The proteolytic system of starter and non-starter bacteria: Components and their importance for cheese ripening

Inf. Dairy Journal 5 (1995) 917-994 0 1995 Elsevier Science Limited Printed in Ireland. All rights reserved 0958-6946/95/$9.50 + .OO 0958-6946(95)00041-O

The Proteolytic System of Starter and Non-Starter Bacteria: Components and Their Importance for Cheese Ripening

Wilhelm

Bockelmann

Federal Dairy Research Centre, Institute of Microbiology, 24103 Kiel, Germany

Hermann-Weigmann-StraBe

1,

ABSTRACT The proteol_vtic system of Lactococcus has been studied in detail for many years, and probably most of the components have been identtfied. More recently there has been an increased interest in the study of the proteolytic enzymes of Lactobacillus species. At the Federal Dairy Research Centre, nine peptidases of a single strain of Lactobacillus delbrueckii subsp. bulgaricus were purified. Biochemicul properties of most of these enzymes were found to be similar compared to Lactococcus enzymes. However, two purified tripeptidases were completely different. Based on these results, a method for the characterization of starter strains has been developed, using nine peptide substrates, which were suitable for assays of single or groups of enzymes. With this method, the proteolytic profiles of peptidasedeficient Lactococcus mutants, provided by the University of Groningen were determined, in order to study the suitability of selected substrates for particular enzymes and to analyse the effect of the loss of certain peptidases on the remaining peptidase activit?;. Starter strains, with known industrial performance, were obtained from a local starter culture supplier. fnitial experiments indicate that the proteolytic profile of starter strains might be correlated to the performance of starters in cheese ripening.

INTRODUCTION The proteolytic system of Lactococcus has been studied for several years and is now well described. It consists of a cell wall bound proteinase and several, probably intracellular peptidases: at least one endopeptidase, an X-prolyldipeptidyl aminopeptidase, a dipeptidase, a prolidase, and at least six aminopeptidases of various specificities. This system was reviewed recently by Tan et al. (1993). More

978

Wilhelm Bockelmann

recently Lactobacillus enzymes were studied in detail and many authors described enzymes that were biochemically similar to Lactococcus enzymes. Due to the large number of reports on purified proteolytic enzymes, a clearer picture of similarities and differences of the proteolytic system of lactic acid bacteria is now possible. References for groups of similar enzymes identified in various species of lactic acid bacteria are listed in Table 1. The importance of proteolytic enzymes of lactic acid bacteria for cheese ripening is obvious. Apart from the cell wall proteinase which is responsible for the initial stage of casein degradation, all or at least some of the known peptidases will also play an important role. There is still no method for a detailed characterization of peptidases of lactic acid bacteria. A protocol will be described here which enables a profile of the various peptidase activities of starter cultures to be produced. This is currently being validated using peptidase deficient Lactococcus mutants, recently supplied by the Department of Genetics of the University of Groningen. In addition, several starter strains used in dairy industry were supplied by a German starter culture supplier. Proteolytic activities were assayed in order to find some correlation between enzymatic activities and performance in cheese ripening.

MATERIAL

AND

METHODS

Bacteria Peptidase deficient Lactococcus mutants were supplied by the Department of Genetics of the University of Groningen. Starter strains of several species of lactic acid bacteria were supplied by Laboratorium Wiesby, Niebtill, Germany. For characterization of proteolytic activities, cells were grown in milk. Proteinase activity was detected with whole cells and calculated by mg dry weight. Peptidase activities were assayed in cell free extracts of mechanically lysed cells and calculated by mg protein. Isolation of peptidases For purification of intracellular peptidases, cell free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 were used. Starting with ammonium sulphate precipitation, anion exchange (DEAE-Sepharose), hydrophobic interaction (Phenyl Sepharose), hydroxy apatite (Biogel HT), metal chelating affinity (Metal Chelating Sepharose), high performance anion exchange (Mono-Q) and HPLCgel filtration (G-3000SW) were used in various combinations as separation principles for chromatography (materials from BioRad, Munchen, Germany; Pharmacia, Freiburg, Germany; TosoHaas. Stuttgart, Germany). Details of purification conditions used can be found in the papers of Kiefer-Partsch et al. (1989), Bockelmann et al. (1991, 92, 95); Wohlrab & Bockelmann (1992, 93, 94). Proteinase and peptidase profiles of lactic acid bacteria Since proteolytic activities are probably regulated by the growth medium, milk was used for growth of cells. Cells were grown at optimum temperatures

skim to a

TABLE 1

Exterkate

Machuga

Bacon et al. (1993)

Bockelmann et al. (1995a) Bockelmann et al. (1991) Lloyd & Pritchard (199 1) Nardi et al. (199 1)

Boven et al. (1988)

PepA

Other AP

PepT

New TP” Pep-X

Pep D

Atlan er al. (1994)

Pep I

“i.e. 29 and 38.8 kDa enzymes from Lactobacillus.

