Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium

Ligninase-I (Mr 42,000-43,000; carbohydrate, 21%) and peroxidase-M2 (Mr 45,000-47,000; carbohydrate, 17%), two representative, hydrogen peroxide-dependent extracellular enzymes produced by ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium BKM-F-1767, were purified and their properties compared. Spectroscopic studies showed that both native enzymes are heme proteins containing protoporphyrin IX. EPR spectroscopy indicated that iron ions are coordinated with the enzymes' prosthetic groups as high-spin ferriheme complexes. We confirmed reports of others that the ligninase-hydrogen peroxide complex (activated enzyme) reverts to its native state on addition of dithionite or one of the enzyme's substrates (e.g., veratryl alcohol); however, we found that the peroxidase-M2-hydrogen peroxide complex required Mn2+ ions to accomplish a similar cycle. The peroxidase oxidized Mn2+ to a higher oxidation state, and the oxidized Mn acted as a diffusible catalyst able to oxidize numerous organic substrates. Unlike ligninase-I which is found free extracellularly, peroxidase-M2 appears to be associated closely with the fungal mycelium. In its peroxidatic reactions, ligninase-I oxidizes a variety of nonphenolic and phenolic lignin model compounds. In the presence of Mn2+, peroxidase-M2 oxidizes numerous phenolic compounds, especially syringyl (3,5-dimethoxy-4-hydroxyphenyl) and vinyl side-chain substituted substrates. Also, the peroxidase-Mn2+ system (without hydrogen peroxide) expresses oxidase activity against NADPH, GSH, dithiothreitol, and dihydroxymaleic acid, forming hydrogen peroxide at the expense of oxygen. Both enzymes were believed to play roles in lignin degradation, and these are discussed.     

Limitations of the lignin peroxidase system of the white-rot fungus, Phanerochaete chrysosporium

The lignin peroxidase enzyme system of the white-rot fungus, Phanerochaete chrysosporium was assayed for its capacity to degrade two recalcitrant aliphatic ether compounds, high-molecular-mass polyethylene glycol (PEG 20 000) and methyl tert-butyl ether. Ligninolytic cultures of Phanerochaete chrysosporium were spiked with each ether compound and incubated in reaction vessels. Separate incubations were conducted in which the ether compounds were present as sole carbon source. Other parameters, such as varying the methyl tert-butyl ether concentration and veratryl alcohol additions were tested. No significant degradation of either compound was observed under any of the conditions tested. Implications of these results are discussed with respect to the oxidative limitations of the lignin peroxidase enzyme system and structural features of substrate molecules that may be requisite for oxidation by this system.     

Suppressive effect of l-phenylalanine on manganese peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract The effect of added l-amino acids and NH₄⁺ on manganese peroxidase activity in ligninolytic cultures of Phanerochaete chrysosporium were investigated. Among 11 amino acids (0.2 mM) tested, including phenylalanine, glutamate, glutamine, histidine, alanine, iso-leucine, ornithine, glycine, aspartate, proline, and arginine, phenylalanine was the most effective in suppression of manganese peroxidase synthesis. However, all the amino acids tested except proline completely suppressed the enzyme synthesis at 2 mM concentration.     

Suppressive effect of l-phenylalanine on lignin peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract Guanosine-5'-diphosphate-3'-diphosphate (ppGpp), an effector for many metabolic pathways, is synthesized by the relA gene product after amino acid limitation. Studies of stringent controlled Escherichia coli CP78 (relA⁺) and relaxed controlled E. coli CP79 (relA⁻) were carried out to test whether these strains differ in the appearance of their cytoplasmic membranes after induction of stringent and relaxed response. Cytoplasmic membrane structures of the cells were investigated by freeze-fracture electron microscopy after cooling the cells. The obtained micrographs showed a net-like distribution of the particles in the cytoplasmic membranes of relaxed controlled cells whereas such a pattern was not detectable in the stringent controlled counterparts.     

