Proteolysis of bovine caseins by cathepsin D: Preliminary observations and comparison with chymosin

I~I. Dairy Journal 5 (1995) 321-336 Elsevier Science Limited Printed in Ireland. 0958-6946/95/$9.50 + .OO

ELSEVIER

0958-6946(94)00010-7

Proteolysis of Bovine Caseins by Cathepsin D: Preliminary Observations and Comparison with Chymosin P. L. H. McSweeney, Department

P. F. Fox

of Food Chemistry, University College, Cork, Ireland

& N. F. Olson Department

of Food Science, University of Wisconsin, Madison, WI 53706, USA

(Received 23 August 1993;revised version accepted 8 April 1994)

ABSTRACT Proteolysis of a,~-. as2, /I- and rc-caseins by bovine cathepsin D (E.C. 3.4.23.5), an indigenous acid proteinase in milk, was studied by reversed-phase HPLC and ureaPAGE. The results indicate that cathepsin D hydrolyzed all casein fractions and was more active on Q- than on /I-casein; a,,-casein was optimally hydrolyzed at pH 4.0. Proteolysis of p-casein was more sensitive to inhibition by NaCI than was a,,-casein. Comparison of the proteolysis of individual caseins with that by chymosin (E.C. 3.4.23.4) showed that the hydrolysis of u,t-casein by the two enzymes was very similar, but the specificities on cc,rcasein differed. The initial stage of B-casein hydrolysis by cathepsin D was similar to that by chymosin and HPLC profiles showed a number of peptides in common. Although cathepsin D hydrolyzed u-casein slowly in solution and the HPLC projZes of hydrolysates were similar to those produced by chymosin, cathepsin D showed poor milk clotting ability.

INTRODUCTION The presence of indigenous proteolytic activity in milk has been recognized for many years. The principal indigenous proteinase in milk is plasmin (fibrinolysin, E.C. 3.4.21.7), which is optimally active at -pH 7.5, and has been studied extensively (see Visser, 1981; Grappin et al., 1985; Grufferty and Fox, 1988; Fox, 1989; Fox and Law, 1991). The presence of a second indigenous proteinase in milk with an acid pH optimum (-pH 4.0) is also recognized (Kaminogawa and Yamauchi, 1972, 1978; Kaminogawa et al., 1980). Kaminogawa and 321

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Yamauchi (1972) described some properties of this ‘milk acid protease’ and suggested that it was similar to the lysosomal proteinase, cathepsin D (E.C. 3.4.23.5). Recently, the zymogen, procathepsin D, has been demonstrated in milk by Larsen et al. (1993) who found that procathepsin D could not autoactivate to cathepsin D at acid pH but could form pseudocathepsin D, by cleavage at Leu2sLeu27 in its propart. The action of plasmin on bovine caseins has been the subject of many investigations (see above references), but the action of the indigenous acid proteinase on caseins has received little attention. It was reported (Kaminogawa and Yamauchi, 1972) that the acid proteinase preferentially hydrolyzed a,t-casein and that its specificity on asI-, /I- and K-caseins was similar to that of chymosin (Kaminogawa et al., 1980). The importance of plasmin in cheese ripening has been demonstrated (Farkye and Fox, 1990, 1991, 1992) and the possible significance of milk acid proteinase in cheese ripening was discussed by Kaminogawa and Yamauchi (1972) and Kaminogawa et al. (1980). Noomen (1978) suggested that the indigenous acid proteinase may play a minor role in cheese ripening and that the presence of CQcasein fragment 24-199 (a,i-I-casein) in aseptic, chemically-acidified, rennet-free cheese may be due to its action. Cathepsin D is a lysosomal enzyme (Go11 et al., 1983). Its physiological role involves intracellular digestion and turnover of proteins. While the enzyme has received little attention in the context of dairy foods, it has been investigated extensively in relation to meat and muscle biology where it may be involved in post mortem changes (Dutson, 1983), including the proteolysis of collagen (Scott and Pearson, 1978) and myofibrillar proteins (Matsumoto et al., 1983; Zeece et al., 1986; Schwartz and Bird, 1977). Cathepsin D is susceptible to autolysis (Lah and Turk 1982). The presence of many enzymes in milk is accidental (Walstra and Jenness, 1984). This may also be the case with cathepsin D which plays no known physiological role in milk. Lysosomes in leucocytes may be a source of cathepsin D in milk. Lysosomal proteinases play a role in involution of the uterus post partum and of the mammary gland at the end of lactation, when extensive turnover of tissue occurs (Pitt, 1975). Lysosomes are present in the mammary gland and the concentration of lysosomal enzymes increases at the onset of pregnancy (Pitt, 1975). However, variations in the concentration of cathepsin D in milk with the stage of lactation are unknown. However, Larsen et al. (1993) speculated that procathepsin D may be secreted into milk, perhaps as a result of the large amount of protein synthesis which occurs during lactation. The objectives of the present study were to characterize the action of cathepsin D on bovine caseins and to compare its action with that of chymosin.

