Lignin Peroxidase Isozyme Research Articles

Selective Adsorption of Lignin Peroxidase on Lignin for Biocatalytic Conversion of Poplar Wood Biomass to Value-Added Chemicals.

Lignin-first biorefineries aim to maximize the valorization of lignin by prioritizing its conversion over other biomass components. This study investigates the selective adsorption of lignin peroxidase (LiP) isozymes from white rot fungi Phanerochaete chrysosporium on lignin, aiming to enhance the biocatalytic conversion of poplar wood biomass into value-added chemicals. The research focuses on the adsorption characteristics of ten recombinant LiP isozymes, particularly PcLiP03, which exhibited the highest adsorption capacity on lignin and negligible adsorption on cellulose. Adsorption isotherms and structural analyses revealed that hydrophobic interactions significantly contribute to the selective adsorption of PcLiP03. Applying PcLiP03 in a continuous stirring cell system effectively converted lignin in unpretreated poplar wood to 2,6-dimethoxy-1,4-benzoquinone under ambient and mild conditions, highlighting its potential for selective lignin depolymerization in integrated biorefineries. This work underscores the importance of enzyme-substrate interactions and offers a promising approach for more efficient and sustainable biomass utilization.

Experimental and theoretical insights into the effects of pH on catalysis of bond-cleavage by the lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

BackgroundLignin peroxidases catalyze a variety of reactions, resulting in cleavage of both β-O-4′ ether bonds and C–C bonds in lignin, both of which are essential for depolymerizing lignin into fragments amendable to biological or chemical upgrading to valuable products. Studies of the specificity of lignin peroxidases to catalyze these various reactions and the role reaction conditions such as pH play have been limited by the lack of assays that allow quantification of specific bond-breaking events. The subsequent theoretical understanding of the underlying mechanisms by which pH modulates the activity of lignin peroxidases remains nascent. Here, we report on combined experimental and theoretical studies of the effect of pH on the enzyme-catalyzed cleavage of β-O-4′ ether bonds and of C–C bonds by a lignin peroxidase isozyme H8 from Phanerochaete chrysosporium and an acid stabilized variant of the same enzyme.ResultsUsing a nanostructure initiator mass spectrometry assay that provides quantification of bond breaking in a phenolic model lignin dimer we found that catalysis of degradation of the dimer to products by an acid-stabilized variant of lignin peroxidase isozyme H8 increased from 38.4% at pH 5 to 92.5% at pH 2.6. At pH 2.6, the observed product distribution resulted from 65.5% β-O-4′ ether bond cleavage, 27.0% Cα-C1 carbon bond cleavage, and 3.6% Cα-oxidation as by-product. Using ab initio molecular dynamic simulations and climbing-image Nudge Elastic Band based transition state searches, we suggest the effect of lower pH is via protonation of aliphatic hydroxyl groups under which extremely acidic conditions resulted in lower energetic barriers for bond-cleavages, particularly β-O-4′ bonds.ConclusionThese coupled experimental results and theoretical explanations suggest pH is a key driving force for selective and efficient lignin peroxidase isozyme H8 catalyzed depolymerization of the phenolic lignin dimer and further suggest that engineering of lignin peroxidase isozyme H8 and other enzymes involved in lignin depolymerization should include targeting stability at low pH.

Unassisted solar lignin valorisation using a compartmented photo-electro-biochemical cell

Lignin is a major component of lignocellulosic biomass. Although it is highly recalcitrant to break down, it is a very abundant natural source of valuable aromatic carbons. Thus, the effective valorisation of lignin is crucial for realising a sustainable biorefinery chain. Here, we report a compartmented photo-electro-biochemical system for unassisted, selective, and stable lignin valorisation, in which a TiO2 photocatalyst, an atomically dispersed Co-based electrocatalyst, and a biocatalyst (lignin peroxidase isozyme H8, horseradish peroxidase) are integrated, such that each system is separated using Nafion and cellulose membranes. This cell design enables lignin valorisation upon irradiation with sunlight without the need for any additional bias or sacrificial agent and allows the protection of the biocatalyst from enzyme-damaging elements, such as reactive radicals, gas bubbles, and light. The photo-electro-biochemical system is able to catalyse lignin depolymerisation with a 98.7% selectivity and polymerisation with a 73.3% yield using coniferyl alcohol, a lignin monomer.

