PROTEOLYSIS BY LACTOBACILLUS CASEI

Transcription

1 PROTEOLYSIS BY LACTOBACILLUS CASEI II. PEPTIDASE AcIvrry1 ERLING BRANDSAETER2 AND F. E. NELSON Iowa Agricultural Experiment Station, Ames, Iowa A study of the proteinase activity of Lactobacillus casei grown in milk and of cell-free extract of L. cawi has been reported on in a previous paper (Brandsaeter and Nelson, 1956). Whereas the proteinases of L. casei have been studied to some extent earlier, very little has been done to characterize the peptidases of this organism. Other lactobacilli hydrolyze some peptides (Christophersen and Thiele, 1953, and Berger et al. 1938a). A survey by Berger et ca. (1938a), as well as more recent work (van der Zant and Nelson, 1954, Zimmerman, 1950 and Dudani, 1950), indicates that most peptidases produced by members of the family Lactobacteriaceae have maximum activity between 7.0 and 8.5. Metal activation of bacterial peptidases has been reported by several workers (Berger et al., 1938b, Dudani, 1950, Zimmerman, 1950 and van der Zant and Nelson, 1954). Manganese and magnesium and in some instances cobalt are most effective activators of peptidases. In the present work the characteristics of the peptidases of L. casei have been studied. Special emphasis has been placed on the effect of metallic ions on the activity of these peptidases. EXPERIMENTAL METHODS The preparation of cell-free extract of L. ca8ei strain 25 was carried out as described in a previous paper (Brandsaeter and Nelson, 1956). Activity of the cell-free extracts was tested on M/30 glycylglycine, diglycylglycine, glycyl-lleucine, glycyl-l-tyrosine, glycyl-i-tryptophan and carbobenzoxyglycyl-l-phenylalanine, m/15 glycyl-dl-alanine, DL-alanylglycine, DL-alanylglycylglycine, DL-leucylglycine and DL-leucylglycylglycine, and m/7.5 DL-alanyl-DL-alanine. Composite buffer (equimolar concentrations of 1 Journal paper No. J-2871 of the Iowa Agricultural Experiment Station, Ames, Iowa. Project No Present address: Agricultural College of Norway, Vollebekk, Norway. Received for publication December 8, 1955 acetate, phosphate and borate) was added to give a 0.05 M concentration in the substrate solution. Merthiolate was added as a preservative to all substrate solutions at a rate of 2.5 mg per 100 ml. All substrates were stored at 2 C. Usually 0.2 ml of cell-free extract was added to 2.5 ml of substrate at the desired and incubated 1 hr at 45 C. Hydrolysis of the peptides was determined by titration of the carboxyl groups with ethanolic KOH, using thymolphthalein as an indicator (Grassman and Heyde, 1929). RESULTS The effect of temperature on peptidase activity by cell-free extract of L. casei 25 at 7.0 was tested on four different dipeptides. Maximum peptidase activity under these conditions was at 45 to 50 C (figure 1). The effect of on the enzymatic hydrolysis of three dipeptides and two tripeptides was tested with the results shown in figure 2. Maximum hydrolysis was in the range of 7.0 to 8.0. Generally the maximum rate of hydrolysis of the glycyl peptides occurred at a higher level than the maximum rate of hydrolysis of the alanyl and the leucyl peptides. DL-alanyl-DLalanine was hydrolyzed rapidly over a wide range. Glycylglycine and diglycylglycine were hydrolyzed rather slowly, but over a wide range. DL-alanylglycylglycine was hydrolyzed rapidly between 's 6.0 and 7.0, but a sharp decline in rate of hydrolysis was observed at the higher levels. Effect of metauic ions. The following metal salt solutions in 0.05 M concentrations were used: CoSO4-7H20, ZnSO4-7H20, NiSO4-7H20, CuSO4.5H20, MnSO4-4H20 and MgSO4-7H20. The desired salt solution in 0.2-ml quantity was incubated with 1.8 ml of cell-free extract at 45 C for 15min; for the control, 0.2 mlof distilled water was used instead of the metal salt solution. The ion-treated cell-free extract in 0.2-ml quantity was added to 2.5 ml of substrate, giving a con- 73

