A new approach for the purification and characterisation of PA49.5, the main prebiotic of Lactococcus lactis subsp. cremoris

International Journal of Food Microbiology 126 (2008) 186–194

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

A new approach for the purification and characterisation of PA49.5, the main prebiotic of Lactococcus lactis subsp. cremoris Étienne Dako a,⁎, Christopher K. Jankowski a, Anne-Marie Bernier c, Alain Asselin b, Ronald E. Simard b a b c

École des sciences des aliments, de nutrition et d'études familiales, Faculté des sciences de la santé et des services communautaires, Université de Moncton (N.-B) Canada, E1A 3E9 Faculté des Sciences de l'Agriculture et de l'Alimentation, Université Laval, Ste-Foy (QC) Canada Collège universitaire de Saint-Boniface, Winnipeg, (MB) Canada

A R T I C L E

I N F O

Article history: Received 3 October 2007 Received in revised form 16 May 2008 Accepted 17 May 2008 Keywords: Autolysis Probiotic Prebiotic Lactic acid bacteria Purification Electrophoresis Fatty acids

A B S T R A C T The main autolysin PA49.5, an enzyme that hydrolyzes or destroys the components of a biological endogenous cell or a tissue, was purified 3045 times from the homogenate of a whole cell extract of Lactococcus lactis subsp. cremoris ATCC 9596 (Mc5), with a recovery yield of 52%. The purification of the protein was carried out through a micro-purification technique using SDS-BigCHAP polyacrylamide gel electrophoresis and concentrated with a Microcon-10 filtration system. SDS-polyacrylamide gel electrophoresis of the purified enzyme confirmed the presence of only one band having a molecular weight of 49.5 kDa. In view of its insolubility, PA49.5 contained in the cell extract precipitate was solubilized in the presence of 0.1% (w/v) of BigCHAP, a non-ionic detergent. Higher concentrations of this detergent completely inhibited the activity of solubilized PA49.5 or prevented its solubilization. The optimal pH and temperature for PA49.5 enzymatic activity are 7.5 and 45 °C respectively. In addition 0.1% or less of PA49.5 significantly increased Mc5 lysis. We observed 55% more lysis with 0.25 μg of purified PA49.5 compared to the control. Gas chromatography analysis of the components of the crude cell extract, of the precipitate and of the supernatant indicates the presence of at least 6 fatty acids. The long-chained fatty acids (e.g. C18:0 and C18:3) detected represent 81.65% of the precipitate from which PA49.5 was purified. Of these two acids, the C18:0 (stearic acid) alone represents 47.40% of the precipitate. Mc5 releases proteins at the beginning (major peak) and at the end (moderate peak) of the exponential stage of growth. Analysis by denaturing polyacrylamide gel electrophoresis with Mc5 cell walls incorporated as the autolysin's substrate identified a band corresponding to PA49.5 in the second peak of protein secretion. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Autolysins are hydrolases implicated in the synthesis of peptidoglycans (responsible for the rigidity of the bacterial wall), the division of daughter cells, genetic transformations and the degradation of the bacterial wall (Rogers et al., 1984; Tomasz, 1984; Dako et al., 1995; Chapot-Chartier, 1996; Smith et al., 2000). Recent post-electrophoresis assays of autolytic activity have facilitated the detection of autolysins in cell wall extracts (Shockman et al., 1967; Hinks et al., 1978; Potvin et al., 1988; Leclerc and Asselin, 1989; Lortal et al., 1997; Dako et al., 2003b). However, the purification of these enzymes remains a difficult stage because these autolysins are generally insoluble due to their association with certain lipophilic components such as teichoic or lipoteichoic acid (Brown et al., 1970; Brown, 1972; Herbold and Glaser, 1975a,b; Fisher et al., 1980a,b). The use of surfactants or detergents is therefore necessary to break the links which binds the enzyme to these components. Among the more commonly used detergents, sodium dodecyl sulphate

⁎ Corresponding author. Tel.: +1 506 858 4080; fax: +1 506 858 4283. E-mail address: [email protected] (É. Dako). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.05.029