Recombinant lignin peroxidase-catalyzed decolorization of melanin using in-situ generated H2O2 for application in whitening cosmetics

Lignin peroxidase has high potential as ingredient in skin whitening cosmetics due to its high redox potential to oxidize recalcitrant melanin. Currently crude mixtures of lignin peroxidase from fungal fermentation are usually applied to cosmetics due to the intrinsic difficulties of expression and purification. However, the present study focused on heterologous expression and purification of lignin peroxidase isozyme H8 (LiPH8) from Phanerochaete chrysosporium and was further used for melanin decolorization. Results revealed that the optimum pH for melanin decolorization using LiPH8 was obtained at pH 4.0. The intermittent feeding of hydrogen peroxide (H2O2) was effectively elevating melanin decolorization efficiency up to 73%, since excessive H2O2 inactivated LiPH8. For cosmetic application, intermittent feeding of H2O2 is not feasible, thus glucose oxidase (GOx) from Aspergillus niger was employed for in-situ generation of H2O2. By optimizing the GOx and glucose concentrations, a melanin decolorization efficiency up to 63.3 ± 2.4% was obtained within 1 h and continued to 84.0 ± 1.8% in 8 h. Conclusively, lignin peroxidase-catalyzed decolorization of melanin with in-situ generated H2O2 revealed a promising approach for whitening cosmetics applications.

In silico-designed lignin peroxidase from Phanerochaete chrysosporium shows enhanced acid stability for depolymerization of lignin

BackgroundThe lignin peroxidase isozyme H8 from the white-rot fungus Phanerochaete chrysosporium (LiPH8) demonstrates a high redox potential and can efficiently catalyze the oxidation of veratryl alcohol, as well as the degradation of recalcitrant lignin. However, native LiPH8 is unstable under acidic pH conditions. This characteristic is a barrier to lignin depolymerization, as repolymerization of phenolic products occurs simultaneously at neutral pH. Because repolymerization of phenolics is repressed at acidic pH, a highly acid-stable LiPH8 could accelerate the selective depolymerization of recalcitrant lignin.ResultsThe engineered LiPH8 was in silico designed through the structural superimposition of surface-active site-harboring LiPH8 from Phanerochaete chrysosporium and acid-stable manganese peroxidase isozyme 6 (MnP6) from Ceriporiopsis subvermispora. Effective salt bridges were probed by molecular dynamics simulation and changes to Gibbs free energy following mutagenesis were predicted, suggesting promising variants with higher stability under extremely acidic conditions. The rationally designed variant, A55R/N156E-H239E, demonstrated a 12.5-fold increased half-life under extremely acidic conditions, 9.9-fold increased catalytic efficiency toward veratryl alcohol, and a 7.8-fold enhanced lignin model dimer conversion efficiency compared to those of native LiPH8. Furthermore, the two constructed salt bridges in the variant A55R/N156E-H239E were experimentally confirmed to be identical to the intentionally designed LiPH8 variant using X-ray crystallography (PDB ID: 6A6Q).ConclusionIntroduction of strong ionic salt bridges based on computational design resulted in a LiPH8 variant with markedly improved stability, as well as higher activity under acidic pH conditions. Thus, LiPH8, showing high acid stability, will be a crucial player in biomass valorization using selective depolymerization of lignin.

Purification and Characterization of Lignin Peroxidase Isozymes from Humicola grisea (Traaen) and Its Application in Bioremediation of Textile Dyes

PEROXIDASES have numerous important applications in industries and biodegradation of organopollutants , so there is a need to explore more and more sources of enzymes with different characteristics. In the present study, lignin peroxidases (LiPs) producing by fungus Humicola grisea were extracted and purified using gel filtration and ion exchange chromatography. Protein and heme protein were eluted in different peaks about 6 of these peaks had LiP activity and this is an indication that LiP in H. grisea was isoenzymes. Four of these isoenzymes were characterized regarding their molecular mass, optimum temperature and optimum pH for activity, substrate concentration and effect of some metal ions. The four purified isoenzymes have maximum activities at different temperatures, where optimum temperature for H2 and H4 isoenzyme were at 60 oC, and the optimum temperatures for H1 and H3 were at 40 oC and 30 oC, respectively. The maximum activities of the four isoenzymes were within the acidic range. The different LiP isoenzymes showed different Km and Vmax values representing the varied affinity of the four isoenzymes using veratryl alcohol as a substrate. Km values for H1, H2, H3 and H4 were 5.26, 5.0, 1.3 and 1.4, respectively. While Vmax values for the same isoenzymes were 1.25, 0.8, 0.87 and 1.1, respectively.

