Calnexin overexpression increases manganese peroxidase production in Aspergillus niger

Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for peroxidase biosynthesis has been proposed to be an important bottleneck. In this work, we analyzed the role of two components of the secretion pathway, the chaperones calnexin and binding protein (BiP), in the production of a fungal peroxidase. Expression of the Phanerochaete chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted in an increase in the expression level of the clxA and bipA genes. In a heme-supplemented medium, where MnP was shown to be overproduced to higher levels, induction of clxA and bipA was also higher. Overexpression of these two chaperones in an MnP-producing strain was analyzed for its effect on MnP production. Whereas bipA overexpression seriously reduced MnP production, overexpression of calnexin resulted in a four- to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin overexpression had no synergistic effect on MnP production. The possible function of these two chaperones in MnP maturation and production is discussed.     

Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Pleurotus sajor-caju

Pleurotus sajor-caju, strain Pl-27, produces manganese-dependent peroxidase (MnP) and laccase, but not lignin peroxidase, when grown on a defined medium with glucose as sole carbon source. MnP activity was detected in fungal cultures supplemented with both high (26 mM-N) and low (2.6 mM-N) nutrient nitrogen although higher specific activity values were recorded under the latter conditions. Conversely, laccase production was not influenced by nutrient nitrogen levels under the growth conditions adopted. Both the titre and time of appearence of MnP were also affected by the concentration of Mn in the culture medium with highest enzyme levels recorded in cultures supplemented with 15 ppm Mn. Two MnP and five laccase isoforms were identified by FPLC and gel electrophoresis.     

Studies on the Production of Fungal Peroxidases in Aspergillus niger

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.     

Effect of various synthetic dyes on the production of manganese-dependent peroxidase isoenzymes by immobilized <Emphasis Type="Italic">Irpex lacteus</Emphasis>

The effect of manganese and selected synthetic dyes on the production of manganese-dependent peroxidase (MnP) by Irpex lacteus immobilized on polyurethane foam was studied. In the cultures grown in a medium containing 65 μM Mn (II), up to three various isoenzymes of MnP were resolved by isolectrofocusing, with pI values within the range of 3.50–6.04. In the cultures grown in a medium containing 2.9 mM Mn (II), two new MnP isoforms (pI 3.28, 3.75) were produced. The addition of structurally different synthetic dyes, an azo dye Reactive Orange 16 (RO16), an anthraquinonic dye Remazol Brilliant Blue R (RBBR), and a triphenylmethane dye Bromophenol Blue (BPB), to the fungal cultures grown in the presence of high manganese inhibited the production of low pI MnP isoforms. However, in the presence of BPB a new MnP isoform with pI 5.67 was detected. BPB was found to induce MnP isoforms which are more effective in RBBR decolorization in vitro than the low pI isoforms present in the control cultures.     

Identification of a potential fungal species by 18S rDNA for ligninases production

Fungal species for ligninases production was investigated by 18S ribosomal DNA sequence analysis. Two primer sets were chosen to amplify a major part of the 18S rDNA, which resulted in intense PCR product of approximately 550–820 bp in size per sample. The results suggest that the 18S rDNA-based approach is a useful tool for identification of unknown potential fungal species for ligninases production. The isolated fungal species produces mainly manganese peroxidase (MnP). The enzyme oxidized a variety of the usual MnP substrates, including lignin related polyphenols. Time course studies showed that maximum production of ligninolytic enzymes MnP (64 IU L^?1), lignin peroxidase (26.35 IU L^?1), and laccase (5.44 IU L^?1), respectively, were achieved after 10 days of cultivation under optimum conditions. Furthermore, the biological decolorization of Remazol Brilliant Blue R dye following 10 days of cultivation was 94 %. NCBI BLAST was used to search for closest matched sequences in the GenBank database and based on sequence homology the first BLAST hit was Dothioraceae sp. LM572 with accession number EF060858.1.     