MATERIALS

AND METHODS

Whole casein was prepared from a sample of bulk bovine milk, obtained from the University of Wisconsin-Madison Dairy Plant, by isoelectric precipitation. Casein was fractionated by ion-exchange chromatography on DEAE-cellulose (DE-52, Whatman, Maidstone, Kent, UK) using 1OmM imidazole buffer, pH 7.0, containing 4.5 M urea and 0.1% (v/v) 2-mercaptoethanol (Creamer, 1974);

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proteins were eluted using a linear NaCl gradient (WI.5 M). Fractions corresponding to usI-, cls2-,/3- and rc-caseins were pooled, dialysed and freeze dried. Bovine cathepsin D (E.C. 3.4.23.5) containing -10 units mg-’ protein was obtained from Sigma Chemical Co. (St. Louis, MO; 1 unit of cathepsin D activity will produce an increase in A2s0 of 1.Omin-’ mL-’ at pH 3.0 at 37°C measured as trichloroacetic acid-soluble products using haemoglobin as substrate). Recombinant chymosin B (E.C. 3.4.23.4, produced by Kluyveromyces marxianus var. lack), was obtained from Gist Brocades Ltd., Delft, the Netherlands, and was dialyzed against H20, freeze dried and resuspended in 100mM K-phosphate buffer, pH 6.5, prior to use (-70 units mL_‘; 1 chymosin unit [CU] mL-’ is the activity necessary to coagulate 10 mL bovine milk, pH 6.5, in 100 s at 30°C). The action of cathepsin D on micellar casein was studied in skim milk diluted 1:6 with water, adjusted to pH 5.5 with 1 M HCI and containing 0.05% (w/v) NaNs. Cathepsin D was added (2 x 10-l U mL-‘) and the sample were incubated at 37°C. Samples were taken periodically for analysis by urea-PAGE. The study was carried out at pH 5.5, which was close to the lowest pH at which the individual caseins were soluble at 30°C and, therefore, the closest possible to the pH optimum of cathepsin D, i.e., -4.0; pH 5.5 is also close to the pH of fresh cheese curd. The optimum pH for hydrolysis of a,‘-casein by cathepsin D was determined by dissolving ol,,-casein (1 mgmL-‘) in Hz0 containing 0.05%, w/v, NaNs and adjusting the pH to values in the range 2-7 with 1 M HCl. Cathepsin D was added (1 x 1O-3U mL-‘) and the mixture incubated at 37°C for 12 h. Because protein precipitation occurred in the pH range 4-5, enzyme was added to these samples prior to pH adjustment. The influence of NaCl on the hydrolysis of Qand /I-caseins was studied by dissolving whole Na caseinate (5 mgmL-‘) in 1OOmM Na acetate buffer, pH 5.5, containing 0.05% NaNs and NaCl at concentrations from 0 to 20% (w/v). Cathepsin D was added (2 x 10-l UmL-‘) and the mixture incubated at 37°C for 1 or 3 h; sub-samples were taken for ureaPAGE. Solutions of cr,,-casein, fi-casein or K--casein (5mgmL-‘) were prepared by dissolving the caseins in 100mM Na acetate buffer, pH 5.5, containing 0.05% NaNs. Cathepsin D was added at the following concentrations: cr,,-casein, 1 x lop2 UmL-‘; /I?-casein, 2 x 10-l UmL-‘; k--casein, 2.5 x 10-l U mL_‘; the samples were incubated at 37°C. Because of poor solubility of crs2-casein in this buffer it was dissolved in 1OOmM Na phosphate buffer, pH 6.5, containing 0.05% NaNs and 4 x 10-l U mL-’ cathepsin D was added. Chymosin was added at the following concentrations (expressed as chymosin units): a,‘-casein, 4 x 10m2; a,2-casein, 1.0; /?-casein, 3 x 10-l; K-casein, 1 x 10p2. (Amounts of chymosin and cathepsin D were chosen so as to yield approximately equal rates of hydrolysis.) Aliquots were taken periodically for analysis by urea-PAGE or reversed-phase HPLC. Samples for urea-PAGE were prepared by the addition of an equal volume of double-strength sample buffer (McSweeney et al., 1993). Discontinuous alkaline urea-PAGE was performed according to the method of Andrews (1983) (12.5% T, 4% C, pH8.9) with direct staining using Coomassie Brilliant Blue G250 (Blakesley and Boezi, 1977). Samples for reversed-phase HPLC were heated at 100°C for 10 min to inactivate the proteinases and pH 4.6insoluble peptides were precipitated by addition of a Na acetate buffer, as described by McSweeney et al. (1993). For rc-casein, the reaction was stopped by