Improvement of catalytic performance of lignin peroxidase for the enhanced degradation of lignocellulose biomass based on the imbedded electron-relay in long-range electron transfer route.

BackgroundAlthough lignin peroxidase is claimed as a key enzyme in enzyme-catalyzed lignin degradation, in vitro enzymatic degradation of lignin was not easily observed in lab-scale experiments. It implies that other factors may hinder the enzymatic degradation of lignin. Irreversible interaction between phenolic compound and lignin peroxidase was hypothesized when active enzyme could not be recovered after the reaction with degradation product (guaiacol) of lignin phenolic dimer.ResultsIn the study of lignin peroxidase isozyme H8 from white-rot fungi Phanerochaete chrysosporium (LiPH8), W251 site was revealed to make the covalent coupling with one moiety of monolignolic radical (guaiacol radical) by LC-MS/MS analysis. Hypothetical electron-relay containing W251 residue was newly suggested based on the observation of repressed radical coupling and remarkably lower electron transfer rate for W215A mutant. Furthermore, the retardation of the suicidal radical coupling between the W251 residue and the monolignolic radical was attempted by supplementing the acidic microenvironment around the W251 residue to engineer radical-robust LiPH8. Among many mutants, mutant A242D showed exceptional catalytic performances by yielding 21.1- and 4.9-fold higher increases of kcat and kcat/KM values, respectively, in the oxidation of non-phenolic model lignin dimer.ConclusionsA mechanism-based suicide inhibition of LiPH8 by phenolic compounds was firstly revealed and investigated in this work. Radical-robust LiPH8 was also successfully engineered by manipulating the transient radical state of radical-susceptible electron-relay. Radical-robust LiPH8 will play an essential role in degradation of lignin, which will be consequently linked with improved production of sugars from lignocellulose biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0664-1) contains supplementary material, which is available to authorized users.

Accelerated degradation of lignin by lignin peroxidase isozyme H8 (LiPH8) from Phanerochaete chrysosporium with engineered 4-O-methyltransferase from Clarkia breweri

Free-hydroxyl phenolic units can decrease or even abort the catalytic activity of lignin peroxidase H8 during oxidation of veratryl alcohol and model lignin dimers, resulting in slow and inefficient lignin degradation. In this study we applied engineered 4-O-methyltransferase from Clarkia breweri to detoxify the inhibiting free-hydroxyl phenolic groups by converting them to methylated phenolic groups. The multistep, enzyme-catalyzed process that combines 4-O-methyltransferase and lignin peroxidase H8 suggested in this work can increase the efficiency of lignin-degradation. This study also suggests approaching the field of multi-enzyme in vitro systems to improve the understanding and development of plant biomass in biorefinery operations.

A calmodulin inhibitor, W-7 influences the effect of cyclic adenosine 3', 5'-monophosphate signaling on ligninolytic enzyme gene expression in Phanerochaete chrysosporium

The capacity of white-rot fungi to degrade wood lignin may be highly applicable to the development of novel bioreactor systems, but the mechanisms underlying this function are not yet fully understood. Lignin peroxidase (LiP) and manganese peroxidase (MnP), which are thought to be very important for the ligninolytic property, demonstrated increased activity in Phanerochaete chrysosporium RP-78 (FGSC #9002, ATCC MYA-4764™) cultures following exposure to 5 mM cyclic adenosine 3', 5'-monophosphate (cAMP) and 500 μM 3'-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition. However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels. Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX. These results suggest that cAMP signaling functions to increase the transcription of LiP and MnP through the induction of cam transcription.

