Appl Microbiol Biotechnol (1991) 35:222-227 017575989100118D
App//ed Microb/okgy Biotechnology © Springer-Verlag 1991
Action of a cell wall proteinase from Lactococcus lactis subsp, cremoris S K l l on bovine asl-casein Julian R. Reid, Christopher H. Moore, Graeme G. Midwinter, and Graham G. Pritchard Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand Received 19 November 1990/Accepted 15 January 1991
Summary. The cell wall-associated proteinase from Lactococcus lactis subsp, cremoris S K l l was partially purified and incubated with a~a-easein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the amino acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of a~-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the as~-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No clear consensus sequence of amino acid residues surrounding the cleavage sites could be identified.
Introduction The level of free amino acids and small peptides in milk is inadequate to account for the rapid growth of lactococci to high cell densities indicating that degradation of milk proteins by starter proteinases and peptidases must be occurring (Mills and Thomas 1978). The initial step in proteolysis involves the action of a cell wall-bound proteinase (Exterkate 1975; Mills and Thomas 1978; Hugenholz et al. 1987) that is capable of degrading one or more of the casein components to oligopeptides, which are then hydrolysed further by a series of peptidases (Thomas and Pritchard 1987). Cell-wallbound proteinases from several different strains of lactococci have been purified in recent years (Geis et al. 1985; Exterkate and de Veer 1987, 1989; Monnet et al. 1987; Bockelmann et al. 1989) and their properties investigated. The proteinases from different strains of lactococci have been classified according to various criteria: pH Offprint requests to: G. G. Pritchard
and temperature optima (Exterkate 1976), immunological cross-reactivity (Hugenholz et al. 1984) and specificity towards the casein components of bovine milk (Visser et al. 1986). On the basis of the specificity of action in hydrolysing casein, two distinct patterns of action are currently recognised which have been designated P~ and Pro-type proteinases (Exterkate and de Veer 1989). The P~-type (or HP-type) proteinase preferentially degrades //-casein. The particular peptide bonds cleaved in the fl-casein molecules by PI-type proteinases have been determined for the enzymes from Lactococcus lactis subsp, lactis NCDO 763 (Monnet et al. 1986, 1989) from L. lactis subsp, cremoris HP (Visser et al. 1988) and from L. lactis subsp, cremoris AC1 (Monnet et al. 1989). While there are some apparent differences between the proteinases from these strains with respect to the particular bonds in//-casein cleaved in long-term digests (Monnet et al. 1989), a remarkably consistent pattern has been found for the initial sites of cleavage. These sites are mainly located in the C-terminal 53-residue region of the fl-casein molecule. The P~-type proteinases so far isolated from L. lactis subsp, cremoris have little or no effect on as-casein. The proteinase from L. laetis subsp, lactis NCDO 763 was shown to catalyse limited degradation of both a, and x-casein (Monnet et al. 1989) but the nature of the products was not described. Gel electrophoresis of casein digests has indicated that the Pro-type (of AMl-type) of proteinase degrades //-casein at sites different from those characteristic of the P~-type and also significantly digests as- and x-casein (Visser et al. 1986) but the identity of the peptide products of digestion has not been previously reported. The present paper describes the sites of cleavage in asacasein by a Pni-type proteinase from L. lactis subsp, eremoris SK11.