Purification and properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium

An intracellular aryl-alcohol dehydrogenase (previously referred to as aryl-aldehyde reductase) was purified from the white-rot fungus Phanerochaete chrysosporium. The enzyme reduced veratraldehyde to veratryl alcohol using NADPH as a cofactor. Other aromatic benzaldehydes were also reduced, but not aromatic ketones. Methoxy-substituted rings were better substrates than hydroxylated ones. The enzyme was also able to reduce a dimeric aldehyde (4-benzyloxy-3-methoxybenzaldehyde). The highest reduction rate was measured when 3,5-dimethoxybenzaldehyde was used as a substrate. On SDS/PAGE the purified enzyme showed one major band with a molecular mass of 47 kDa, whereas gel filtration suggested a molecular mass of 280 kDa. Polyclonal antibodies raised against the gel purified 47-kDa protein were able to immunoprecipitate the aryl-alcohol dehydrogenase indicating that its activity possibly resides entirely in this protein fragment. The pI of the enzyme was 5.2 and it was most active at pH 6.1. The aryl-alcohol dehydrogenase was partially inhibited by typical oxidoreductase inhibitors.     

Functional diversity of cytochrome P450s of the white-rot fungus Phanerochaete chrysosporium

The functional diversity of cytochrome P450s (P450s) of the white-rot basidiomycete, Phanerochaete chrysosporium, was studied. A series of compounds known to be P450 substrates of other organisms were utilized for metabolic studies of P. chrysosporium. Metabolic conversions of benzoic acid, camphor, 1,8-cineol, cinnamic acid, p-coumaric acid, coumarin, cumene, 1,12-dodecanediol, 1-dodecanol, 4-ethoxybenzoic acid, and 7-ethoxycoumarin were observed with P. chrysosporium for the first time. 1-Dodecanol was hydroxylated at seven different positions to form 1,12-, 1,11-, 1,10-, 1,9-, 1,8-, 1,7-, and 1,6-dodecandiols. The effect of piperonyl butoxide, a P450 inhibitor, on the fungal conversion of 1-dodecanol was also investigated, indicating that hydroxylation reactions of 1-dodecanol were inhibited by piperonyl butoxide in a concentration-dependent manner. With 11 substrates, 23 hydroxylation reactions and 2 deethylation reactions were determined and 6 products were new with the position of hydroxyl group incorporated. In conclusion, fungal P450s were shown to have diverse and unique functions.     

Decolourisation of synthetic and spentwash melanoidins using the white-rot fungus Phanerochaete chrysosporium JAG-40

Phanerochaete chrysosporium JAG-40 was isolated from the soil samples saturated with spilled molasses collected from a sugar mill. This isolate decolourised synthetic and natural melanoidins present in spentwash in liquid fermentation; up to 80% in 6 days at 30 degrees C under aerobic conditions. A large inoculum size stimulated fungal biomass production, but this gave less decolourisation of pigment; 5% w/v (dry weight) mycelial suspension was found optimum for maximum decolourisation in melanoidin medium supplemented with glucose and peptone. Gel-filtration chromatography showed that larger molecular weight fractions of melanoidin were decolourised more rapidly than small molecular weight fractions.     

Biosorption of 2,4-dichlorophenol by immobilized white-rot fungus Phanerochaete chrysosporium from aqueous solutions

The fungus Phanerochaete chrysosporium was immobilized in several polymer matrices: Ca-alginate, Ca-alginate-polyvinyl alcohol (PVA) and pectin, and was then used as a biosorbent for removing 2,4-dichlorophenol (2,4-DCP) in wastewater. Immobilization of P. chrysosporium onto pectin was less efficient than that onto other matrices because of its poor mechanical strength and low adsorption efficiency. Ca-alginate immobilized fungal beads with biocompatibility exhibited good mechanical strength and adsorption efficiency over 60%. Among the different biomass dosages in Ca-alginate immobilized fungal beads, 1.25% (w/v) was the optimum. The adsorption data of 2,4-DCP on the blank Ca-alginate beads, free, and immobilized fungal biomass could be described by the Langmuir and Freundlich isotherms very well. Desorption operation was efficiently completed by using distilled water as eluant, and the desorption efficiency reached 82.16% at an optimum solid/liquid ratio of 14.3. The consecutive adsorption/desorption cycles studies employing the Ca-alginate immobilized fungal beads demonstrated that the immobilized fungal biomass could be reused in five cycles without significant loss of adsorption efficiency and adsorbent weight.     

