Production and Characterization of Recombinant Phanerochaete chrysosporium Cellobiose Dehydrogenase in the Methylotrophic Yeast Pichia pastoris

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.     

Cellobiose dehydrogenase from Phanerochaete chrysosporium is encoded by two allelic variants.

The complete nucleotide sequences of two alleles of cellobiose dehydrogenase, cdh-1 (3,627 bp) and cdh-2 (3,623 bp), from Phanerochaete chrysosporium OGC101 are reported. The nucleotide sequences of cdh-1 and cdh-2 exhibit 97% similarity. A total of eighty-six point mutations between cdh-1 and cdh-2 are observed. Both alleles have 14 exons, and the introns are located at exactly the same positions. The translation products of these alleles have identical amino acid sequences. Restriction fragment length polymorphism analyses of homokaryotic derivatives show segregation of the CDH alleles.     

Cellobiose oxidase from Phanerochaete chrysosporium can be cleaved by papain into two domains

Cellobiose oxidase from the white rot fungus Phanerochaete chrysosporium has been purified to homogeneity by a new method. The enzyme has been cleaved by papain into two fragments: one containing the heme group and one containing the flavin group. The flavin fragment can oxidize cellobiose and is reoxidized by oxygen. Cellobiose oxidase binds to cellulose to approximately the same extent as cellobiohydrolase I. The cellulose-binding site is located on the flavin domain. The enzyme cannot be totally displaced from cellulose by cellobiose, and it is still active when adsorbed to cellulose. The possible role of the enzyme in lignocellulose degradation is discussed.     

Cellobiose oxidase from Phanerochaete chrysosporium. Stopped-flow spectrophotometric analysis of pH-dependent reduction.

Cellobiose oxidase (CBO) from Phanerochaete chrysosporium can utilize dichlorphenol-indophenol (Cl2Ind) and cytochrome c as effective electron acceptors for the oxidation of cellobiose. However, the pH dependencies of activity for these electron acceptors are significantly different. Both compounds act as effective electron acceptors at pH 4.2, whereas only dichlorophenol-indophenol is active at pH 5.9. To explain this discrepancy, the pH dependencies of the reduction rates of FAD and heme, respectively, in CBO by cellobiose have been investigated by stopped-flow spectrophotometry. Both FAD and heme are reduced with a high rate constant at pH 4.2. In contrast, at pH 5.9, only FAD reduction is fast, while the reduction of the heme is extremely slow. As a conclusion, the reduction of cytochrome c by CBO is dependent on heme, which functions at a lower pH range compared to reduction of FAD.     

培养于天然冷杉木片的黄孢原毛平革菌木质素过氧化物酶基因表达的RT-PCR分析

利用RT-PCR方法分析了生长于冷杉木片上的黄孢原毛平革菌木质素过氧化物酶基因lipA2(GLG3)、lipC1(GLG2)、lipC2(GLG5)、lipD2(GLG1)、lipE(LPO811)的表达。结果发现在不同的培养时间里仅有特定的基因表达,在第2周时仅有lipA2(GLG3)基因表达,在第4周时未检测到任何基因的表达,在第6周时lipD2(GLG1)和lipC1(GLG2)基因表达,在第8周时仅有lipA2(GLG3)基因表达。这些结果说明,在冷杉木片上培养的黄孢原毛平革菌的lip基因表达具有明显的时间特异性,并且与限定培养基中得到的结果明显不同。     

Penetrability of White Rot-Degraded Pine Wood by the Lignin Peroxidase of Phanerochaete chrysosporium

The penetration of enzymes into wood cell walls during white rot decay is an open question. A postembedding immunoelectron microscopic technique was the method of choice to answer that question. Infiltration of pine wood specimens with a concentrated culture filtrate greatly improved the labeling density and, thereby, reproducibility. Characterization of the concentrated culture filtrate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) revealed three closely spaced proteins of molecular weights about 42,000 showing immunoreactivity against anti-lignin peroxidase serum. It was shown by immunogold labeling that lignin peroxidase of Phanerochaete chrysosporium is located on the surface of the wood cell wall or within areas of heavy attack. It did not diffuse into undecayed parts of the cell wall. The reasons for preventing lignin peroxidase from penetrating wood cell walls during white rot decay are discussed.     