223
Materials and methods Organisms, culture conditions and harvesting. L. lactis subsp, cremoris SKI 12 (deVos et al. 1984) was obtained from the starter collection of the New Zealand Dairy Research Institute, Palmerston North, New Zealand (isolate number 5221). Cultures for enzyme isolation were grown in 3.5 1 skim-milk medium containing glycerophosphate as a buffer (Mills and Thomas 1978) for 16 h at 22 ° C. The culture was neutralised to pH 7.0, 210 ml of 25% (w/v) sodium citrate were added and the cells sedimented by centrifugation. The cells were washed twice with sodium acetate/phosphate buffer (containing 46 mM sodium acetate and 4 mM sodium dihydrogen phosphate, pH 6.4) at 4 ° C. Solubilization and purification of proteinase activity. Proteinase activity was solubilised by incubation of the cells in 50 mM sodium phosphate buffer, pH 6.4, at 30 ° C for two successive 1 h periods (Mills and Thomas 1978). The same buffer (but at 10 mM) was used for subsequent steps in purification and all steps were earried out at 4 ° C. The pooled supernatants from the two incubations were cooled on ice, diluted with an equal volume of cold water and loaded onto a column (4 cm x 14 cm) of DEAE-cellulose. The column was washed with 200 ml buffer and eluted with a linear NaC! gradient (0-0.8 M). This procedure yields a single peak of proteinase activity separated from lysine aminopeptidase and X-prolyl dipeptidyl aminopeptidase activity. The pooled, concentrated proteinase fractions were further purified by gel permeation on a column (3 cm x 75 era) of Sephacryl $300 (Pharmacia, Uppsala, Sweden) eluting with buffer at a flow rate of 19 ml h -~. The concentrated, pooled fractions from the Sephacryl column were then passed through Superose 6 (Pharmacia LCC 500 PLUS FPLC using a Superose-6 HR 10/30 column) eluting with 10 mM phosphate buffer (pH 6.4) containing 50 mM NaCI at 0.5 mlmin -~. A single major protein peak with a shoulder was obtained from the Superose 6 column; proteinase activity corresponded to the main protein peak. The proteinase was used immediately for digestion studies since the instability of the proteinase by autoproteolysis has been well established (Exterkate and deVeer 1985; Bockelmann et al. 1989). Proteinase activity was monitored ftuorimetrically during the purification procedure using fluorescein isothiocyanate-labelled fl-casein according to the method of Twining (1984). Lysine aminopeptidase and X-prolyl dipeptidyl aminopeptidase were determined using the 7-amido-4methylcoumarin derivatives of lysine and glycyl-proline, respectively, according to the procedure of Meyer and Jordi (1987).
HPLC analysis. Samples (200 Ixl) of the ultrafiltered TFA-soluble peptides were analysed by reverse-phase HPLC (chromatograph: Spectra Physics SP8800, San Jose, Calif., USA; column: Vydac 218TP C18, Alltech Associates, Deerfield, II1., USA, 25 cm x 0.46 cm). Buffer A contained 0.1% TFA in distilled water; buffer B contained 0.08% TFA in acetonitrile. Peptides were eluted with a linear gradient from 0% B to 50% B at 1 ml min-1 over 50 rain. Peptides were detected by the absorbance at 220 nm, collected and dried under vacuum. Peak areas were determined using an integrator (Spectra Physics SP4400). Peptide identification. Peptides were completely sequenced by the automated Edman method using a gas-phase protein sequencer (Applied Biosystems 470A, Foster City, Calif., USA, with a 120A PTH analyser). Peptide sequences were matched to the known primary structure of asl-casein (Mercier et al. 1971). The identity of the peptides was confirmed by comparing the mass calculated from the sequence with that determined by mass spectrometry. Mass spectra were determined using a VG 70-250 double focussing magnetic sector mass spectrometer (VG Analytical, Manchester, UK) fitted with a liquid secondary mass spectrometry ion source and caesium ion gun. Peptides were analysed in a matrix of acidified glycerol.