Proteomic and metabolomic analyses of the white-rot fungus Phanerochaete chrysosporium exposed to exogenous benzoic acid

Intracellular processes of the white-rot basidiomycete Phanerochaete chrysosporium involved in the metabolism of benzoic acid (BA) were investigated at the proteome and metabolome level. Up-regulation of aryl-alcohol dehydrogenase, arylaldehyde dehydrogenase, and cytochrome P450s was observed upon addition of exogenous BA, suggesting that these enzymes play key roles in its metabolism. Intracellular metabolic shifts from the short-cut TCA/glyoxylate bicycle system to the TCA cycle and an increased flux in the TCA cycle indicated activation of the heme biosynthetic pathway and the production of NAD(P)H. In addition, combined analyses of proteome and metabolome clearly indicated the role of trehalose as a storage disaccharide and that the mannitol cycle plays a role in an alternative energy-producing pathway.     

Fractionation of subcellular membranes of the secretory pathway from the peroxidase-producing white-rot fungus Phanerochaete chrysosporium

Abstract Mycelia from the basidiomycete Phanerochaete chrysosporium , producing lignin and manganese peroxidases, were homogenized and fractionated on a sucrose gradient. The main subcellular fungal membrane fractions were successfully separated. Lipid composition analyses of the isolated membranes as well as associated marker enzymes distribution gave evidence to similarities with membranes originating from plants. Lignin and manganese peroxidases were investigated by immunodetection in subcellular fractions. Our results show that lignin and manganese peroxidases are mainly associated with Golgi apparatus vesicles and, to a lesser extent, with endoplasmic reticulum and light density vesicles, but not with plasma membranes.     

A novel enzymatic decarboxylation of oxalic acid by the lignin peroxidase system of white-rot fungus Phanerochaete chrysosporium

Oxidation of veratryl alcohol by lignin peroxidase (LiP) was potently inhibited by oxalic acid. The inhibition analysis with Lineweaver-Burk plots clearly showed that the type of inhibition is non-competitive. The enzymatic oxidation of veratryl alcohol in the presence of 14C-oxalic acid yielded radioactive carbon dioxide. The results indicate that the apparent inhibition of LiP is caused by reduction of the veratryl alcohol cation radical intermediate back to the substrate level by oxalate, which is concomitantly oxidized to carbon dioxide.     

Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium

Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin----14CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H2O2-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.     

Characterization of a hydroxyl-radical-producing glycoprotein and its presumptive genes from the white-rot basidiomycete Phanerochaete chrysosporium

During wood decay, the white-rot basidiomycete Phanerochaete chrysosporium secretes low-molecular-mass glycoproteins that catalyze a redox reaction between O(2) and electron donors to produce hydroxyl radical. This reaction accounts for most of the hydroxyl radical produced in wood-degrading cultures of P. chrysosporium. In combination with phenol oxidases, hydroxyl radical is believed to play a role in lignin degradation. The secreted glycoproteins also reduce Fe(III) to Fe(II) and strongly bind Fe(II). The partially purified glycoproteins contain 1-amino-1-deoxy-2-ketose (ketoamine) produced by the condensation of side-chain amino groups and carbohydrate. cDNAs and two putative genes encoding these glycoproteins, glp1 and glp2, have been isolated and sequenced. The 875bp glp1 and 864bp glp2 are found on scaffold 2 of the P. chrysosporium genome. These presumptive genes each consist of seven introns and eight exons. The latter encode a predicted protein of 138 amino acids and a 22-amino-acid signal sequence for secretion. The predicted protein sequences are nearly identical to N-terminal and internal sequences obtained from the partially purified glycoprotein. The molecular masses of the deduced mature proteins, 13,981 (glp1) and 13,970 (glp2), coincide with the molecular mass of the glycoprotein as determined by tricine-SDS-PAGE.     

