EXTRACELLULAR PROTEINASE OF STREPTOCOCCUS LACTIS'

JOURnNAL OF BACTERIOLOGY Vol. 87, No. 1, pp. 49-53 January, 1964,Copyright 1964 by the American Society for Microbiology Printed in U.S.A. EXTRACELLULAR PROTEINASE OF STREPTOCOCCUS LACTIS' W. T. WILLIAMSON, S. B. TOVE, AND M. L. SPECK Departments of Food Science and Animal Science, North Carolina State of the University of North Carolina at Raleigh, North Carolina ABSTRACT WILLIAMSON, W. T. (North Carolina State of the University of North Carolina, Raleigh), S. B. TovE, AND M. L. SPECK. Extracellular proteinase of Streptococcus lactis. J. Bacteriol. 87 :49-53. 1964.-Streptococcus lactis as shon to produce an extracellular proteolytic enzyme(s). A 12-fold purification of the proteinase as obtained from the cell-free culture medium by ammonium sulfate fractionation, calcium phosphate gel treatment, and chromatography on diethylaminoethyl cellulose. Only 32% of the activity as destroyed by heating at 98 C for 1 hr. The requirement of free sulfhydryl groups for enzyme activity as indicated by inhibition ith p-chloromercuric benzoate. Diisopropyl fluorophosphate did not inhibit the proteinase. The to closely related lactic streptococci, Streptococcus lactis and S. cremoris, are the predominant species in dairy starter cultures. They are believed to be the sources of proteinases important in the hydrolysis of milk proteins during the manufacture of cultured-milk products. Intracellular proteolytic enzymes of these bacteria ere studied by Baribo and Foster (1952), and Van der Zant and Nelson (1954). Sasaki and Nakae (1959) reported that S. lactis produces an extracellular proteinase. Extracellular enzymes produced by the lactic streptococci may have an important role in the industrial usage of these cultures. In the present investigation, an extracellular proteinase produced by S. lactis as purified, and certain of its properties ere studied. MATERIALS AND METHODS S. lactis E as transferred eekly in litmus milk, ith 1% inoculum, incubation at 22 C for 14 to 16 hr, and intervening storage at 2 C. I Contribution from the Departments of Food Science and Animal Science, North Carolina Agricultural Experiment Station, Raleigh. Published ith the approval of the Director of Research as paper no. 1676 of the Journal Series. Received for publication 5 August 1963 The medium used for the groth of S. lactis as a modification of that used by Niven (1944). Casein hydrolysate as replaced by unhydrolyzed casein in a concentration of 2 mg/ml of medium. The medium as adjusted to ph 7. and 15-liter volumes ere autoclaved in carboys for 35 min at 121 C. After sterilization, the medium as tempered to 32 C. The culture of S. lactis used for inoculum as gron at 32 C in 15 ml of broth hich had the folloing composition: Tryptone, 1. g; yeast extract, 1. g; K2HPO4,.2 g; glucose,.1 g; distilled ater to 1 ml. The cells ere ashed in sterile.5 M phosphate buffer (ph 7) before being added to the carboy of medium. During the groth of the organism in the carboys, the liquid medium as maintained at ph 6.5 to 7. by the addition of sterile 1% NaOH. Cell groth as folloed turbidimetrically at 65 m,u, ith the uninoculated broth as the blank. At the end of the logarithmic phase of groth, the cells ere centrifuged from the medium at 27, X g ith a continuous-flo refrigerated centrifuge at 3 C, and the cell-free medium as stored at 1 C for future use. Assay of proteolytic activity. The assay as based on the hydrolysis of hemoglobin or serum albumin, as measured by the increase in absorption at 28 m,u after precipitation of the protein by trichloroacetic acid. Samples of the enzyme studied (.5 to 2. ml) ere pipetted into a series of test tubes. The volumes ere adjusted to 2 ml ith.5 M phosphate buffer (ph 7.), mixed, and preincubated at 37.5 C in a ater bath for 5 min. A solution of the protein substrate as preincubated separately at the same time. The hemoglobin and albumin substrates ere prepared by suspending 1 g of hemoglobin substrate poder (Worthington Biochemical Corp., Freehold, N.J.), or 1 g of three-times crystallized bovine albumin (Nutritional Biochemicals Corp., Cleveland, Ohio) in 1 ml of distilled ater; 1-mi volumes of the substrate solution ere 49

