Article pp.193-205 du Vol.24 n°3 (2004)

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ARTICLE ORIGINAL ORIGINAL PAPER

Casein phosphatase activities in some lactic acid bacteria

P. Jolivet^1*, S. Guizelin^1,2, E. Seznec^1,2, J.-M. Soulié²

RÉSUMÉ

Mise en évidence d’activités caséine phosphatases chez plusieurs sou- ches de bactéries lactiques

Des activités caséine phosphatases endocellulaires et pariétales, mesurées par la déphosphorylation d’un substrat spécifique ([³²P]caséine), ont été retrouvées chez plusieurs souches de bactéries lactiques largement utilisées en industrie fromagère (trois souches de Lactococcus lactis, deux souches de Lactobacillus helveticus et une souche de Streptococcus thermophilus).

À l’inverse, la souche testée de Lactobacillus plantarum ne présentait aucune activité phosphatase notable. La souche de L. lactis ayant les meilleures acti- vités au cours de sa croissance a été retenue pour les études de caractérisa- tion enzymatique. L’activité endocellulaire était une phosphatase alcaline (pH optimum de 8) fortement dépendante du magnésium et inhibée par 20 mM de fluorure de sodium. L’activité pariétale consistait en une phosphatase acide (pH optimum de 4,5) très peu affectée par la présence de magnésium et de NaF. Ces deux enzymes étaient différemment inhibées par d’autres composés du lait ou du fromage (phosphate, calcium, chlorure de sodium), l’activité pariétale étant toujours moins affectée que l’activité endocellulaire.

Les deux enzymes ont déphosphorylé de manière significative de la caséine totale à pH 6,6, pH initial du lait et peuvent donc apparaître comme des sources potentielles d’activités phosphatases en technologie fromagère.

Mots clés

activité caséine phosphatase, bactéries lactiques, caséines, déphosphorylation.

SUMMARY

Intracellular and cell-wall casein phosphatase activities were identified in 6 strains of lactic acid bacteria (three strains of Lactococcus lactis, two

1. Laboratoire de Chimie Biologique, INRA, INA PG, Centre de Biotechnologies Agro-Industrielles, F-78850 Thiverval-Grignon, France.

2. Laboratoire SOREDAB S.A., La Tremblaye, F-78125 La Boissière École, France.

*Correspondence : [email protected].

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strains of Lactobacillus helveticus and one strain of Streptococcus ther- mophilus) habitually used in cheese making. At the opposite, the tested strain of Lactobacillus plantarum did not exhibited significant activity. Casein phosphatase activity was assayed through the dephosphorylation of a specific substrate : [³²P]casein. One strain of L. lactis that appeared to have the most significant activity during growth was chosen for further investigation.

Intracellular activity was an alkaline phosphatase (optimum pH 8) which proved strongly dependent on the presence of magnesium and was highly inhibited with 20 mM sodium fluoride. Cell-wall activity was an acid phosphatase (optimum pH 4.5) only marginally affected by magnesium and NaF.

Both enzymes were differentially inhibited by phosphate, calcium and sodium chloride which are present in high concentration in milk and cheese, the cell-wall one being always less affected. Both enzymes were able to significantly dephosphorylate total casein at pH 6.6, physiological pH of milk, and then could be potential sources of phosphatases for cheese processing.

Key words

casein phosphatase activity, lactic acid bacteria, caseins, dephosphorylation.

1 – INTRODUCTION

Cheese making is a complex process involving enzymes from milk, rennet and microorganisms and leading to major modifications of milk components.

Caseins are phosphoproteins and their degradation by proteinases produces phosphopeptides which are particularly resistant to further hydrolysis due to the protective effect of the phosphate residues. Casein phosphopeptides have anti- cariogenic properties and as metal ion complexes they have potential as dietetic supplements to increase the bioavailability of calcium, iron and other essential metal ions (HYNEK et al., 1999). However, some casein phosphopeptides are not cleaved into smaller peptide fragments, which can lead to the accumulation of long peptides which have negative influence on final sensory properties of cheese. The combined action of proteinases and phosphatases is thus required for flavour development during cheese ripening and for the release of free ami- noacids (AKUZAWA and FOX 1998). Phosphatases have an important role in the ripening of hard cheeses like Gruyère, Appenzeller, Emmental, Parmigiano Reg- giano and Grana Padano (DENONI et al., 1997) and may be involved in the early stages in cheese making in other technologies. Although both acid and alkaline phosphatase activities are present in cheese, the former are probably more active during ripening due to their relatively low optimum pH. Dephosphorylation could modify the cheese-making qualities of caseins such as calcium-binding capacity, micelle microstructure, rennet coagulation, curd strength, syneresis or susceptibility to proteolysis (for a review see CHARDOT et al., 1999). It has been confirmed that after its dephosphorylation, β-casein digestion with plasmin is accelerated and induces the attack of a new peptide bond (JOLIVET et al., 2000).