(SDS) is the most widely used (Coyette and Shockman, 1973; Foster, 1992; Chang et al., 1994; Tan et al., 1995; Buist et al., 1995). SDS easily solubilizes the autolysins, hence its utilization is preferred to solubilize and purify these lytic enzymes (Brown, 1972). While the preliminary treatment of cell extracts with SDS allows the solubilization of these enzymes (Shockman et al. (1967), a major disadvantage remains in that it is very denaturing. During the purification of a protein, it is useful to be able to follow its activity, if necessary, at every stage. The main disadvantage of the use of SDS in the extraction process is that it does not allow the assay of enzyme activity because it readily denatures the proteins. To improve the assay of the autolytic activity using denaturing polyacrylamide gel electrophoresis (PAGE) alternative non-adulterating detergents were evaluated (CHAPS, Big CHAPS). These detergents are used in the solubilization of proteins strongly associated with hydrophobic compounds and they have two characteristics in common: i) they are easily dialyzed due to the presence of the cholanoic acid group; ii) they do not affect the activity of the proteins when used in low concentrations (Marshak et al., 1996). CHAPS (MW:615 Da) is classified as a zwitterion because of its positive and negative charge while BigCHAP (MW: 862 Da) is non-ionic. The aim of solubilization with a detergent is to increase the

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purification of an active protein while maintaining its catalytic activity. Thus, the detergent utilized should permit the assay of enzymatic activity and favour its purification (Helenius et al., 1979). In a previous study it was shown that 0.1% of CHAPS in a solution containing 3% (v/v) whole cell crude protein extract allowed the complete solubilization of the principal autolysin (PA49.5) which was initially insoluble (Dako et al., 2003b). However, this same study reported that 0.1% (w/v) of CHAPS did not allow the elimination of substances responsible for the insolubility of PA49.5, since these substances were also found in the supernatant. The dialysis of these supernatants followed by lyophilization facilitated the formation of the insoluble PA49.5—lipophilic substances complexes (Dako et al., 2003b). The purification of the autolysins from lactic acid bacteria and particularly of Lactococcus is very difficult. In fact, autolysins are endogenous lytic enzymes widely associated with the cell wall (Shockman et al., 1967) and particularly with certain cellular compounds such as teichoic acid (Brown et al., 1970) and polysaccharides (Lemee et al., 1995). The main polysaccharide in Lactococcus subsp., Lactobacillus subsp. and Leuconostoc subsp. is dextran which is hydrolysed at its α-1–6 bonds (Kenne and Lindberg, 1983). Some of these autolysins were purified from proteins separated in polyacrylamide gel (Chan and Glaser,1972; Rogers et al.,1984; Shockman and Höltje,1994; Østlie et al.,1995; Dako et al., 2003b) or by cloning (Buist et al., 1995). The objectives of this study were to develop a new technique to solubilize and to purify the main autolysin (PA49.5) of Lactococcus lactis subsp. cremoris ATCC 9596 (Mc5) and to characterize this autolysin in terms of optimal pH and temperature. The analysis by gas chromatography of fatty acids from crude extract, of the precipitate and of the supernatant was utilized to identify and quantify the lipidic components responsible for the insolubility of PA49.5 found primarily in the precipitate. The release of hydrolases during the growth of Mc5 was equally studied. Mc5 is in fact rich in enzymes such as aminopeptidases and carboxypeptidases that act in the maturation of cheeses (Dako et al., 1995, 2003b). The presence of PA49.5 in this bacterium provides an added interest especially into its purification in order to better understand its mode of action and the spontaneous bacterial lysis attributed to this autolysin that releases the cheese maturation enzymes. 2. Materials and methods 2.1. Bacterial strain and culture conditions L. lactis subsp. cremoris ATCC 9596 (Mc5) came from the collection from the Centre STELA (Laval University, Quebec, QC) and was cultivated in M-17 media (Difco, Montreal, QC) at 37 °C. It was cultivated twice in the same medium before being used according to the method described by Dako et al. (1995). 2.2. Purification steps of PA49.5 from Mc5 2.2.1. Step 1: 1st crude extract 2.2.1.1. Autolysin extraction in the presence of lithium chloride (LiCl). The method developed by Brown (1973) for the extraction of autolysins from Bacillus subtilis was modified for our study. We cultivated 20 L of Mc5 in M-17 culture broth. The whole cells were recuperated by centrifugation (15 000 ×g/15 min/4 °C) at the end of the exponential stage of growth after several washings with physiological solution (0.85% NaCl). Whole cells (5% w/v) were resuspended in desorption solution (50 mM Tris–HCl buffer, pH 7.5 and 5 M LiCl) and incubated at 4 °C under gentle agitation (1000 rpm) for 1 h. After incubation, the extract was centrifuged at 15 000 ×g/15 min/4 °C. The supernatant was recuperated and an equal volume of water was added to prevent the inactivation or the denaturation of the proteins by the high concentration of LiCl. The mixture was then dialyzed (4 × 8 h) in 20 L of 10 mM of Tris–HCl buffer, pH 7.5 or in deionized, sterilized water. The final volume of the dialyzate was 8 L.