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the addition of an equal volume of 16% trichloroacetic acid (TCA); the 8% TCA-soluble material thus obtained was used for analysis. Reversed-phase HPLC was performed using a Beckman HPLC system (Beckman, San Ramon, CA, consisting of an autosampler, model 507, programmable solvent module, model 126AA, and programmable detector, model 166, interfaced with a personal computer using Beckman System Gold software). A LiChrosorb RP-18 (5 pm) column (Metachem Technologies Inc., Redondo Beach, CA or Merck, Darmstadt, Germany) was used with an Adsorbosphere Cis guard column (Alltech Associates, Deerfield, IL). Elution was by means of a gradient using 0.1% trifluoroacetic acid (TFA) in Hz0 (Solvent A) and 0.1% TFA in acetonitrile (Aldrich Chemical Co., Milwaukee, WI) (Solvent B). The eluent consisted of 100% A for 5 min followed by a gradient from 0 to 50% B at a rate of 1.65% B min-‘. The influence of cathepsin D on the rennetability of milk was determined by preincubating reconstituted (12%, w/v, in 0.01 M CaC12) low heat skim milk powder (INRA, Poligny, France), containing -1 mgmL-’ thymol (final pH -6.4) with cathepsin D (0.5 U mL_‘) at 30°C. Ahquots (1 mL) were taken periodically and their rennet clotting time (RCT) determined by the addition of 100 PL of diluted (1: 100 with water) Hansen’s standard calf rennet (Chr. Hansens Ltd., Reading, UK). RCT was determined according to a standard method (IDF, 1987). RESULTS AND DISCUSSION Urea polyacrylamide gel electrophoretograms of micellar casein (in diluted skim milk) hydrolysed by cathepsin D are shown in Fig 1; a,i-casein was completely degraded within 3 h, while there was still some residual /I-casein after 24 h. Thus, cr,,-casein in micelles was preferentially degraded by cathepsin D, in agreement with the specificity of the acid proteinase isolated from bovine milk (Kaminogawa and Yamauchi, 1972, 1978). Electrophoretograms of cc,i-casein hydrolyzed by cathepsin D at pH values in the range 2-7 indicated that a,,-casein was optimally hydrolyzed at about pH4 (results not shown). Kaminogawa and Yamauchi (1972) found that the milk acid proteinase isolated by them had maximum activity at pH 4.0. Likewise, Schwartz and Bird (1977) reported that cathepsin D optimally degraded native myosin and actin at this pH, although Matsumoto et al. (1983) found cathepsin D to be maximally active on the myosin heavy chain at pH 3.4. The influence of NaCl on the hydrolysis of a,~- and p-caseins is shown in Fig 2. The hydrolysis of /3-casein by cathepsin D was more susceptible to inhibition by NaCl than that of a,i-casein (under the conditions of this study, hydrolysis of /I-casein was strongly inhibited by lo%, w/v, NaCl; after 3 h a,,-casein was completely hydrolyzed in the presence of IO%, w/v, NaCl and slight hydrolysis occurred in the presence of 20%, w/v). Fox and Walley (1971) reported that the hydrolysis of /I-casein by chymosin was more strongly inhibited by NaCl than that of cr,i-casein. Low concentrations of NaCl (1%) appeared to increase the activity of cathepsin D on both IQ- and p-caseins (Fig. 2); Fox and Walley (1971) found that a,,-casein was optimally degraded by chymosin in the presence of 5% (w/v) NaCl.