5 WILLIAMSON, TOVE, AND SPECK bj. BACTERIOL. added, ith stirring, and the assay tubes ere incubated at 37.5 C for 6 min. To stop the reaction, 3 ml of 5% trichloroacetic acid ere added ith stirring. After 2 min, the material as filtered and the clear filtrates ere read in a Beckman model DU spectrophotometer at a avelength of 28 m,u against ater as a blank. Results are reported as increase in optical density. One unit of activity as equal to a change in optical density of.28 per hr under the above conditions of assay. Purification of enzyme from cell-free broth. All manipulations in the enzyme purification ere carried out at to 2 C. Unless otherise specified,.5 M phosphate buffer (ph 7.) as used throughout purification. Protein determinations ere made by the Folin-Ciocalteau phenol method as described by Lory et al. (1951), ith vitamin-free casein (Nutritional Biochemicals Corp.) used for the standard curve. Precipitation ith 6% saturated ammonium sulfate (step 1). The cell-free broth as brought to 6% saturation ith solid ammonium sulfate and constant stirring. After complete addition of the ammonium sulfate, the mixture as alloed to stand overnight. The precipitate as collected ith a continuous-flo centrifuge at 27, X g. The supernatant fluid as discarded, and the precipitate as taken up in phosphate buffer and dialyzed 24 hr against this buffer. Purification ith 4% saturated ammonium sulfate (step 2). The crude extract from above as made up to 4% saturation by addition of solid ammonium sulfate ith stirring. After standing overnight, the precipitate as removed by centrifugation for 2 min at 6,7 X g and discarded. The supernatant fluid as dialyzed 24 hr against phosphate buffer. Purification ith 45% saturated ammonium sulfate (step 3). The dialyzed supernatant fluid from the preceding step as sloly brought to 45% saturation by the addition of nine parts of saturated ammonium sulfate and one part of 2 M phosphate buffer (ph 7.) hile the mixture as gently agitated ith a magnetic stirrer. The mixture as alloed to stand overnight. After centrifugation for 2 min at 6,7 x g, the precipitate as taken up in phosphate buffer and the supernatant fluid as discarded. The suspended precipitate as dialyzed against distilled ater. Calcium-phosphate gel absorption and elution (step 4). Calcium-phosphate gel, prepared according to Keilin and Hartree (1938), contained 35.7 mg/ml, and as added in incremental amounts to the dilute enzyme extract previously adjusted to ph 5.5 ith a.2 M acetate buffer. For each 1 ml of enzyme suspension, 6.14 mg of gel ere added. After the addition of each increment, the suspension as stirred for 15 min and then centrifuged for 5 min at 18, X g. The first increment of gel as discarded. The second and third increments of gel ere combined and stirred for 15 min ith 2 ml of cold ater. The gel as centrifuged from the ater and resuspended in.2 M phosphate buffer (ph 7.) to elute the enzyme. The mixture as stirred for 15 min and the gel as centrifuged out and discarded. The enzyme as dialyzed against distilled ater. Chromatography on diethylaminoethyl (DEAE) cellulose (step 5). The enzyme as purified on DEAE cellulose by chromatographic separation as described for horse serum by Sober et al. (1956). Gradient elution as carried out ith a mixing chamber in the folloing sequence of solutions:.5 M NaH2PO4, ph 7;.2 M NaH2PO4, ph 6.;.5 M NaH2PO4;.5 M NaH2PO4 +.5 M NaCl; and.5 M NaH2PO4 +.1 M NaCL. RESULTS Results of purification methods. Results of the enzyme purification are shon in Table 1. Initial investigation had demonstrated that there as a direct proportionalitv beteen protein hydrolysis and the volume of the cell-free broth assayed. The proteolytic activity, hoever, as very dilute; therefore, ammonium sulfate precipitation as used in the first step to concentrate the enzyme. The specific activity of the fraction as loer than that of the original cell-free broth, and a large portion of the enzyme as lost during this fractionation step. From this point on, fractionation ith ammonium sulfate, adsorption and elution from calcium phosphate gel, and chromatography on DEAE cellulose resulted in 12- fold purification ith 13% recovery. The enzyme as lyophilized and stored at -2 C. An attempt as made to concentrate the enzyme by precipitation at ph 5. to bypass the loss of activity at the initial step, but the precipitate collected did not demonstrate any proteinase activity. Effect of ph on activity. The effect of ph on activity as determined by assaying in to buffers. Buffer-substrate solutions, prepared ith.1 M tris(hydroxymethyl)aminomethane (tris)-phos-