Involvement of an Extracellular Glucan Sheath during Degradation of Populus Wood by Phanerochaete chrysosporium

Observations by transmission electron microscopy of wood samples of Populus tremula inoculated with the white rot fungus Phanerochaete chrysosporium showed that, at certain stages of their growth cycle, hyphae were encapsulated by a sheath which seems to play an active role in the wood cell wall degradation. Chemical and immunochemical techniques and C nuclear magnetic resonance spectroscopy were applied to demonstrate the beta-1,3-1,6-d-glucan nature of the sheath. Double-staining methods revealed the interaction between the extracellular peroxidases involved in lignin degradation and the glucan mucilage. The glucan was also shown to establish a material junction between the fungus and the wood cell wall. It was concluded that, by means of these interactions, the sheath provides a transient junction between the hyphae and the wood, thus establishing a point of attachment to the site of the degradation. The association of peroxidases to the glucan matrix is in favor of the role of the sheath as a supporting structure. Furthermore, that the sheath was hydrolyzed during the attack demonstrated its active role both in providing the H(2)O(2) necessary to the action of peroxidases and in providing a mode of transport of the fungal enzymes to their substrates at the surface of the wood cell wall.     

Impact of Phanerochaete chrysosporium on the Functional Diversity of Bacterial Communities Associated with Decaying Wood

Bacteria and fungi naturally coexist in various environments including forest ecosystems. While the role of saprotrophic basidiomycetes in wood decomposition is well established, the influence of these fungi on the functional diversity of the wood-associated bacterial communities has received much less attention. Based on a microcosm experiment, we tested the hypothesis that both the presence of the white-rot fungus Phanerochaete chrysosporium and the wood, as a growth substrate, impacted the functional diversity of these bacterial communities. Microcosms containing sterile sawdust were inoculated with a microbial inoculum extracted from a forest soil, in presence or in absence of P. chrysosporium and subsequently, three enrichment steps were performed. First, bacterial strains were isolated from different microcosms previously analyzed by 16S rRNA gene-based pyrosequencing. Strains isolated from P. chrysosporium mycosphere showed less antagonism against this fungus compared to the strains isolated from the initial forest soil inoculum, suggesting a selection by the fungus of less inhibitory bacterial communities. Moreover, the presence of the fungus in wood resulted in a selection of cellulolytic and xylanolytic bacterial strains, highlighting the role of mycospheric bacteria in wood decomposition. Additionally, the proportion of siderophore-producing bacteria increased along the enrichment steps, suggesting an important role of bacteria in iron mobilization in decaying-wood. Finally, taxonomic identification of 311 bacterial isolates revealed, at the family level, strong similarities with the high-throughput sequencing data as well as with other studies in terms of taxonomic composition of the wood-associated bacterial community, highlighting that the isolated strains are representative of the wood-associated bacterial communities.     

Respiratory pathways and oxygen toxicity in Phanerochaete chrysosporium

Phanerochaete chrysosporium maintained on glucose as the carbon source contained severely impaired mitochondria that were characterised by the loss of both succinate dehydrogenase and cytochrome oxidase activities. These cells maintained a constant value for energy charge using anaerobic metabolism. Cells with these properties express lignin peroxidase when supplied with a pure oxygen atmosphere, which may reflect a response to accumulating reactive oxygen species. Cells maintained on cellulose retained fully functional mitochondria, but expressed lignin peroxidase without being exposed to a pure oxygen atmosphere. In the cells maintained on cellulose, mitochondrial function may be limited by the supply of glucose, leading to the accumulation of reactive oxygen species.     