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p-CN

fl-I-CN

as,-CN cq,-CNf24-

199

(G,-I-CN)

Fig. 1. Urea-polyacrylamide gel electrophoretograms (12.5% T, 4% C, pH 8.9) of Na. caseinate (lane C), micellar casein (lane I) and micellar casein hydrolysed by bovine cathepsin D (2 x 10-l U mL_‘) at 37°C in 100 mM Na acetate buffer, pH 5.5, for 0, 0.25, 0.5, 1, 3, 6, 12 or 24 h (lanes 2-9).

Kaminogawa et al. (1978, 1980) found that there were similarities between the action of milk acid proteinase and chymosin on aSI-, /I- and rc-caseins. A peptide with an electrophoretic mobility identical to the primary fragment produced from a,,-casein by chymosin (i.e., cc,,-CN f24-199) was produced by the action of milk acid proteinase (Kaminogawa et al., 1980). These authors also demonstrated the production of a peptide with an electrophoretic mobility similar to b-1 (/I-CN fl189/192), the primary large peptide produced from /Scasein by chymosin, when /?-casein was incubated with milk acid proteinase. It was, therefore, decided to compare proteolysis of bovine caseins by cathepsin D with that by chymosin. Electrophoretograms of a,,-casein hydrolyzed by cathepsin D were very similar to those of chymosin-hydrolyzed cr,r-casein (Fig. 3), confirming the findings of Kaminogawa et af. (1980) with respect to the formation of aSI-CN f24-199 and also demonstrating that the peptides produced on further hydrolysis of a,,-casein by chymosin or cathepsin D had identical electrophoretic mobilities. Reversedphase HPLC of the pH4.6-soluble peptides produced from a,,-casein by cathepsin D or chymosin were also very similar (Fig. 4a, b); the pH 4.6-soluble peptides produced from cc,,-casein by chymosin have been identified (McSweeney et al., 1993). The order in which the pH 4.6-soluble peptides were produced from a,,-casein by the two enzymes was also similar (Fig. 5a, b); cathepsin D produced

Fig. 2. Urea-polyacrylamide gel electrophoretograms (conditions see Fig. 1) of Na caseinate (lane C), Na caseinate control (lanes 1, 8) and Na caseinate (5 mg mL_‘) hydrolysed by bovine cathepsin D (2 x 10-l UmL-‘) at 37°C in 100 mM Na acetate buffer, pH 5.5, in the presence of 0, 1, 2.5, 5, 10 or 20% (w/v) NaCl for 1h (lanes 2-7) and for 3 h (lanes 9-14).

q, -CNf24. .199 tsr -I-C1 U)

%I-CN

$-I-CN

fi-CN

Fig. 3. Urea-polyacrylamide gel electrophoretograms (conditions see Fif. I) of Na caseinate (lane C) and cc,l-casein (5 mg mL_‘) in 100 mM Na acetate buffer, pH 5.5, hydrolysed at 37°C by chymosin (4 x lo- U mL_‘) for 0, 1, 3, 6, 12 or 24 h (lanes 1-6) or bovine kathepsin D (1 x 10e2 UmL-‘) for 0, 1, 3, 6, 12 or 24 h (lanes 7-12).

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Fig. 4. Reversed-phase HPLC chromatograms of pH 4.6-soluble peptides produced from bovine cl,,-casein (5 mg mL-‘) by (A) chymosin or (B) bovine cathepsin D in 100 mM Na acetate buffer, pH 5.5, for 6 h at 37°C. The identity of peptides is as follows:- (1) a,,-casein f 154-159, (2) f 150-153, (3) f 157-164, (4) f 154164, (5) f 150-156, (6) f l-23, (7) f 2440, (8) f 165-199 (McSweeney et al., 1993). ----- Acetonitrile gradient.

