Proteolytic specificity of elastase on bovine beta-casein

The lysosomes of the principal somatic cell type recruited on mastitic infe ction, polymorphonuclear leucocytes (PMN), contain a wide range of hydrolyt ic enzymes which aid in the destruction of ingested bacteria. These enzymes are of increasing interest in terms of milk quality, but little is known a bout their activity on the caseins. The objective of this study was to dete rmine the cleavage specificity of elastase, one of the principal PMN protei nases, on beta-casein. beta-casein (5 mg ml(-1)) was dissolved in 0.1 M pho sphate buffer, pH 7.5 and 1.76x10(-3) U ml(-1) of elastase were added. Samp les were taken over a 24 h period and analysed by urea polyacrylamide gel e lectrophoresis and high performance liquid chromatography. Peptides were id entified by N-terminal sequencing and mass spectrometry. Elastase cleaved p -casein at several sites including Ile(26)-Asn(27), Gln(40)-Thr(41), IIe(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), Al-1 01-Met(102), Glu(108)-Met(109), Phe(52)-Thr(120), Glu(131)-Asn(132), Leu(16 3)-Ser(164), Ala(189)-Phe(190), Phe(190)-Leu(191) and Pro(204)-Phe(205) Som e 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 beta-casein and it is therefore possible that indigen ous elastase in milk may be of significance to the proteolysis of milk prot eins. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.