Possible origins of phosphatases in cheese are from bovine milk and/or from the cheese microflora (LARSEN and PARADA 1988). PELLEGRINO et al. (1995, 1997)

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suggested a reactivation, in the outer part of cheeses, of milk alkaline phosphatase inactivated by pasteurization. On the other hand, LORIENT and LINDEN

(1976) concluded that milk alkaline phosphatase would possess a very low activity in raw milk due to the presence of endogenous inhibitors (phosphate ions, lactose, β-lactoglobulin). Bovine milk contains also a heat-stable acid phosphatase which is capable of dephosphorylating phosphoproteins, including the caseins. The purification of this enzyme was reported by BINGHAM and ZIT- TLE (1963). Protein phosphatases from micro-organisms have also to be considered. Acid or alkaline phosphatase activities were detected in whole cells and crude enzyme extracts from lactic acid bacteria (ANDREWS and ALICHANIDIS 1975

; LARSEN and PARADA 1988 ; KYRIAKIDIS et al., 1993 ; BIANCHI-SALVADORI et al., 1995 ; HYNEK et al., 1999), yeasts and fungi involved in cheese technology (BAY- LISS et al., 1948 ; ADLER, 1978 ; ALTIKRETE et al., 1984). More recently, acid phosphatases were purified and characterized from the cell membrane fraction of Lactococcus lactis ssp. lactis (AKUZAWA and FOX 1998) and from a cell-free extract of Lactobacillus plantarum (MAGBOUL and McSWEENEY 1999). In Yarrowia lipolytica and Kluyveromyces marxianus, neutral casein phosphatases were rec- ognized besides acid and alkaline activities (JOLIVET et al., 1997, 1998, 2001). In these yeasts, there are both intracellular and exocellular casein phosphatases.

The major protein phosphatase from Y. lipolytica has been used to dephosphorylate phosvitin and casein and the resulting modifications in functional properties have been described (QUEIROZ-CLARET et al., 1997 ; QUEIROZ-CLARET et al., 1998).

The purpose of this work was to search for the presence of casein phosphatases in several species of lactic acid bacteria (lactococci and lactobacilli) commonly used in the cheese making process and assess some of the enzyme characteristics with respect to their potential involvement in milk transforma- tions. Cell-wall proteinases of the tested strains were characterized but very few is known about their phosphatase activities.

2 – MATERIALS AND METHODS

2.1 Strains and culture conditions

Lactococcus lactis subsp. cremoris Wg2, L. lactis subsp. cremoris MG1363, L. lactis subsp. lactis B2136, Lactobacillus plantarum B1407 were maintained by SOREDAB S.A.S. Streptococcus thermophilus (B05 Ch. Hansen) was amplified from the commercial supply, Lactobacillus helveticus CNRZ 223 and Lb. helveti- cus ATCC 12046 were a generous gift from S. Lortal and J.-L. Maubois (Labora- toire de Recherches de Technologie Laitière, INRA, Rennes). Lactococci and S.

thermophilus were anaerobically grown in M17 broth (Biokar) at 30˚C for lacto- cocci and at 42˚C for S. thermophilus. Lactobacilli were grown in MRS broth.

Cells of Lb. plantarum were anaerobically incubated at 30˚C and cells of Lb. hel- veticus at 37˚C in shaken Erlenmeyer flasks. For all strains, sampling was carried out at regular intervals for 18-24 h. Growth was monitored by cell number determination, optical density measurement at 650 nm and pH decrease.

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2.2 Preparation of enzyme fractions

The cells were harvested by centrifugation at 4˚C (4800 x g for 15 min). One volume of cells was suspended in three volumes of 50 mM sodium acetate buffer (pH 6.6) with three volumes of glass beads (diameter 0.1 mm, Braun). Cell disruption was performed in a FastPrep FP120 (Savant Instruments, NY) for three cycles with 10 s of disruption at maximal speed (6.5 m s^-1) and 2 min of cooling on ice. The ground cell suspension was centrifuged (20 000 x g for 15 min at 4˚C) and the supernatant contained the soluble intracellular enzymes.

