Purification and Characterization of Milk Lipase. I. Purification

Abstract

Milk lipase was isolated successfully from elarifier sediment. The method involves preparation of acetone powder of the sediment, extraction of the enzyme with water, and fractional precipitation with ammonium sulfate and acetone. The enzyme with some minor contaminants selectively precipitates between 35 and 45% saturation of ammonium sulfate and at 45% (v/v) concentration of acetone. Finally, the enzyme is purified by chromatography on a Sephadex G-50 column. The purity and homogeneity of the enzyme was confirmed with the ultracentrifugal and gel electrophoretic techniques. The specific activity of the purest lipase fraction had a purification of about 88-fold on the nfilk protein basis, with an over-all yield of about 22%. The purification was 250-fold on the basis of milk solids and 2,600-fold on the basis of milk. The purified enzyme gave a typical protein ultraviolet absorption spectrum with maximum absorption at 277 m/z and minimum at 251 m~, with A:~/A~sl ratio being 2.34. Chemical analysis showed that the enzyme contained 49.63% carbon, 7.51% hydrogen, 14.33% nitrogen, ]..04% sulfur, and 0.26% phosphorus.