Relative Stability of Recombinant Versus Native Peroxidases fromPhanerochaete chrysosporium

Nie, Guojun; Reading, N. Scott; Aust, Steven D.

doi:10.1006/abbi.1999.1180

Cited by 51 publications

(27 citation statements)

References 41 publications

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“…This type of covalent posttranslational modification has been confirmed with isoenzyme H4 (14), which possesses 55 potential sites for O-glycosylation, as opposed to only 4 sites for N-glycosylation. In turn, MnP1 from C. subvermispora possesses 56 potential sites for O-glycosylation and seven potential sites for N-glycosylation.…”

Section: Vol 67 2001mentioning

confidence: 74%

“…Several expression vectors were prepared with MnP1 cDNA from C. subvermispora (formerly MnP13-1) (14). All constructs were made by the PCR overlap extension technique (7) by using proofreading polymerase Pfu (Stratagene, La Jolla, Calif.), and the junctions of fusions were sequenced.…”

Section: Methodsmentioning

confidence: 99%

“…In turn, MnP1 from C. subvermispora possesses 56 potential sites for O-glycosylation and seven potential sites for N-glycosylation. Nie et al (14) showed that O-glycans play a role in the thermal stability of peroxidases from P. chrysosporium. O-Glycans might also have this role in H4, LiP2, or LiP8, since N-glycans represent only a small fraction of the total associated glycans.…”

Section: Vol 67 2001mentioning

confidence: 99%

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Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

Larrondo

Lobos

Stewart

et al. 2001

Appl Environ Microbiol

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We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the ␣-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.

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Section: Vol 67 2001mentioning

confidence: 74%

Section: Methodsmentioning

confidence: 99%

Section: Vol 67 2001mentioning

confidence: 99%

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Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

Larrondo

Lobos

Stewart

et al. 2001

Appl Environ Microbiol

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“…This difference could be the result of a higher degree of glycosylation of the recombinant enzyme and apparently has no major effect on the activity of rMnP. MnP is both N-and O-glycosylated, although Nie et al (29) have showed that glycosylation is not essential for the enzyme activity of MnP. Overglycosylation has also been observed in the production of recombinant chloroperoxidase (Conesa et al, unpublished) and phytase (50) in Aspergillus.…”

Section: Discussionmentioning

confidence: 99%

Studies on the Production of Fungal Peroxidases in Aspergillus niger

Conesa

Hondel

Punt

2000

Appl Environ Microbiol

127

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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.

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“…Fungal peroxidases are posttranscriptionally modified, thus the recombinant system must be able to promote proper folding, which depends on the correct coordination of the prosthetic heme group, formation of disulfide bridges and inclusion of structural calcium ions [Conesa et al, 2002b]. Glycosylation appears to enhance stability and solubility, but has no effect on activity [Nie et al, 1999]. Although homologous production may be an alternative, no overproduction has been achieved so far.…”

Section: Heterologous Expressionmentioning

confidence: 99%

Fungal Enzymes for Environmental Purposes, a Molecular Biology Challenge

2008

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In their capacity to transform xenobiotics and polluting compounds, fungal peroxidases and their use in the environmental field have a recognized and important potential. However, both fundamental and practical issues, such as enzyme stability and availability, have delayed the development of industrial applications. Three main protein engineering challenges have been identified: (1) Enhancement of operational stability, specifically hydrogen peroxide stability in the case of fungal peroxidases. (2) Increase of the enzyme redox potential in order to widen the substrate range. (3) Development of heterologous expression and industrial production. The bottlenecks, advances and strategies that have been proven successful are discussed.

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Relative Stability of Recombinant Versus Native Peroxidases fromPhanerochaete chrysosporium

Cited by 51 publications

References 41 publications

Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

Studies on the Production of Fungal Peroxidases in Aspergillus niger

Fungal Enzymes for Environmental Purposes, a Molecular Biology Challenge

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