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Effect of Dephosphorylation of Casein on Its Coagulation and Proteolysis by Chymosint

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Effect of Dephosphorylation of Casein on Its Coagulation and Proteolysis by Chymosint Agric. Biol. Chem., 42 (5), 1031-1035,1978 Effect of Dephosphorylation of Casein on Its Coagulation and Proteolysis by Chymosint Kunio YAMAUCHIand Yoshiki YONEDA* Department of Agricultural Chemistry, The Universityof Tokyo, Bunkyo-ku, Tokyo 113 Received December 19, 1977 Whole casein dephosphorylated almost completely with a bovine spleen phosphoprotein phosphatase was prepared and its coagulation by chymosin and its proteolysis by chymosin and other proteases were compared with those of native whole casein. Dephosphorylated casein coagulated more slowly than native casein in the calcium caseinate system containing 20 mm calcium. The yield of coagulated protein was the same for both caseins. Calcium content of the precipitate from dephosphorylated casein was less than one fourth of that from native casein. Dephosphorylated casein gave a much softer gel than native casein when com- pared in the sol system prepared by Grindrod et al. General proteolysis of casein with chymo sin at pH 6.8 and with pepsin at pH 1.8 was accelerated and that with trypsin at pH 8.2 was retarded by dephosphorylation of casein. Effect of enzymatic dephosphorylation of dephosphorylated a,,-casein form micelles in bovine casein on its properties has been the presence of ƒÈ-casein and Cal+. They ob- studied by several investigators.'-" The served that the micelles formed from de authors" have reported about the effect of phosphorylated a„-casein are larger in size enzymatic dephosphorylation on the interac and fewer in number than those from intact tion of calcium ions with whole and a,,- as,-casein. Yoshikawa et al." reported that the caseins. The binding of calcium ions to the calcium-dependent precipitation of ƒÀ-casein is phosphate groups of a„-casein did not seem completely lost when the phosphate groups essential for precipitation. However, sedi are removed. The modification of casein by mentability and turbidity of the micelle dis phosphatases has recently been reviewed by persion prepared from dephosphorylated whole Bingham." casein were not the same as those of the micelle On the other hand, the coagulation of casein dispersion from native whole casein, suggest- by chymosin (rennin; EC 3.4 23.4) has been ing that the phosphate groups are related to extensively investigated from various view- the size and structure of casein micelles. points."' As calcium involves the step of the Ando et al.8) made electron microscopic ob coagulation and phosphate groups of casein servation on the changes of casein micelles by participate in the binding of calcium to casein, progressive dephosphorylation with a phos some investigators have tried to know the phoprotein phosphatase. They observed that effect of dephosphorylation of casein on its the surface of the micelles ruined and the coagulation by chymosin. Hsu et al." re- micelles aggregated with the progress of ported that the rennet-coagulation time of dephosphorylation. partially dephosphorylated casein increased to Bingham et al." showed that both a,,- and a maximum at 40-50" dephosphorylation. Bargoni12) reported that the casein dephos ' An abstract of this paper was presented at the phorylated with a phosphoprotein phosphatase Annual Meeting of the Agricultural Chemical Society was not coagulated by chymosin. Although of Japan at Fukuoka, April 2, 1970. * Present address: Research Laboratory of Meiji the method used by Bargoni was different from Milk Products Co., Ltd. that by Hsu et al., the results obtained by both 1032 K. YAMAUCHI and Y. YONEDA workers seem conflicting. Coagulation test was also performed using the caseinate sol in semblance of skim milk.") 1.3 g of We have studied on the effect of enzymatic native or dephosphorylated casein was dissolved by dephosphorylation of whole casein on the addition of the four salt solutions described by Grindrod chymosin-coagulation time and the properties et al.16> As pH of the final sol was slightly lower than of the coagulated protein. The effect of de 6.8, the pH was adjusted to 6.8 with a small amount of concentrated NaOH solution and the volume was phosphorylation on the proteolysis of the made up to 50 ml with water. To 20 ml of the casei casein by chymosin and some other proteases nate sol in a 50 ml-beaker, 4 ml of 0.059 M CaCl2 was also investigated. The casein dephos solution was added. After preincubation at 30°C phorylated almost completely by a bovine for 15 min, 2 ml of chymosin solution (40 ƒÊg chymosin spleen phosphoprotein phosphatase was used per ml) was added and coagulation time was measured. in this study. Curd tension was determined at about 10 min after the addition of chymosin by a curd tension meter (Iio Denki Co.). MATERIALS AND METHODS 4. Determination of non protein nitrogen (NPN) 1. Casein and dephosphorylated casein. Whole released by chymosin and other proteases. First, the casein and dephosphorylated whole casein were pre effect of dephosphorylation of casein on the primary pared as described previously.2> Bovine spleen phos phase of its coagulation by chymosin was evaluated by phoprotein phosphatase prepared by the method of determining the release of non protein nitrogen (NPN). Revel and Racket") was used for dephosphorylation. 