Kaminogawa

Prolidase

et al. (1984)

& Ives (1984)

& de Veer (1987)

Chapot-Chartier

PepC

et al. (1990)

et al. (1993)

& Exterkate

et al. (198 1)

Baankreis

Hwang

(1991)

Bockelmann et al. (1995b) Booth er al. (1990) Mayo et al. (1991) Tan et al. (1992)

Bosman

Sahlstrom

Niven (1991)

er al. (1994)

et al. (1994)

& Bockelmann

et al. (1989)

Gilbert

et al. (1994)

Rabier & Desmazeaud

Khalid & Marth (1990) Meyer & Jordi (1987) Zevaco et al. (1990)

Mierau

Wohlrab

Neviani

(1973)

(1994)

Bockelmann er al. (1992) Tan & Konings (1990)

Blanc et al. (1993) Khalid & Marth (1990)

Arora & Lee (1992) Exterkate et al. (1992) van Alen-Boerrigter et al. (199 1)

PepN

Fernandez

Monnet ef al. (1995) Yan et al. (1987b)

Mierau et al. (1993) Yan et al. (1987a)

Kunji et al. (1993) Tan et al. (1991)

Pep0

et al. (1993)

Chandan ef al. (1987) Martin-Hernandez et al. (1994) Visser et al. (1991)

Bockelmann et al. (1989) Geis et a/. (1985) Visser et al. (1988)

Argyle ef al. (1976) Coolbear et al. (1992b) Reid et al. (1991)

CEP

Studies on the Proteolytic Enzymes of Lactic Acid Bacteria (references)

et al. (1994)

& Bockelmann

(1993)

& Bockelmann

Klein er al. (1994)

Wohlrab

(1992)

Kiefer-Partsch er al. (1989) Myakawa et al. (1991)

Wohlrab

Eggimann & Bachmann (1980) Tsakalidou & Kalantzopoulos (1992)

Pritchard

Coolbear et al. (1992a) Nissen-Meyer et al. (1992) Yamamoto er al. (1993)

< 2 ;;:

S 2.

x

g 4

980

WilheIm Bockelmann

culture pH of 5.2 and centrifuged. For analysis of peptidases, cells were lysed with glass beads, and cell free extracts were obtained after centrifugation. Since most of the proteinase activity remained with the particulate fraction after lysis, proteinase assays were performed with intact cells which were resuspended in buffer after harvesting. Based on basic results on purified peptidases, several peptide substrates were selected for the quantitative determination of peptidases. Most peptides contained terminal leucine which was liberated in the assays. Free leucine was oxidized by amino oxidase, followed by oxidation of the calorimetric dye odianisidine with hydrogen peroxide and peroxidase (Wohlrab & Bockelmann, 1993). The resulting brown colour was measured at 436 nm. Amino acid or peptide derivatives (nitroanilides) were used according to Bockelmann et al. (1991). For the detection of endopeptidase activities the described assays were not suitable because no amino acids were released from oligopeptides. By capillary electrophoresis, the degradation of suitable oligopeptides (metenkephalin, bradykinin) could be detected [Bockelmann et al., 1995(c)]. Separations were performed within 6-9 min. For the quantification of proteinase activity, a fluorimetric assay was established. Total casein was labelled with the fluorescent dye fluorescein isothiocyanate (FITC). After incubation with whole cells, intact caseins and large acid insoluble degradation products were precipitated with 5% (w/v) trichloroacetic acid (TCA). Liberated TCA-soluble, fluorescent peptides were detected in a fluorimeter with excitation at 350 nm and emission at 550nm. An alternative proteinase assay which detects intact caseins was developed. Using capillary electrophoresis with urea-containing electrophoresis buffers, the degradation of single casein components could be assayed.

