Identification of a specific manganese peroxidase among ligninolytic enzymes secreted by Phanerochaete chrysosporium during wood decay.

The specific enzymes associated with lignin degradation in solid lignocellulosic substrates have not been identified. Therefore, we examined extracts of cultures of Phanerochaete chrysosporium that were degrading a mechanical pulp of aspen wood. Western blot (immunoblot) analyses of the partially purified protein revealed lignin peroxidase, manganese-dependent peroxidase (MnP), and glyoxal oxidase. The dominant peroxidase, an isoenzyme of MnP (pI 4.9), was isolated, and its N-terminal amino acid sequence and amino acid composition were determined. The results reveal both similarities to and differences from the deduced amino acid sequences from cDNA clones of dominant MnP isoenzymes from liquid cultures. Our results suggest, therefore, that the ligninolytic-enzyme-encoding genes that are expressed during solid substrate degradation differ from those expressed in liquid culture or are allelic variants of their liquid culture counterparts. In addition to lignin peroxidase, MnP, and glyoxal oxidase, xylanase and protease activities were present in the extracts of the degrading pulp.     

黄孢原毛平革菌木素降解酶系的研究进展

黄孢原毛平革菌木素降解酶系主要由木素过氧化物酶、锰过氧化物酶和乙二醛氧化酶组成。由于该酶系特殊的降解机制,除了木质素,它能降解许多种类的有机污染物,因此在环保方面有巨大的应用前景。本文主要综述了国内外对该酶系的研究进展。     

Characterization of reactions catalyzed by manganese peroxidase from Phanerochaete chrysosporium

Manganese peroxidase (MnP) is one of two extracellular peroxidases believed to be involved in lignin biodegradation by the white-rot basidiomycete Phanerochaete chrysosporium. The enzyme oxidizes Mn(II) to Mn(III), which accumulates in the presence of Mn(III) stabilizing ligands. The Mn(III) complex in turn can oxidize a variety of organic substrates. The stoichiometry of Mn(III) complex formed per hydrogen peroxide consumed approaches 2:1 as enzyme concentration increases at a fixed concentration of peroxide or as peroxide concentration decreases at a fixed enzyme concentration. Reduced stoichiometry below 2:1 is shown to be due to Mn(III) complex decomposition by hydrogen peroxide. Reaction of Mn(III) with peroxide is catalyzed by Cu(II), which explains an apparent inhibition of MnP by Cu(II). The net decomposition of hydrogen peroxide to form molecular oxygen also appears to be the only observable reaction in buffers that do not serve as Mn(III) stabilizing ligands. The nonproductive decomposition of both Mn(III) and peroxide is an important finding with implications for proposed in vitro uses of the enzyme and for its role in lignin degradation. Steady-state kinetics of Mn(III) tartrate and Mn(III) malate formation by the enzyme are also described in this paper, with results largely corroborating earlier findings by others. Based on a comparison of pH effects on the kinetics of enzymatic Mn(III) tartrate and Mn(III) malate formation, it appears that pH effects are not due to ionizations of the Mn(III) complexing ligand.     

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 manganese peroxidase and laccase in laboratory-scale bioreactors by Phanerochaete chrysosporium

The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) in laboratory-scale bioreactors was studied. One bioreactor was filled with cubes of polyurethane foam and the other with cubes of nylon sponge, in order to determine the more suitable carrier to produce high ligninolytic enzyme activities by this fungus. Both cultivations were carried out in batch. Manganese-dependent peroxidase activities about 600 U l^у were achieved in the bioreactor filled with cubes of nylon sponge, while up to 500 U l^у were detected in that filled with cubes of polyurethane foam. Furthermore, quite high levels of laccase appeared in both cultures: maximum activities of 114 U l^у and 62 U l^у were obtained on nylon and polyurethane supports, respectively.     

Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium.

The promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was used to drive expression of mnp1, the gene encoding Mn peroxidase isozyme 1, in primary metabolic cultures of Phanerochaete chrysosporium. A 1,100-bp fragment of the P. chrysosporium gpd promoter region was fused upstream of the mnp1 gene to construct plasmid pAGM1, which contained the Schizophyllum commune ade5 gene as a selectable marker. pAGM1 was used to transform a P. chrysosporium ade1 auxotroph to prototrophy. Ade+ transformants were screened for peroxidase activity on a solid medium containing high carbon and high nitrogen (2% glucose and 24 mM NH4 tartrate) and o-anisidine as the peroxidase substrate. Several transformants that expressed high peroxidase activities were purified and analyzed further in liquid cultures. Recombinant Mn peroxidase (rMnP) was expressed and secreted by transformant cultures on day 2 under primary metabolic growth conditions (high carbon and high nitrogen), whereas endogenous wild-type mnp genes were not expressed under these conditions. Expression of rMnP was not influenced by the level of Mn in the culture medium, as previously observed for the wild-type Mn peroxidase (wtMnP). The amount of active rMnP expressed and secreted in this system was comparable to the amount of enzyme expressed by the wild-type strain under ligninolytic conditions. rMnP was purified to homogeneity by using DEAE-Sepharose chromatography, Blue Agarose chromatography, and Mono Q column chromatography. The M(r) and absorption spectrum of rMnP were essentially identical to the M(r) and absorption spectrum of wtMnP, indicating that heme insertion, folding, and secretion were normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidative degradation of high molecular weight chlorolignin by manganese peroxidase of Phanerochaete chrysosporium

Phanerochaete chrysosporium was able to degrade high molecular weight chlorolignins (Mr greater than 30,000) from bleach plant effluents, although a direct contact between ligninolytic enzymes and chlorolignin was prevented by a dialysis tubing. In the absence of the enzymes, Mn3+ depolymerized chlorolignin when complexed with lactate causing the color, chemical oxygen demand (COD) and dry weight to decrease by 80%, 60% and 40%, respectively. Manganese peroxidase effectively catalyzed the depolymerization of chlorolignin in the presence of Mn2+ and H2O2. It can be concluded from these results that manganese peroxidase plays the major role in the initial breakdown and decolorization of high molecular weight chlorolignin in bleach plant effluents by P. chrysosporium in vivo.     

