Abstract
A novel type of ascorbate oxidase was purified 420-fold from the cytosolic fraction of the mycelia of Pleurotus ostreatus with an overall yield 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 consists of two subunits with a molecular mass of 46 kDa. The N-terminal amino acid sequence of the enzyme was Asp-Val-Lys-Thr-Leu-Gln-Glu-His-Leu-Gln-Leu-Ala-Leu-Met-Val-. The enzyme was optimally active at pH 5.2, monitored at 37 degrees C. The enzyme had affinity toward L-ascorbic acid, D-ascorbic acid, L-erythroascorbic acid, and D-erythroascorbic acid. Under optimal conditions, the Km value of the enzyme toward L-ascorbic acid 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 reduced form, and alpha and beta bands at 558 and 527 nm only in its reduced form, respectively. On the basis of spectral changes after treatment with cyanide and carbon monoxide, the enzyme is a hemoprotein, quite similar to b-type cytochrome, and contains 2 mol of heme per molecule. The reaction catalyzed by the enzyme was L-ascorbic acid + O2 --> dehydro-L-ascorbic acid + H2O2.
Highlights
L-Ascorbic acid is oxidized by a successive reversible oneelectron transfer process with a free radical intermediate, and the ascorbate redox system consists of L-ascorbic acid, L-ascorbyl free radical, and dehydro-L-ascorbic acid [1]
We report the purification and characterization of a novel type of an intracellular heme-containing ascorbate oxidase from an oyster mushroom, Pleurotus ostreatus
The activity of ascorbate oxidase was overlapped with that of laccase, because laccase occurs in the mycelial crude extract of P. ostreatus and can oxidize ascorbic acid
Summary
Materials—L-Ascorbic acid and D-ascorbic acid were purchased from Merck, Sepharose CL-6B, reactive red 120-Sepharose CL-4B, Sephadex G-200, S Sepharose CL-6B, and molecular mass markers for high performance gel filtration chromatography from Sigma, Protein PAK SW300 and Pico-Tag column from Waters, poly(vinylidene difluoride) membrane from Millipore, and molecular mass standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from Boehringer Mannheim. Microorganism and Growth Conditions—The mycelium of white-rot fungus Pleurotus ostreatus, a kind gift from the Korean Forest Research Laboratory, was grown in complete medium containing 2% malt extract (w/v), 0.5% peptone (w/v), 0.5% yeast extract (w/v), and 1% glucose (w/v). It was cultivated for 4 days at 28 °C in a 500-ml Erlenmeyer flask containing 200 ml of medium in a reciprocally shaking water bath operating at 100 rpm. Enzyme Assay—The activity of ascorbate oxidase was determined using 0.1 M sodium phosphate, citrate buffer (pH 5.4) containing 0.5 mM EDTA, according to the method of Oberbacher and Vines [16]. The starting cell mass was 100 g by wet weight
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