RESULTS

AND

DISCUSSION

Purification of proteolytic enzymes To date, more than 12 different proteolytic enzymes have been purified from lactic acid bacteria (Table 2). The proteolytic system of Lactococcu.s has been studied in detail. For most of the enzymes isolated from various lactococcal strains, corresponding enzymes were detected in a single strain of Lactobacillus delbrueckii subsp. bulgaricus and in other species of Lactobacillus, suggesting a general importance of the identified enzymes for all lactic acid bacteria (see Table 1). Cell wall bound proteinase (PrtP) The lactococcal cell wall bound proteinase was one of the first enzymes that was purified. Based on specificity of action, two types of proteinases were observed, a proteinase cleaving a,-caseins and l3-casein (e.g. strains AMl, SK1 l), and a proteinase with a preferential cleavage of D-casein (strains ACl, Wg2, NCD0763 and others). However, some proteinases were purified which had intermediate specificities. Analysis of the genes showed homologies of more than 98% for the

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TABLE 2 Proteolytic Enzymes of a Single Strain of Lactobacillus delbrueckii subsp. bulgaricus; compared to known Lactococcal Enzymes. Biochemically Similar Enzymes are Listed in One Column Lactobacillus

Lactococcus

Cell wall proteinase (> 145 kDa) PrtP Endopeptidase (70 kDa) Pep0 Aminopeptidase (95 kDa) PepN Aminopeptidase (54 kDa) PepC Dipeptidase (5 1 kDa) Prolidase (44 kDa) (95 kDa) XPDAP (PepX) (85 kDa) ??? tripeptidase (52-55 kDa) PepT Tripeptidase I (29 kDa) ??? Tripeptidase II (39 kDa) ??? Detected iminopeptidase (50 kDa) Pep1 Detected aminopeptidase PepA ??? aminopeptidase (45 kDa) PepP Aminopeptidase (32 kDa) ???

two described types of lactococcal proteinases, differences in specificity were largely due to point mutations in the gene (Tan et al., 1993). Due to the instability of the proteinases in solution, published differences in the molecular masses were probably caused by different solubilization and purification methods. Washing of lactococcal cells with calcium free buffer produced an unstable 145 kDa enzyme (Geis et al., 1985) while treatment of lactococcal cells with lysozyme released a more stable 180 kDa form of the proteinases (Coolbear et al., 1992b). Similar enzymes were also detected in Lactobacillus (Argyle et al., 1976; Chandan et al., 1987; Yamamoto et al., 1993; Martin-Hernandez et al., 1994). All enzymes were serine type proteinases with an acidic pH-optimum. Published molecular masses ranged from 80 to 200 kDa. X-prolyldipeptidyl

aminopeptidases

(PEP-X)

X-prolyldipeptidyl aminopeptidases were purified from cell free extracts of several species of lactic acid bacteria (see Table 1). Purification of the X-prolyldipeptidyl aminopeptidase from Lb. delbrueckii subsp. bulgaricus B14 and Lb. acidophilus (Bockelmann et ccl., 1991) showed that these enzymes were easily accessible for purification because of their affinity to zinc. X-prolyldipeptidyl aminopeptidases could be purified by a combination of anion exchange and metal chelating affinity chromatography with immobilized Zn*+ only, since most proteins of cell free extracts did not bind to the immobilized zinc ligand. All known X-prolyldipeptidyl aminopeptidases (from Lactococcus, Lactobacillus, Streptococcus thermophilus) have similar properties. They are serine type enzymes, have a broad pH optimum around pH 7, and temperature optima between 4045°C. X-pro dipeptides are preferably cleaved from the N-terminus

982

Wilhelm Bockelmann

of oligopeptides, however, the enzymes also display low activities towards Nterminal dipeptides of different kinds (e.g. Ala-Ala-p-NA). In comparison to Lactobacillus, Lactococcus enzymes have a smaller apparent molecular mass on SDS-PAGE (85 kDa vs 95 kDa). N-terminal amino acid sequences of enzymes from Lb. acidophilus and Lb. delbrueckii subsp. lactis were nearly identical (Bockelmann et al., 1991). There was also some sequence similarity compared to Lactococcus. Three of the nine N-terminal amino acid were identical (Met 1, Asn,, Vals). In four positions an exchange of a single nucleotide can cause the observed differences (Phe3/Tyr3, HisS/GlnS, Phe6/Tyr6, SerT/AlaT) (Bockelmann et al., 1991).

