lnternathmalJournalofk)md Micnshioh~gy, 15 (1992) 87-98
117
(t!l 1992 Elsevier Science Publishers B.V. All rights resetwed 0168-16(15/92/$05.00 FOGD 1111462
Purification of X-prolyl dipeptidyl aminopeptidase from Lactobacillus casei subspecies M. El Abboudi ~, M. El Soda 2, S. Pandian ~, R.E. Simard i and N.F. Olson "~ ('entre th' recherche S'II'~LA, Facuhd th,s sciences ~h' I'agricuhure et de I'alimentation, Unirersitc ~Lat'al, S~e-I"oy (Qtu;hec), Cana&t; ' Facuhy o]'Agricuhun', Unirersity o]'Alexandria, Ah,xandria, ED,pt; attd ~ Center for Daio' Research, University of Wis,'onsht, Madison, WI, USA (Received 5 July 1991; accepted 26 December 1991)
Prolyl dipeptidylaminopeptidases from two subspecies of Lactolmcillus casei were purified and biochemically characterized. L. casei ssp. casei UL21 (a debittering strain)and L. casei ssp. rhamnosus UL26 (a non-debittering strain) were the source bacteria for this study. Punfie...... v i n e s from both the sources was effeetcd by a gel filtration step through S e p h a c ~ by ion-exchange chromatography through DEAE Sephacel. This rendered an electrop, : neous enzyme preparalion. The purified enzymes fr,.ml both the sources showed similar i ,tL~ ~ re o~ optimum (45°(") and pH optimum (7.11). Their activity profiles on various substrates and the inhibition by different inhibitors were also found to be simihr, indicating that this enzyme i~ . ~aps not significantly involved in the debinering process during the maturation of cheese. Key words: X-Prolyl dipeptidyl aminopeptidase: Lactobacillus casei; Purification
Introduction Dipeptidyl peptidase (EC 3.,4.14.1), which catalyses sequenlial release of Nterminal dipeptides, was described recently in a large number of lactic acid bacteria. The first evidence for the presence of dipeptidyl peptidase in dairy bacteria organisms was reported by Casey and Meyer (1985). These authors detected dipeptidyl amin~ peptidase (DAP) activity in cellular extracts of 21 lactic acid bacteria. Purification and characterization of the enzymes from Lactobacillus lactis and Streptococcus thermophilus was then accomplished by Meyer and Jordi (1987). Both enzymes had a molecular weight (M r) of about 165 kDa; and isoelectric pH near 4.5 and were shown to be constituted of two subunits. The L. lactis enzyme had an optimum pH of 7.0 and an optimum temperature of 50-55°C. The Correspondence address: S. Pandian, Centre de Recherche STELA, Facult6 des sciences de I'agriculture et de l'alimentation, Universit6 Laval, Ste-Foy (Quebec), Canada G I K 7P4.