0.5 ml of chymosin solution (40 ƒÊg chymosin per ml) The rate of dephosphorylation was about 98%. The was added to 5 ml of 2 % caseinate or dephosphory yield of dephosphorylated casein was 17.8 g from 21 g lated caseinate solution (pH 6.8) at 30°C. After of native casein. proper time, 5.5 ml of 24% trichloroacetic acid was added to stop the reaction. The mixture was filtered 2. Chymosin and other proteases. Chymosin was after standing for one hr at room temperature. Nitro purified from Hansen's rennet tablets by DEAE cellu gen in the filtrate was determined by the micro. lose chromatography.14> Crystalline porcine pepsin Kjeldahl method. was purchased from Sigma Chemical Co. and crystal- Next, the effect of dephosphorylation of casein on line bovine trypsin was a product of Sanko Junyaku its general proteolysis by chymosin and other proteases Co. was investigated. One ml of chymosin solution 3. Coagulation test and analysis of eoagulum. (10 mg chymosin) was added to 25 ml of 2% native or Native or dephosphorylated casein was dissolved with dephosphorylated caseinate solution (pH 6.8) at 37°C. diluted NaOH to give a solution of about pH 7.0. At proper intervals, 1-ml aliquots were removed and A suitable amount of CaC12 solution was added and mixed with 4 ml of 15 % trichloroacetic acid. After the final pH was adjusted to 6.8 with a small amount standing for one hr at room temperature, the samples of diluted NaOH solution. The final protein con were filtered and nitrogen in the filtrate was deter- centration was made to 2 % by addition of water. mined. Similar experiments were performed with Ten parts of native or dephosphorylated casein trypsin (0.5 mg per 26 ml of the reaction mixture) at solution containing CaCl2 were taken into a test tube, pH 8.2 and with pepsin (0.5 mg per 26 ml of the reac preincubated at 30°C for 10 min, and then mixed with tion mixture) at pH 1.8. The substrate for pepsin was one part of diluted chymosin solution (40 ƒÊg chymosin prepared by dissolving native or dephosphorylated casein with diluted HCL per ml of water) at the same temperature. Coagula tion time was measured. Test was repeated at least twice. Whenever the coagulated protein was to be separated, the content was allowed to stand at 30°C for RESULTS AND DISCUSSION additional 30 min after the biginning of coagulation, 1. Effect of dephosphorylation on coagulation and then centrifuged at 2000 rpm for 5 min. The supernatant was passed through Toyoroshi No. 5A time paper. Nitrogen and calcium contents of the filtrate Dephosphorylated casein was coagulated by were determined and subtracted from those of the chymosin in the presence of suitable amounts original casein solution to obtain yield of the coagu of calcium. Time of the coagulation was lated protein and calcium content retained in the about twice long as that of native casein when coagulum. Nitrogen was determined by the micro- Kjeldahl method and calcium by the method of compared in the presence of 20 mm CaCl2 at Kamal.ll> pH 6.8 (Table I). However, opposite result Coagulation and Proteolysis of Dephosphorylated Casein 1033 caseins. The amount of calcium precipitated with dephosphorylated casein was much less than that with native casein. The result is consistent with that obtained by high speed centrifugaion of native and dephosphorylated casein micelles." It is concluded that phos phate groups of casein are not essential for coagulation. However, it was observed that the coagulum from dephosphorylated casein was soft and fragile as compared to that from native casein. 3. Effect of dephosphorylation on liberation of NPN from casein by chymosin Liberations of 12% trichloroacetic acid FIG. 1. Effect of Calcium Concentration on Coagu lation Time.•›•\•› soluble nitrogen (NPN) from native and dephosphorylated caseins by chymosin are , native casein; •---0, dephosphorylated casein. Calcium caseinate solution of pH 6.8 was used. compared in Fig. 2. NPN content at 0 min Chymosin concentration is ten times as much as that (blank value) of native casein was slightly used in Table I. higher than that of dephosphorylated casein. The rate of net increase of NPN by the action TABLE I. COMPARISON OF COAGULATION OF NATIVE AND DEPHOSPHORYLATED CASEINS of chymosin was the same for native and IN CALCIUM CASEINATE SYSTEM BY dephosphorylated caseins. Therefore, the CHYMOSIN primary enzymatic phase of casein coagulation 2 % calcium caseinate solution (pH 6.8) containing by chymosin (specific liberation of NPN from 20 mm CaCl2 was used.
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