PYRANOSONE DEHYDRATASE FROM THE BASIDIOMYCETE PHANEROCHAETE-CHRYSOSPORIUM - IMPROVED PURIFICATION, AND IDENTIFICATION OF 6-DEOXY-D-GLUCOSONE AND D-XYLOSONE REACTION-PRODUCTS

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 Phanero chaete chrysosporium under agitated conditions. The enzymes were purif ied from mycelial extracts of the fungus and separated from each other by hydrophobic interaction chromatography on Phenyl-Sepharose and Phe nyl-Superose. Two pyranosone dehydratase activity peaks, PD I and PD I I, were resolved. The major PD I fraction, consisting about 74% of the total dehydratase activity, was further purified by anion exchange ch romatography on Mono Q to yield apparently pure enzyme as judged by SD S-PAGE and gel filtration on Superose 12. Isoelectric focusing indicat ed 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 pu rification was 60.7-fold with 50% yield. The enzyme acted on several o sones (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-glycosdiulo ses), new enzymatically produced sugar derivatives. The latter labile compounds were trapped as diphenylhydrazine or o-phenylenediamine deri vatives and spectroscopically identified. The analogous D-glucosone de hydration 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 som e heavy metal ions (Hg2+, Cu2+).