88
corresponding values for Str. thennophih~s were pH 6.5 to 8.2 and 45°C. The enzymes seem to be serine proteases and are significantly more active on X-prolyl peptides than other peptide substrates. The cell wall proteinase system of Luctococcus h.'tis ssp. cremoris was found to cont~fin ~l metal independent X-prolyl dipeplidyl peptidase (Kicfer-Partsch et al., 1989). The enzyme is a serine proteinasc of about 9() kDa showing optimal activities at a tcmpcra;.ui~c t,i 45-5U~t_: a , d ct pH 7.0. Since d'~en, a gleat deal of attention has been given to this class of en~'mes and several reports describing their purification and characterization appeared recently. Khalid and Marth (1990) purified the enzyme from L. helceth'us CNRZ 32. The enzyme shows a M~ of 72 kDa. Optimal activity by the purified enzyme was observed at pH 7.0 ~md at 40°C. Serinc seems to be involved in the catalytic action of the enzyme since diisopropyl fluorophosphate (DFP) was an cffcctivc inhibitor. Booth et al. (19911) described the purification of a DAP from the cytoplasm of Lactococcus lactis ssp. cremoris. This enzyme had a M r of !17 kDa, with a pH optimum bctwccn 6.{1 and 9.1). Phenyl-methylsulfonylfluoride (PMSF) was an effective inhibitor. An X-prolyl dipeptidyl peptidase has also beer, purified from Lactococcus lactis ssp. lactis by ion exchange chromatography and gel filtration (Zevaco et al., 1990). The enzyme was shown to be a 190 kDa dimer, and its optimal activity occurred at pH 7.5 at 40-45°C. This enzyme was inhibited by the presence of inhibitors specific for scrine proteinases. Prolyldipeptidyl peptidase was also detected in L. delbrueckii ssp. bulgaricus (Arian et al., 1990). In a comparative study between several cheese-related microorganisms, El Soda et al. (1991)detected X-prolyl dipeptidyl peptidase activity in Lactobacillus casei, Propionibacterium shermanii and Pediococcus sp. On the other hand, Leuconostoc mesenteroides ssp. mesenteroides and Bret'ibacterium linens lacked the activity. The partially purified enzymes from L. casei, shermanff and t'ediococcus sp. showed an optimum pH close to neutrality and optimum temperature of 40-50°C. The enzyme from these three microorganisms was strongly inhibited by the scrine proteinase inhibitor, PMSF. Bocketman and Teuber (1990) compared X-prolyl dipeptidyl peptidases of several lactobacilli, lactococci and streptococci. All enzymes displayed rather similar properties and they all belonged to the group of serine proteinases. Lactococcus and streptococcus enzymes were however distinguished from iactobacillus enzymes by a lower molecular weight and different substrate specificity. A dipeptidyl aminopeptidase hydrolysing X-prolyl aminomethylcoumarin substrates was purified from Lactococcus lactis ssp. lactis H1 by Lloyd and Pritchard (1991). The active enzyme showed a M~ of approximately 150 kDa and was inhibited by PMSF. The authors also showed that the enzyme is chromosomally enct~ded. This group of enzymes, widely spread in lactic acid bacteria, probably plays a role in nitrogen nutrition of these fastidious organisms. The possible role of the enzyme during the ripening process is not fully understood Booth et al. (1990), however, suggested that they may release either single proline residues or prolyl-
~9 proline sequences from oligopeptides and allowing aminopeptidase action to proceed. Considering its significant role during cheese ripening, some attention was also rce~ ntly given to the genetics of the enzyme. The cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidasc gene from Lactoc, lactis ssp. cremoris (Mayo et al., 1991) and Lactoe. lactis ssp. lactis (Nardi el al., 1991) have been reported. The Pep X P gene from Lactoc. lactis ssp. lactis was shown to be 99% homologous to the Pep X P gene from Lactoc. lactis ssp. cremoris (Nardi et al., 1991). In the present investigation, the purification and characterization of the X-proiyl dipeptidyl aminopeptidase (DAP) from debittering and non-debittering strains of L. casei subspecies are described. A comparative study of some of the biochemical properties of these enzymes is also presented.
