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Archives of Microbiology

, Volume 160, Issue 1, pp 27–34 | Cite as

Pyranosone dehydratase from the basidiomycete Phanerochaete chrysosporium: improved purification, and identification of 6-deoxy-d-glucosone and d-xylosone reaction products

  • Jiří Gabriel
  • Jindřich Volc
  • Petr Sedmera
  • Geoffrey Daniel
  • Elena Kubátová
Original Papers
  • 54 Downloads

Abstract

Pyranose oxidase and pyranosone dehydratase (aldos-2-ulose dehydratase), enzymes which convert in coupled reactions d-glucose to β-pyrone cortalcerone, peaked coincidently during idiophasic growth of Phanerochaete chrysosporium under agitated conditions. The enzymes were purified from mycelial extracts of the fungus and separated from each other by hydrophobic interaction chromatography on Phenyl-Sepharose and Phenyl-Superose. Two pyranosone dehydratase activity peaks, PD I and PD II, were resolved. The major PD I fraction, consisting about 74% of the total dehydratase activity, was further purified by anion exchange chromatography on Mono Q to yield apparently pure enzyme as judged by SDS-PAGE and gel filtration on Superose 12. Isoelectric focusing indicated microheterogeneity of the protein by the presence of at least five protein bands with pI 5.1–5.3. PD II had a pI of 5.75. Overall PD I purification was 60.7-fold with 50% yield. The enzyme acted on several osones (glycosuloses), with the preferred substrate being d-glucosone. d-Xylosone and 6-deoxy-d-glucosone were dehydrated at C-3-C-4 to give the corresponding 5-hydroxy-2,3-dioxoalcanals (4-deoxy-2,3-glycosdiuloses), new enzymatically produced sugar derivatives. The latter labile compounds were trapped as diphenylhydrazine or o-phenylenediamine derivatives and spectroscopically identified. The analogous d-glucosone dehydration product did not accumulate due to its further transformation. pH optimum of PD I activity was 6.0 and its pH stability was optimal at pH 7-11. The enzyme was sensitive to Me2+ chelating agents and some heavy metal ions (Hg2+, Cu2+).

Key words

Pyranosone dehydratase Aldos-2-ulose dehydratase Pyranose oxidase Glucosone Xylosone Phanerochaete chrysosporium 

Abbreviations

DMAB

3-dimethylaminobenzoic acid

DTT

dithiothreitol

MBTH

3-methyl-2-benzothiazolinone hydrazone-hydrochloride

PD

pyranosone dehydratase

PMSF

phenylmethylsulfonyl fluoride

POD

pyranose oxidase

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Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Jiří Gabriel
    • 1
  • Jindřich Volc
    • 1
  • Petr Sedmera
    • 1
  • Geoffrey Daniel
    • 2
  • Elena Kubátová
    • 1
  1. 1.Institute of MicrobiologyAcademy of Sciences of Czech RepublicPragueCzech Republic
  2. 2.Department of Forest ProductsThe Swedish University of Agricultural SciencesUppsalaSweden

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