Bovine Milk Lipase. I. Isolation from Skimmilk

A procedure was developed for the isolation of milk lipase from skimmilk: Fresh skimmilk is coagulated with rennet, separating curd, and whey by centrifugation, with most of the lipase accompanying the curd. The lipase is solubilized from the curd by maceration in 1 m NaCl and centrifugation. The supernatant is half-saturated with (NH 4 ) 2 SO 4 , the precipitate collected, dissolved, dialyzed, and fractionated on DEAE-cellulose. The lipase-rich fraction is made to 30% (v/v) with dimethylformamide and then to half-saturation with (NH 4 ) 2 SO 4 . Under these conditions, the lipase remains soluble and is finally purified by filtration through Sephadex G-200. The homogeneity of the preparation was established by ultracentrifugation and starch gel electrophoresis. Specific activity was about 500 times that of skimmilk, with an over-all yield of 10%. The sedimentation coefficient was calculated to be 7.5 S, and the molecular weight about 210,000. With milk fat as substrate, pH optimum was 9.2 and temperature optimum 37C.