Materials and Methods
Bacterial strabts The strains used in this study were L. casei ssp. casei UL21 (a debittering strain) and L. casci ssp. rhamnosus UL26 (a non-debittering strain). The strains were stored in sterile skimmed milk at -30°C. Cultication of the microorganisms and preparation of the crude cell free extract An active overnight subculture of the strains in MKS medium was used to inoculate (2%) fresh MRS medium. After 12 h of growth at 37°C the culture was centrifuged at 10000 × g for 15 min at 4°C. The cell pellet was washed twice in 0.01 M potassium phosphate buffer (pH 7.0). Disintegration of the cells was accomplished as described previously (El Abboudi et al., 1990); the cells were ground in a mortar with 2 volumes of Alumina powder (Sigma #A-2039). Enzyme assay Dipeptidyl aminopeptidase activity was assayed spectrophotometrically at 410 nm by assessing the degree of hydrolysis of Arg-Pro p-nitroanilidc as previously described by El Soda and Desmazeaud (1982). Protein estimatio, was determined with the method of Lowry et al. (1951) using bovine serum albumin as standard. One unit of enzymatic activity was defined as that amount of enzyme producing an increase of 0.01 unit of absorbance at. 41(1 nm per min at 37°C. Specific activity was defined as the number of activity units per mg of protein. Polyacrylamide gel electrophoresis The method of Davis (1964) for the non-denaturing polyacrylamide gel electrophoresis was utilized. The final acrylamide concentration was 7% (w/v) alad 0.I M phosphate buffer (pH 7.0) was utilized as the electrophoresis buffer to assure enzyme stability in the gel. The gels were silver stained by the method of Morrissey (1981). Sodium dodecyl sulfate-polyacrylamid,: gel electrophoresis (SDS-PAGE) was done on 12% running gel and 4% stacking gel by the method of Laemmli
911
(1970). Dipeptidyl aminopeptidase activity was detected on the gels after clectrophoresis using Gly-Pro/3-naphthylamidcs as described by Miller and MacKinnon (1974).
Purij~catton g[' the dipeptidyl amhuqn:otidase For gel filtration the crudc cell frcc extract of L. easel ssp. casei UL21 and L. casei ssp. rhamnosus UL26 eont~i~i~g ie,:pectively 71.7 and 113 m g / m l protein were applied to a Pharmacia LK?~ c:~iamn (26/1001 of Sephacryl $300 SF equilibrated with 0.(ll M potassium pho:,phate buffer, pH 7.0. Elution was performed at a flow rate of 30 ml/h. The pooled active fractions af~?~ ,:el filtration were lyophilizcd in a Virtis lyophilizcr and rcsuspcnded in 0.0t ~,i phos h?.te buffer, pH 7.0. This solution was then applied to a DEAE Sephacel ion cxch:mger (26/4(I; Pharmaci~a LKID equilibrated with the samc buffer. Elution from ti~c column was accomplish¢5 using a 0.01 M-0.5 M linear concentration gradie,t of phosphate buffer, pH 7.g, at a flow rate of 20 ml/h. The activc fracti:ms from DEAE ion exchange were then pooled and dialyscd against 0.01 M phosphate buffer ovcrnight and s;orcd at -20°C. Effect of bzhibitors on enzyme acticity The enzyme was prcincubated in (1.01 M phosphate buffer (pl-I 7.0~ for 1 h with pnenylmethylsulfonylfluorid¢ (OMSF), p-hydroxymercurybenzoate (PHMB), 1:10 phcnanthroline or ethylenediami.nc ~et~~acetic acid (EDTA) at a concentration of cithcr 0.1 mM or 1.0 mM at 3 , C . Enzyme activity was measured using the standard enzyme assay procedure, and thc rate of hydrolysis of Arg-Pro pnitroanilide in the absence of the inhibitors was taken as 100% enzyme activity. Effect of metal ions on enz3,me acticity The dialyscd enzyme was preincubated for 1 h in the presence or absence of various divalent cations: MnCI 2, MgCI_, and CoCI 2 at a final cemcentration of 1 and 10 mM in phosphate buffer, pH 7.0. The remaining activity was assayed at 37°C for 15 min using Arg-Pro p-nitroanilide as a substrate. The rate of hydrolysis of Arg-Pro p-nitroanilide in thc absence of metal ions was considered as 100% enzyme activity.
Results
Purification of the dipeptMyl amhlopeptMase from L. casei subspecies The gel filtration profiles (Fig. 1) showed that the DAP from the two strains was cluted at approximately the same fracas.ion which indicated a close similarity in their molecular weights. The ion cxchar~ge chromatography profiles (Fig. 2) show that the DAP from L. casei ssp. casei was eluted at a m~larity of 0.10 while it was 0.13 in the case of L. casei ssp. rhamnosus which is another confirmation for the resemblance of the enzyme in the two L. casei subspecies.