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2.2.2. Step II: 2nd crude extract The final dialyzate (8 L) was lyophilized and suspended in 285 mL of 10 mM of Tris–HCl buffer, pH7.5 (thus concentrated 28 times) and corresponds to the whole cell autolysin crude extract (ACE), (285 mL of ACE). The protein in the ACE extract was quantified according to the method of Lowry et al. (1951). All assays were performed in triplicate. 2.2.3. Step III: optimization of the conditions of solubilization of PA49.5 2.2.3.1. Successive precipitations of the whole cell autolysin crude extract (ACE). A 142.5 mL aliquot of ACE (concentrated 28 times) was precipitated a minimum of five times by centrifugation (15 000 ×g/ 15 min/4 °C), followed by the suspension of the precipitate in 10 mM of Tris–HCl buffer, pH7.5 and the incubation of the extract for 10 min at 4 °C under constant agitation (1000 rpm). The cell extract supernatant (SCE) and the cell extract final precipitate (PCE) were measured. The final precipitate was used to purify the PA49.5. 2.2.3.2. Effect of BigCHAP and heating on the solubility of PA49.5. An aliquot of 30 μL of PCE was suspended in 1 mL of water containing respectively 0.08; 0.1; 0.2; 2; and 4% (w/v) of BigCHAP and 0,1% of CHAPS (as control). The non-heated samples were conserved at 4 °C for 12 min. The heated samples were incubated at 100 °C/2 min and cooled for 10 min at 4 °C. The solutions were centrifuged at 15 000 ×g/ 15 min/4 °C and the precipitate resuspended in 30 μL of water. One (1) μL of resuspended precipitate was taken, as well as 34 μL of supernatant for each treatment. PA49.5 activity was verified by electrophoresis in a denaturing polyacrylamide gel containing 0.2% (w/v) of Mc5 cell walls extract as the substrate. 2.2.3.3. Preparation of crude cell walls for incorporation into SDS-PAGE. The method of Leclerc and Asselin (1989) was modified as follows: Bacterial cells were harvested at the exponential phase of growth and centrifuged at 15 000 ×g for 15 min at 4 °C. The pellet was washed with 100 mL of 10 mM Tris–HCl, pH 7.5 for 30 min at 4 °C. The pellet was lyophilised and 1 g of cells was suspended in 100 mL of 2% (w/v) of SDS. The suspension was shaken at 1000 rpm for 30 min on a rotary shaker at room temperature (RT) and sonicated on ice for 5 min at the maximum power setting (Sonic Dismembrator, Quigley-Rochester Inc.). The extract was heated at 100 °C for 15 min and centrifuged at 12 000 ×g for 15 min at room temperature. The pellet was resuspended in 100 mL of 0.1% (v/v) purified Triton X-100 (Dako et al., 2003a) and incubated for 30 min at RT with gentle shaking to remove SDS and membranes. The suspension was centrifuged as above and the pellet washed 4 times every 30 min in 100 mL of distilled water. The final pellet was lyophilised and Table 1 Composition of SDS and SDS-BigCHAP polyacrylamide gels SDS-PAGE

SDS-BigCHAP PAGE

15% Polyacrylamide

15% Polyacrylamide

Separation gel Bis-Acryl. 30–0.8% Tris–HCl. 3 M pH8.9 H2O SDS 10% BigCHAP 10% Temed A.P. (10%)

Vol. (mL) 3.500 1.250 3.600 0.100 0.000 0.015 0.075

Separation Gel Bis-Acryl 30–0.8% Tris–HCl. 3 M pH8.9 H2O SDS 30% BigCHAP 15% Temed A.P. (10%)

Vol. (mL) 3.500 1.250 3.600 0.033 0.067 0.015 0.075

Concentration gel Bis–Acryl 30–0.8% Tris–HCl. 0.5 M pH6.7 H2O SDS 30% BigCHAP 15% Temed ⁎A.P. (10%)

Vol. (mL) 1.600 1.250 7.000 0.100 0.000 0.020 0.075

Concentration gel Bis-Acryl 30–0.8% Tris–HCl. 0.5 M pH6.7 H2O SDS 30% BigCHAP 15% Temed A.P. (10%)

Vol. (mL) 1.600 1.250 7.000 0.033 0.067 0.020 0.075

⁎AP: Ammonium persulfate.