General aminopeptidases Two general aminopeptidases (PepN, PepC) were found in Lactococcus. Corresponding enzymes with similar biochemical properties were also found in several Lactobacillus species and S. thermophilus which shows the general importance of these aminopeptidases for lactic acid bacteria (see Table 1). The PepN-like aminopeptidase from Lb. delbrueckii subsp. bulgaricus B14 (Bockelmann et al., 1992) cleaved Lys-p-NA and Leu-Gly-Gly among other substrates. The enzyme was metal dependent, completely inhibited by EDTA and reactivated by Mn2+. Activity was severely inhibited by 0.1 mM Zn2+ and Cu2+, but the enzyme was relatively insensitive to NaCl (50% residual activity at 2M NaCl). The native enzyme was probably a monomer with an apparent molecular mass of approximately 95 kDa on SDS-PAGE. Peptidases with similar biochemical properties (metal dependent, pH optimum pH 7.0, molecular mass approximately 95 kDa) have also been found in Lb. casei subsp. casei (Arora & Lee, 1992) Lb. helveticus (Blanc et al., 1993, Khalid & Marth, 1990) Lb. lactis (Eggimann & Bachmann, 1980) and Streptococcus thermophilus (Tsakalidou & Kalantzopoulos, 1992). In contrast to similar biochemical properties, the Nterminal amino acid sequence (10 residues) of the bulgaricus B14 aminopeptidase bore no similarities to the lactococcal PepN (Tan & Konings, 1990) except for an N-terminal alanine residue. PepC was first purified from Lactococcus lactis subsp. cremoris AM2 (Neviani et al., 1989). A PepC-like aminopeptidase was purified also from Lb. delbrueckii subsp. bulgaricus B14. Purification of the Lactobacillus enzyme was easy because the enzyme was eluted later (0.3 M NaCl) than most other proteins in the first purification step (anion exchange chromatography; Wohlrab & Bockelmann, 1993). With a second step of FPLC anion exchange chromatography, electrophoretic purity could be achieved. SDS-PAGE in the absence of B-mercaptoethanol resulted in a single band at 220 kDa (54 kDa under reducing conditions). In contrast to the PepN of Lb. bulgaricus, Lys-pNA was not cleaved by the PepC-like enzyme. Suitable substrates were di- and tripeptides of a wide range (highest activity with Leu-Gly) with optimal activity observed at 50°C and pH 7.0. Aminopeptidase activity was increased by reducing agents and EDTA but inhibited by most divalent cations (except Ca2+ and Mg2+) and thiol group inhibitors (e.g. p-hydroxymercuribenzoate). In contrast to the Lactobacillus enzyme, the lactococcal enzyme was reported to form hexameric complexes (300 kDa) and had a lower temperature optimum

Dair_vbacteria: proteolysis and cheese ripening

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(40°C).

Apart from these differences the enzymes compared well, for example their insensitivity towards EDTA which was only found for PepC. Recently a PepC-like enzyme was also found in Lb. helveticus by Fernandez et al. (1994). The finding of a PepC-like enzyme in Lb. bulgaricus and helveticus suggests that it, like Pep-N, may be present in other lactic acid bacteria also. A third general aminopeptidase was purified from cell free extracts of Lb. delbrueckii subsp. bulgaricus B14 (Wohlrab & Bockelmann, 1994). On SDSPAGE a single protein band with a molecular mass of “32 kDa was observed. By HPLC gel filtration, a molecular mass of “260 kDa was estimated. Optimum aminopeptidase activity was obtained at pH 6.5 and 45°C. The enzyme was activated by Mg2+, Co2+ and Zn2+ at concentrations up to 0.1 mM (hisher concentrations of these metal ions inhibited peptidase activity), while Ca + increased enzyme activity at concentrations up to 10 mM. Metal chelating agents inhibited enzyme activity strongly whereas thiol group- and serine enzyme inhibitors, as well as reducing agents, had no significant effect on peptidase activity. The EDTA-inhibited enzyme activity could be reactivated by Co2+ and Zn2+. Preferred substrates among the hydrolyzed di- and tripeptides were Leu-Leu-Leu, Leu-Gly-Gly, Leu-Ala-Pro, and Leu-Leu, while amino acid- and peptide- nitroanilides were cleaved at a lower rate. The Nterminal amino acid sequence (15 amino acids) showed no terminal methionine and had no similarities to other known N-terminal sequences of aminopeptidases of lactic acid bacteria and resembles no other of the known peptidases of lactic acid bacteria. However, an oligonucleotide probe made for the Lactobacillus enzyme gave positive signals with Lactococcus DNA in dot blot hybridization experiments under stringent washing conditions (unpublished data), suggesting that this type of aminopeptidase might also be present in Lactococcus. Endopeptidase (PepO) An endopeptidase was purified from cell free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14. Unlike most other proteins it did not bind to immobilized Cu*+ on metal chelating affinity chromatography, which was therefore used to remove impurities contaminating enzyme extracts after three purification steps. It was characterized by a molecular mass of 70 kDa, cleaved metenkephalin into Tyr-Gly-Gly and Phe-Met and bradykinin at Pro7-Phes. It was inhibited by the classical agents for serine- and metal-dependent enzymes (i.e. diiso ropyl fluorophosphate and EDTA, respectively) and by Cu’+, Cd”, and Hg ?+ , but activated significantly by Co’+. It resembled the lactococcal endopeptidase (PepO) purified by Tan et al. (1991), but the temperatureand pH optima of the Lactobacillus enzyme were considerably higher (47°C vs. 40°C pH 7.7 vs. pH 6.0). A second endopeptidase found in Lactococcus, cleaving bradykinin at Phes-Serb (LEP I; Yan et al.. 1987b; Monnet et al., 1994) could not be detected in the same Lactobacillus strain. Dipeptidase (PepD) A dipeptidase was purified from cell free extracts of Lb. delbrueckii subsp. bulgaricus B14 by anion exchange- and metal chelating (immobilized Cu’+) affi-

