Rapid and Large Scale Isolation of Chymosin (Rennin) by Pepstatin-aminohexylagarose. Hideyuki KOBAYASHI and Kazuo MURAKAMI

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1 Agric. Biol. Chem., 42(12), , 1978 Rapid and Large Scale Isolation of Chymosin (Rennin) by Pepstatin-aminohexylagarose Hideyuki KOBAYASHI and Kazuo MURAKAMI Institute of Applied Biochemistry, The University of Tsukuba, Ibaraki-ken , Japan Received May 11, 1978 A rapid and large scale isolation of chymosin was achieved by an affinity column including pepstatin-aminohexylagarose with a column of DEAE-cellulose. The affinity column with 3 ml wet gel made it possible to isolate 90 mg pure chymosins (1, II and III) from 20 g crude rennet tablet. Isoelectric points of chymosin-i, -II and -III were 4.6, 4.6 and 4.5, respectively. Each of chymosins was homogeneous electrophoretically and chromatographically. Chymosin (EC ) is the predominant milk-clotting enzyme in the fourth stomach of the calf. The use of this enzyme in cheese making has been known since ancient times. Calf chymosin had been purified by conven tional processes and crystallized by several in vestigators1) in the past. However, heterogeneity of crystalline chymo sin has been observed by free boundary electro phoresis,2) by paper electrophoresis,3) by im muno electrophoresis4) and by polyacrylamide gel electrophoresis.5) Foltmann6,7) successful ly fractionated crystalline chymosin by chro matography on DEAE-cellulose and obtained three active fractions which were designated chymosin-a, -B and -C. He found no indi cation that chymosin-a and -B were hetero geneous but considered chymosin-c to be a mixture containing some degradation products. Thus the isolation of chymosins by conven tional procedures is a long and complicated process bedeviled with problems of autodigestion. We had succeeded in purifing pressor enzyme, renin (EC ) from hog8,9) and human10) kidneys very effectively by an affinity column, pepstatin-aminohexylagarose. How ever, pepstatin11) an N-acylated pentapeptide is not only an inhibitor of renin but also a general inhibitor12) of acid proteases from various sources. The purpose of this investigation is to invent a rapid and facile method for isolation of calf chymosin in the native form by the pepstatinaminohexylagarose. MATERIALS AND METHODS Enzyme. One commercial rennet tablet (Chr. Hansen Laboratory, Denmark) was dissolved in 50 ml each of 0.01 M acetate buffer, ph 5.5 and centrifuged at 2000 x g for 20 min. The supernatant was used as a crude enzyme solution. Chymosin assay. Chymosin was assayed by clotting of skim milk. Ten grams of skim milk powder (Mori naga-nyugyo Co.) were suspended in 100 ml of 0.01 M calcium chloride solution and used as a substrate. To 5 ml of the substrate was added 0.5 ml of chymosin solution which could clott the milk at 35 C between 1 to 3 min. The chymosin solution contained approximately 0.5 mg protein in the crude preparation or 10 `15ƒÊg pure chymosin. The moment when the thin film of milk breaks into visible particles was noted as the clotting time. The ph of the reaction mixture was 6.3. Chymosin activities are calculated by following equation. Soxhlet unit (S.U.)=2400/T ~5/0.5 D where, T: clotting time (second) D: dilution factor of enzyme Affinity column. The affinity gel, in which pepstatin was coupled to aminohexylagarose was prepared by the previously described method.8) One ml of the affinity gel contained 1.5 to 2 y moles of covalently bound pep statin which was determined by amino acid analysis of the gel. Acetylation of free amino groups on pepstatinaminohexylagarose gel was performed at room tempera ture for 16 hr in the presence of an excess of acetic acid