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Table 2 The GC results for the fatty acid standard (GL-60, diluted 1/10) Fatty acid (common name)

Chain (shorthand designation)

(%) Wt

Retention time

Butyric acid Caproic acid Caprylic acid Capric acid Lauric acid Myristic acid Myristolic acid Palmitic acid Palmitolic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Arachidic acid

C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0

4 2 1 3 4 10 2 25 5 10 25 3 4 20

2.540 4.032 5.543 6.954 8.342 10.277 10.671 13.230 13.605 16.977 17.416 18.223 19.466 22.312

resuspended as a 2% (w/v) cell wall suspension in water containing 0.02% (w/v) sodium azide and stored at 4 °C. 2.2.4. Step IV: micro-purification 2.2.4.1. Purification of PA49.5 by elution from a SDS-polyacrylamide gel and a SDS-BigCHAP gel. To confirm that the excised band corresponded to PA49.5, the solubilized protein in BigCHAP was analysed by 2 dimensional gel electrophoresis using a modified method of Aoshuang et al. (2005). SDS (33%) was added to permit the migration of the proteins protected by BigCHAP. The gels were prepared as described in Table 1. The extraction of the band containing PA49.5 was carried out according to a modification of the technique described by Sheer (1994). 2.2.4.2. Purification of PA49.5 by the technique of micro-purification in polyacrylamide gel. The acrylamide gel was coloured in 0.1% (w/v) of Coomasie Bleu (bright R-250) or silver nitrate according to the method of Dako et al. (2003b). The 49.5 and 40 kDa bands were excised from the gel using a scalpel and placed in micro-centrifuge tube. The blue dye was removed by the addition of the washing buffer (50% (v/v) methanol). The sample was sonicated for 15–20 min at 45 °C until all the gel remnants were well suspended. The washing solution was eliminated by centrifugation (1000 rpm/3 min) and 50–100 μL of extraction buffer (100 mM NaCO3, 8 M urea, 0.1% (w/v) BigCHAP and/or SDS, 2 mM DTT) was added. The suspension was incubated for 30 min at 45 °C. Using a mortar, the gel remnants were homogenized and incubated in a bainmarie for 2–3 h at 45 °C or overnight. The Micropure 0.22 μ separator was placed in the Microcon-10 equipped with a membrane to prevent the protein of 40 and/or 49.5 kDa from passing through. Then 100 μL of the washing solution was added to the Micropure and the gel puree was introduced using a clean, sterile pipette. The tube was rinsed with 100 μL of washing buffer, to recuperate all of the gel remnants that were then transfered to the Micropure. Centrifugation was done (15 000 ×g/ 30 min/4 °C) until all the liquid in the separator was totally eliminated. The protein was retained at the surface of the Microcon-10 membrane. Washing solution (100 μL) was added to eliminate all traces of the extraction buffer by centrifugation (14 000 ×g/70 min/4 °C). The extract of autolysin was then resuspended in 400 μL of 10 mM Tris–HCl, pH 7.5 and centrifuged at 13 000 g for 30 min at 4 °C. To recuperate the concentrated purified autolysin, the Microcon-10 was inverted into a new microfuge tube and centrifuged at 1000 g for 3 min. Purified PA40 and PA49.5 activity was verified by SDS-PAGE in the presence of the cell wall extract of Mc5 substrate. 2.3. Estimation of the specific activity of PA49.5 during the purification stages A mixture of 100 μL of 10 mM of Tris–HCl buffer, pH 7.5, with 280 μL of water was preheated for 10 min at 4 °C. An aliquot of 10 μL of the purified

autolysin PA49.5 and 10 μL of Mc5 wall extract substrate were added to the preheated mixture and everything was heated for 5 min at 45 °C. After incubation, the reaction was stopped by adding 600 μL of deionized, sterilized water. The reduction of absorbance was recorded at 450 nm during the incubation at 45 °C according to the method described by Rogers et al. (1984). One unit of the enzyme activity represents a change in absorbance (A450) of 0.001 in 1 min, for 1 mL of enzyme. T:U: ¼

