Purification of human milk bile salt-activated lipase

Abstract

A modified procedure for a large-scale purification of human milk bile salt-activated lipase (BAL) has been devised. An initial step used cholate-Sepharose affinity chromatography for the partial purification of the enzyme followed by the removal of cholate with a Bio-Rex 5 anionexchange resin. The final step of purification used heparin-Sepharose affinity chromatography. This procedure of purification resulted in a 50-fold purification of BAL from human skim milk and a specific activity of 50–60μmol/min/mg with p-nitrophenyl acetate as substrate. From 450 ml of human skim milk, approximately 30 mg of purified enzyme could be obtained. The N-terminal-region amino acid sequence of the purified BAL was determined as follows: Ala-Lys-Leu-Gly-Ala-Val-Tyr-Thr-Glu-Gly-Lys-Phe-Val-Glu-Gly-Val-Asn-Lys-Lys-Leu-Gly-Leu-. Despite the finding that BAL interacts with heparin-Sepharose, soluble heparin had no effect on BAL activity. The possible physiological role of BAL-heparin interaction has also been discussed.