The main components of bovine whole casein were characterized by electrofocusing; pi values of a s r , /?-, K-, y r , y 2 -and y 3 -caseins were determined. A further identification of casein components was achieved by a 2-dimensional electrophoresis study. 2-Dimensional patterns of y-caseins obtained from a hydrolysate of yS-cascin by bovine plasmin are in good agreement with those of y-caseins naturally present in whole casein.

SUMMABYIsoelectric focusing and 2-dimensional electrophoresis were used to study the development of the pH 4·6-insoluble fraction during Camembert cheese ripening. Modifications of this fraction were due mainly to the action of 5 proteinases: rennet (chymosin + bovine pepsin), plasmin and Penicillium caseicolum aspartyl-and metalloproteinases. Rennet was inactive on β-casein, but acted very early on αs1-casein. However, rennet and P. caseicolum aspartyl-proteinase had a very similar action on the latter substrate, which prevented clear definition of the respective actions of these proteinases on αs1-casein after 7 d of ripening. Plasmin action on β-casein was important from 21 and 35 d of ripening at the surface and in the centre of the cheese respectively, suggesting an important influence of pH changes during maturation. The respective activities of the metallo-and aspartyl-proteinases of P. caseicolum were characterized and followed using β-casein degradation products as markers. The metallo-proteinase activity was detectable immediately after the development of the Penicillium (7 d), while that of the aspartyl-proteinase was observed 3 d later. Thereafter, the amount of β-casein degradation peptides resulting from the metalloproteinase decreased while that resulting from the aspartyl-proteinase increased, suggesting a more important role of the latter enzyme.

The results reported in the present paper and N-terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species.Bovine gastricsin was prepared from commercial extracts of adult bovine vells by a procedure involving DEAE-cellulose chromatography, gel filtration on Sephacryl S-200 and a further DEAE-cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Its molecular weight was estimated by gel filtration to be 32 800.Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pl values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin-A-Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin-A -Sepharose 4B.Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins.Bovine gastricsin which is inactivated by reaction with diazoacetyl-~~-nor~eucine methyl ester and with 1,2-epoxy-(p-nitrophenoxy)propane in a 1 : I and 1 : 2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N-acetyl-L-phenylalanyl-L-diiodotyrosine. Its specific clotting activity with x-casein as substrate is only half of that of chymosin and pepsin A.

The aspartyl proteinases of Penicillium caseicolum and P. roqueforti acted identically on /f-casein; both enzymes split at least 3 bonds: Lys 29 -Ile 30 , Lys 97 -Val 98 and Lys 99 -Glu 100 . From a sl -casein, these proteinases released 6 main degradation products which arose from the splitting of 4 bonds; P. roqueforti aspartyl proteinase was found to cleave 1 bond at a higher rate than P. caseicolum aspartyl proteinase. A hypothetical sequential hydrolysis mechanism of a sl -casein by these 2 enzymes is proposed from a study of the degradation by isoelectric focusing and by 2-dimensional electrophoresis.Proteinases play a fundamental role in the dairy industry. Fox (1981) distinguishes 3 proteinase classes which can be involved; these are the indigenous proteinases naturally occurring in milk (e.g. plasmin), the endogenous proteinases coming from microorganisms and the exogenous proteinases which are added to the milk (e.g. milk-clotting enzymes). The in vitro and in situ proteolytic action of some of these proteinases on caseins, particularly those of bovine pepsins A and chymosin, is now well-known (for a recent review, see Visser, 1981). However, the enzymes of microorganisms have not been studied extensively and the characterization of their proteolytic activities on casein is still ill-defined. This communication reports a comparison of the action of the aspartyl proteinases of Penicillium caseicolum and P. roqueforti on ft-and a sl -caseins as measured by isoelectric focusing (IEF) and 2-dimensional electrophoresis. In addition the 3 main bonds cleaved by P. caseicolum aspartyl proteinase on /J-casein were identified. These 2 mould enzymes, which belong to the exocellular proteolytic system (Lenoir et al. 1979;Zevaco et al. 1973) play an important role during Camembert-type cheese ripening (P. caseicolum) and blue cheese ripening (P. roqueforti). MATERIALS AND METHODS Preparation of caseins and proteinasesWhole casein, purified a sl B-and /?A 2 -caseins were prepared according to Mercier et al. (1968). P. caseicolum aspartyl proteinase (a gift from Dr Lenoir) was prepared as described elsewhere (Lenoir et al. 1979). P. roqueforti aspartyl proteinase was prepared according to Zevaco et al. (1973).

SummaryUsing polyacrylamide gel electrophoresis, 2-dimensional gel electrophoresis and electrofocusing, it has been shown that 4 basic fragments were produced by action of plasmin on buffalo β-casein. These fragments were present in whole buffalo casein prepared from bulk milk. Their mol. wts, 20000, 16000 and 11000 (2 components) were determined by SDS-gradient polyacrylamide gel electrophoresis. It is suggested that 3 of them are homologous to bovine γ1-, γ2- and γ3-caseins respectively, while the last one (mol. wt 16000), which has no counterpart in plasmin digest of bovine β-casein, could arise from a cleavage at bond 68–69.