T. U. S. A. Q. P.

A450 1 min 1 mL  ;  0; 001 t ðminÞ V ðmLÞ

S:A: ¼

T:U: Q :P:

Total Unit (U) Specific Activity (U mg− 1) Quantity of Protein (mg)

2.4. Effect of β-mercaptoethanol on the activity of purified PA49.5 and on the lysozyme (poultry egg white) activity β-mercaptoethanol was used to verify the presence of disulphide bridges or cystine residues in the PA49.5 molecule. Lysozyme, a hydrolase containing 4 disulphide bridges, was used as the control. Two solutions (1) and (2) corresponding respectively to (0.08 μg/μL of lysozyme) and (0.04 μg/μL lysozyme, 0.01 μg/μL of PA49.5) were tested in absence of the β-mercaptoethanol as controls. A concentration of 2% β-mercaptoethanol was applied to a mixture (3) containing 0.04 μg/μL of lysozyme and 0.01 μg/μL of PA49.5. Activity was detected by denaturing SDS-PAGE as previously described (Dako et al., 2003b) using 0.2% of autoclaved wall extract of Mc5 (121 °C/20 min) as a substrate. The proteins in the gel were reactivated by incubation of the gel in a 10 mM sodium acetate buffer pH 5.0 containing 1% (v/v) purified Triton X-100, for 3 to 24 h at 45 °C. Prestained standard SDS-PAGE low range molecular weight markers (Bio–Rad, CA) were used. 2.5. Effect of pH on the lytic activity of purified PA49.5 We added 10 μL of purified PA49.5 to 1 mL of 10 mM of Tris–HCl buffer at different pH (4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8; 8.5; 9; 9.5; 10) containing 10 μL of autoclaved (121 °C/20 min) Mc5 wall extract as the substrate. The suspension was incubated at 37 °C for 60 min. The effect of the pH on enzyme activity was determined by spectrophotometry by measuring the decrease in the optical density (650 nm) every 30 s over a 60 min period. The rate of autolysis was defined according to the method of Dako et al. (2003a). 2.6. Effect of temperature on the autolysis in the presence of purified PA49.5 We added 10 μL of purified PA49.5 to 1 mL of 10 mM of Tris–HCl buffer, pH 7.5 containing 10 μL of autoclaved Mc5 cell wall extract (121 °C/20 min) as the substrate. The suspension was incubated for 5 to 10 min at different temperatures (25, 30, 35, 37, 40, 45, 50 and 55 °C). The effect of temperature was measured by spectrophotometry by recording the decrease in optical density at 650 nm. The rate of autolysis was defined according to the method of Dako et al. (2003a). The control was evaluated by the absence of PA49.5. 2.7. Quantification and analysis of esterified free fatty acids by gas chromatography (GC) 2.7.1. Gas chromatography equipment and programming Hewlett-Packard 5890A chromatograph with a gas support (H2, 55 Kpa (8 psi)), Hewlett-Packard 3392 A integrator, capillary column model DB-225 (0.25 mm inside diameter), liquid thickness in column (0.25 μ), “splitless” type injector at 230 °C, flame ionization detector (FID) at 250 °C, H2:20 psi.

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Fig. 1. Effect of BigCHAP on the solubilization of PA49.5. The samples were treated in the presence of 0.08; 0.1; 0.2; 2; 4% (w/v) BigCHAP and 0.1% (w/v) CHAPS (control) with or without heating. Denaturing PAGE of the precipitate (PCE) and the supernatant (SCE) with 0.2% Mc5 cell wall extracts. (a) Lanes 1, 2, 1′ and 2′: PCE and the SCE respectively of the two control ACE and 0.1% (w/v) of CHAPS not heated; lanes 3, 4, 5 and 3′, 4′ and 5′: PCE and the SCE respectively, without heating with BigCHAP (0.08; 0.1; 0.2%) (w/v). Lanes 7, 8, 9 and 7′, 8′, and 9′: PCE and the SCE respectively of the same concentrations, heated. Lanes 6 and 6′: PCE and the SCE, heated, with 0.1% of CHAPS. (b) Lanes 1 and 1′: PCE and the SCE of the control. Lanes 2 and 3: PCE with heat with 2 and 4% (w/v) of BigCHAP. Lanes 4 and 5: PCE not heated with 2 and 4% (w/v) of BigCHAP. Lanes 2′, 3′ 4′, 5′ SCE from 2; 3, 4 and 5). Mr: molecular weight marker.