2 74 BRANDSAETER AND NELSON [vol. 72 TEMPERATURE Figure 1. Effect of temperature on peptidae / A activity at 7.0 by cell-free extract of Lactobacil-. a a I lus caei. (Reaction time 1 hr) centration of metallic ions in the reaction mixture Figure S. Effect of on peptidase activity of approximately m. The tests were run by cell-free extract of Lactobacillus casei. (Test at 45 C for 1 hr, and the results obtained are run at 45 C for 1 hr) shown in table 1. Cobalt had the greatest effect on the enzymatic hydrolysis of peptides; activation strongly by cobalt at 7.7. Zinc activated the was strongest at acidic 's, except for glycyl- hydrolysis of some peptides. Nickel slightly glycine whose hydrolysis also was activated activated the hydrolysis of glycyl-dl-alanine TABLE 1 Effect of metallic ione on peptidase activity by cell-free extract of Lactobacillus ca8ei* Substrate Per cent Hydrolysis in Presence of the Following lonst None Co+ Znt Ni4+ CuW Mn+ Mg+ Glycyl-DL-alanine Glycyl-DL-alanine Glycyl-L-leucine _- - Glycyl-L-leucine Glycyl-L-tyrosine Glycyl-L-tryptophan Glycylglyciney Diglycylglycinė DL-Alanylglycine DL-Leucylglycne _ DL-Leucylglycine DL-Alanyl-DL-alanine DL-Alanyl-DL-alanine DL-Alanylglycylglycine DL-Leucylglycylglycine Cbot Glycyl-L-phenylalanine _ Cbo Glycyl-L-phenylalanine * 0.2 ml cell-free extract in 2.5 ml of substrate, incubated at 45C for 1 hr. t Ion concentration in digestion mixture was M. t Cbo - carbobenzoxy.

3 PROTEOLYSIS BY LACTOBACILLUS CASEI. II 75 - Figure 8. Effect of cobalt and zinc on the enzymatic hydrolysis of peptides at various levels (Metal ion concentration in mixture m) Figure 4. Effect of cobalt and zinc on the enzymatic hydrolysis of peptides at various levels. Metal ion concentration in reaction mixture m. (Reaction time 1 hr) at 6.0 and 8.0, and strongly inhibited at different levels on the enzymatic hydrolysis the hydrolysis of DL-alanylglycine, DL-leucylglycine and DL-alanylglycylglycine at 7.0, hydrolysis at the lower levels, resulting in a of peptides. Cobalt in all cases activated the but had little effect on the hydrolysis of the other maximum rate of hydrolysis when tested at peptides tested. Copper and manganese were 5.5. Hydrolysis of glycyl-ityrosine (figure inhibitory or had no effect in all cases tested. 3A), glycylglycine (figure 3B) and glycyl-dlalanine (figure 4A) in the presence of cobalt had a Magnesium inhibited the hydrolysis of glycylglycine and diglycylglycine, but otherwise had second optimum at 7.5 or higher. This second little effect. optimum was not found with DL-alanylglycine In figures 3 and 4 are presented representative (figure 4B), hydrolysis of this peptide being data showing the effect of cobalt and zinc ions strongly inhibited by cobalt at 6.5 and above.