()I
A 4
1,0
0.8 3 A A
E t-
0.6
E cO
O OO
2
,
117
(t!l 1992 Elsevier Science Publishers B.V. All rights resetwed 0168-16(15/92/$05.00 FOGD 1111462
Purification of X-prolyl dipeptidyl aminopeptidase from Lactobacillus casei subspecies M. El Abboudi ~, M. El Soda 2, S. Pandian ~, R.E. Simard i and N.F. Olson "~ ('entre th' recherche S'II'~LA, Facuhd th,s sciences ~h' I'agricuhure et de I'alimentation, Unirersitc ~Lat'al, S~e-I"oy (Qtu;hec), Cana&t; ' Facuhy o]'Agricuhun', Unirersity o]'Alexandria, Ah,xandria, ED,pt; attd ~ Center for Daio' Research, University of Wis,'onsht, Madison, WI, USA (Received 5 July 1991; accepted 26 December 1991)
Prolyl dipeptidylaminopeptidases from two subspecies of Lactolmcillus casei were purified and biochemically characterized. L. casei ssp. casei UL21 (a debittering strain)and L. casei ssp. rhamnosus UL26 (a non-debittering strain) were the source bacteria for this study. Punfie...... v i n e s from both the sources was effeetcd by a gel filtration step through S e p h a c ~ by ion-exchange chromatography through DEAE Sephacel. This rendered an electrop, : neous enzyme preparalion. The purified enzymes fr,.ml both the sources showed similar i ,tL~ ~ re o~ optimum (45°(") and pH optimum (7.11). Their activity profiles on various substrates and the inhibition by different inhibitors were also found to be simihr, indicating that this enzyme i~ . ~aps not significantly involved in the debinering process during the maturation of cheese. Key words: X-Prolyl dipeptidyl aminopeptidase: Lactobacillus casei; Purification
Introduction Dipeptidyl peptidase (EC 3.,4.14.1), which catalyses sequenlial release of Nterminal dipeptides, was described recently in a large number of lactic acid bacteria. The first evidence for the presence of dipeptidyl peptidase in dairy bacteria organisms was reported by Casey and Meyer (1985). These authors detected dipeptidyl amin~ peptidase (DAP) activity in cellular extracts of 21 lactic acid bacteria. Purification and characterization of the enzymes from Lactobacillus lactis and Streptococcus thermophilus was then accomplished by Meyer and Jordi (1987). Both enzymes had a molecular weight (M r) of about 165 kDa; and isoelectric pH near 4.5 and were shown to be constituted of two subunits. The L. lactis enzyme had an optimum pH of 7.0 and an optimum temperature of 50-55°C. The Correspondence address: S. Pandian, Centre de Recherche STELA, Facult6 des sciences de I'agriculture et de l'alimentation, Universit6 Laval, Ste-Foy (Quebec), Canada G I K 7P4.