Intact cells were suspended in 50 mM acetate buffer (200 mg cells in 1 ml buffer) to test cell-wall enzyme activity. The cell integrity was checked by the measurement of intracellular lactate dehydrogenase activity in the presence of 4 mM sodium pyruvate and 0.2 mM NADH. Cell integrities of L. lactis B2136 and Lb. helveticus CNRZ 223 were estimated to be near 97.5% and 92.5%

respectively.

2.3 Determination of casein phosphatase activity

Casein phosphatase activity was assayed through the dephosphorylation of [³²P]casein as described earlier (JOLIVET et al., 2001). [³²P]casein was obtained through the reaction of dephosphorylated casein (Sigma-Aldrich) and [γ-³²P]ATP (Amersham Pharmacia Biotech, France, 54 kBq nmol^-1) in the presence of rabbit cAMP-dependent protein kinase and 25 nM cAMP. Labelled casein was sepa- rated from excess [γ-³²P]ATP by gel filtration. Casein dephosphorylation was carried out by incubating aliquot enzyme fractions at 30˚C with 6 – 11 µM [³²P]casein. Proteins were precipitated with cold 20% (w/v) trichloroacetic acid (TCA) and the radioactivity was counted in the supernatant through liquid scin- tillation (Tricarb 1500, Packard Instrument). Dephosphorylation reaction was also carried out in the presence of proteinase inhibitors (1 µM pepstatin, 0.2 mM phenylmethylsulfonyl fluoride, 4 mg L^-1 leupeptin) to check that proteolysis of the labelled casein by contaminant proteinases did not interfere with the dephosphorylation assay. The activities were expressed either as ³²P released or as total phosphorus hydrolysed from casein taking into account the specific radioactivity of [³²P]casein.

The effect of pH on the casein phosphatase activities was investigated in 50 mM sodium acetate, 2-(N-morpholino)ethanesulfonic acid or triethanolamine buffers encompassing the pH range from 3.2 to 9.0. The effect of temperature was analysed in acetate buffer (pH 6.6).

2.4 Dephosphorylation of caseins

Dephosphorylation experiments were carried out at 30˚C by incubating solu- tions of commercial bovine αs casein, β casein or total casein (Sigma-Aldrich) with cell-free extract or cell suspension in acetate buffer (pH 6.6). The final casein concentration was approximately 60 µM. Cell-free extract or cell suspension was added to obtain a final concentration corresponding to respectively 2.2 or 3.7% (w/v) equivalent fresh matter.

Free phosphate released during the reaction was measured by the method of BAYKOV et al. (1988) adapted by QUEIROZ-CLARET and MEUNIER (1993).

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Reaction medium aliquots were directly mixed with colour reagent (malachite green) and absorbance was measured at 630 nm in a double-beam spectro- photometer (Uvikon 941, Bio-Tek Kontron) after 15 min incubation. The amount of released phosphate was determined from a calibration curve, using KH₂PO₄ as standard. The dephosphorylation yield was calculated taking into account a mean molecular mass of 24 kDa for αs casein and β casein and 23.4 kDa for total casein. The total phosphate content of caseins was determined as reported by ECKMAN and JÄGER (1993) with malachite green reagent after 15 min hydrolysis at 100˚C with 2 N NaOH and was found to be 9 mol of phosphate/mol of casein for αscasein, 5 mol P/mol for β casein and 5.5 mol P/ mol for total casein.

3 – RESULTS AND DISCUSSION

3.1 Expression of casein phosphatase activities during growth of lactic acid bacteria

The growth of 7 strains of lactic acid bacteria was monitored for 18-24 h by cell number determination, optical density measurement and pH decrease. For each strain, the three sampling times were defined as exponential phase (T1, between 5 to 6 h), early stationary phase (T2, 7-9 h) and late stationary phase (T3, 14-16 h). Casein phosphatase activity in the cell-free extracts expressed as [³²P] casein dephosphorylation was determined. Cell-wall activity was also assayed after suspending washed cells in acetate buffer. Table 1 illustrated the results obtained with one series of experiments. Casein phosphatase activities were highly dependent on strains and furthermore, large differences appeared between genera, species and subspecies.