Programming: 100 °C up to 200 °C (35 °C/min) with a departure time of 2 min and final time of 10 min. 2.8. Extraction and analysis of fatty acids The fatty acids contained in the crude extract (ACE), the precipitate (PCE) and the supernatant (SCE) of ACE were analysed by gas chromatography. To analyse fatty acids by GC they were first transformed into volatile methylated esters. A 500 μL aliquot of each sample was lyophilized and suspended in 200 μL of BF-3 methanol to methylate the free fatty acids and the triglycerides. After incubation (65 °C for 15 min), the suspension was added to 200 μL of water and 200 μL of hexane. It is necessary to avoid producing emulsions that trap air bubbles that potentially inhibit the action of hexane.

Anhydrous sodium sulfate (Na2SO4) was added to capture the traces of water. After centrifugation at 1200 rpm, the hexane supernatant containing the methylated fatty acids was recuperated and a sample was placed in an autosample vial and analysed by gas chromatography. Fatty acids were quantified by weighing a sample before and after hexane evaporation. The GL-60 fatty acid standard (Sigma, Canada) was diluted 1/10 before use (Table 2). 3. Results 3.1. Effect of BigCHAP on the solubilization of PA49.5 Fig. 1 shows the effect of BigCHAP, with and without heating, on the solubility of PA49.5. PA49.5 is present in the precipitate of the two

Fig. 2. Purification of PA49.5 and PA40 of Lactococcus lactis subsp. cremoris by the micro-purification technique starting from SDS-PAGE and SDS-BigCHAP-PAGE (1:3) (See Table 1 for more details). Mr: molecular weight marker.

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Fig. 3. Electrophoretic profile of PA49.5 purified from an SDS-BigCHAP polyacrylamide gel (15%) (a) after colouring with silver nitrate, (b) activity of the band of purified PA49.5 from lane 2 in a) transferred in a polyacrylamide gel (10%) containing SDS-BigCHAP in the presence of autoclaved Mc5 cell wall extract as substrate. Each well contains 6 μl (2.5 μg) purified PA49.5. Control (PCE) contains 11.2 μg of total proteins (6 μl). Mr: molecular weight markers.

controls, notably ACE (Fig. 1a, lane 1) and the sample treated with 0.1% (w/v) CHAPS without heating (lane 2). A weak activity essentially attributable to the dilution factor is nevertheless observed in the two supernatants (1′, 2′). On the other hand, by heating (100 °C/2 min) in the presence of 0.1% (w/v) of CHAPS, PA49.5 is entirely solubilized (lane 6′). The intensity of its activity is however weaker than that obtained with BigCHAP. Without heating very little PA49.5 is found in the precipitate with 0.08; 0.1 and 0.2% BigCHAP (lanes 3, 4, 5). AT these concentrations BigCHAP most of this protein is present in the supernatant (lanes 3′, 4′, 5′). The same result is obtained with heating (7′, 8′) with the exception of the supernatants with 0.2% (w/v) of BigCHAP (lane 9′). The results obtained with 0.2% BigCHAP in the absence of heat show that this concentration certainly allows the solubilization of PA49.5 but it becomes inhibitive to this autolysin when heated (lane 9′). In moving from 0.2 to 2% and to 4% (w/v) BigCHAP (Fig. 1b), it becomes practically impossible to solubilize PA49.5 even with the thermic treatment (lanes 4′, 5′). It is noted as well that these concentrations inhibit the small quantity of PA49.5 in a solution. BigCHAP allows, in weak concentrations and in the absence of thermic treatment, the significant solubilization of PA49.5. When used in high concentrations, BigCHAP induces the opposite effect by precipitating the protein instead of solubilizing it. Moreover, one notices a reduction in the intensity of autolytic activity (lanes 2, 3, 4, 5, Fig. 1b) with the exception of the control (lane 1, Fig. 1b). The artifact observed at the bottom of the gel (Fig. 1), is due to the absence of dialysis. Its presence affects neither the migration of autolysins nor the intensity of the autolytic bands. This phenomenon indicates that denaturing PAGE is not affected by the presence of BigCHAP. For the continuation of this study, 0.1% (w/v) of BigCHAP was therefore used.