Transformations of arylpropane lignin model compounds by a lignin peroxidase of the white-rot fungus Phanerochaete chrysosporium

Various lignin model compounds of the O-arylpropane type were oxidized with purified lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium, and oxidation products were identified by gas-chromatography/mass-spectroscopy procedures. Our results are in accord with the theory that lignin peroxidase catalyzes one-electron oxidations of its substrates with formation of cation radicals, and that these radicals undergo degradative reactions that are predictable from a knowledge of cation radical and oxygen chemistry. Cation radicals formed from O-arylpropane model compounds appeared to undergo the following types of degradative transformations: addition of water to ring-centered radicals, followed by proton loss yielding quinones and alcohols; nucleophilic attack by hydroxy functions on propanoid moieties giving cyclic ketals as intermediates which decompose to yield side chain migration products; transfer of the charge of a radical from a ring to the associated alkyl moiety through an ether bond, with loss of a proton from the latter, forming a new carbon-centered radical. The new alkyl-centered radicals apparently were able to abduct dioxygen to form peroxyl radicals which decomposed giving a variety of oxidation products and probably superoxide anion. Specific examples of the above transformations are presented, and their relevance to lignin degradation is discussed.     

Decolourization of olive mill waste-waters by the white-rot fungus Phanerochaete chrysosporium: involvement of the lignin-degrading system

Summary The decolourization of olive mill waste-waters (OMW) by Phanerochaete chrysosporium was investigated. OMW decolourization occurred during the primary phase of growth when glycerol was used as the carbon source, and during secondary metabolism in nitrogen-limited cultures. The decolourization was found to be extensive (74% of colour removal, 80% of chemical oxygen demand removal) when the cultures were supplement d with veratryl alcohol and flushed with O₂. The biodegradation system was repressed with glutamate as a nitrogen source. These results suggest that all or part of the lignin-degrading system of P. chrysosporium played a role in biodegradation of OMW. The decolourization of OMW corresponds to depolymerization of high-molecular-mass aromatics combined with mineralization of a wide range of monoaromatic compounds. Correspondence to: S. Sayadi     

Regulation of intracellular cyclic AMP levels in the white-rot fungus Phanerochaete chrysosporium during the onset of idiophasic metabolism

Adenylate cyclase activity in Phanerochaete chrysosporium was present in cell fractions sedimenting at 1,000xg, 15,000xg, and in the 150,000xg supernatant. A small amount of activity in the 1,000xg pellet could be solubilised by treatment with Triton X-100, and the enzyme in all fractions required an ATP-Mn²⁺ substrate. Adenylate cyclase activity in the 150,000xg pellet was low (0.003 nmol/mg protein·min) and may have resulted from contamination by other fractions. Highest adenylate cyclase specific activity (0.37 nmol/mg protein ·min) was recorded in the 150,000xg supernatant at the onset of idiophasic metabolism. During this growth phase, adenylate cyclase activity also increased in the 1,000xg pellet and was maximally 4.5-fold greater than that in primary phase cultures. No significant cAMP-phosphodiesterase activity could be detected during growht in any of the cell fractions or in the growth medium with either Mn²⁺, Mg²⁺, or Ca²⁺ as added cations. The extracellular cAMP concentration increased logarithmically during primary growth; however, in cultures in idiophasic metabolism cAMP levels remained constant and relatively low. We suggest that excretion into the medium is the principal means by which intracellular cAMP levels are decreased in P. chrysosporium.Abbreviation EB extraction buffer