VOL. 87, 1964 EXTRACELLULAR PROTEINASE OF S. LACTIS 51 phate and.1 M tris-citrate buffer, ranged from ph 5 to 1. The optimal activity as found at ph 8.5 in both buffers, although the shape of the to curves differed (Fig. 1). A sharp ph activity curve as observed ith the tris-citrate, hereas a broad optimal ph as observed ith the tris-phosphate. Effect of substrate conc ntration on activity. The effect of substrate concentration on activity in hemoglobin and albumin substrates is presented in Fig. 2. Saturation of the enzyme as obtained ith 1 mg of each substrate per tube. A linear relationship as observed ith added levels of each substrate up to 1 mg per tube. Slightly greater activity as observed hen the proteinase as assayed against the albumin substrate. Temperature stability of enzyme. The enzyme suspension as placed in a test tube and then stirred in a boiling-ater bath. After 5 min, the contents of the tube reached 98 C; thereafter, at 5-min intervals, a.5-ml sample as removed and immediately placed in a cold test tube hich contained.5 ml of ater, and the mixture as returned to an ice bath. After exposure to heat, 2 ml of.1 M tris-citrate buffer (ph 8.5) containing 1 mg of hemoglobin ere added to each tube for assay of activity. The effect of heating the enzyme at 98 C for 1 hr is shon in Fig. 3. The activity of the enzyme solution shoed a slight increase hen heated for 15 to 2 min, but, upon continued heating, the activity declined gradually, and at the end of the 6-min heating period the activity had decreased only 32%. Results of inhibitor studies. Both p-chloromercuric benzoate (PCMB) and sodium fluoride inhibited the activity of the enzyme. Sodium fluoride inhibited 26%, and PCMB 15%, hen the substrate concentration as 1 mg per tube. Both inhibitors employed appeared noncompetitive by Lineeaver-Burke plots (Fig. 4). Diisopropyl fluorophosphate as also studied at concentrations from 17 to 1-4 M in the same manner as the above inhibitors, and no inhibition as observed. Effect of reducing substance on activity. Because the studies ith the inhibitors indicated SH groups ere involved in the activity of the enzyme, the effect of three reducing agents as studied to determine hether reducing compounds ould increase the activity of the proteinase. Both sodium thioglycolate and sodium sulfite increased the activity of the enzyme at a concentration of.1 M, but no effect as dem- TABLE 1. Summary of purification procedure Proteinase Step Total vol Units Specific per ml activity ml Cell-free medium 3, 1.66 6% (NH4)2SO4 1,225 6.31.53 4% (NH4)2SO4 1,35 4.62 3.3 45% (NH4)2SO4 9 5. 22.3 Ca3(P4)2 gel 5 3.87 51.6 DEAE cellulose 3 3.4 8.95 E.4 N /.3- C, c.) z.2 >-.1 >- - < ck ph FIG. 1. Effect of ph on proteinase activity hen assayed in to buffers against hemoglobin substrate. The assays ere incubated at 37.5 C ith 1 mg per tube of substrate. onstrated by L-cysteine at this concentration (Table 2). Neither sodium sulfite nor sodium thioglycolate produced any effect at.1 M concentrations. When the cysteine concentration as increased to.1 M, an increase in activity as obtained, hereas sodium thioglycolate as inhibitory at this concentration. DISCUSSION Several investigators (Van der Zant and Nelson, 1953a; Morgan and Nelson, 1951; Williamson and Speck, 1962) observed proteolytic activity early in the groth period of S. lactis. Because of the early proteolytic activity, Van der Zant and Nelson (1953b) suggested that an extracellular proteolytic enzyme as produced by the organism. More direct evidence for the existence of an extracellular proteinase as obtained by Sasaki and Nakae (1959), ho observed hydrolysis of milk proteins by the cell-free

52 WILLIAMSON, TOVE, AND SPECK J. BAcTzRioL: E 3 the activity from the large volume of cell-free broth. Because of this large initial loss, it is possible that other proteolytic enzymes may have been present in the cell-free broth. Under the conditions of assay, enzyme ac- 14 12 4 X 1-3 PCMB.2 - I C) z d.1 // o o ALBUM IN /.-. HEMOGLOBIN - 6 NOIN. 4 2 2 4 6 8 1 12 SUBSTRATE (MG/ TUBE) FIG. 2. Effect of substrate concentrations on proteinase activity hen assayed for 6 min at 87.5 C and ph 8.5 in.1 M tris-citrate buffer. 14 NaF 5 X 1-3 12 E N -6 NO IN. z.4 C- Q2 O i. I I I,I I 1 2 3 4 5 6 LENGTH OF HEATING (MINUTES) FIG. 3. Activity of the proteinase after heating for different times at 98 C. filtrate of S. lactis. Confirmation of an extracellular proteinase produced by S. lactic is provided by our findings, and a procedure is described hereby a 12-fold purification of the enzyme as obtained. Unfortunately, 75% of the proteolytic activity of the cell-free broth as lost at the initial step primarily aimed at concentrating -.2 2.4.6 I /SUBSTRATE FIG. 4. Plots of the reciprocals of substrate concentrations vs. the reciprocals of activity (optical density increase) hen the proteinase as assayed in the presence of 4 X 1-8 M PCMB and 5 X 1-3 X NaF. The assays ere incubated at 87.5 C for 6 min at ph 8.5 in tris-citrate buffer. TABLE 2. Effect of reducing compounds on activity of the proteinase* Reducing agent Concn (X) of reducing agent.1.1.1 Na thioglycolate 1.7.385.328 Na2SOa 2..845.33 L-Cysteine.372.49.315 * Results are expressed as optical density. A control, ith no reducing agent, had an optical density of.332.