984

Wilhelm Bockelmann

nity chromatography and repeated high performance anion exchange chromatography (Wohh-ab & Bockelmann, 1992). The native dipeptidase was probably a monomer (51 kDa by SDS-PAGE and HPLC gel filtration) and was most active at pH 7 and 50°C. Reducing agents increased enzyme activity while metal chelating agents had an inhibitory effect. EDTA-inhibited enzyme activity could be restored by Co2+ and Mn2+. Enzymes with similar properties were also found in Lactococcus lactis subsp. cremoris (Hwang et al., 1982; van Boven et al., 1988) and Streptococcus thermophilus (Rabier & Desmazeaud, 1973). The dipeptidases exhibited a specificity toward dipeptides with a large, hydrophobic amino acid residue at the N-terminal end. Peptides containing proline were not cleaved. All mentioned enzymes displayed high activities and stability up to pH 9-10, while temperature optima were around 50°C. Above this temperature the enzymes, except the Streptococcus dipeptidase, became rapidly inactivated (Streptococcus: 70°C 75% residual activity). The affinity for Leu-Leu was low (Km 0.56 mM for Lactobacillus and 1.6 mM for Lactococcus; van Boven et al., 1988). Both Lactococcus enzymes differed from the Lactobacillus enzyme in their sensitivitiy to reducing agents. The Lactococcus enzymes were strongly inhibited by DTT and D-mercaptoethanol while the activity of the Lactobacillus peptidase was increased by these reagents at the same concentrations. Tripeptidases Specific detection of tripeptidases in chromatographic fractions was difficult, since suitable substrates (Leu-Gly-Gly, Leu-Leu-Leu, Met-Gly-Gly) were also cleaved by the known three aminopeptidases at the same position, and leucine or methionine were liberated by all enzymes. Therefore, different substrates were used on the same chromatographic fractions to distinguish between the different enzymes. Leu-Trp-Met-Arg was cleaved by all known aminopeptidases, L-Lys-pNA was cleaved by all aminopeptidases except PepC of Lb. bulgaricus. The first enzyme that was purified from cell free extracts of Lb. delbrueckii subsp. bulgaricus B14 had a molecular mass of 29 kDa ([Bockelmann et al., 1995(a)]. A second tripeptidase of 38.8 kDa was subsequently purified ([Bockelmann et al., 1995(b)], which for the first time showed the presence of two tripeptidases in a single strain of lactic acid bacteria. Only tripeptides were cleaved by both enzymes. The 29 kDa enzyme did not cleave tripeptides containing a proline residue in any position. However, proline containing peptides were cleaved to some extent by the 38 kDa enzyme, when the proline residue was at the N- or C- terminus of the tripeptide. A typical PepX substrate like Ala-Pro-Leu was not cleaved by either enzyme. The temperature optimum of the 38.8 kDa tripeptidase was higher than that of the 29 kDa enzyme (55°C vs 40°C), but both tripeptidases had the same pH optimum, which, at pH 6.0, was lower than that for the other known peptidases of the same strain. The tripeptidases from Lb. delbrueckii subsp. bulgaricus B14 were completely different from tripeptidases purified from Lactococcus strains (Bosman et al., 1990; Bacon et al., 1993). The lactococcal tripeptidase (PepT) had a molecular mass of approximately 48852 kDa, and showed highest activity at pH 7.5 and 55°C. A lactococcal-type enzyme could not be detected in Lactobacillus deibrueckii subsp. bulgaricus, confirming the data of Tan et al. (1993) whereby