Effects of Nitrogen Supplements on Degradation of Aspen Wood Lignin and Carbohydrate Components by Phanerochaete chrysosporium

A supplement of KH₂PO₄, MgSO₄, CaCl₂, trace elements, and thiamine accelerated the initial rate of aspen wood decay by Phanerochaete chrysosporium but did not increase the extent of lignin degradation. Asparagine, casein hydrolysate, and urea supplements (1% added N) strongly inhibited lignin degradation and weight loss. The complex nitrogen sources peptone and yeast extract stimulated lignin degradation and weight loss. Albumen and NH₄Cl had intermediate effects. Conversion of [¹⁴C]lignin to ¹⁴CO₂ and water-soluble materials underestimated lignin degradation in the presence of the complex N sources. The highest ratio of lignin degradation to total weight loss and the largest increase in cellulase digestibility occurred during the decay of unsupplemented wood. Rotting of aspen wood by P. chrysosporium gives smaller digestibility increases than have been found with some other white-rot fungi.     

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.     

Metabolism of cyanide by Phanerochaete chrysosporium

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.     

Cellobiose Dehydrogenase, an Active Agent in Cellulose Depolymerization

The ability of cellobiose dehydrogenase purified from Phanerochaete chrysosporium to modify a Douglas fir kraft pulp was assessed. Although the addition of cellobiose dehydrogenase alone had little effect, supplementation with cellobiose and iron resulted in a substantial reduction in the degree of polymerization of the pulp cellulose. When the reaction was monitored over time, a progressive depolymerization of the cellulose was apparent with the concomitant production of cellobiono-1,5-lactone. Analysis of the reaction filtrates indicated that glucose and arabinose were the only neutral sugars generated. These sugars are derived from the degradation of the cellobiose rather than resulting from modifications of the pulp. These results suggest that the action of cellobiose dehydrogenase results in the generation of hydroxyl radicals via Fenton's chemistry which subsequently results in the depolymerization of cellulose. This appears to be the mechanism whereby a substantial reduction in the degree of polymerization of the cellulose can be achieved without a significant release of sugar.     

Cellobiose dehydrogenase (cellobiose oxidase) from Phanerochaete chrysosporium as a wood-degrading enzyme. Studies on cellulose,xylan and synthetic lignin

Degradation of carboxymethylcellulose (CMC), xylan and synthetic lignin was studied in a cellobiose dehydrogenase system, that reduced Fe(III) to Fe(II) with cellobiose as electron donor, which in the presence of hydrogen peroxide degraded all the above representatives of the main wood components, probably by forming Fenton's reagent. The production of hydroxyl radicals was shown by benzoate decarboxylation. For the CMC and xylan studies viscometry was used, while lignin degradation was studied by measuring the passage of ¹⁴C-labelled synthetic lignin (DHP) through membranes of different molecular-mass cut-off. The possible participation of cellobiose dehydrogenase, Fe(III) and hydrogen peroxide in wood degradation by white-rot and brown-rot fungi is discussed.     

Genetic Recombination in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

Heterokaryons made from auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium were induced to fruit. The isolation of wild-type and double-mutant phenotypes from these crosses indicated that genetic recombination had occurred. Cytological studies demonstrated that more than 90% of the basidiospores from the wild-type and auxotrophic strains and from forced heterokaryons were binucleate. Colonies of the wild-type strain of P. chrysosporium arising from single, predominantly uninucleate conidia were all capable of producing fruit bodies and basidiospores.     

Semi-continuous ligninase production using foam-immobilised Phanerochaete chrysosporium

Summary The immobilisation of Phanerochaete chrysosporium in cubes of polyurethane foam enables ligninase to be produced on a semi-continuous basis. At each successive harvest, cultures are purged with oxygen and ligninase activity induced with veratryl alcohol. Using 200 ml of a five-fold dilution of the batch culture medium in 1l flasks, harvests of ligninase with the same apparent protein profile by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are obtained every 24 to 48 h.It is also possible to store cubes of foam containing pellets of P. chrysosporium so that further yields of ligninase comparable to fresh cultures can be produced within 48 h when desired.