Proteolytic specificity of elastase on bovine β-casein

The lysosomes of the principal somatic cell type recruited on mastitic infection, polymorphonuclear leucocytes (PMN), contain a wide range of hydrolytic enzymes which aid in the destruction of ingested bacteria. These enzymes are of increasing interest in terms of milk quality, but little is known about their activity on the caseins. The objective of this study was to determine the cleavage specificity of elastase, one of the principal PMN proteinases, on β-casein. β-Casein (5 mg ml−1) was dissolved in 0.1 M phosphate buffer, pH 7.5 and 1.76×10−3 U ml−1 of elastase were added. Samples were taken over a 24 h period and analysed by urea polyacrylamide gel electrophoresis and high performance liquid chromatography. Peptides were identified by N-terminal sequencing and mass spectrometry. Elastase cleaved β-casein at several sites including Ile26-Asn27, Gln40-Thr41, Ile49-His50, Phe52-Ala53, Gln56-Ser57, Leu58-Val59, Asn68-Ser69, Val82-Val83, Val95-Ser96, Ser96-Lys97, Lys97-Val98, Ala101-Met102, Glu108-Met109, Phe119-Thr120, Glu131-Asn132, Leu163-Ser164, Ala189-Phe190, Phe190-Leu191 and Pro204-Phe205. Some of these sites are also cleaved by chymosin, plasmin or the cell envelope-associated proteinase of Lactococcus. The results show that elastase has a broad specificity on β-casein and it is therefore possible that indigenous elastase in milk may be of significance to the proteolysis of milk proteins.