Ligninolytic enzymes of the fungus Irpex lacteus ( Polyporus tulipiferae): isolation and characterization of lignin peroxidase

The extracellular ligninolytic enzymatic activity of the fungus Irpex lacteus ( Polyporus tulipiferae) grown in nonimmersed liquid culture under nitrogen limitation was characterized. Laccase (LAC), manganese-dependent lignin peroxidase (MNP) and lignin peroxidase (LIP) activities were detected and their formation followed a temporal sequence peaking at day 4, 7 and 11, respectively. Three LIP isozymes, designated LIP1, LIP2 and LIP3, were isolated from manganese-depleted cultures, and exhibited molecular masses of 41, 41 and 44 kDa, respectively, pIs of 5.0, 4.9 and <4.6, respectively, and to 4–5% glycosylation. All isozymes were active towards a range of phenolic substrates, with veratryl alcohol being the preferred one. The kinetics constants obtained for veratryl alcohol and H 2O 2 were comparable to those of LIPs from other white-rot fungi. The N-terminal sequences of LIP1 and LIP2 were identical and presented 64% similarity with LIP3, and were closely related to the amino acid sequences of LIP isozymes from Phlebia radiata (LIG III), Trametes versicolor (LIG C) and Phanerochaete chrysosporium (GLG2, GLG3 and GLG4). Sequence alignment with other reported LIPs revealed fewer identical or similar residues. Taken together, these data suggest that despite similarity in catalytic properties, kinetics parameters and spectral characteristics, there seems to be high degree of diversity among LIPs of different white-rot fungi.

Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L.

The site-directed mutations H82A and Q222A (residues near the heme access channel), and W171A and F267L (residues near the surface of the protein) were introduced into the gene encoding lignin peroxidase (LiP) isozyme H8 from Phanerochaete chrysosporium. The variant enzymes were produced by homologous expression in P. chrysosporium, purified to homogeneity, and characterized by kinetic and spectroscopic methods. The molecular masses, the pIs, and the UV-vis absorption spectra of the ferric and oxidized states of these LiP variant enzymes were similar to those of wild-type LiP (wtLiP), suggesting the overall protein and heme environments were not significantly affected by these mutations. The steady-state and transient-state parameters for the oxidation of veratryl alcohol (VA) by the H82A and Q222A variants were very similar to those of wtLiP, demonstrating that these residues are not involved in VA oxidation and that the heme access channel is an unlikely site for VA oxidation. In contrast, the W171A variant was unable to oxidize VA, confirming the apparent essentiality of Trp171 in VA oxidation by LiP. The kinetic rates of spontaneous LiP compound I reduction in the absence of VA were similar for W171A and wild-type LiP, suggesting that there may not be a radical formed on the Trp171 residue of LiP in the absence of VA. For the F267L variant, both the K(m app) value in the steady state and the apparent dissociation constant (K(D)) for compound II reduction were greater than those for wtLiP. These results indicate that the site including W171 and F267, rather than the heme access channel, is the site of VA binding and oxidation in LiP. Whereas Trp171 appears to be essential for VA oxidation, it apparently is not independently responsible for the spontaneous decomposition of oxidized intermediates. The nearby Phe267 apparently is also involved in VA binding.

Identification of genes for lignin peroxidases and manganese peroxidases in ectomycorrhizal fungi.

• Genes for ligninolytic enzymes, normally associated with white-rot fungi, are shown to be widespread in a broad taxonomic range of ectomycorrhizal (ECM) fungi. • ECM fungi were screened for lignin peroxidase (LiP) and manganese peroxidase (MnP) genes by PCR using primers specific for known isozymes in the white-rot fungus Phanerochaete chrysosporium, with DNA sequencing used to confirm the identity of the amplified fragments. • Genes for LiPs were detected in ECM fungi representing the orders Agaricales, Aphyllophorales, Boletales, Cantharellales, Hymenochaetales, Sclerodermatales, Stereales and Thelephorales. MnP genes were detected in only Cortinarius rotundisporus and three ECM Stereales taxa. • The presence of genes for decomposer activities supports putative evolutionary relationships between ECM and saprotrophic fungi. Expression of the lignolytic genes may facilitate ECM fungal access to nutrients associated with dead plant material in soil and potentially a supplementary carbon supply. Strict functional boundaries between ECM and decomposer fungi may be less clear-cut than previously thought.