2 2228 H. KOBAYASHI and K. MURAKAMI and water soluble carbodiimide at ph 4.7. Coupling efficiency of the acetyl group was checked by a color test of free amino group with 2,4,6-trinitrobenzensulfonate.14) The acetylation of the free amino groups of pepstatin-aminohexylagarose gel did not modify either the binding or the elution conditions of rennin. Therefore all experiments in the present investigation were performed on the affinity gel without acetylation. All the procedure of the chymosin purification was carried out at 4-6 C unless otherwise stated. Polyacrylamide gel electrophoresis was run in a 6 cm gel containing 7.5% polyacrylamide with 2.5 cross-linkage at ph 9.5 with a constant current of 3 ma per gel for 3 hr with the omission of the stacking gel. Protein bands were stained by coomassie brilliant blue R solution. SDS-polyacrylamide gel electrophoresis was run in 7 cm gel containing 10 % polyacrylamide with 2.7 % cross linkage with a constant current of 7 ma per tube. The gels contained 0.1 % SDS. Isoelectric focusing was run in 7.5 cm gel containing 5 % polyacrylamide with 4.8% cross linkage and 2 ampholine with a constant voltage of 200 V for 5 hr. After run, the gel was sliced into small fragments with 2.5 mm width. Each fragment was extracted with 0.5 ml deionized water and its chymosin activity was determined as described above. Protein bands on the polyacrylamide gel was stained by coomassie brilliant blue G 250. RESULTS Chymosin was inhibited by a low concen tration of pepstatin as shown in Fig. 1. Con centration of pepstain indicating 50% inhibi tion was 5 to 6 x 10-6 M which was similar to FIG. 2. Affinity Chromatography of the Crude Chymosin Solution on Pepstatin-aminohexylagarose. One and nine-tenth liters of the crude chymosin solu tion containing 20 g of the rennet tablet was applied to an affinity column (1.2 x 3 cm) equilibrated with 0.01 M acetate buffer ph 5.5. The flow rate was 8 nil/ hr and approximately 12 ml fractions were collected. œ \ œ, absorbance at 280 nm; \, milk clotting activity. The other details are in the text. that in renin inhibition.13) As shown in Fig. 2, the chymosin in the crude preparation was adsorbed on the affinity col umn at ph 5.5 completely. The column was thoroughly washed with the same buffer until no further protein was eluted, then washed with the starting buffer containing 0.5 M NaCl to elute non-chymosin proteins bound to the column. The chymosin fraction was eluted by stepwise lowering of ph to 3.0 with 0.1 M FIG. 1. Inhibition of Chymosin by Pepstatin. To 2.5 ml of the crude enzyme solution were added 0.25 ml of pepstatin solution in a mixture of dimethyl formamide: methanol (1: 5) and 2.5 ml of the substrate solution. The enzyme and the substrate solutions were prepared as described in MATERIALS AND METHODS. FIG. 3. Gel Filtration of Chymosin after the Affinity Column (Fig. 2) on. a Sephadex G-100 (1.5 x 90 cm) in 0.01 M Acetate, ph 5.5. The flow rate was 6 ml/hr and 2 nil fractions were collected. œ \ œ absorbance at 280 nm; \, milk clotting activity.

3 Affinity Column of Chymosin 2229 ponents are called chymosin-i, -II and -III hereafter. Figure 6 shows that each of chymosin-i, -II and -III gives a discrete band with very slightly different distance of migration on a polyacryl amide gel electrophoresis at ph 9.5 indicating their electrophoretical homogeneity and simi larity. Isoelectric focusing in the ph 4-6 FIG. 4. SDS-polyacrylamide Gel Electrophoresis of Chymosin after the Affinity Column in Fig. 2. acetic acid. The active fractions from the affinity column showed a symmetrical profile on a Sephadex G-100 as shown in Fig. 3 and a discrete protein band on the SDS-electro phoresis as shown in Fig. 4. However, Fig. 5 revealed that the chymosin fraction after the affinity column was adsorbed on a DEAE-cellulose at ph 5.8, eluted by a very shallow linear gradient of salt concen tration on a DEAE-cellulose and separated into three active components. The three com FIG. 6. Polyacrylamide Gel Electrophoresis of Chymosin after the Affinity Column (Fig. 2) and of Chymosin-I, -II and -III after a Column of the DEAEcellulose (Fig. 3). Mix: a mixture of chymosin I, -11 and -III after the affinity column. FIG. 5. Ion Exchange Chromatography of Chymosin after the Affinity Column (Fig. 2) on DEAF-cellulose. The chymosin fractions from the affinity column indicated by the bracket in Fig. 2 were concen trated by pressure filtration and dialyzed against 0.2 M phosphate buffer, ph 5.8 overnight. The dialyzed sample was applied to a DEAF-cellulose column (0.9 x 20 cm, Whatman DE-52). The chymosin was eluted by a linear gradient generated between 300 ml each of the initial buffer and of 0.3 M phosphate buffer, ph 5.6. The flow rate was 9 ml/hr and 2.7 ml fractions were collected. œ \ œ, absorbance at 280 nm; \, milk clotting activity; \, phosphate concentration.