Lactobacillus plantarum B1407 displayed only minor casein phosphatase activ- ities (table 1). An acid phosphatase from a cell-wall extract of one strain of Lb.

plantarum was reported by MAGBOUL and McSWEENEY (1999). However, the activity of this enzyme towards sodium caseinate was very low (3% dephosphorylation for 17 h) when it quickly hydrolysed a range of other phosphate esters. Our specific casein phosphatase assay involving the dephosphorylation of [³²P] casein shows, without ambiguity, the existence of only low activity of casein phosphatase in Lb. plantarum. At the opposite, Lb. helveticus CNRZ 223 and ATCC 12046 exhibited a high intracellular activity and a significant cell-wall activity.

The three tested Lactococcus lactis strains displayed casein phosphatase activities but with different patterns (table 1). L. lactis MG1363 appeared to express both significant intracellular and cell-wall activities and to maintain con- stant activities throughout its growth. Taking into account the specific radioactivity of [³²P] casein, intracellular phosphatase could release about 3 nmol total P min^-1 g^-1 fresh cells under our experimental conditions (pH 6.6, 30˚C, 6 - 11 µM casein). The activities recovered from L. lactis B2136 per g of fresh cells were always lower than those of MG1363 but this strain showed the higher productivity with more than twice fresh matter (about 4 g for 500 mL of culture) and therefore an higher total casein phosphatase activity.

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Table 1

Casein phosphatase activity measured in the course of growth^a of 7 strains of lactic acid bacteria.

Tableau 1

Détermination de l’activité caséine phosphatase au cours de la croissance de 7 souches de bactéries lactiques.

Streptococcus thermophilus B05 expressed a transitory activity during the exponential phase (table 1). Moreover, since its growth was rather low, total casein phosphatase activity recovered was low. Acid phosphatase activity previ- ously detected by LARSEN and PARADA (1988) in one strain of S. thermophilus was also very low with regard to the other micro-organisms considered.

We attempted to characterize the two forms of casein phosphatase activities from Lactococcus lactis B2136 with respect to pH, temperature, some known effectors, kinetic parameters and substrate assays. As production of phosphatases was higher from strain B2136, this strain was chosen for this purpose and compared in some instances with Lactobacillus helveticus CNRZ 223.

3.2 Characterization of casein phosphatases

The activity determined through the dephosphorylation of [³²P] casein is indeed due to phosphatase activity and not to an interference of proteinase activity leading to the release of TCA-soluble peptides. In the presence of proteinase inhibitors, casein phosphatase activity is completely active (96 % ± 10).

Strain Casein phosphatase activity^b (pmol ³²P min^-1 g^-1 fresh matter) Intracellular activity^c Cell-wall activity^d

T1 T2 T3 T1 T2 T3

Lactococcus lactis Wg2 26.5 26.3 19.3 18.1 14.1 14.3

Lactococcus lactis MG1363 29.7 30.5 29.6 25.7 22.5 22.4

Lactococcus lactis B2136 17.8 14.3 20.6 26.7 11.2 27.3

Lactobacillus plantarum B1407

00.3 00.3 00.3 20.1 10.1 20.7

Lactobacillus helveticus CNRZ 223

44.0 26.0 26.0 29.6 12.6 25.7

Lactobacillus helveticus ATCC 12046

26.9 09.8 21.9 29.2 19.3 28.2

Streptococcus thermophilus B05

19.1 09.9 07.9 30.5 11.0 26.4

a during the growth, samples were taken at the exponential phase (T1), early stationary phase (T2) and late stationary phase (T3) determined for each strain.

b phosphatase activity was determined through the dephosphorylation of [³²P] casein and expressed as ³²P released during the reaction. The results were reported to the weight of fresh cells.

c extraction was performed with 1 volume fresh cells and 3 volumes acetate buffer.

d 200 mg fresh cells were suspended into 1 ml acetate buffer.

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The effect of pH on casein phosphatase activities is illustrated in figure 1.

Intracellular activity from L. lactis B2136 was an alkaline phosphatase with its maximal activity near pH 8. At the opposite, cell-wall activity from L. lactis and Lb. helveticus showed an optimum pH near 4.5. In the case of Lb. helveticus CNRZ 223, a slight contamination with intracellular phosphatase released during cell preparation, buffer resuspension and the dephosphorylation reaction was noticed, due to the lower cell integrity.