4 76 BRANDBAETER AND NELSON [vol. 72 GLYCYL- LTYROSINE a U) IL a 6a 0 IL o so U) z z ei to Figure 6. The effect of various concentrations of cobalt on the hydrolysis of peptides at various levels. (Reaction time 1 hr) TABLE 2 Effect of Co++ and Zn+ on hydrolysis of DL-alanylglycine Per cent Hydrolysis in Presence of: No dndoa Co" Zn ~~~~Co++ X Co++ X ions M x loris + Zn+n + ~~~~ x Zn5+ it Zinc did not have any effect on the hydrolysis of the peptides at 7.0 or below, but hydrolysis of glycyl-l-tyrosine, glycyl-dl-alanine and DLalanylglycine was activated by zinc at the higher levels. The effect of various concentrations of cobalt on the enzymatic hydrolysis at different levels was tested on DL-alanylglycine (figure 5A) and on glycyl-l-tyrosine (figure 5B). The activation by cobalt at 5.5 decreased with decreasing cobalt ion concentration. The lowest concentration used actually inhibited hydrolysis at 5.5. The hydrolysis of DL-alanylglycine at reactions of 6.5 or above and the hydrolysis of glycyl- L-tyrosine at reactions of 7.0 or above were inhibited by all three cobalt concentrations used. An expenment was carried out by adding both cobalt and zinc to the reaction mixture of DLalanylglycine and cell-free extract. The test was run at 45 C for 1 hr and at 's 5.5 and 7.0. The results shown in table 2 are average values of duplicate runs. The effect of cobalt was partly eliminated in the presence of m zinc. When A-A ol-alanylglycine AT 7.0 1B- OL-ALANYLGLYCINE AT 5.7+Co+ C-C GLYCYL-OL-ALANINE AT 0o 0 0-D GLYCYL-DL-ALANINE AT 5.7.Co+ AT to Ow Z 0w~~~ w> w ItL cn 0 Za E-E DL-ALANYL-OL-ALANINE r -,.-,"i U &O 5 ao OF HEATED ENZYME Figure 6. The effect of heating to 61.7 C for 2 min at different levels on peptidase activity by cell-free extract of Lactobacillus casei. (Heated enzyme tested at 45 C for 1 hr)

5 1956] PROTEOLYSIS BY LACTOBACILLUS CASEI. II 77 equimolar concentrations of zinc and cobalt were used in the reaction mixture, the effect of cobalt was eliminated completely. The effect of heating to 61.7 C for 2 min at different levels on the peptidase activity of the cell-free extract of L. casei is shown in figure 6. Maximum stability occurred at 6.0. The enzyme was inactivated rapidly by heat below 5.5 and above 6.5. Enzyme heated at 6.0 retained 88 per cent of initial activity when tested on DL-alanylglycine and DL-alanyl- DL-alanine at 7.0 and on glycyl-dl-alanine and glycyl-l-tyrosine at 8.0 in the absence of cobalt. Less than 75 per cent of the activity on three of the same peptides tested at 5.7 in the presence of cobalt Was retained under the same conditions. Attempts to separate the various peptidase activities by adsorption on alumina gel, followed by step-wise elution, and by electrophoresis in a starch bed were not uniformly successful. However, indications of some approaches which might prove fruitful in the future were obtained. DISCUSSION Several di- and tri-peptides were hydrolyzed by the cell-free extract of L. casei strain 25. The temperature optima for hydrolysis of some of these peptides varied slightly, but not enough to serve as a basis for assuming that different enzyme systems were involved. The optima for hydrolysis and the rates of hydrolysis varied greatly for the different peptides, but in a different manner than what van der Zant and Nelson (1954) observed for hydrolysis of the same peptides by cell-free extract of Streptococcus lactis. The variations of optima possibly are due not only to the presence of different enzyme systems but also to the enzyme-substrate combination. Two or more enzyme systems with overlapping specificities also might be responsible for the hydrolysis of the various substrates. In this case the observed optima might be somewhere between the optima for hydrolysis by each of the enzyme systems present. Manganese and magnesium ions have been reported by many workers (Dudani, 1950, Smith and Spackman, 1955, van der Zant and Nelson, 1954 and Zimmerman, 1950) as being activators of different peptidases. In the present study these ions had little effect on the hydrolysis of peptides. Cobalt and to some extent zinc were found to have the greatest effect, both activating and inhibiting the hydrolysis of peptides. Strong activation by cobalt at 5.5 is of interest because this is in the neighborhood of the usually found in ripening Cheddar cheese. Dudani (1950) also found that cobalt strongly activated the enzymatic hydrolysis of glycyl-l-leucine at 5.0 by cell-free extract of Streptococcus liquefaciens. However, Dudani did not find that cobalt inhibited the hydrolysis of DL-alanylglycine at 7.0 or higher, as was the case in the present study. The cobalt ion is, according to Smith (1948), an exceedingly specific activator for the hydrolysis of glycylglycine; hydrolysis of other peptides containing aliphatic amino acids is not influenced by cobalt. This statement does not seem to hold for the peptidases from the cell-free extract of L. casei, although there are some indications that the effect of cobalt depends on the presence of at least one glycine unit in the peptide. The effect also seems to depend on whether the glycine is at the carboxy or at the amino terminal of the peptide. Hydrolysis of glycylglycine was strongly activated by cobalt at levels ranging from 5.0 to 8.5, whereas hydrolysis of DLalanyl-DL-alanine was not affected by cobalt ions at any of the levels tested. In the present study, zinc activated the hydrolysis of some peptides at 7.0 or above. Activation by zinc at 8.0 also was reported by Berger et al. (1938a) in a study of peptidases from Leuconostoc mesenteroides. The heat inactivation studies indicate that the peptidase activities at 5.7 and in the presence of cobalt were less stable than the peptidase activities at 7.0 to 8.0 and in the absence of cobalt. These results may indicate the presence of at least two different enzyme systems. Based on the variation in optima and on metal ion activation, the hydrolysis of DL-alanylglycine and glycyl-dl-alanine probably are catalyzed by two different enzyme systems. Possibly a third enzyme system was responsible for the hydrolysis of glycylglycine at 7.7 in the presence of cobalt. There also were some indications that one enzyme system was responsible for the activity on a variety of peptides at 5.5 in the presence of cobalt. However, the results obtained are not conclusive and the studies on this phase need to be extended.