VOL. 87, 1964 EXTRACELLULAR PROTEINASE OF S. LACTIS 53 tivity as found to be optimal at about ph 8.5. This value agrees ith observations on the proteolysis of milk by S. lactis (Van der Zant and Nelson, 1953b) and the hydrolysis of milk proteins by cell-free culture filtrates of this organism (Sasaki and Nakae, 1959). The purified enzyme is similar to trypsin in its alkaline ph optimum and in its remarkable heat stability (Northrup, 1932). On the other hand, it differs from trypsin in that it as not inhibited by diisopropyl fluorophosphate (Balls and Jansen, 1952); this indicates the probable absence of a serine residue at the active center. In contrast, the inhibition by PCMB suggests that a free sulfhydryl group(s) is required for activity. The limited data suggest noncompetitive inhibition by PCMB, and indicate that the sulfhydryl group(s) also is not at the active center. ACKNOWLEDGMENT This study as supported in part by American Dairy Association grant no. 397. LITERATURE CITED BALLS, A. K., AND E. F. JANSEN. 1952. Stoichiometric inhibition of chymotrypsin. Advan. Enzymol. 13:321-343. BARIBO, L. E., AND E. M. FOSTER. 1952. The intracellular proteinases of certain organisms from cheese and their relationship to the proteinases in cheese. J. Dairy Sci. 35:149-16. KEILIN, D., AND B. HARTREE. 1938. On the mechanism of the decomposition of hydrogen peroxide by catalase. Proc. Roy. Soc. (London) Ser. B 124:397-45. LOWRY,. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement ith the Folin phenol reagent. J. Biol. Chem. 193:265-275. MORGAN, M. E., AND F. E. NELSON. 1951. The distribution of certain amino acids in soluble fractions of milk cultures of Streptococcus lactis. J. Dairy Sci. 34:446-456. NIVEN, C. F., JR. 1944. Nutrition of Streptococcus lactis. J. Bacteriol. 47:343-35. NORTHROP, J. H. 1932. Crystalline trypsin. IV. Reversibility of the inactivation and denaturation of trypsin by heat. J. Gen. Physiol. 16:323. SASAKI, R., AND T. NAKAE. 1959. Studies on the distribution and properties of lactic acid bacteria in co's milk in Japan. III. Characteristics of proteolytic enzymes produced by some lactic acid bacteria. Japan. J. Zootech. Sci. 3:7-1. SOBER, H. A., F. J. GUTTER, M. M. WYCKOFF, AND E. A. PETERSON. 1956. Chromatography of proteins. II. Fractionation of serum protein on anion-exchange cellulose. J. Am. Chem. Soc. 78:756-763. VAN DER ZANT, W. C., AND F. E. NELSON. 1953a. Proteolysis by Streptococcus lactis gron in milk ith and ithout controlled ph. J. Dairy Sci. 36:114-1111. VAN DER ZANT, W. C., AND F. E. NELSON. 1953b. Characteristic of an endocellular proteolytic enzyme system of Streptococcus lactis. J. Dairy Sci. 36:1212-1222. VAN DER ZANT, W. C., AND F. E. NELSON. 1954. Characteristics of some endocellular peptidases of Streptococcus lactis. J. Dairy Sci. 37:795-84. WILLIAMSON, W. T., AND M. L. SPECK. 1962. Proteolysis and curd tension in milk associated ith accelerated starter culture groth. J. Dairy Sci. 45:164-169.