Results and discussion T h e p r o g r e s s o f the a s - c a s e i n d i g e s t i o n is r e v e a l e d b y S D S - P A G E (Fig. 1). T h e ors-casein u s e d ( S i g m a ) contained two components both of which were degraded by proteinase. Chromatographic analysis had shown t h a t t h e c a s e i n was a l m o s t e n t i r e l y a , l - c a s e i n . Since all of the oligopeptide products sequenced from the HPLC f r a c t i o n s (see b e l o w ) c o u l d b e i d e n t i f i e d in t h e as~-casein s e q u e n c e a n d n o n e f r o m a n y o t h e r c a s e i n seq u e n c e , it s e e m s p r o b a b l e t h a t b o t h t h e m a j o r a n d m i nor components of the casein substrate are variants of a s l - c a s e i n . T h e c a s e i n was a l m o s t c o m p l e t e l y d e g r a d e d to l o w e r m o l e c u l a r m a s s p r o d u c t s o v e r the 48 h d i g e s -
Hydrolys& of casein. Four millilitres of as-casein (10 mg/ml from Sigma, St Louis, Mo., USA) and 0.9 ml partially purified enzyme solution (115 Ixg protein ml-1), were incubated at 24 ° C. Samples (400 Ixl) for HPLC analysis were taken at 0, 10, 30 min, 1, 2, 4, 6, 10, 24 and 48 h and the reaction stopped by addition of trifluoroacetic acid (TFA, 1% final concentration). After centrifugation to remove TFA-insoluble peptides, the supernatant was ultrafiltered using a Centricon 10 microcentrator (Mr cutoff 10 kDa). A further series of samples (50 p.1) for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were withdrawn at the same times and the reaction stopped by boiling with 50 ~tl of 0.125 M TRIS/HC1 (pH 6.8) containing 4% (w/v) SDS, 20% (v/ v) glycerol and 10% (v/v)fl-mercaptoethanol. SDS-PAGE analysis. An aliquot (10 ~tl) of each digested sample was fractionated by SDS-PAGE according to the method of Laemmli (1970) using a vertical gel electrophoresis unit (Pharmacia GE/4) maintained at 20 ° C. The running gel contained 15% (w/v) acrylamide in 0.39 M TRIS/HC1 (pH 8.8); the stacking gel 5% (w/v) acrylamide in 0.06 M TRIS/HCI (pH 6.7). The reservoir buffer (pH 8.3) contained 0.025 M TRIS and 0.19 M glycine. Gels were stained in 0.125% Coomassie Blue R250 in methanol:acetic acid:water (5:1:4) and destained in the same solvent (but using the proportions 3 : 1 : 10, respectively).
Fig. 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showing hydrolysis of asl-casein by the partially purified proteinase from Lactococcus lactis subsp, cremoris S K l l : lanes 1 and 12, standard proteins; lanes 2-11, O, 10, 30 min, 1, 2, 4, 6, 10, 24 and 48 h digest samples, respectively. Standard proteins: a, triose phosphate isomerase (26 kDa); b, soya bean trypsin inhibitor (20 kDa); c, myoglobin (17 kDa); d, cytochrome c (12.4 kDa); e and f, cyanogen bromide fragments of myoglobin (8 and 6 kDa, respectively). Arrow indicates start of resolving gel
224
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App//ed Microb/okgy Biotechnology © Springer-Verlag 1991
Action of a cell wall proteinase from Lactococcus lactis subsp, cremoris S K l l on bovine asl-casein Julian R. Reid, Christopher H. Moore, Graeme G. Midwinter, and Graham G. Pritchard Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand Received 19 November 1990/Accepted 15 January 1991
Summary. The cell wall-associated proteinase from Lactococcus lactis subsp, cremoris S K l l was partially purified and incubated with a~a-easein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the amino acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of a~-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the as~-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No clear consensus sequence of amino acid residues surrounding the cleavage sites could be identified.
Introduction The level of free amino acids and small peptides in milk is inadequate to account for the rapid growth of lactococci to high cell densities indicating that degradation of milk proteins by starter proteinases and peptidases must be occurring (Mills and Thomas 1978). The initial step in proteolysis involves the action of a cell wall-bound proteinase (Exterkate 1975; Mills and Thomas 1978; Hugenholz et al. 1987) that is capable of degrading one or more of the casein components to oligopeptides, which are then hydrolysed further by a series of peptidases (Thomas and Pritchard 1987). Cell-wallbound proteinases from several different strains of lactococci have been purified in recent years (Geis et al. 1985; Exterkate and de Veer 1987, 1989; Monnet et al. 1987; Bockelmann et al. 1989) and their properties investigated. The proteinases from different strains of lactococci have been classified according to various criteria: pH Offprint requests to: G. G. Pritchard
and temperature optima (Exterkate 1976), immunological cross-reactivity (Hugenholz et al. 1984) and specificity towards the casein components of bovine milk (Visser et al. 1986). On the basis of the specificity of action in hydrolysing casein, two distinct patterns of action are currently recognised which have been designated P~ and Pro-type proteinases (Exterkate and de Veer 1989). The P~-type (or HP-type) proteinase preferentially degrades //-casein. The particular peptide bonds cleaved in the fl-casein molecules by PI-type proteinases have been determined for the enzymes from Lactococcus lactis subsp, lactis NCDO 763 (Monnet et al. 1986, 1989) from L. lactis subsp, cremoris HP (Visser et al. 1988) and from L. lactis subsp, cremoris AC1 (Monnet et al. 1989). While there are some apparent differences between the proteinases from these strains with respect to the particular bonds in//-casein cleaved in long-term digests (Monnet et al. 1989), a remarkably consistent pattern has been found for the initial sites of cleavage. These sites are mainly located in the C-terminal 53-residue region of the fl-casein molecule. The P~-type proteinases so far isolated from L. lactis subsp, cremoris have little or no effect on as-casein. The proteinase from L. laetis subsp, lactis NCDO 763 was shown to catalyse limited degradation of both a, and x-casein (Monnet et al. 1989) but the nature of the products was not described. Gel electrophoresis of casein digests has indicated that the Pro-type (of AMl-type) of proteinase degrades //-casein at sites different from those characteristic of the P~-type and also significantly digests as- and x-casein (Visser et al. 1986) but the identity of the peptide products of digestion has not been previously reported. The present paper describes the sites of cleavage in asacasein by a Pni-type proteinase from L. lactis subsp, eremoris SK11.