6 78 BRANDSAETER AND NELSON [VOL. 72 Cell-free extracts of Lactobacillus cawei strain 25 contain high peptidase activity, with optima ranging from 7.0 to 8.0. The hydrolysis of most peptides was strongly activated by cobalt ions at 5.5 to 6.0. Variations in optima, metal ion activation and heat stability indicate that different peptidase systems were involved. REFERENCES BERGER, J., JOHNSON, M. J. AND PETERSON, W. H. 1938a The proteolytic enzymes of bacteria. I. The peptidases of Leuconostoc mesenteroides. J. Biol. Chem., 124, BERGER, J., JOHNSON, M. J. AND PETERSON, W. H. 1938b The proteolytic enzymes of bacteria. II. The peptidases of some common bacteria. J. Bacteriol., 38, BRANDSAETER, E. AND NELSON, F. E Proteolysis by Lactobacillus casei. I. Proteinase activity. J. Bacteriol., 72, CHRISTOPHERSEN, J. AND THIELE, H tjber den Einfluss von Substrat und Temperatur auf die proteolytische Aktivitat einiger Kasebakterien. Kieler Milchw. Forschungsber., 4, DUDANI, A. T Proteolytic and coagulating enzymes of enterococci. Ph.D. Thesis. Ames, Iowa, Iowa State College Library. GEAS5MAN, W. AND HEYDE, W Alkalimetrische Mikrobestimmung der Aminos8uren und Peptiden. Z. physiol. Chem. (Hoppe- Seyler's), 183, SMITH, E. L Studies on dipeptidases. III. Hydrolysis of methylated peptides. The role of cobalt in the action of glycylglycine dipeptidase. J. Biol. Chem., 176, SMITH, E. L. AND SPACKMAN, D. H Leucine aminopeptidase. V. Activation, specificity, and mechanism of action. J. Biol. Chem., 212, VAN D5ER ZANT, W. C. AND NELSON, F. E Characteristics of some endocellular peptidases of Streptococcus lacti8. J. Dairy Sci., 37, ZIMMERMAN, H. K Proteolytic enzymes of ripening Cheddar cheese. Ph.D. Thesis. Ames, Iowa, Iowa State College Library. Downloaded from on December 14, 2018 by guest