Lignin Peroxidase Isoenzymes Research Articles

Characterization of Lignin Peroxidase from the Suspected Novel Strain <i>Phanerochaete chrysosporium</i> ITB Isolate

This study was aimed to characterize lignin peroxidase (LiP) obtained from Phanerochaete chrysosporium ITB isolate. The characterizations included molecular weight, the pH and optimum working temperature of the crude extract of the enzyme, the temperature stability, the effect of metal ions and inhibitors, their precipitation with ethanol, and the storage stability. The LiP of P. chrysosporium ITB isolates was 34 kDa. The crude extract of LiP displays high activity at pH between 3 until 5 and 26–32 °C, has good thermal stability at 26–32 °C for 20 h. The activity is affected by Pb2+, K+, Co2+, Fe2+, Cd2+, Mg2+, and Cu2+ EDTA, Na+, Cr3+, Hg2+, NaN3, Ni2+, and Ca2+ ions, is not affected by Mn2+ and Zn2+ ions, precipitated with the optimum ethanol at 64% ethanol saturation which results in an increase in specific activity of 2.3 times. The crude extract storage at 0 °C is more stable than the precipitate resulting from ethanol precipitation and resuspension from ethanol precipitation. These results strengthen that LiP from P. chrysosporium is another LiP isoenzyme that can be used for bioremediation processes. Unfortunately, the concentration using the ethanol precipitation method has not been effective, so further studies using other methods should be required.

Lignin peroxidase isoenzyme: a novel approach to biodegrade the toxic synthetic polymer waste

ABSTRACTFungal metabolites are playing an immense role in developing various sustainable waste treatment processes. The present study aimed at production and characterization of fungal lignin peroxidase (EC 1.11.1.14) with a potential to degrade Polyvinyl Chloride. Optimization studies revealed that the maximum enzyme production occurred at a temperature 25°C, pH 5 in the 4th week of the incubation period with fungal strain. Enzyme assay was performed to find out the dominating enzyme in the culture broth. The molecular weight of the enzyme was found to be 46 kDa. Partially purified lignin peroxidase from Phanerocheate chrysosporium was used for the degradation of PVC films. A significant reduction in the weight of PVC film was observed (31%) in shake flask experiment. FTIR spectra of the enzyme-treated plastic film revealed structural changes in the chemical composition, indicating a specific peak at 2943 cm−1 that corresponded to alkenyl C–H stretch. Moreover, deterioration on the surface of PVC films was confirmed by Scanning Electron Microscopy tracked through activity assay for the lignin peroxidase. Extracellular lignin peroxidases from P. chrysosporium play a significant role in the degradation of complex polymeric compounds like PVC.

Factors Affecting the Induction of Lignin Peroxidase in Manganese-Deficient Cultures of the White Rot Fungus <i>Phanerochaete chrysosporium</i>

The lignin peroxidase (LIP) production and regulation, in manganese ions (Mn2+) deficient cultures of the white rot fungus Phanerochaete chrysosporium, is still not clearly understood. Mn2+ deficiency is correlated to low levels of manganese containing superoxide dismutase (MnSOD). In this work, we show that despite the low activity level of MnSOD in Mn2+-deficient cultures, the presence of H2O2 is essential for the expression of the lip-H2 gene, which encodes for the major LIP isoenzyme produced (LIP-H2). Thus, the H2O2 present in Mn2+-deficient cultures is probably produced by other mechanisms rather than dismutation of superoxide ions by MnSOD. Glyoxal oxidase gene (glox) expression was significantly higher than MnSOD (MnSOD1) and cellobiose dehydrogenase (cdh1) expression in Mn2+-deficient cultures, indicating its clear involvement in H2O2 production in those cultures. Glyoxal oxidase may compensate the absence of MnSOD activity in Mn2+-deficient cultures. The high levels of reactive oxygen species (ROS) needed for the enhancement of LIP expression in Mn2+-deficient cultures were not directly correlated to the protein kinase C (PKC) activity involved in signal transduction pathway. High level of oxidative stress was observed in MnSOD silenced mutants, grown in the presence of Mn2+, indicating that oxidative stress in Mn2+-deficient cultures was caused by low levels of MnSOD rather than the deficiency in Mn2+. The results of this work can further contribute to the understanding of LIP regulation in Mn2+-deficient cultures.