223
Materials and methods Organisms, culture conditions and harvesting. L. lactis subsp, cremoris SKI 12 (deVos et al. 1984) was obtained from the starter collection of the New Zealand Dairy Research Institute, Palmerston North, New Zealand (isolate number 5221). Cultures for enzyme isolation were grown in 3.5 1 skim-milk medium containing glycerophosphate as a buffer (Mills and Thomas 1978) for 16 h at 22 ° C. The culture was neutralised to pH 7.0, 210 ml of 25% (w/v) sodium citrate were added and the cells sedimented by centrifugation. The cells were washed twice with sodium acetate/phosphate buffer (containing 46 mM sodium acetate and 4 mM sodium dihydrogen phosphate, pH 6.4) at 4 ° C. Solubilization and purification of proteinase activity. Proteinase activity was solubilised by incubation of the cells in 50 mM sodium phosphate buffer, pH 6.4, at 30 ° C for two successive 1 h periods (Mills and Thomas 1978). The same buffer (but at 10 mM) was used for subsequent steps in purification and all steps were earried out at 4 ° C. The pooled supernatants from the two incubations were cooled on ice, diluted with an equal volume of cold water and loaded onto a column (4 cm x 14 cm) of DEAE-cellulose. The column was washed with 200 ml buffer and eluted with a linear NaC! gradient (0-0.8 M). This procedure yields a single peak of proteinase activity separated from lysine aminopeptidase and X-prolyl dipeptidyl aminopeptidase activity. The pooled, concentrated proteinase fractions were further purified by gel permeation on a column (3 cm x 75 era) of Sephacryl $300 (Pharmacia, Uppsala, Sweden) eluting with buffer at a flow rate of 19 ml h -~. The concentrated, pooled fractions from the Sephacryl column were then passed through Superose 6 (Pharmacia LCC 500 PLUS FPLC using a Superose-6 HR 10/30 column) eluting with 10 mM phosphate buffer (pH 6.4) containing 50 mM NaCI at 0.5 mlmin -~. A single major protein peak with a shoulder was obtained from the Superose 6 column; proteinase activity corresponded to the main protein peak. The proteinase was used immediately for digestion studies since the instability of the proteinase by autoproteolysis has been well established (Exterkate and deVeer 1985; Bockelmann et al. 1989). Proteinase activity was monitored ftuorimetrically during the purification procedure using fluorescein isothiocyanate-labelled fl-casein according to the method of Twining (1984). Lysine aminopeptidase and X-prolyl dipeptidyl aminopeptidase were determined using the 7-amido-4methylcoumarin derivatives of lysine and glycyl-proline, respectively, according to the procedure of Meyer and Jordi (1987).
