scholarly journals Isolation and Purification of Pyranose 2-Oxidase from Phanerochaete chrysosporium and Characterization of Gene Structure and Regulation

2004 ◽  
Vol 70 (10) ◽  
pp. 5794-5800 ◽  
Author(s):  
Theodorus H. de Koker ◽  
Michael D. Mozuch ◽  
Daniel Cullen ◽  
Jill Gaskell ◽  
Philip J. Kersten
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Solid Substrate ◽  
Culture Conditions ◽  
Lignocellulose Degradation ◽  
Supporting Evidence ◽  
Isolation And Purification ◽  
Lignin Peroxidases ◽  
Liquid Chromatography Tandem Mass ◽  
Glyoxal Oxidase

ABSTRACT Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrate culture using mild extraction conditions and was purified. 13C-nuclear magnetic resonance confirmed production of d-arabino-hexos-2-ulose (glucosone) from d-glucose with the oxidase. Peptide fingerprints generated by liquid chromatography-tandem mass spectrometry of tryptic digests and analysis of the corresponding cDNA revealed a structurally unusual sequence for the P. chrysosporium POX. Relatively high levels of pox transcript were detected under carbon-starved culture conditions but not under nutrient sufficiency. This regulation pattern is similar to that observed for lignin peroxidases, manganese peroxidases, and glyoxal oxidase of P. chrysosporium, supporting evidence that POX has a role in lignocellulose degradation.

scholarly journals Structure, Organization, and Transcriptional Regulation of a Family of Copper Radical Oxidase Genes in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

2006 ◽  
Vol 72 (7) ◽  
pp. 4871-4877 ◽  
Author(s):  
Amber Vanden Wymelenberg ◽  
Grzegorz Sabat ◽  
Michael Mozuch ◽  
Philip J. Kersten ◽  
Dan Cullen ◽  
...  
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Extracellular Enzymes ◽  
Draft Genome ◽  
Defined Medium ◽  
White Rot ◽  
Lignocellulose Degradation ◽  
Significant Similarity ◽  
Secretion Signal ◽  
Liquid Chromatography Tandem Mass ◽  
Transcript Profiles

ABSTRACT The white rot basidiomycete Phanerochaete chrysosporium produces an array of nonspecific extracellular enzymes thought to be involved in lignin degradation, including lignin peroxidases, manganese peroxidases, and the H2O2-generating copper radical oxidase, glyoxal oxidase (GLX). Preliminary analysis of the P. chrysosporium draft genome had identified six sequences with significant similarity to GLX and designated cro1 through cro6. The predicted mature protein sequences diverge substantially from one another, but the residues coordinating copper and constituting the radical redox site are conserved. Transcript profiles, microscopic examination, and lignin analysis of inoculated thin wood sections are consistent with differential regulation as decay advances. The cro2-encoded protein was detected by liquid chromatography-tandem mass spectrometry in defined medium. The cro2 cDNA was successfully expressed in Aspergillus nidulans under the control of the A. niger glucoamylase promoter and secretion signal. The recombinant CRO2 protein had a substantially different substrate preference than GLX. The role of structurally and functionally diverse cro genes in lignocellulose degradation remains to be established.

scholarly journals Expression of Phanerochaete chrysosporium Genes Encoding Lignin Peroxidases, Manganese Peroxidases, and Glyoxal Oxidase in Wood

1998 ◽  
Vol 64 (9) ◽  
pp. 3536-3538 ◽  
Author(s):  
Bernard J. H. Janse ◽  
Jill Gaskell ◽  
Masood Akhtar ◽  
Daniel Cullen
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Lignin Peroxidase ◽  
Ligninolytic Enzymes ◽  
Wood Chips ◽  
Mrna Levels ◽  
Defined Media ◽  
Magnetic Capture ◽  
Lignin Peroxidases ◽  
Genes Encoding ◽  
Glyoxal Oxidase

ABSTRACT Expression of Phanerochaete chrysosporium genes encoding ligninolytic enzymes was assessed in wood. Poly(A) RNA was extracted from colonized wood chips by magnetic capture, and specific transcripts were quantified by competitive reverse transcriptase PCR. mRNA levels varied substantially among lignin peroxidase genes, and transcript patterns were dramatically different from those in previous studies with defined media.

scholarly journals Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main α-galactosidase

1999 ◽  
Vol 339 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Harry BRUMER ◽  
Paul F. G. SIMS ◽  
Michael L. SINNOTT
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Substrate Inhibition ◽  
Enzyme Reaction ◽  
Lignocellulose Degradation ◽  
K Value ◽  
Softwood Pulp ◽  
Leaving Groups ◽  
Kinetics Of ◽  
Optimal Ph

The main α-galactosidase was purified to homogeneity, in 30% yield, from a solid culture of Phanerochaete chrysosporium on 1 part wheat bran/2 parts thermomechanical softwood pulp. It is a glycosylated tetramer of 50 kDa peptide chains, which gives the N-terminal sequence ADNGLAITPQMG(?W)NT(?W)NHFG(?W)DIS(?W)DTI. It is remarkably stable, with crude extracts losing no activity over 3 h at 80 °C, and the purified enzyme retaining its activity over several months at 4 °C. The kinetics of hydrolysis at 25 °C of various substrates by this retaining enzyme were measured, absolute parameters being obtained by active-site titration with 2´,4´,6´-trinitrophenyl 2-deoxy-2,2-difluoro-α-d-galactopyranoside. The variation of kcat/Km for 1-naphthyl-α-d-galactopyranoside with pH is bell-shaped, with pK1 = 1.91 and pK2 = 5.54. The αD(V/K) value for p-nitrophenyl-α-d-glucopyranoside is 1.031±0.007 at the optimal pH of 3.75 and 1.114±0.006 at pH 7.00, indicating masking of the intrinsic effect at optimal pH. There is no α-2H effect on binding galactose [αD(Ki) = 0.994±0.013]. The enzyme hydrolyses p-nitrophenyl β-l-arabinopyranoside ∼ 510 times slower than the galactoside, but has no detectable activity on the α-d-glucopyranoside or α-d-mannopyranoside. Hydrolysis of α-galactosides with poor leaving groups is Michaelian, but that of substrates with good leaving groups exhibits pronounced apparent substrate inhibition, with Kis values similar to Km values. We attribute this to the binding of the second substrate molecule to a β-galactopyranosyl-enzyme intermediate, forming an E•βGal•αGalX complex which turns over slowly, if at all. 1-Fluoro-α-d-galactopyranosyl fluoride, unlike α-d-galactopyranosyl fluoride, is a Michaelian substrate, indicating that the effect of 1-fluorine substitution is greater on the first than on the second step of the enzyme reaction.

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