Proteolytic specificity of elastase on bovine β-casein

Abstract

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 Ile 26-Asn 27, Gln 40-Thr 41, Ile 49-His 50, Phe 52-Ala 53, Gln 56-Ser 57, Leu 58-Val 59, Asn 68-Ser 69, Val 82-Val 83, Val 95-Ser 96, Ser 96-Lys 97, Lys 97-Val 98, Ala 101-Met 102, Glu 108-Met 109, Phe 119-Thr 120, Glu 131-Asn 132, Leu 163-Ser 164, Ala 189-Phe 190, Phe 190-Leu 191 and Pro 204-Phe 205. 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.