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Process for production of ascorbate oxidase
| Details |
Inventors: Matsumura, Eiji; Ishikawa, Hidehiko; Misaki, Hideo;
Assignee: Toyo Jozo Kabushiki Kaisha (Shizuoka, JP)
Primary Examiner: Shapiro; Lionel M.
Assistant Examiner:
Attorney, Agent or Firm: Young & Thompson
Ascorbate oxidase is produced by extraction from plants of the genus Sechium, particularly the species thereof which is Sechium edule Sw. The crushed plant tissues are extracted with an aqueous alkaline solvent, preferably at about pH 11. The extract is then subjected to centrifugation, concentration under vacuum, salting out with ammonium sulfate and solvent fractionation with acetone, to prepare crude ascorbate oxidase, which is then further purified by dialysis, ion exchange chromatography, adsorption chromatography and gel filtration. The ascorbate oxidase thus obtained has an optimum pH of about 7, a km value of 0.3 mM, an isoelectric point of around pH 6.3 and a molecular weight of about 100,000. |
|
DETAILED DESCRIPTION What is claimed is: 1.
A process for the production of ascorbate oxidase, comprising contacting plant tissue of the species Sechium edule Sw.
with an aqueous alkaline medium as an extracting solvent, and separating ascorbate oxidase from the solvent solution thereof thus produced.
2.
A process as claimed in claim 1, in which the pH of said aqueous alkaline medium is about 11.
Description:
The present invention relates to a process for the production of ascorbate oxidase, and ascorbate oxidates thus produced.
Ascorbate oxidase catalyzes a reaction which produces one mole of dehydroascorbic acid and one mole of water from one mole of ascorbic acid and 1/2 mole of oxygen, and falls in the enzyme classification EC 1.
10.
3.
3.
L-Ascorbate: oxygen oxidoreductase; common name: ascorbate oxidase.
Such enzymes have been prepared from pericarps of genus Cucurbita and genus Cucumis and have been reported to exist in several seedlings [Enzyme Handbook, pages 158-159, 1966, Asakura Publ.
Co.
].
Ascorbate oxidase of genus Cucurbita and genus Cucumis has a molecular weight of about 140,000 and exists mainly in pericarps of the said plants.
The sarcocarp thereof has only a trace of activity, and so the preparation of ascorbate oxidase has been only from the separated pericarps, with the result that the yield was quite low.
Ascorbate oxidase in seedlings has been detected by observing an oxidative action of seedlings extract on ascorbic acid.
However, substantial and discrete enzyme isolation and the preparation of ascorbate oxidase have not been confirmed.
We have examined extensively the potential sources of ascorbate oxidase of various origins, such as green peppers (Capsicum grossum), oranges (Citrus sinensis), sweet potatoes (Ipomea batatas), potatoes (Solanum tuberosum) and goosefoot (Chenopodium ambrosioides var.
anthelminticum), and no ascorbate oxidase activity was found.
We have now found that not only the pericarps but also the whole plants of Sechium eduleSw
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Molecular Biology 
