Purification, Characterization, and Molecular Cloning of a Pyranose Oxidase from the Fruit Body of the Basidiomycete,Tricholoma matsutake

Abstract

A new H(2)O(2)-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H(2)O(2) and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V(max), K(m), and k(cat) for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50 degrees C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.

Pyranose 2-oxidase from Phanerochaete chrysosporium—Expression in E. coli and biochemical characterization

“…The respective gene had previously been identified in the P. chrysosporium genome as encoding the P2Ox enzyme purified from mycelium grown on wheat bran supplemented with corn steep liquor (De Koker et al, 2004). cDNA was synthesized from mRNA obtained under carbon-limiting conditions, as this was shown to promote transcription of the p2ox-gene (De Koker et al, 2004), and ligated into an expression plasmid for E. coli, a host-vector system that was already shown to work effectively for other, related pyranose oxidase enzymes (Bastian et al, 2005;Heckmann-Pohl et al, 2006;Kotik et al, 2004;Maresova et al, 2005;Takakura and Kuwata, 2003). We were able to obtain yields of 4500 U of P2Ox activity and 270 mg of active P2Ox protein per liter of production medium in shake-flask cultures without optimization of the expression system and cultivation conditions.…”

“…Previous attempts at determining the N-termini of purified pyranose oxidases have yielded ambiguous results, with the first amino acid obtained in sequencing being as far as 66 amino acid residues into the conceptual translations of the respective ORFs (Artolozaga et al, 1997;Nishimura et al, 1996;Takakura and Kuwata, 2003). It has been speculated that processing of P2Ox, i.e., removal of a putative (unidentified) signal sequence is responsible for these discrepancies (Nishimura et al, 1996;Takakura and Kuwata, 2003).…”

“…These include Coriolus (Trametes) versicolor (Nishimura et al, 1996), Trametes hirsuta (Christensen et al, 2000), Tricholoma matsutake (Takakura and Kuwata, 2003), Peniophora sp. (Bannwarth et al, 2004), Trametes multicolor (synonymous for T. ochracea) (Kujawa et al, 2006;Vecerek et al, 2004) and Trametes pubescens (Maresova et al, 2005).…”

Isolation and Purification of Pyranose 2-Oxidase fromPhanerochaete chrysosporiumand Characterization of Gene Structure and Regulation

“…gi|31044224|) and Tricholoma matsutake (TmPOX; accession no. gi|25553433| [44]). Grey shaded amino acids indicate homology with PcPOX; black shaded amino acids with white typeface indicate CvPOX-GMC consensus sequences (see the text).…”

Bioconversion of d-glucose into d-glucosone by Glucose 2-oxidase from Coriolus versicolor at Moderate Pressures

“…Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) is synthesized by several white rot fungi such as Coriolus versicolor and Phanerochaete chrysosporium and plays an important role in lignin biodegradation [1][2][3][4][5]. Traditional carbohydrate chemistry requires multiple and complex steps of activation and protection chemistry but it lack stereo-specificity in such reactions.…”