Southern blot screening for lignin peroxidase and aryl-alcohol oxidase genes in 30 fungal species

Screening to detect genes encoding lignin peroxidase (LiP) and aryl-alcohol oxidase (AAO) has been carried out with 30 fungal strain using DNA probes from genes lpo of Phanerochaete chrysosporium (encoding LiP isoenzyme H8) and aao of Pleurotus eryngii. Evidence for the presence of genes closely related to lpo was found in Bjerkandera adusta, Fomes fomentarius, Ganoderma applanatum, Ganoderma australe, Lentinula degener, Peniophora gigantea, P. chrysosporium, Phanerochaete flavido-alba and Trametes versicolor, whereas the gene aao was detected in Pleurotus species and B. adusta. The presence of both genes was only detected in B. adusta. These results suggest that different enzymatic system, formed by enzymes encoded by different genes, are responsible for lignin degradation by white-rot fungi.

Reversible alkaline inactivation of lignin peroxidase involves the release of both the distal and proximal site calcium ions and bishistidine co-ordination of the haem

Phanerochaete chrysosporium lignin peroxidase isoenzyme H2 (LiP H2) exhibits a transition to a stable, inactive form at pH 9.0 with concomitant spectroscopic changes. The Söret peak intensity decreases some 55% with a red shift from 408 to 412 nm; the bands at 502 nm and 638 nm disappear and the peak at 536 nm increases. The EPR spectrum changes from a signal typical of high spin ferric haem to an exclusively low spin spectrum with g=2.92, 2.27, 1.50. These data indicate that the active pentaco-ordinated haem is converted into a hexaco-ordinated species at alkaline pH. Room temperature near-IR MCD data coupled with the EPR spectrum allow us to assign the haem co-ordination of alkali-inactivated enzyme as bishistidine. Re-acidification of the alkali-inactivated enzyme to pH 6 induces further spectroscopic changes and generates an irreversibly inactivated species. By contrast, a pH shift from 9.0 to 6.0 with simultaneous addition of 50 mM CaCl(2) results in the recovery of the initial activity together with the spectroscopic characteristics of the native ferric enzyme. Incubating with 50 mM CaCl(2) at a pH between 6.0 and 9.0 can also re-activate the enzyme. Divalent metals other than Ca(2+) do not result in restoration of activity. Experiments with (45)Ca indicate that two tightly bound calcium ions per enzyme monomer are lost during inactivation and reincorporated during subsequent re-activation, consistent with the presence of two structural Ca(2+) ions in LiP H2. It is concluded that both the structural Ca(2+) ions play key roles in the reversible alkaline inactivation of LiP H2.

A Method of Fast Separation of Lignin Peroxidases Using Convective Interaction Media Disks

The HPLC separation of lignin peroxidase isoenzymes using Convective Interaction Media disks containing quaternary amine and diethylaminoethyl ion-exchange active groups is proposed. In contrast to standard HPLC procedures the separation can be performed within a few minutes without considerably affecting the separation resolution. The method is reproducible and gives a linear response of integrated peak area to protein concentration for all measured isoenzymes. The separation resolution is retained unchanged by applying crude culture filtrate instead of a sample previously frozen and dialyzed. The optimized method might therefore be used for on-line monitoring of lignin peroxidase isoenzyme composition during fermentation. On the other hand, the proposed method is comparable in time to the original method of lignin peroxidase activity measurement (proposed by Tien and Kirk), providing additionally the isoenzyme composition.

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 fungusPhanerochaete 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 inEscherichia coli.The kcatfor veratryl alcohol oxidation was 11 s−1, Kmfor veratryl alcohol ∼0.49 mM, and Kmfor hydrogen peroxide ∼25 μM at pH 2.3. The Kmfor veratryl alcohol was higher and Kmfor 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 kcatwas ∼2.6 × 102s−1at 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.