4 2230 H. KOBAYASHI and K. MURAKAMI FIG. 7. Isoelectric Focusing of Chymosin-I, -II and -III after a Column of the DEAE-cellulose (Fig. 3). œ \ œ, ph value; \, milk clotting activity. Protein bands on the polyacrylamide gel were shown in the center of each figure. TABLE I. a Protein concentration was determined by an absorbance at 280 nm with an extinction coefficient, E1% 1cm = 15 (unpublished our data).b Twenty grams of Chr. Hansen rennet tablet. range revealed that the isoelectric points were 4.6, 4.6 and 4.5 for chymosin-i, -II and -III respectively as shwon in Fig. 7. In each in stance only one major!protein peak coinciding with the chymosin activity was noted. As indi cated in Table I, a 6 fold purification was achieved at a yield of 60 % by the affinity chro matography to obtain the chymosin prepa ration which had almost the same specific ac tivity as the pure chymosin. A gel filtration on the Sephadex G-100 for isolation of chymo sin was omitted because no purification was achieved by this step. DISCUSSION All data in the present investigation show chymosin-i, -II and -III after chromatography on DEAE-cellulose are homogenous electro phoretically and chromatographically, and their molecular properties are very similar in each other. Heterogeneity of chymosin was disclosed only by a delicate elution condition with a very shallow linear gradient of phosphate concen tration from 0.2 M to 0.3 M on a DEAE-cellu lose but not by a gel filtration on Sephadex G- 100 and polyacrylamide gel electrophoresis with and without SDS.

5 Affinity Column of Chymosin 2231 Other investigators1) had also failed to dis close heterogeneity of chymosin by gel filtra tion, by ultracentrifugation or by movingboundary electrophoresis. To detect heterogeneity of chymosin prepa ration after the affinity column, we used the same elution condition on the DEAE-cellulose as Foltmann.7) An elution profile of chymosin on the DEAF-cellulose in Fig. 5 was quite similar to the one of crystalline chymosin preparation obtained by Foltmann1,7) after his conventional and time consuming method. Judged from their elution profile on the DEAE-cellulose and their specific activity, chymosin-i, -II and -III in the present investi gation are very similar to Foltmann's chymo sin-a, -B and -C although our affinity chro matographic method is entirely different from his method except for the final chromatography on DEAF-cellulose. Chymosin-III was a minor component with a low specific activity. There is no internationally agreed unit for milk-clotting activity and different samples of milk powder show variation in clotting ac tivity. Thus it is difficult to compare specific activity of chymosins in different laboratories exactly. However, our final preparation of chymosin has almost the same specific activity as Foltmann's preparation," if there is no variation of clotting activity due to the milk powder used in both laboratories. As a proportion of chymosin-i, -II and -III did not change during our purification, autodigestion or degradation of chymosin does not seem to occur in the course of purification. The small affinity column with 3 ml wet gel made it possible to isolate 90 mg pure chymo sins (I, II and III) from the 20 g crude rennet tablet by only two steps instead of the conven tional 5 to 6 steps.') Moreover, the affinity column does not need to be equilibrated with a starting buffer precisely, because of its strong affinity to chymosin. Thus the isolation pro cedure including the affinity column is suitable for rapid and large scale purification of chymo sin. A yield of 60% by the affinity chromato graphy is somewhat low probably due to denatutation of chymosin at ph 3.0 during elution or to failure of elution of chymosin like enzymes such as pepsin in the crude prepa ration. We15) have a preliminary evidence that porcine pepsin and some microbial milk clotting enzymes were adsorbed on the affinity column completely and did not be eluted at all. Thus, pepstatin-aminohexylagarose could also be used for a separation of chymosin from several acid proteases. The detail of this ex periment will be published in another paper. REFERENCES 1) B. Foltmann, "Milk Proteins," Vol. II, ed. by H. A. Mckenzie, Academic Press Inc., New York, N.Y., 1971, p ) C. A. Ernstrom, J. Dairy Sci., 41, 1663 (1958). 3) T. A. J. Payens, 16th Intern. Dairy Congr., B, 410 (1962). 4) R. Schober, N. Heimburger and I. Printz, Milch wissenschaft, 15, 506 (1960). 5) N. Asato and A. G. Rand, Anal. Biochem., 44, 32 (1971). 6) B. Foltmann, Acta Chem. Scand., 14, 2059 (1960). 7) B. Foltmann, "Methods in Enzymology," Vol. 19, ed. by S.P. Colowick and N.Q. Kaplan, Academic Press Inc., New York, N.Y., 1970, p ) K. Murakami and T. Inagami, Biochem. Biophys. Res. Commun., 62,757 (1975). 9) T. Inagami and K. Murakami, J. Biol. Chem., 252, 2978 (1977). 10) K. Murakami and T. Inagami, Circ. Res., 41, Suppl, 11,4 (1977). 11) H. Umezawa, T. Aoyagi, H. Morishima, M. Matsuzaki, H. Hamada and T. Takeuchi, J. Antibiot., 23,259 (1970). 12) T. Aoyagi, S. Kunimoto, H. Morishima, T. Take uchi and H. Umezawa, ibid., 24, 687 (1971). 13) K. Murakami and T. Inagami, Biochem. Biophys. Res. Commun., 54,482 (1973). 14) P. Cuatrecasas, J. Biol. Chem., 245,3059 (1970). 15) K. Murakami and H. Kobayashi, unpublished data.