compared to the SDS gel that required staining with silver nitrate. One also notices that the combination of SDS and BigCHAP did not influence the migration of either band. The preliminary treatment with 0.1% (w/v) BigCHAP allowed for the protection of the solubilized PA49.5 against the denaturing effect of SDS since the protein retained its activity. The results from Figs. 2 and 3(a,b) confirm that this method allows the purification of the autolysin of Lactococcus. Additionally, it was observed that with 2.5 μg of purified PA49.5, the band intensity is 3 to 5 times superior to that of the control (Fig. 3b). Table 3 presents the results of the purification of PA49.5 from Mc5 cell wall extracts at each of the four stages of the procedure. The enzyme is purified by a factor of 3 045 times with a final yield of 52%. At the beginning of purification, the first crude extract contained 1700 units of enzymes. During each subsequent step total enzyme activity was lost, with 880 units remaining after the micropurification step. The specific enzyme activity is defined as the ratio of the total units of enzyme activity to the total mass of proteins. The specific activity of PA49.5 increased from 0.85 to 2588 units of PA49.5/mg of protein since contaminating proteins are eliminated at each stage (Table 3). Thus, the mass of total proteins recuperated decreased from 2000 mg to 0.34 mg. This new approach to purification has therefore allowed us to recuperate 52% of the protein (880/1700 × 100), which is equivalent to 880 units at the end of purification from 1700 units of enzyme initially present in the extract. This corresponds to a purification level of 3045 times, or 2588 U mg− 1.

3.2. Comparative study of PAGE with SDS or SDS/BigCHAP for the purification of PA49.5

Steps

The results for SDS-PAGE and SDS/BigCHAP-PAGE show the same electrophoretic profile (Fig. 2). The presence of the band of ∼40 kDa indicates that the technique of micro-purification used here, is applicable to the isolation of any autolysin. The protein of 40 kDa caught our attention since it was present in most of the stages of purification. The intensity of the bands is a lot stronger with BigCHAP than with SDS (Fig. 2). Moreover, after colouring with Coomasie blue, one can already clearly detect the two bands isolated using the SDS-BigCHAP gel as

1 1st Crude extract 8000.00 2000.00 brut (RE) 2 2nd Crude extract 285.00 530.01 3 Successive 6.00 4.51 precipitations) 4 Micro-purification 0.50 0.34

Table 3 Purification steps of PA49.5 from Mc5 Volume

Total Total Specific protein(†) activity(‡) activity

(ml)

(mg)

Unit (U)

Total Purification Yield factor

(U mg− 1) (%)

No of times

1700.00

0.85

100

1

1550.00 1314.00

2.92 291.35

91 77

3 343

880.00

2588.00

52

3045

†: Protein concentration was determined by the method of Lowry et al. (1951). ‡: Enzymatic activities determined in steps 1. 2 and 3 were underestimated (PA49.5 still insoluble).

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Fig. 4. Effect of β-mercaptoethanol on egg white lysozyme and purified PA49.5 activity. The activity was detected by denaturing SDS-PAGE (10%) containing 0.2% of autoclaved Mc5 wall extract as a substrate.

3.3. Effect of β-mercaptoethanol on the activity of purified PA49.5 Fig. 4 shows that the activity profile of purified PA49.5 was not modified by the presence of 2% of β-mercaptoethanol (sample 3, in duplicate). However, the lysozyme of egg whites (control) is totally inhibited in its presence, when compared with the samples untreated with β-mercaptoethanol.

autoclaved Mc5 cell membranes alone (controls) with maximal lysis activity of 25% observed after incubation for 5 min at 45 °C. The effect of temperature is particularly more evident in the presence of purified PA49.5 than in its absence. These experiments were all performed in buffer at pHop. 3.6. Release of total proteins and autolysins during growth

The optimal temperature of PA49.5 is 45 °C, where it attains a maximum lysis of 80% after 5 min of incubation (Fig. 6). We also observed a lysis of about 66% at 37 °C just as in Fig. 5. The data in Fig. 6 show that temperature also has an effect on the autolysis of

