Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1977-06-15
1979-10-30
Shapiro, Lionel M.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
435803, 435191, C07G 7026
Patent
active
041727630
ABSTRACT:
Bovine milk xanthine oxidase, referred to herein as XO, is isolated and purified from raw whole milk by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Sodium salicylate, ethylenediaminetetraacetate (EDTA), and a 0.2 M phosphate buffer are added to fresh milk. After incubation at 40.degree.-45.degree. C. for 105 min., the mixture is adjusted to 1-2% with Triton X-100 and allowed to incubate for 15 min. The mixture is cooled to 4.degree. C., followed by a 2-step fractionation of the proteins with ammonium sulfate. The crude enzyme is isolated as a red-brown precipitate which is dissolved in 0.1 M Tris/CaCL.sub.2 buffer and stored for from 12 to 168 hours at -20.degree. C. The isolated enzyme is purified by column chromatography (Sephadex G-75, Sephacryl S-200, Sepharose 6B, and Sephadex G-75). The final stage of purification is accomplished by passing the enzyme preparation through a DEAE-Sephadex A-50 column in a continuous linear salt gradient from 0.005 M to 0.1 M pyrophosphate buffer.
The purified enzyme is not denatured and has, on the average, a constant E.sub.280 /E.sub.450 ratio of 4.1, and one symmetric peak by gel chromatography. Analysis by polyacrylamide disc gel electrophoresis demonstrates a single band, while commercially available XO shows 7-14 bands, only one of which contains XO activity (the rest being impurities). The average yield of the final product is about 21% which is 110% higher than the yield of less pure XO obtained using the best prior art.
REFERENCES:
waud et al., Archives of Biochemistry & Biophysics, vol. 169, pp. 695-701, (1975).
Methods in Enzymology, vol. II, 1955, pp. 482-485.
Shapiro Lionel M.
University of Delaware
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