Dairy bacteria: proteolysis and cheese ripening

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antibodies raised against the lactococcal PepT were not reacting with enzyme preparations from Lactobacillus delbrueckii subsp. bulgaricus. This could indicate a clear difference between Lactococcus and Lactobacillus delbrueckii in tripeptidase activities. Prolidase A dipeptidase cleaving dipeptides of the X-Pro type (prolidase) was purified recently from the same strain of Lactobacillus delbrueckii subsp. bulgaricus. Like most other known peptidases (except tripeptidases), the prolidase is quite similar to a lactococcal enzyme (Kaminogawa et al., 1984). It was a metalloenzyme with an apparent molecular mass of approximately 44 kDa. The enzyme showed remarkable instability in purified preparations and to date a detailed biochemical characterizationhas not been possible. Table 2 gives a summary on purified proteolytic enzymes from lactic acid bacteria. Results on purified Lactobacillus enzymes were obtained from a single strain. Seven enzymes were biochemically similar to known Lactococcus enzymes. One aminopeptidase and two tripeptidases from Lactobacillus have not yet been found in Lactococcus while the lactococcal-type tripeptidase, PepT, has not yet been identified in Lactobacillus. Characterization

of proteolytic properties

A protocol for the detailed characterization of the proteolytic system of lactic acid bacteria is currently being developed (Table 3). Casein was selected for proteinase assays, eight peptide substrates were selected to assay single or groups

TABLE 3 Characterization of Proteolytic Activities from Lactic Acid Bacteria. Several Substrates are used in Three Different Assays to Quantify Proteinases and Peptidases

Substrate

Enzyme(s)

assayed

spectrophotom. test (oxid. of free leucine and o-dianisidine) XPDAP Ala-Pro-Leu aminopeptidases Leu-Gly-Pro tripeptidases. aminopeptidases Leu-Gly-Gly dipeptidase, aminopeptidases Leu-Gly Leu-Pro prolidase Asp-Phe aminopeptidase (PepA) proline-iminopeptidase Pro-Leu Capillary electrophoresis [Abs.zaa,,] proteinase(s) Native casein endopeptidase Metenkephalin, bradykinin Fluorimetric assay FITC-casein proteinase(s)

986

Wilhelm Bockelmann

of peptidases. Since there were overlapping activities found for the known peptidases (determined with purified enzyme preparations), it will probably not be possible to find a specific substrate for each enzyme. To determine the specificity of the selected substrates, peptidase-deficient Lactococcus mutants were provided by the University of Groningen, Department of Genetics. The use of stable mutants, completely lacking one or more peptidases, is probably the only way to show the suitability of substrates used for characterization. Analysis of the suitability of peptidase substrates using these peptidase mutants is currently being undertaken. Proteinase detection For detection of proteinases, caseins labeled with radioactive or fluorescent dyes are generally used. The main disadvantage of these assays is that they rely on the liberation of small, acid soluble peptides which necessitate long incubation times of 1 h or more. As an alternative, a proteinase assay was developed based on the method of De Jong et al. (1993) using analytical capillary electrophoresis (CE). In citrate buffer pH 2.5, containing urea and methylhydroxypropylcellulose as additives, casein components were separated within 22min and could be quantitied separately, which gives information on both activity and specificity (Fig. 1). Depending on the strain, incubation times of less than 1 h could be used for proteinase detection with whole cells of lactic acid bacteria. The existence of two l3-casein peaks in commercial Hammarsten-casein (Fig. 1) is due to the existence of different genetic variants of B-casein in the preparations (DAi & 13A2). They differ in a single amino acid (BA,:H~s~~, bA2:Pi0,,). In comparison to HPLC, CE offers some advantages when separating caseins. Running costs are minimal compared to HPLC. There is no contact of mechanical parts with electrophoresis buffers containing high concentrations of urea which has to be used for solubilization of caseins. Glass capillaries used for separations are not gel filled, which reduces the danger of blocked capillaries. The necessary high levels of salt in buffers, however, led to some problems during separations. The electrical current occasionally increased to a level that lead to interruptions of CE runs and a steady baseline could not be obtained (Fig. 1). However, since the drift of the baseline (due to the high amount of urea) was reproducible, quantification of casein peaks was still possible. Peptidases Only natural peptide substrates were selected for the assay of peptidases, since they are closest to casein derived peptides and convenient chromogenic peptide derivatives are not suitable for some of the enzymes (dipeptidases, tripeptidases). Peptides with terminal leucine (or phenylalanine) were selected since these amino acids can be used in combination with amino acid oxidase and peroxidase to oxidize the dye o-dianisidine to its brown, oxidized form. For quantification of endopeptidase activity, however, this assay was not suitable and in this case analytical capillary electrophoresis was used to detecte cleavage of oligopeptides. For some peptidases more or less specific substrates are available. Cleavage of Leu-Pro signified prolidase activity, while Ala-Pro-Leu was mainly cleaved by Xprolyl-dipeptidyl-aminopeptidases, Asp-Phe by the PepA aminopeptidase and