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Fig. 5. Changes in the areas of the HPLC peaks corresponding to the major pH 4.6-solu-

ble peptides produced from ol,i-caseinin solution in 100 mM Na acetate buffer, pH 5.5, by (A) chymosin or (B) bovine cathepsin D. (W) a,,-casein f l-23, (0) f 165-199, (0)f 157164, (o)f 2440, (A) f 150-153 and (a) f 154-159.

the peptide a,,-casein f165-199 more quickly than did chymosin and the concentration of the peptide a,,-CN fl-23 decreased on extended incubation. Electrophoretograms of cathepsin D hydrolysates of crsrcasein differed markedly from those of chymosin hydrolysates (Fig. 6). The major urea-PAGEdetectable peptide which accumulated in the chymosin hydrolysate (electrophoretic mobility of 4.8 cm) did not appear in electrophoretograms of cathepsin D hydrolysates. A group of peptides (mobilities 6.2-6.7 cm) were detectable in the cathepsin D hydrolysates but not in those produced by chymosin. However, HPLC profiles of the pH 4.6-soluble peptides produced from a,2-casein by cathepsin D or chymosin had a number of peptides with similar elution times (Fig. 7a, b), although some of these peaks may have originated from a,,-casein, present as a contaminant (McSweeney, 1993). Proteolysis of fl-casein by cathepsin D was similar in some respects to that by chymosin (Fig. 8). Like with chymosin, /I-1(/I-CN fl-189/192; electrophoretic mobility 3.6 cm) was the primary product produced from fi-casein by cathepsin D; P-11 (P-CN fl-165/167; electrophoretic mobility 4.7 cm) was also formed but did not accumulate in cathepsin D hydrolysates, in contrast to those produced by chymosin. Bands with electrophoretic mobilities in the region of 5.5-6.4 cm were present in both hydrolysates, but differences between the specificities of the two enzymes were apparent in this region of the electrophoretogram. HPLC

Fig. 6. Urea-polyacrylamide gel electrophoretograms (conditions see Fig. 1) of Na caseinate (lane C) and ctS2-casein(5 mgmL_‘) in 100 mM Na phosphate buffer, pH 6.5, hydrolysed by chymosin (1 UmL-‘) for 0, 1, 3, 6, 12 or 24 h (lanes l-6) or bovine cathepsin D (4 x 10-l UmL-‘) for 0, 1, 3, 6, 12 or 24 h (lanes 7-12) at 37°C.

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Fig. 7. Reversed-phase HPLC chromatograms of pH 4.6~soluble peptides produced from bovine cc,*-casein (5 mg mL-‘) by chymosin (A) or bovine cathepsin D (B) in 100 mM Na phosphate buffer, pH 6.5, for 24 h at 37°C and from bovine p-casein (5 mg mL_‘) by chymosin (C) or bovine cathepsin D (D) in 100 mM Na acetate buffer, pH 5.5 for 6 h. ----- Acetonitrile gradient.

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HPLC elution profiles of the 8% trichloroacetic acid-soluble peptides produced from K-casein (5 mg mL_‘) by chymosin (A) or bovine cathepsin D (B) in 100 mM Na acetate buffer, pH 5.5, at 37°C for 20min (GMP = K-casein fl06169). ----- Acetonitrile gradient.

of the pH4.6-soluble peptides in hydrolysates of p-casein by cathepsin D or chymosin had a number of peaks in common (Fig. 7c, d), but differed in respect to the peptide eluting at c. 27 min and those in the region from 3G35 mm. Production of a peptide with an electrophoretic mobility identical to that of para-lc-casein by acid milk proteinase was shown by Kaminogawa et al. (1980). The HPLC profile of the chymosin hydrolysate of u-casein contained only one peptide peak (elution time -32min, Fig. 9a), which, based on other studies in this laboratory and elsewhere is the glycomacropeptide, K-CN fl06-169. At relatively high levels of enzyme, cathepsin D produced a peptide with a similar elution time (Fig. 9b), indicating cathepsin D hydrolyses rc-casein slowly at Phelos-Metlos. Preliminary experiments indicated that cathepsin D had poor milk clotting properties (it failed to coagulate milk in 2.5 h at an enzyme concentration of 1 U mL_‘). The influence of cathepsin D on the rennetability of milk by chymosin is shown in Fig. 10. Preincubation of milk with cathepsin profiles

334

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et al.