Figure 1

pH dependence of intracellular casein phosphatase activity from Lactococcus lactis B2136 ( ) and cell-wall casein phosphatase activity from Lactococcus lactis B2136

( ) and Lactobacillus helveticus CNRZ 223 (∆).

Effet du pH sur l’activité caséine phosphatase endocellulaire de Lactococcus lactis B2136 ( ) et les activités pariétales de Lactococcus lactis B2136 ( ) et Lactobacillus

helveticus CNRZ 223 (∆).

The optimum temperature was between 40-50˚C for intracellular activity of L. lactis and cell-wall activities of L. lactis and Lb. helveticus. As shown in figure 2, L. lactis cell-wall activity was significant over a wide temperature range.

phosphatase activity (% of maximal activity) 120

100

80

60

40

20

0

2 3 4 5 6

pH

7 8 9 10

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Figure 2

Temperature dependence of intracellular casein phosphatase activity from Lactococcus lactis B2136 ( ) and cell-wall casein phosphatase activity from

Lactococcus lactis B2136 ( ) and Lactobacillus helveticus CNRZ 223 (∆).

Effet de la température sur l’activité caséine phosphatase endocellulaire de Lactococcus lactis B2136 ( ) et les activités pariétales de Lactococcus lactis

B2136 ( ) et Lactobacillus helveticus CNRZ 223 (∆).

The addition of magnesium acetate proved that intracellular casein phosphatase activities were strictly dependent on the presence of magnesium (table 2). The same result was observed when magnesium acetate was replaced with magnesium chloride (data not shown). In contrast, cell-wall activities were much less dependent on magnesium (table 2). As intracellular activities were very low in the absence of added magnesium, the effect of all other compounds was assayed in the presence of 20 mM magnesium acetate. Intracellular activities were also dependent on the presence of fluoride and once more cell-wall activities were much less dependent (table 2). A NaF concentration of 20 mM is known to inhibit most phosphatases but higher concentrations are necessary in the case of acid phosphatases. This has been observed by KYRIAKIDIS et al.

120

100

80

60

40

20

0

Activity (% of maximal activity)

0 10 20 30 40 50 60 70

Temperature (°C)

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(1993) on acid phosphatases from lactic acid bacteria. The effect of other compounds, known to be phosphatase effectors or present in milk or cheese in high concentrations was studied on Lactococcus lactis B2136 phosphatase activities (table 3). Micromolar concentrations of zinc and vanadate are known to highly inhibit phosphotyrosyl phosphatase activity (BRAUTIGAN and SHRINER, 1988). The results show that no phosphotyrosyl phosphatase activity was present in L. lac- tis besides alkaline intracellular phosphatase and acid cell-wall phosphatase. In the case of intracellular phosphatase, apparent K_i for phosphate and calcium was respectively 7.8 mM ± 1.2 and 5.5 mM ± 1.5. These values are in the same range of the concentrations of free phosphate and calcium in milk. It is obvious that cell-wall phosphatase activity was less affected by phosphate, calcium and sodium chloride.

Table 2

Effect of magnesium acetate and sodium fluoride on phosphatase activities from Lactococcus lactis and Lactobacillus helveticus.

Tableau 2

Effet de l’acétate de magnésium et du fluorure de sodium sur les activités phosphatases de Lactococcus lactis et Lactobacillus helveticus.

Lactococcus lactis B2136 Lactobacillus helveticus CNRZ 223 Compound Intracellular

activity Cell-wall activity Intracellular

activity Cell-wall activity

%

activity^a SD^b %

activity^a SD^c %

activity^a SD^c Mg acetate

20 mM

100 100 100 100

Mg acetate 0 mM

4 3 61 7 24 6 81 10

NaF 20 mM^d

11 5 83 10 25 5 86 11

a Activity in the presence of 20 mM magnesium was taken as 100.

b Average and standard deviation from 6 separate experiments.

c Average and standard deviation from 9 separate experiments.

d NaF effect was tested in the presence of 20 mM magnesium acetate.

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Table 3

Effects of various agents on intracellular and cell-wall phosphatase activities from Lactococcus lactis B2136.

Tableau 3

Effet de différents composés sur les activités phosphatases endocellulaire et pariétale de Lactococcus lactis B2136.