Oxidation of Crocein Orange G by lignin peroxidase isoenzymes. Kinetics and effect of H2O2.

The ligninolytic enzyme system of Phanerochaete chrysosporium is able to decolorize several recalcitrant dyes. Three lignin peroxidase isoenzymes, LiP 3.85, LiP 4.15, and LiP 4.65, were purified by preparative isoelectric focusing from the carbon-limited culture medium of P. chrysosporium. Based on amino terminal sequences, the purified isoenzymes correspond to the isoenzymes H8, H6, and H2, respectively, from the N-limited culture. The purified isoenzymes were used for decolorization of an azo dye, Crocein Orange G (COG). According to the kinetic data obtained, the oxidation of COG by lignin peroxidase appeared to follow Michaelis-Menten kinetics. Kinetic parameters for each isoenzyme were determined. The inactivating effect of ascending H2O2 concentrations on COG oxidation is shown to be exponential within the used concentration range. The best degree of decolorization of 100 microM COG was obtained when the H2O2 concentration was 150 microM. This was also the lowest H2O2 concentration for maximal decolorization of 100 microM COG, regardless of the amount of lignin peroxidase used in the reaction.

Purification of Major Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium by Chromatofocusing

The basidiomycete Phanerochaete chrysosporium produces several isoforms of lignin peroxidase, which catalyzes the oxidative depolymerization of lignin. To date, ion-exchange chromatography and preparative isoelectric focusing (IEF) have been commonly used for isolation of lignin peroxidase isoenzymes. In this work we have purified major lignin peroxidases to high purity by a one-step chromatographic method, chromatofocusing. The purified isoenzymes were identified by analytical IEF using isoenzymes purified by preparative IEF as standards. The specific activities and spectral properties of the isoenzymes were comparable with the previously published data. The predominant isoenzyme under the growth conditions used was LiP 4.65. Almost 50% of the lignin peroxidase activity applied into the column was recovered in the LiP 4.65 fraction. The total recovery of the lignin peroxidase activity was over 80%.

Secretion of Ligninolytic Enzymes and Mineralization of 14 C-Ring-Labelled Synthetic Lignin by Three Phlebia tremellosa Strains

Production of ligninolytic enzymes by three strains of the white rot fungus Phlebia tremellosa (syn. Merulius tremellosus) was studied in bioreactor cultivation under nitrogen-limiting conditions. The Mn(II) concentration of the growth medium strongly affected the secretion patterns of lignin peroxidase and laccase. Two major lignin peroxidase isoenzymes were expressed in all strains. In addition, laccase and glyoxal oxidase were purified and characterized in one strain of P. tremellosa. In contrast, manganese peroxidase was not found in fast protein liquid chromatography profiles of extracellular proteins under either low (2.4 muM) or elevated (24 and 120 muM) Mn(II) concentrations. However, H(2)O(2)- and Mn-dependent phenol red-oxidizing activity was detected in cultures supplemented with higher Mn(II) levels. Mineralization rates of C-ring-labelled synthetic lignin (i.e., dehydrogenation polymerizate) by all strains under a low basal Mn(II) level were similar to those obtained for Phanerochaete chrysosporium and Phlebia radiata. A high manganese concentration repressed the evolution of CO(2) even when a chelating agent, sodium malonate, was included in the medium.

Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium

The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.

An improved method for the purification of lignin peroxidases from phanerochaete chrysosporium INA-12: properties of two major isoforms

1. 1. Phanerochaete chrysosporium INA-12 secretes several lignin peroxidase isoenzymes. This paper reports an improved procedure for the purification of the different isoforms compared to those previously described. 2. 2. Lignin peroxidases are first concentrated and prefractionated on fast-flow ion-exchangers which avoid concentration by ultrafiltration and dialysis. 3. 3. Further purification is achieved by hydrophobic interaction chromatography and anion-exchange FPLC. 4. 4. Two major forms were purified to homogeneity. Kinetic measurements and protein characterization (isoelectric points, phosphate content) suggest that they are similar to those produced by P. chrysosporium BKM strain.

