reaction with compound II, the competition between these two pathways depending on the stability of the radical cation.