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     

Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes

AIMS: Poly(ethylene glycol) (PEG) and some substances similar to PEG in chemical structure were tested as stimulators of ligninolytic enzyme production in shaken culture of Phanerochaete chrysosporium. METHODS AND RESULTS: The substances that caused high enzymatic activity were linear polymers [poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol) and poly(vinyl alcohol)] and cyclic polymers (crown ether). They can have terminal groups other than -OH [PEG (di)methyl ether, PEG sulphate, PEG derivative with the amino group and xanthate]. The maximum lignin peroxidase activities were compared with the surface pressure caused by the stimulator. Addition of polymers composed of charged monomer units did not increase the enzymatic activity and the fungi did not grow at all on addition of polymers having a fixed positive charge. CONCLUSIONS: Lignin peroxidase activity was increased after the addition of polymers with uncharged monomer units. It was higher and its maximum was reached in a shorter time on addition of polymers with higher molecular weights. SIGNIFICANCE AND IMPACT OF STUDY: Beside Tweens there are several polymers that stimulate ligninolytic enzyme production in shaken culture of P. chrysosporium. Their characteristics are: similarity to PEG in chemical structure, having uncharged monomer units and high molecular weight.     

Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae.

A manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3' untranslated region of the glucoamylase gene of Aspergillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies.     

Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris

A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C.     

Heterologous expression of athermostable manganese peroxidase from Dichomitus squalens in Phanerochaete chrysosporium

Dichomitus squalens belongs to a group of white-rot fungi which express manganese peroxidase (MnP) and laccase but do not express lignin peroxidase (LiP). To facilitate structure/function studies of MnP from D. squalens, we heterologously expressed the enzyme in the well-studied basidiomycete, Phanerochaete chrysosporium. The glyceraldehyde-3-phosphate-dehydrogenase (gpd) promoter of P. chrysosporium was fused to the coding region of the mnp2 gene of D. squalens, 5 bp upstream of the translation start site, and placed in a vector containing the ural gene as a selectable marker. Purified recombinant protein (rDsMnP) was similar in kinetic and spectral characteristics to both the wild-type MnPs from D. squalens and P. chrysosporium (PcMnP). The N-terminal amino acid sequence of the rDsMnP was determined and was identical to the predicted sequence. Cleavage of the propeptide followed a conserved amino acid motif (A-A-P-S/T) in both rDsMnP and PcMnP. However, the protein from D. squalens was considerably more thermostable than its P. chrysosporium homolog with half-lives 15- to 40-fold longer at 55 degrees C. As previously demonstrated for PcMnP, addition of exogenous MnII and CdII conferred additional thermal stability to rDsMnP. However, unlike PcMnP, ZnII also confers some additional thermal stability to rDsMnP at 55 degrees C. Some differences in the metal-specific effects on thermal stability of rDsMnP at 65 degrees C were noted.     

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l^–1 and 356 U l^–1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l^–1 min^–1 using the extracellular culture filtrate.     

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.     

Continuous production of lignin peroxidase by Phanerochaete chrysosporium

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

The ligninolytic activities of Lentinus edodes and Phanerochaete chrysosporium

Summary Two important lignin-degrading fungi with existing or potential applications in the production of food, feed and/or fiber products from wood are Lentinus edodes (Berk.; Sing.=Lentinula edodes [Pegler]) and Phanerochaete chrysosporium (Burds). This study discusses their relative ability to degrade lignin and the factors controlling their ligninolytic activity (synthetic ¹⁴C-lignin¹⁴CO₂). Ligninolytic activity in P. chrysosporium is known to develop after the fungus ceases vegetative growth, and to require both O₂ and an exogenous carbon source such as glucose. It has an extracellular ligninase in high titer which is assayed by the oxidation of veratryl alcohol to veratraldehyde. Here, P. chrysosporium was found to have a high capacity for lignin degradation (it was not easily saturated with lignin). Certain inorganic elements, including Fe²⁺, Ca²⁺ and Mo⁶⁺, were found to stimulate its ligninolytic activity. Calcium addition was required, with 40 ppm Ca²⁺ giving the highest activity. As in P. chrysosporium, ligninolytic activity in L. edodes was found to require both O₂ and an exogenous carbon source. However, in contrast to P. chrysosporium, L. edodes was only moderately ligninolytic, had a lower capacity for lignin degradation (was more easily saturated with lignin), and showed maximal activity only during the vegetative growth period. Also in contrast to P. chrysosporium, ligninolytic activity in L. edodes was not stimulated by Ca²⁺. Instead, manganese was required, with 10 ppm Mn²⁺ giving optimal activity. An extracellular ligninase capable of oxidizing veratryl alcohol to veratraldehyde was not detected in L. edodes.