|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1992 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1991 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Ascorbate oxidase (EC 126.96.36.199), obtained from various plant origin such as cucumber, was used as analytical enzyme in the clinical field. But cucumber ascorbate oxidase was unstable enzyme, more stable enzyme was a desired.
A screening test was undertaken to isolated microorganisms, that produced ascorbate oxidase. The enzyme activity was found in a culture filtrate of fungal strain (HI-25), newly isolated from a soil sample. Based on the morphological characteristics, this isolate was identified as Acremonium sp. From the examinations of cultural conditions for enzyme production were found ; strain HI-25 was aerobically cultured by a jar fermenter at 25 ﾟC in a medium containing 5% glycerol, 2% defeatted soybeans, 0.1% monosodium L-glutamate, 0.1% KH_2PO_4, 0.02% MgSO_4/7H_2O, and 0.01% KCl, pH 6.0. After cultivation, an ascorbate oxidase was purified from the culture filtrate by an ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and Butyl-Toyopearl, and gel
filtration twice on Sephadex G-100. The purification was 850-fold with an activity yield of 8.8%. The purified enzyme gave a single band on SDS polyacrylamide gel electrophoresis, and had a molecular weight of 80,000 by SDS polyacrylamide gel electrophoresis and 76,000 by native gel filtration. This enzyme was most active at pH 4.0, 45ﾟC, and was most stable between pH 6.0 -10.0 and at temperature below 60ﾟC. The presence of copper and carbohydrate were demonstrated in the purified ascorbate oxidase preparation ; 4 gram atom Cu/mol, carbohydrate content 14.1%. The enzyme was inhibited by various compound that form a complex with mwtal ions. The reaction product from L-ascorbic acid was identified as dehydroascorbic acid. The stoichiometric investigations on the oxidation reaction revealed that this enzyme belong to EC 188.8.131.52 group, using molecular oxygen as an electron acceptor. The Km value from L-ascorbic acid was calculated to be 0.19 mM. And this enzyme did not oxidize other than ascorbic acid dericatives.
Application of Acremonium sp. ascorbate oxidase was investigated. To estimate ascorbic acid by measuring the dissolved oxygen with oxygen electrode, a good correlation between oxygen comsumption and ascorbic acid concentration was observed. The application of the enzyme to diagnostic reagent was confirmed in the human serum cholesterol estimation.