Dairy bacteria: proteolysis and cheese ripening

1

987

casein (blank)

a82

14

16

18

20

22

24

20

22

24

casein digest (2h) a Sl \ a 82 10 0 -10 14

16

18

Retention time [min] Fig. 1. Quantification of proteinase activity using native casein separated by capillary electrophoresis. Identification of the peaks was done by comparison of commercially available total casein with purified casein components, supplied by the Department of Food Chemistry of the University College Cork, Ireland.

by the Pep1 iminopeptidase. The degree of cleavage by more than one selected peptidase could only be determined by the use of appropriate peptidase deficient mutants. In case of Ala-Pro-Leu it was shown that the loss of PepX reduced the specific activity of cell free extracts considerably. However, activity towards this substrate was not zero, residual specific activity on Ala-Pro-Leu being approximately 10% of a wild type strain. An explanation could be cleavage of the N-terminal alanine by PepP, described to be specific for X-Pro-Pro-Y-Z peptides (Monnet, 1993). By subsequent cleavage of the terminal proline of the remaining Pro-Leu by PepI, leucine can be liberated which can be spectrophotometrically detected. The suitability of tripeptides with a different N-terminal amino acid (e.g. Gly-Pro-Leu) or of peptides with chromogenic groups (GlyPro-NA, Ala-Pro-NA) will be tested with PepX deficient mutants in the near future. Pro-Leu

Wilhelm Bockelmann

988

Metenkephalin was used to detect endopeptidase activity. It was cleaved into a tripeptide and a dipeptide (Tyr-Gly-Gly and Phe-Met) by the purified endopeptidase. In cell free extracts other peptides were liberated from metenkephalin, probably due to the activities of aminopeptidases, tripeptidase(s) and a dipeptidase (Fig. 2). Seven degradation products were separated (Gly-Gly, Tyr-Gly, TyrGly-Gly/Phe-Met, Gly-Phe-Met, Gly-Gly-Phe-Met, Met, and Tyr/Phe). The

a.

1. Gly-Gly

0.01

.

2. Tyr-Gly 3. Tyr-Gly-Gly I Phe-Met 4. Gly-Phe-Met 5. Gly-Gly-Phe-Met 6. Tyr-Gly-Gly-Phe-Met 7. Met 8. Tyr / Phe

0

--f-0.00

b.

8

c. 8

7 4 I

J

2

6 4 8 Retention time [min]

10

Fig. 2. Capillary electrophoresis of metenkephalin degradation by cell free extracts of a PepX deficient mutant (a) and a wild type strain (b) of Lactococcus lactis subsp. cremoris, and a wild type strain (c) of Lactobacillus deibrueckii subsp. bulgaricus B14. Peak 2 (TyrGly) was not detected in assays of the PepX deficient mutant, indicating that liberation of Tyr-Gly was due to the action of the X-prolyl-dipeptidyl-aminopeptidase. The non-detection of Tyr-Gly in bulgaricus assays might be explained by higher dipeptidase activity since the concentrations of free tyrosine is considerably higher (peak 8).