3.3 Dephosphorylation of commercial caseins

The time-course of commercial casein dephosphorylation by casein phosphatases from Lactococcus lactis B2136 was analysed through the determination of released phosphate at pH 6.6, which is the pH of milk but not the optimal pH for either of the intracellular or cell-wall phosphatase activities. To define the optimal casein concentration, kinetic parameters of intracellular and cell-wall enzymes from L. lactis were first determined through the dephosphorylation of [³²P] casein. Reac- tion time was chosen to be 10 min from the determination of the linear portion of the hydrolysis curve. Measurements used 8 different substrate concentrations (0- 25 µM). K_m and V_max were calculated by non linear-fitting of the Michaelis equation to the experimental data (table 4). K_ms for casein were low for both forms of the activities and much lower than casein concentration in milk. The ratio V_max/K_m may be used to assess the catalytic efficiency of an enzyme. It was similar for both forms of the casein phosphatase activities in our experimental conditions. αs casein, β casein and total casein concentrations were set to 60 µM and the dephosphorylation reaction was carried out at 30°C for 24 h. The action of intracellular phosphatase activity led to a 50% dephosphorylation yield of total casein after 8 h when only a 28% yield was obtained with cell-wall activity (figure 3). This result could be due to the fact that pH 6.6 is slightly less favourable for the cell-wall activity as compared to the intracellular activity (figure 1). Nevertheless these rates were sufficient to lead to a significant casein dephosphorylation. In the case of αscasein, maximal dephosphorylation yield with intracellular and cell-wall phosphatase activities was 30% and

% activity^a

Compound Intracellular activity Cell-wall activity

CaCl₂ 5 mM 70 93

10 mM 33 76

NaH₂PO₄ 10 mM 49 99

NaCl 0.5% 67 nd^b

1% 42 79

2% 32 46

Zn acetate 0.1 mM 72 91

Na vanadate 0.2 mM 108 108

a Activity in the presence of 20 mM magnesium was taken as 100 for each activity. The average of relative standard deviation obtained from 2 or 3 separate experiments was 7.5%.

b not determined.

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18%, respectively. In the case of β casein, this yield reached 36 and 53%, respectively. As it has been observed by LORIENT et al. (1976) using milk alkaline phosphatase, αscasein could be dephosphorylated to a less degree than β casein. It is likely that the phosphate groups are more accessible in β casein structure.

Table 4

Kinetic parameters of intracellular and cell-wall casein phosphatases from Lactococcus lactis B2136 determined with [³²P] casein as substrate.

Tableau 4

Paramètres cinétiques des activités phosphatases endocellulaire et pariétale de Lactococcus lactis B2136 déterminés avec l’utilisation

du substrat marqué [³²P] caseine.

Figure 3

Time-course of total casein (60 µM) dephosphorylation by intracellular ( ) or cell-wall ( ) casein phosphatase activity from Lactococcus lactis B2136.

Cinétique de déphosphorylation de la caséine totale (60 µM) par les caséine phosphatases endocellulaire ( ) ou pariétale ( ) de Lactococcus lactis B2136.

K_m^a

(µM) SD

V_max^a (nmol tot P g^-1 fresh

matter min^-1)

SD V_max/K_m

Intracellular activity

18.6 0.4 39.8 2.2 2.1

Cell-wall activity

18.5 0.7 16.3 0.6 1.9

a K_m and V_max were determined from non linear fitting of Michaelis equation to the experimental data.

60

Dephosphorylation yield (%) 50

40

30

20

10

0

0 5 10

Time (h)

15 20 25 30

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4 – CONCLUSION

Intracellular and cell-wall casein phosphatases were identified in the three tested strains of Lactococcus lactis and the two tested strains of Lactobacillus helveticus. Two forms of phosphatase activities from Lactococcus lactis subsp.

lactis B2136 were characterized as an alkaline intracellular enzyme and an acid cell-wall one. These two activities were able to significantly dephosphorylate total casein, αs casein and β casein. It is likely that intracellular enzymes were not released during the first stages of cheese process. More, since cell-wall activity was only moderately affected by free phosphate and calcium concentrations, and owing its optimum at lower pH and its higher heat tolerance which is of advantage during processing and progressed cheese ripening, this isoform could be preferentially used in the casein dephosphorylation in the course of milk technology (prematuration, cheese making and ripening). The purification of cell-wall casein phosphatase activity from Lactococcus lactis is in progress and the enzyme could be used to dephosphorylate caseins in milk.

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