Engineering of a Manganese-Binding Site in Lignin Peroxidase Isozyme H8 from Phanerochaete chrysosporium

A Mn2+-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 Mn2+ oxidation were compared to those of native recombinant enzyme and to fungal Mn peroxidase isozyme H4, respectively. The mutated enzyme was able to oxidize Mn2+ 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 Km for Mn2+ was determined to be 1.57 mM and the kcat = 5.45 s−1. These results indicate that the mutated lignin peroxidase is less effective in Mn2+ 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.

Crystal structures of pristine and oxidatively processed lignin peroxidase expressed in Escherichia coli and of the W171F variant that eliminates the redox active tryptophan 171. Implications for the reaction mechanism

The heme enzyme lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium contains a solvent exposed redox active tryptophan residue (Trp171) that carries a unique hydroxy group stereo-specifically attached to its C β atom. A Trp171Phe mutant has no activity at all towards the substrate veratryl alcohol. The mechanism of veratryl alcohol oxidation involving β-hydroxy-Trp171 is largely unknown. Here, we present the first crystal structures of LiP isozyme H8 at high resolution in its pristine non-hydroxylated form, of the C β-hydroxylated form, and of the Trp171Phe mutant using recombinantly expressed and refolded protein produced from Escherichia coli. As a consequence, all structures are unglycosylated. Structural changes in response to the mutation are marginal and allow us to attribute the complete lack of activity exclusively to the absence of the redox active indole side-chain. The origin of the stereospecificity of the Trp171 hydroxylation can be explained on structural grounds. A reaction mechanism involving Trp171 is proposed and the possible function of the modification is discussed. Another important result regarding the ongoing debate on the co-ordination state of the heme iron in the resting state is that the iron is six co-ordinate in all cases the data being collected at room temperature. The mean distance from the iron to the distal water ligand is 2.18(±0.08) Å. The radical scavenger orcinol was found to decrease radiation damage to the crystals, during data collection at room temperature.

MnII Is Not a Productive Substrate for Wild-Type or Recombinant Lignin Peroxidase Isozyme H2

The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter was used to drive the homologous expression of the lignin peroxidase (LiP) isozyme H2 gene in primary metabolic cultures of Phanerochaete chrysosporium. The molecular mass, pI, and optical absorption spectra of purified recombinant LiPH2 (rLiPH2) were essentially identical to those of wild-type LiPH2 (wtLiPH2). wtLiPH2 was prepared by growing cells in the absence of MnII, conditions under which P. chrysosporium manganese peroxidase (MnP) is not expressed, ensuring that wtLiPH2 was not contaminated with MnP. The kinetics of veratryl alcohol (VA) oxidation were essentially identical for rLiPH2 and wtLiPH2. The rLiPH2, wtLiPH2, and wild-type LiP isozyme H8 (wtLiPH8) enzymes were used to reexamine previous claims that LiPH2 can oxidize MnII at a rate sufficient to promote catalytic turnover of the enzyme. Our results demonstrate that rLiPH2, wtLiPH2, and LiPH8 do not turn over under steady-state conditions, when MnII is the sole reducing substrate. Furthermore, transient-state kinetic analyses show that the reduction rate of the catalytic intermediate, LiP compound I, by VA was at least 2 × 103-fold higher than the rate of reduction in the presence of MnII. No reduction of LiP compound II was observed in the presence of MnII. In contrast to previous claims, these data strongly suggest that MnII is not a productive substrate for LiPH2 or LiPH8.

Extracellular Mannose-6-phosphatase of Phanerochaete chrysosporium: A Lignin Peroxidase-Modifying Enzyme

