Abstract
Ascorbate oxidase (AsA oxidase) [EC 1. 10. 3.3] was purified from the spores of Myrothecium verrucaria IAM 5063. To purify this enzyme, the extract from the spores was chromatographed on DEAESephadex A-50, Sepharase 6B and CM-Sephadex C-50. In this way, the refative activity of AsA oxidase was increased by as much as 700 times.
This AsA oxidase was capable of oxidizing L-ascorbic acid (L-AsA) to form dehydroascorbic acid. Its activity was maximal in the pH range af 6-7 at approximately 30°C. A kinetic study showed that, under these optimal conditions, the Km value of the reaction was 3.0mM. The molecular weight of the enzyme was found to be approximated at 380K, which is abaut 2 times that of AsA oxidase of vegetable origin.
The exclusive singularity of the present mold AsA oxidase toward L-AsA was noted as a distinct difference from vegetable. AsA oxidase.
Mold AsA oxidase was even inhibited by D-iso ascorbic acid. The mechanism of this inhibition was found to be non-competitive on the basis of a Lineweaver-Burk plot.
The addition of Cu2+ increased the activity of AsA oxidase by a factor of 5.7.
A sigmoidal relationship was observed between the Cu2+ concentration and AsA oxidase activity, indicating the enzyme to passibly be a kind of cupper-binding enzyme.