Manganese Peroxidase, Produced by Trametes versicolor during Pulp Bleaching, Demethylates and Delignifies Kraft Pulp

Previous work has shown that Trametes (Coriolus) versicolor bleaches kraft pulp brownstock with the concomitant release of methanol. In this work, the fungus is shown to produce both laccase and manganese peroxidase (MnP) but not lignin peroxidase during pulp bleaching. MnP production was enhanced by the presence of pulp and/or Mn(II) ions. The maximum level of secreted MnP was coincident with the maximum rate of fungal bleaching. Culture filtrates isolated from bleaching cultures produced Mn(II)- and hydrogen peroxide-dependent pulp demethylation and delignification. Laccase and MnP were separated by ion-exchange chromatography. Purified MnP alone produced most of the demethylation and delignification exhibited by the culture filtrates. On the basis of the methanol released and the total and phenolic methoxyl contents of the pulp, it appears that MnP shows a preference for the oxidation of phenolic lignin substructures. The extensive increase in brightness observed in the fungus-treated pulp was not found with MnP alone. Therefore, either the MnP effect must be optimized or other enzymes or compounds from the fungus are also required for brightening.     

Ligninolytic Enzymes of the White Rot Basidiomycete Trametes trogii

The white rot fungus Trametes trogii strain BAFC 463 produced laccase, manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, as well as two hydrogen peroxide‐producing activities: glucose oxidizing activity and glyoxal oxidase. In high‐N (40 mM N) cultures, the titres of laccase, MnP and GLOX were 27 (6.55 U/ml), 45 (403.00 mU/ml)and 8 (32,14 mU/ml) fold higher, respectively, than those measured in an N‐limited medium. This is consistent with the fact that the ligninolytic system of T. trogii is expressed constitutively. Lower activities of all the enzymes tested were recorded upon decreasing the initial pH of the medium from 6.5 to 4.5. Adding veratryl alcohol improved GLOX production, while laccase activity was stimulated by tryptophan. Supplying Tween 80 strongly reduced the activity of both MnP and GLOX, but increased laccase production. The titre of MnP was affected by the concentration of Mn in the culture medium, the highest levels were obtained with 90 μM Mn (II). LiP activity, as CDH activity, were detected only in the mediumsupplemented with sawdust. In this medium, laccase production reached a maximum of 4.75 U/ml, MnP 747.60 mU/ml and GLOX 117.11 mU/ml. LiP, MnP and GLOX activities were co‐induced, attaining their highest levels at the beginning of secondary metabolism, but while MnP, laccase, GLOX and CDH activities were also present in the primary growth phase, LiP activity appears to beidiophasic. The simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus makes it an attractive microorganism for future biotechnological applications.     

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     

Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity

Summary The effect of various carbon and nitrogen sources on laccase, manganese-dependent peroxidase (MnP), and peroxidase production by two strains of Pleurotus ostreatus was investigated. The maximal laccase yield of P. ostreatus 98 and P. ostreatus 108 varied depending upon the carbon source from 5 to 62 U l⁻¹ and from 55 to 390 U l⁻¹, respectively. The highest MnP and peroxidase activities were revealed in medium supplemented by xylan. Laccase, MnP, and peroxidase activities of mushrooms decreased with supplementation of defined medium by inorganic nitrogen sources. Peptone followed by casein hydrolysate appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, their positive effects on enzyme accumulation were due to a higher biomass production. The secretion of MnP and peroxidase by P. ostreatus 108 was stimulated with supplementation of casein hydrolysate to the control medium since the specific MnP and peroxidase activities increased 15-fold and 3.5-fold, respectively.     

Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.     

Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization

The main manganese peroxidase (MnP) isoenzyme of Agaricus bisporus ATCC 62459 produced in lignocellulose-containing cultures was isolated, cloned and sequenced. In liquid medium, where MnP was previously detected only in trace amounts, the production of MnP was enhanced by rye and wheat bran supplements. The pI (3.25) and N-terminal amino acid sequence (25 aa) of the enzyme from bran-containing cultures were identical to those reported from compost-isolated MnP1. MnP1 is a 328-aa long polypeptide preceded by a 26-aa leader peptide. The nucleotide sequence and putative amino acid sequence of MnP1 reveal its similarity to Pleurotus ostreatus MnP3 (62.5%), Lepista irina versatile peroxidase (VP) (61.8%) and Pleurotus eryngii VPs VPL2 and VPL1 (61.9% and 61.2%, respectively). The intron-exon structure resembles that of P. ostreatus MnP1 and P. eryngii VPL1. Despite the sequence similarity to VPs, in the A. bisporus MnP1 sequence, alanine (A163) is present instead of tryptophane (W164), distinguishing it from the veratryl alcohol oxidising P. eryngii VPLs. The MnP sequence can be used as a tool to examine the pattern of ligninolytic gene expression during the growth and fruiting of A. bisporus to optimise compost composition, fungal growth and mushroom production.     