88
corresponding values for Str. thennophih~s were pH 6.5 to 8.2 and 45°C. The enzymes seem to be serine proteases and are significantly more active on X-prolyl peptides than other peptide substrates. The cell wall proteinase system of Luctococcus h.'tis ssp. cremoris was found to cont~fin ~l metal independent X-prolyl dipeplidyl peptidase (Kicfer-Partsch et al., 1989). The enzyme is a serine proteinasc of about 9() kDa showing optimal activities at a tcmpcra;.ui~c t,i 45-5U~t_: a , d ct pH 7.0. Since d'~en, a gleat deal of attention has been given to this class of en~'mes and several reports describing their purification and characterization appeared recently. Khalid and Marth (1990) purified the enzyme from L. helceth'us CNRZ 32. The enzyme shows a M~ of 72 kDa. Optimal activity by the purified enzyme was observed at pH 7.0 ~md at 40°C. Serinc seems to be involved in the catalytic action of the enzyme since diisopropyl fluorophosphate (DFP) was an cffcctivc inhibitor. Booth et al. (19911) described the purification of a DAP from the cytoplasm of Lactococcus lactis ssp. cremoris. This enzyme had a M r of !17 kDa, with a pH optimum bctwccn 6.{1 and 9.1). Phenyl-methylsulfonylfluoride (PMSF) was an effective inhibitor. An X-prolyl dipeptidyl peptidase has also beer, purified from Lactococcus lactis ssp. lactis by ion exchange chromatography and gel filtration (Zevaco et al., 1990). The enzyme was shown to be a 190 kDa dimer, and its optimal activity occurred at pH 7.5 at 40-45°C. This enzyme was inhibited by the presence of inhibitors specific for scrine proteinases. Prolyldipeptidyl peptidase was also detected in L. delbrueckii ssp. bulgaricus (Arian et al., 1990). In a comparative study between several cheese-related microorganisms, El Soda et al. (1991)detected X-prolyl dipeptidyl peptidase activity in Lactobacillus casei, Propionibacterium shermanii and Pediococcus sp. On the other hand, Leuconostoc mesenteroides ssp. mesenteroides and Bret'ibacterium linens lacked the activity. The partially purified enzymes from L. casei, shermanff and t'ediococcus sp. showed an optimum pH close to neutrality and optimum temperature of 40-50°C. The enzyme from these three microorganisms was strongly inhibited by the scrine proteinase inhibitor, PMSF. Bocketman and Teuber (1990) compared X-prolyl dipeptidyl peptidases of several lactobacilli, lactococci and streptococci. All enzymes displayed rather similar properties and they all belonged to the group of serine proteinases. Lactococcus and streptococcus enzymes were however distinguished from iactobacillus enzymes by a lower molecular weight and different substrate specificity. A dipeptidyl aminopeptidase hydrolysing X-prolyl aminomethylcoumarin substrates was purified from Lactococcus lactis ssp. lactis H1 by Lloyd and Pritchard (1991). The active enzyme showed a M~ of approximately 150 kDa and was inhibited by PMSF. The authors also showed that the enzyme is chromosomally enct~ded. This group of enzymes, widely spread in lactic acid bacteria, probably plays a role in nitrogen nutrition of these fastidious organisms. The possible role of the enzyme during the ripening process is not fully understood Booth et al. (1990), however, suggested that they may release either single proline residues or prolyl-
~9 proline sequences from oligopeptides and allowing aminopeptidase action to proceed. Considering its significant role during cheese ripening, some attention was also rce~ ntly given to the genetics of the enzyme. The cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidasc gene from Lactoc, lactis ssp. cremoris (Mayo et al., 1991) and Lactoe. lactis ssp. lactis (Nardi el al., 1991) have been reported. The Pep X P gene from Lactoc. lactis ssp. lactis was shown to be 99% homologous to the Pep X P gene from Lactoc. lactis ssp. cremoris (Nardi et al., 1991). In the present investigation, the purification and characterization of the X-proiyl dipeptidyl aminopeptidase (DAP) from debittering and non-debittering strains of L. casei subspecies are described. A comparative study of some of the biochemical properties of these enzymes is also presented.
