A HEME-CONTAINING ASCORBATE OXIDASE FROM PLEUROTUS-OSTREATUS

A novel type of ascorbate oxidase was purified 420-fold from the cytos olic fraction of the mycelia of Pleurotus ostreatus with an overall yi eld of 13%. The molecular mass of the native enzyme determined by high performance gel permeation chromatography was 94 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme co nsists of two subunits with a molecular mass of 46 kDa. The N-terminal amino acid sequence of the enzyme was -Thr-Leu-Gln-Glu-His-Leu-Gln-Le u-Ala-Leu-Met-Val-. The enzyme was optimally active at pH 5.2, monitor ed at 37 degrees C. The enzyme had affinity toward L-ascorbic acid, D- ascorbic acid, L-erythroascorbic acid, and D-erythroascorbic acid, Und er optimal conditions, the K-m value of the enzyme toward L-ascorbic a cid was 0.48 mM. The absorption spectra of the native enzyme exhibited a Soret maximum at 418 nm in its oxidized form and at 426 nm in its r educed form, and alpha and beta bands at 558 and 527 nm only in its re duced form, respectively, On the basis of spectral changes after treat ment with cyanide and carbon monoxide, the enzyme is a hemoprotein, qu ite similar to b-type cytochrome, and contains 2 mol of heme per molec ule, The reaction catalyzed by the enzyme was L-ascorbic acid + O-2 -- > dehydro-L-ascorbic acid + H2O2.