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appearance of Gly-Gly-Phe-Met, Gly-Gly and Tyr shows the presence of aminopeptidase activities. Phe and Met were probably liberated from Phe-Met by aminopeptidaseand/or dipeptidase activities. The appearance of Tyr-Gly and Gly-Phe-Met in metenkephalin digests signified the presence of dipeptidyl aminopeptidase activity. With the help of a PepX-deficient mutant it was proved that specificity of PepX was not restricted to N-terminal X-Pro residues, and that production of Tyr-Gly from metenkephaline was indeed due to the action of PepX and not to a second general dipeptidyl aminopeptidase (Fig. 2). Alternative substrates which are more specitic for endopeptidase activity are currently being tested. Metenkephalin with a blocked N-terminus (Boc-metenkephalin) could not be used since it was not soluble under assay conditions. An interesting substrate could be the nona-peptide bradykinine which was also cleaved by the purified endopeptidase. Since the N-terminal aminoacids are Arg-Pro-Pro-X-, it should not be degraded by general aminopeptidases. However, it could be a suitable substrate for the PepP (specific for N-terminal X-Pro-Pro-Y-Z) which was recently described (Monnet, 1993). With an endopeptidasedeficient mutant the influence of PepP on the cleavage of bradykinin andderivative peptides is currently under investigation. Another potential endopeptidase substrate could be dansyl-D-Ala-Gly-Phe-(pN02)-Gly, a fluorigenic substrate for enkephalinases (Florentin et al., 1984). The blocked N-terminus makes it inaccessible to aminopeptidases, while cleavage by enkephalinases liberates dansyl-D-Ala-Gly-Phe. This leads to a fluorescence increase, due to the elimination of intramolecular quenching of the fluorophore by the nitrophenyl group. Initial experiments showed that this substrate is also cleaved by Lactococcus and Lnctobacillus endopeptidases. Leu-Gly-Gly and Leu-Gly-Pro were selected for detection of tripeptidaseand aminopeptidase activities, respectively. It would be expected that both substrates are suitable for both types of enzymes. However, proline containing tripeptides were cleaved poorly by purified Lactobacillus tripeptidases, but readily cleaved by purified aminopeptidases. Current studies with aminopeptidaseand tripeptidasenegative mutants should show the extent of cleavage of these substrates by both types of enzymes. An alternative substrate for the detection of aminopeptidases could be Leu-Trp-Met-Arg, which can be assayed with the same method. Potential cleavage of Leu-Trp-Met-Arg by the endopeptidase or X-prolyldipeptidyl aminopeptidase (at Trp?-Met3) would not interfere with aminopeptidase detection, since only the release of leucine is measured (Wohlrab & Bockelmann, 1993). However, a subsequent cleavage of the degradation products (dipeptides) by dipeptidase activity could interfere with aminopeptidase detection. Substrates like Lys-p-NA, Leu-p-NA, Ala-p-NA could also be used for aminopeptidase detection, but aminopeptidase activities are lower on these amino acid derivatives and cannot be directly compared to the activities of other enzymes on natural substrates. Proteolytic

activities of starter strains

In cooperation with a local starter culture supplier, peptidases of 35 starter strains (Lactococcus and Lactobacillus species), some with known industrial performance, were analysed. Activities on Ala-Pro-Leu, Leu-Gly-Gly, Leu-Pro, Leu-Gly-Pro, and Leu-Gly were generally high (lo&500 mUnits mg-’ cell free protein). Activities on Pro-Leu and Asp-Phe were considerably lower (5-50 mUnits rng-’ cell free protein).

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Wilhelm Bockelmann 1600 0

strain 43-9 strain 43-2

1200 .s-$ m 8 m 0 z $

1000 600 600 400 200 0

Fig. 3. Proteolytic activities of three selected strains of Lactococcus lactis. Peptidase activities of cell free extracts are expressed in mUnits mg-’ protein. Proteinase activities of whole cells are expressed as liberated fluorescence intensity/min mg dry weight.

For characterization, all strains were grown in skim milk at their optimum growth temperature and were harvested at pH 5.2. In spite of reproducible growth conditions, all strains showed a marked variation of specific peptidase activities of cell free extracts. Differences could be as high as 40% in two independent growth experiments, to which the variation of the method itself contributed 5-10%. All selected strains grew well in milk, independent of the level of peptidase activities observed. Three Lactococcus strains with known industrial performance were analysed (Fig. 3). Two strains (43-2 and 43-9) were reported to produce a very good flavour. Peptidase activities of these two strains were above the average for the 3.5 strains analysed. A slow acid producing variant (16-3) which initially was thought to produce a good flavour, produced a bitter off-flavour. Peptidase activities of this strain (except prolidase activity towards Leu-Pro) were generally lower than the average for the 35 strains. Analysis of proteinase activity indicated a possible explanation; it was considerably higher compared to the other two strains (Fig. 3). In spite of the lower proteinase activities strains 43-2 and 43-9 also grew well in milk. These preliminary experiments indicate that the proteolytic profiles of starter strains might be correlated to the performance of starters in cheese ripening and might be worth analysing in more detail.

ACKNOWLEDGEMENTS I wish to thank my collegues from the Department of Genetics of the University of Groningen, especially Igor Mierau, for providing a number of peptidase deli-

Dairy bacteria:

cient mutants, Federal Dairy

proteolysis

and cheese

essential to the applied research Research Center in Kiel.

ripening

currently

being undertaken

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at the

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