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0

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D appeared to reduce its RCT, presumably as a result of slow cleavage of rc-casein by cathepsin D. The ability of cathepsin D to hydrolyse aSl- and p-caseins in micelles was demonstrated earlier (Fig. 1) and cathepsin D appeared to be able to hydrolyse the Phe ros-Metles bond in rc-casein in solution (Fig. 9b). However, cathepsin D acted slowly on K-casein in solution and likewise its action in milk (even at the relatively low pH of -6.4 and a high enzyme concentration of 0.5 U mL_‘) was slow, requiring about 6 h to produce a coagulum. This slow action is undoubtedly, in part, due to the pH of milk being unfavourable for the action of cathepsin D (optimum activity at pH 4.0), although even at pH 5.5, cathepsin D was relatively less active on K-casein than on CI,~-or /?-caseins (based on enzyme concentrations necessary to achieve hydrolysis). Thus, it would appear that further study of the action of cathepsin D on the PhereS-Metlob bond of rc-casein is warranted. The results of this investigation indicate that all bovine caseins are susceptible to hydrolysis by cathepsin D in vitro, that a,,-casein is more susceptible to

Proteolysis of bovine caseins by cathepsin D

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proteolysis than /I-casein with maximal hydrolysis rate at pH4.0 and that the proteolysis of /I-casein is more susceptible to inhibition by NaCl than is cc,i-casein. The enzyme should be active under the ionic conditions present in many young cheeses (e.g. Cheddar, pH 5.2 and c. 5% NaCl-in-moisture) but its activity is probably masked by that of chymosin. The specificity of cathepsin D on CI,~-and rc-caseins is similar to that of chymosin, but there were differences between the specificities of the enzymes on /I- or cr,2-caseins. Cathepsin D acted only slowly on rc-casein in milk and showed poor rennet clotting activity.

ACKNOWLEDGEMENTS This study was supported by the Center for Dairy Research, University of Wisconsin-Madison, the Wisconsin Milk Marketing Board and by a bursary in Food Science and Technology from the National University of Ireland.

REFERENCES Andrews, A. T. (1983). Proteinases in normal bovine milk and their action on caseins. J. Dairy Res. 50, 45-55.

Blakesley, R. W. & Boezi, J. A. (1977). A new staining

technique for proteins in polyacrylamide gels using Coomassie Brilliant Blue G250. Anal. Biochem. 82, 58& 81. Creamer, L. K. (1974). Preparation of a,,-casein A. J. Dairy Sci. 57, 341-44. Dutson, T. R. (1983). Relationship of pH and temperature to disruption of specific muscle proteins and activity of lysosomal proteases. J. Food Biochem. 7, 223345. Farkye, N. Y. & Fox, P. F. (1990). Observations on plasmin activity in cheese. J. Dairy Res. 57, 413-18. Farkye, N. Y. & Fox, P. F. (1991). Preliminary study on the contribution of plasmin to proteolysis in Cheddar cheese: cheese containing plasmin inhibitor, 6-aminohexanoic acid. J. Agr. Food Chem. 39,78688. Farkye, N. Y. & Fox, P. F. (1992). Contribution of plasmin to Cheddar cheese ripening: effect of added plasmin. J. Dairy Res. 59, 209-16. Fox, P. F. (1989). Proteolysis during cheese manufacture and ripening. J. Dairy Sci. 72, 1379-1400. Fox, P. F. & Law, J. (1991). Enzymology of cheese ripening. Food Biotechnol. 5, 23962. Fox, P. F. & Walley, B. F. (1971). Influence of sodium chloride on the proteolysis of casein by rennet. J. Dairy Res. 38, 165-70. Goll, D. E., Otsuka, Y., Nagainis, P. A., Shannon, J. D., Sathe, S. K. & Muguruma, M. (1983). Role of muscle proteinases in maintenance of muscle integrity and mass. J. Food Biochem. 7, 137-77. Grappin, R., Rank, T. C. & Olson, N. F. (1985). Primary proteolysis of cheese proteins during ripening. A review. J. Dairy Sci. 68, 531. Grufferty, M. B. & Fox, P. F. (1988). Milk alkaline proteinase. J. Dairy Res. 55, 609-30. IDF (1987). Standard 1 lOA, Determination of chymosin and bovine pepsin contents (chromatographic method). Intern. Dairy Fed., Brussels. Kaminogawa, S. & Yamauchi, K. (1972). Acid protease of bovine milk. Agr. Biol. Chem. 36,2351-56. Kaminogawa, S. & Yamauchi, K. (1978). Degradation of casein components by milk acid protease. Proc. 20th Intern. Dairy Congr., Paris, p. 313.

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