HPLC analysis. Samples (200 Ixl) of the ultrafiltered TFA-soluble peptides were analysed by reverse-phase HPLC (chromatograph: Spectra Physics SP8800, San Jose, Calif., USA; column: Vydac 218TP C18, Alltech Associates, Deerfield, II1., USA, 25 cm x 0.46 cm). Buffer A contained 0.1% TFA in distilled water; buffer B contained 0.08% TFA in acetonitrile. Peptides were eluted with a linear gradient from 0% B to 50% B at 1 ml min-1 over 50 rain. Peptides were detected by the absorbance at 220 nm, collected and dried under vacuum. Peak areas were determined using an integrator (Spectra Physics SP4400). Peptide identification. Peptides were completely sequenced by the automated Edman method using a gas-phase protein sequencer (Applied Biosystems 470A, Foster City, Calif., USA, with a 120A PTH analyser). Peptide sequences were matched to the known primary structure of asl-casein (Mercier et al. 1971). The identity of the peptides was confirmed by comparing the mass calculated from the sequence with that determined by mass spectrometry. Mass spectra were determined using a VG 70-250 double focussing magnetic sector mass spectrometer (VG Analytical, Manchester, UK) fitted with a liquid secondary mass spectrometry ion source and caesium ion gun. Peptides were analysed in a matrix of acidified glycerol.
Results and discussion T h e p r o g r e s s o f the a s - c a s e i n d i g e s t i o n is r e v e a l e d b y S D S - P A G E (Fig. 1). T h e ors-casein u s e d ( S i g m a ) contained two components both of which were degraded by proteinase. Chromatographic analysis had shown t h a t t h e c a s e i n was a l m o s t e n t i r e l y a , l - c a s e i n . Since all of the oligopeptide products sequenced from the HPLC f r a c t i o n s (see b e l o w ) c o u l d b e i d e n t i f i e d in t h e as~-casein s e q u e n c e a n d n o n e f r o m a n y o t h e r c a s e i n seq u e n c e , it s e e m s p r o b a b l e t h a t b o t h t h e m a j o r a n d m i nor components of the casein substrate are variants of a s l - c a s e i n . T h e c a s e i n was a l m o s t c o m p l e t e l y d e g r a d e d to l o w e r m o l e c u l a r m a s s p r o d u c t s o v e r the 48 h d i g e s -
Hydrolys& of casein. Four millilitres of as-casein (10 mg/ml from Sigma, St Louis, Mo., USA) and 0.9 ml partially purified enzyme solution (115 Ixg protein ml-1), were incubated at 24 ° C. Samples (400 Ixl) for HPLC analysis were taken at 0, 10, 30 min, 1, 2, 4, 6, 10, 24 and 48 h and the reaction stopped by addition of trifluoroacetic acid (TFA, 1% final concentration). After centrifugation to remove TFA-insoluble peptides, the supernatant was ultrafiltered using a Centricon 10 microcentrator (Mr cutoff 10 kDa). A further series of samples (50 p.1) for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were withdrawn at the same times and the reaction stopped by boiling with 50 ~tl of 0.125 M TRIS/HC1 (pH 6.8) containing 4% (w/v) SDS, 20% (v/ v) glycerol and 10% (v/v)fl-mercaptoethanol. SDS-PAGE analysis. An aliquot (10 ~tl) of each digested sample was fractionated by SDS-PAGE according to the method of Laemmli (1970) using a vertical gel electrophoresis unit (Pharmacia GE/4) maintained at 20 ° C. The running gel contained 15% (w/v) acrylamide in 0.39 M TRIS/HC1 (pH 8.8); the stacking gel 5% (w/v) acrylamide in 0.06 M TRIS/HCI (pH 6.7). The reservoir buffer (pH 8.3) contained 0.025 M TRIS and 0.19 M glycine. Gels were stained in 0.125% Coomassie Blue R250 in methanol:acetic acid:water (5:1:4) and destained in the same solvent (but using the proportions 3 : 1 : 10, respectively).
Fig. 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showing hydrolysis of asl-casein by the partially purified proteinase from Lactococcus lactis subsp, cremoris S K l l : lanes 1 and 12, standard proteins; lanes 2-11, O, 10, 30 min, 1, 2, 4, 6, 10, 24 and 48 h digest samples, respectively. Standard proteins: a, triose phosphate isomerase (26 kDa); b, soya bean trypsin inhibitor (20 kDa); c, myoglobin (17 kDa); d, cytochrome c (12.4 kDa); e and f, cyanogen bromide fragments of myoglobin (8 and 6 kDa, respectively). Arrow indicates start of resolving gel
224
30rail.L,...,L _-
~
2h
~
~
~h
c 0
M m
1
u Z