The Onset of Lignin-Modifying Enzymes, Decrease of AOX and Color Removal by White-Rot Fungi Grown on Bleach Plant Effluents

Decrease of adsorbable organic chlorine (AOX) is becoming the most important criterion for the efficiency of pulp mill effluent treatment in the 1990s. Two methods, designated MYCOR and MYCOPOR which utilize the white-rot fungus Phanerochaete chrysosporium have earlier been developed for the color removal of pulp mill effluents, but the processes have also a capacity to decrease the amount of chlorinated organic compounds. Lignin peroxidases (ligninases) produced by P. chrvsosporium may dechlorinate chlorinated phenols. In this work possibilities to use selected white-rot fungi in the treatment of E1-stage bleach plant effluent were studied. Phlebia radiata. Phanerochaete chrvsosporium and Merulius (Phlebia) tremellosus were compared in shake flasks for their ability to produce laccase, lignin peroxidase(s) and manganese-dependent peroxidase(s) and to remove color from a medium containing effluent. Softwood bleaching effluents were treated by carrier-immobilized P. radiata in 2 1 bioreactors and a 10 1 BiostatR -fermentor. Dechlorination was followed using Cl ion and AOX determinations. All fungi removed the color of the effluent. In P. radiata cultivations AOX decrease was ca. 4 mg l−1 in one day. Apparent lignin peroxidase activities as determined by veratryl alcohol oxidation method were negligible or zero in a medium with AOX content of ca. 60 mg l−1, prepared using about 20 % (v/v) of softwood effluent. However, the purification of extracellular enzymes implied that large amounts of lignin peroxidases were present in the medium and, after the purification, in active form. Enzyme proteins were separated using anion exchange chromatography, and they were further characterized by electrophoresis (SDS-PAGE) to reveal the kind of enzymes that were present during AOX decrease and color removal. The most characteristic lignin peroxidase isoenzymes in effluent media were LiP2 and LiP3.

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.

Mn(II) Regulation of Lignin Peroxidases and Manganese-Dependent Peroxidases from Lignin-Degrading White Rot Fungi

Two families of peroxidases-lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)-are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of CO(2) from C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of CO(2) release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium, two other species of Phanerochaete, three species of Phlebia, Lentinula edodes, and Phellinus pini.

Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium

Phanerochaete chrysosporium BKM-1767 secretes multiple lignin peroxidase isoenzymes when grown under nitrogen-limited conditions. Here we report the purification of these heme-containing peroxidases, and their physical and catalytic characterization. Ten hemeproteins, designated H1–H10, were separated by anion exchange HPLC. Six of them, H1, H2, H6, H7, H8, and H10, were lignin peroxidases, oxidizing veratryl alcohol in the presence of H 2O 2. The other four (three peaks were resolved) exhibited manganese-dependent peroxidase activity, oxidizing vanillylacetone in the presence of H 2O 2 and Mn +2. The lignin peroxidases have different isoelectric points, between p14.7 and 3.3, and molecular weights between 38 and 43 kDa, determined by SDS-PAGE. All are N- and probably O-glycosylated. Three organic substrates and H 2O 2 were used to compare their kinetic properties: the organic substrates were veratryl alcohol, 1,4-dimethoxybenzene, and the lignin model compound 1-(3,4-dimethoxyphenyl)-2-( o-methoxyphenoxy)-propane-1,3-diol. K M and TN values for each of these substrates varied significantly; e.g. for veratryl alcohol K M values were from 86 to 480 μ m and TN values were from 1.3 to 8.3 s -1. The ranking of the isoenzyme activities differed with the different substrates, suggesting differences in affinities or in active site accessibilities. The K M for H 2 O 2 varied between 13 and 77 μ m. Immunological blot analysis and partial proteolytic digestion patterns showed that the isoenzymes have a high degree of homology. The isoenzyme concentrations in extracellular culture fluid were found to vary relatively and absolutely with culture time. A nomenclature scheme for these 10 hemeproteins has been proposed. This scheme should simplify identification of these proteins in the literature as well as be adaptable to others found in Phanerochaete chrysosporium.