Production and regulation of lignocellulose-degrading enzymes of <Emphasis Type="Italic">Poria</Emphasis>-like wood-inhabiting basidiomycetes

The wood-decomposing fungal species Antrodia macra, A. pulvinascens, Ceriporiopsis aneirina, C. resinascens and Dichomitus albidofuscus were determined for production of laccase (LAC), Mn peroxidase (MnP), lignin peroxidase (LiP), endo-l,4-P-β-glucanase, endo-l,4-β-xylanase, cellobiohydrolase, 1,4-β-glucosidase and 1,4-β-xylosidase. The results confirmed the brown-rot mode of Antrodia spp. which did not produce the activity of LAC and MnP. The remaining species performed detectable activity of both enzymes while no strain produced LiP. Significant inhibition of LAC production by high nitrogen was found in all white-rot species while only MnP of D. albidofuscus was regulated in the same way. The endoglucanase and endoxylanase activities of white-rotting species were inhibited by glucose in the medium while those of Antrodia spp. were not influenced by glucose concentration. The regulation of enzyme activity and bio-mass production can vary even within a single fungal genus.     

Lignin-Modifying Enzymes of the White Rot Basidiomycete Ganoderma lucidum

Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (M_r of 40,000 and 66,000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity (~100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.     

Potential of ligninolytic enzymatic complex produced by white‐rot fungi from genus Trametes isolated from Bulgarian forest soil

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.     

Extracellular Ligninolytic Enzymes in Bjerkandera adusta and Lentinus squarrosulus

Extracellular ligninolytic enzyme activities were determined in two white-rot fungi, Bjerkandera adusta and Lentinus squarrosulus. To investigate the activity of extracellular enzymes, cultures were incubated over a period of 20 days in nutrient rich medium (NRM) and nutrient poor medium under static and shaking conditions. Enzymatic activity was varied with media and their incubation conditions. The highest level of Aryl alcohol oxidase (AAO) was detected under shaking condition of both medium while Manganese peroxidase (MnP) activity was best in NRM under both conditions. AAO is the main oxidases enzyme in B. adusta while laccase plays important role in L. squarrosulus. MnP is the main peroxidase enzyme in both varieties.     

Studies on the production of fungal peroxidases in Aspergillus niger

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.     

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 ¹⁴CO₂ from ¹⁴C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of ¹⁴CO₂ 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.     

Statistical optimization of medium components to increase the manganese peroxidase productivity by Phanerochaete chrysosporium NCIM 1197

The production of manganese peroxidase (MnP) by the white-rot fungus Phanerochaete chrysosporium NCIM 1197 was investigated by the screening and optimization of the media constituents and physiological factors. MnP production by the fungus was used as the response to screen the media constituents with statistically valid Plackett-Burman (P-B) design. Response surface methodology (RSM) was applied to optimize the level of screened media constituents. Amongst the media constituents screened, glucose, maltose, ammonium chloride, and urea were selected as the most important for MnP enhancement. A five-level Central Composite Design (CCD) was used in optimizing the important media constituents for maximizing the MnP production. The optimal medium composition for maximum MnP production was 13.88 mM of glucose, 13.88 mM of maltose, 0.02 mM of ammonium chloride, and 0.02 mM of urea. The final experiment was conducted to validate the model, which was shown to produce 70.20 U/mL of MnP with a predicted value of 66.49 U/mL on the 8^th day of incubation.     

Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor

During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk’s medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn²⁺-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures.