Pyranose oxidase and
pyranosone dehydratase (aldos-2-ulose dehydratase),
enzymes which convert in coupled reactions
D-glucose to beta-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+).