The lignin peroxidase (LIP) isozyme profile of the white-rot fungus Phanerochaete chrysosporium changes markedly with culture age. This change occurs extracellularly and results from enzymatic dephosphorylation of LIP isozymes. In this study, a novel mannose 6-phosphatase (M6Pase) from extracellular culture fluid filtrate of P. chrysosporium, shown to be responsible for the extracellular postranslational modification of LIP, was purified and characterized. In vitro incubation of the purified M6Pase with purified LIP isozyme H2 resulted in its conversion to isozyme H1, with an equimolar release of orthophosphate. Using different sugar phosphates as substrate, the enzyme exhibited narrow specificity, showing activity mostly for mannose 6-phosphate (Km = 0.483 mM). The enzyme displayed a molecular mass of 82 kDa, as determined by gel filtration, and 40.4 and 39.1 kDa, on SDS–PAGE, suggesting that the native form is a dimer. The N-terminal sequence of the enzyme has no homology with that of other reported phosphatases. M6Pase is a metaloprotein with manganese and cobalt as the preferred metal ions. It is N-glycosylated proteins with an isoelectric point of 4.7-4.8 and a pH optimum of 5. Based on its characteristics, M6Pase from P. chrysosporium seems to be a unique phosphatase responsible for posttranslation modification of LIP isozymes.

Transformation of macromolecules from a brown coal by lignin peroxidase

Indirect evidence has suggested that lignin peroxidase (LiP) of the white-rot fungus Phanerochaete chrysosporium catalyses oxidative decolourisation and depolymerisation of macromolecules from brown coal in vivo. In this study we show that LiP catalyses these transformations in vitro. Unmethylated (USC45 coal) and methylated (MWSC6 coal) fractions of solubilised macromolecules (M(r) > 30,000) from a brown coal were treated with a semi-purified preparation of LiP isozymes from P. chrysosporium. Both coal fractions were decolourised, losing between 26% and 39% of their absorbance at both 280 nm and 400 nm, in reactions that had an absolute requirement for H2O2 and veratryl alcohol. Neither coal fraction was transformed when the enzyme was heat-inactivated or in the presence of the LiP inhibitor metavanadate. Gel-permeation chromatography showed that MWSC6 coal but not USC45 was depolymerised and yielded low-molecular-mass (M(r) < 30,000) fragments. Nine monomeric products were identified by GC-MS.

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.

Manganese deficiency can replace high oxygen levels needed for lignin peroxidase formation by Phanerochaete chrysosporium.

The combined effects of Mn and oxygen on lignin peroxidase (LIP) activity and isozyme composition in Phanerochaete chrysosporium were studied by using shallow stationary cultures grown in the presence of limited or excess N. When no Mn was added, LIP was formed in both N-limited and N-excess cultures exposed to air, but no LIP activity was observed at Mn concentrations greater than 13 mg/liter. In oxygen-flushed, N-excess cultures, LIP was formed at all Mn concentrations, and the peak LIP activity values in the extracellular fluid were nearly identical in the presence of Mn concentrations ranging from 3 to 1,500 mg/liter. When the availability of oxygen to cultures exposed to air was increased by growing the fungus under nonimmersed liquid conditions, higher levels of Mn were needed to suppress LIP formation compared with the levels needed in shallow stationary cultures. The composition of LIP isozymes was affected by the levels of N and Mn. Addition of veratryl alcohol to cultures exposed to air did not eliminate the suppressive effect of Mn on LIP formation. A deficiency of Mn in N-excess cultures resulted in lower biomass and a lower rate of glucose consumption than in the presence of Mn. In addition, almost no activity of the antioxidant enzyme Mn superoxide dismutase was observed in Mn-deficient, N-excess cultures, but the activity of this enzyme increased as the Mn concentration increased from 3 to 13 mg/liter. No Zn/Cu superoxide dismutase activity was observed in N-excess cultures regardless of the Mn concentration.

Expression of the Lignin Peroxidase H2 Gene fromPhanerochaete chrysosporiuminEscherichia coli

The DNA sequence for the extracellular lignin peroxidase isozyme H2 fromPhanerochaete chrysosporium,obtained from cDNA clone λML-6, was synthesized by PCR and successfully expressed inEscherichia coliunder control of the T7 promoter. The portion of the cDNA encoding the signal peptide, not found in the mature native enzyme, was not included. Recombinated lignin peroxidase H2 (rLiPH2) was produced in inclusion bodies in an inactive form. Active enzyme was obtained by refolding with glutathione-mediated oxidation in a medium containing urea, Ca2+, and hemin. The recombinant enzyme had spectral characteristics and kinetic properties identical to that of native enzyme isolated fromP. chrysosporium.Surprisingly, rLiPH2, like the native enzyme, also exhibited some manganese peroxidase activity.