Materials and Methods
Bacterial strabts The strains used in this study were L. casei ssp. casei UL21 (a debittering strain) and L. casci ssp. rhamnosus UL26 (a non-debittering strain). The strains were stored in sterile skimmed milk at -30°C. Cultication of the microorganisms and preparation of the crude cell free extract An active overnight subculture of the strains in MKS medium was used to inoculate (2%) fresh MRS medium. After 12 h of growth at 37°C the culture was centrifuged at 10000 × g for 15 min at 4°C. The cell pellet was washed twice in 0.01 M potassium phosphate buffer (pH 7.0). Disintegration of the cells was accomplished as described previously (El Abboudi et al., 1990); the cells were ground in a mortar with 2 volumes of Alumina powder (Sigma #A-2039). Enzyme assay Dipeptidyl aminopeptidase activity was assayed spectrophotometrically at 410 nm by assessing the degree of hydrolysis of Arg-Pro p-nitroanilidc as previously described by El Soda and Desmazeaud (1982). Protein estimatio, was determined with the method of Lowry et al. (1951) using bovine serum albumin as standard. One unit of enzymatic activity was defined as that amount of enzyme producing an increase of 0.01 unit of absorbance at. 41(1 nm per min at 37°C. Specific activity was defined as the number of activity units per mg of protein. Polyacrylamide gel electrophoresis The method of Davis (1964) for the non-denaturing polyacrylamide gel electrophoresis was utilized. The final acrylamide concentration was 7% (w/v) alad 0.I M phosphate buffer (pH 7.0) was utilized as the electrophoresis buffer to assure enzyme stability in the gel. The gels were silver stained by the method of Morrissey (1981). Sodium dodecyl sulfate-polyacrylamid,: gel electrophoresis (SDS-PAGE) was done on 12% running gel and 4% stacking gel by the method of Laemmli
911
(1970). Dipeptidyl aminopeptidase activity was detected on the gels after clectrophoresis using Gly-Pro/3-naphthylamidcs as described by Miller and MacKinnon (1974).
Purij~catton g[' the dipeptidyl amhuqn:otidase For gel filtration the crudc cell frcc extract of L. easel ssp. casei UL21 and L. casei ssp. rhamnosus UL26 eont~i~i~g ie,:pectively 71.7 and 113 m g / m l protein were applied to a Pharmacia LK?~ c:~iamn (26/1001 of Sephacryl $300 SF equilibrated with 0.(ll M potassium pho:,phate buffer, pH 7.0. Elution was performed at a flow rate of 30 ml/h. The pooled active fractions af~?~ ,:el filtration were lyophilizcd in a Virtis lyophilizcr and rcsuspcnded in 0.0t ~,i phos h?.te buffer, pH 7.0. This solution was then applied to a DEAE Sephacel ion cxch:mger (26/4(I; Pharmaci~a LKID equilibrated with the samc buffer. Elution from ti~c column was accomplish¢5 using a 0.01 M-0.5 M linear concentration gradie,t of phosphate buffer, pH 7.g, at a flow rate of 20 ml/h. The activc fracti:ms from DEAE ion exchange were then pooled and dialyscd against 0.01 M phosphate buffer ovcrnight and s;orcd at -20°C. Effect of bzhibitors on enzyme acticity The enzyme was prcincubated in (1.01 M phosphate buffer (pl-I 7.0~ for 1 h with pnenylmethylsulfonylfluorid¢ (OMSF), p-hydroxymercurybenzoate (PHMB), 1:10 phcnanthroline or ethylenediami.nc ~et~~acetic acid (EDTA) at a concentration of cithcr 0.1 mM or 1.0 mM at 3 , C . Enzyme activity was measured using the standard enzyme assay procedure, and thc rate of hydrolysis of Arg-Pro pnitroanilide in the absence of the inhibitors was taken as 100% enzyme activity. Effect of metal ions on enz3,me acticity The dialyscd enzyme was preincubated for 1 h in the presence or absence of various divalent cations: MnCI 2, MgCI_, and CoCI 2 at a final cemcentration of 1 and 10 mM in phosphate buffer, pH 7.0. The remaining activity was assayed at 37°C for 15 min using Arg-Pro p-nitroanilide as a substrate. The rate of hydrolysis of Arg-Pro p-nitroanilide in thc absence of metal ions was considered as 100% enzyme activity.
Results
Purification of the dipeptMyl amhlopeptMase from L. casei subspecies The gel filtration profiles (Fig. 1) showed that the DAP from the two strains was cluted at approximately the same fracas.ion which indicated a close similarity in their molecular weights. The ion cxchar~ge chromatography profiles (Fig. 2) show that the DAP from L. casei ssp. casei was eluted at a m~larity of 0.10 while it was 0.13 in the case of L. casei ssp. rhamnosus which is another confirmation for the resemblance of the enzyme in the two L. casei subspecies.
()I
A 4
1,0
0.8 3 A A
E t-
0.6
E cO
O OO
2
,