During the growth of Mc5, there are two peaks (I and II) of protein release as indicated in Fig. 7, with the majority being released in the beginning of the exponential phase of growth when nutritious elements are still available in the medium. After 6 h of growth there is a decrease in protein release presumably due to the progressive exhaustion of the nutriments in the medium. However, there is a second release of total proteins at the end of the exponential phase of growth. This result demonstrates that during bacterial growth in a medium sufficiently rich in nutriments such as M-17, bacterial growth takes place during specific periods as indicated by the release of certain proteins at the beginning and at the end of the exponential phase of growth. The two protein peaks (I, II, Fig. 7) appearing at 4 and 8 h of incubation time were tested by SDS-PAGE in the presence of substrate for the presence of PA49.5. The results confirm the presence

Fig. 5. Effect of the pH on autolytic activity of purified PA49.5 on Mc5 cell walls. The action of PA49.5 was measured by the decrease of the optical density at 650 nm over a 60 min at 37 °C. Incubation times 2.5 (A); 5.0 (B) and 7.5 min (C) at pH 4 to 10 in 0.5 increments are represented here.

Fig. 6. Effect of temperature on the autolysis of autoclaved Mc5 cell wall in the presence of purified PA49.5. PA49.5 activity is measured by the decrease of the optical density at 650 nm over a 10 min incubation period at pHop 7.5 at both 37 °C and 45 °C. The controls are autoclaved cell walls without the enzyme.

3.4. The effect of pH on the autolytic activity of PA49.5 Optimal enzymatic activity is observed at pH is 7.5 (pHop) with a maximum of 66% lysis after 5 min of incubation time at 37 °C (Fig. 5). To avoid overcrowding on Fig. 5 only the times of 2.5, 5 and 7.5 min are recorded. The optimal incubation time for maximal enzymatic activity is 5 min. The optimal pH was used in subsequent experiments. 3.5. The effect of temperature on the autolytic activity of PA49.5

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Fig. 7. Bacterial growth of Mc5 in M-17 broth and total protein release over the 14 h incubation evaluated by measuring OD at 650 nm every 2 min. The control is represented by the M-17 broth without cells. Peaks I and II were sampled and further tested for the presence of PA49.5.

of a weak band of PA49.5 activity in the second peak (II), while no activity was detected in peak I. 3.7. Analysis of fatty acid content in extraction samples by gas chromatography The analysis of the fatty acids content in the crude extract (ACE), the precipitate (PCE) and the supernatant (SCE) was performed by gas chromatography (GC). The aim of this analysis was to identify fatty acids that could be associated with PA49.5 and thereby be responsible for its insolubility in the precipitate (PCE). The results of the GC analysis indicate the presence of at least six different fatty acids in the three samples (Table 4). In fact, in the crude extract (ACE) the fatty acids C8, C10, C12, C14, C18, C18:2 and C18:3 are present. With the exception of C18:3, the same fatty acids are found in the three extracts. The difference however lies in the level of the fatty acid contents in the three samples. For example, The PCE has twice as much C14 and C12 and 17% more C18:0 than the SCE. The SCE also has more than twice as much C18:2 than does the PCE. Moreover, more long-chained fatty acids are found in the three specimens (ACE: 92.25%; PCE: 80.20%; SCE: 82.58%) than the shortchained (C4-8) or medium-chained (C10; 12; 13) fatty acids. Among the long-chained fatty acids from the crude extract, we found twice as many unsaturated (66.45%) as saturated (33.55%) fats. The PCE had 25% more saturated fats as compared to the SCE. 4. Discussion The choice of detergent used in the extraction and purification or enzymes demands particular attention and must take into account the specific properties of the enzyme (Helenius et al., 1979; Furth et al., 1984; Marshak et al., 1996). The solubilizing effect of SDS has already been confirmed in the literature (Brown et al., 1970; Chan and Glaser, 1972; Brown, 1972; Shockman and Höltje, 1994; Foster,1992; Buist et al., 1995; Valence and Lortal, 1995; Dako et al., 1995; Lortal et al., 1997;). It has been suggested that the only approach to purify an autolysin would be to use SDS (Brown, 1972) however the results of this study show very clearly that BigCHAP is an excellent detergent to solubilize proteins, particularly those that are bound to the cell wall like autolysins. Both detergents solubilize

the PA49.5—lipophylic complexes however SDS is denaturing and difficult to dialyze whereas BigCHAP is not only easily dialyzed but maintains the enzymatic activity of PA49.5 (Marshak et al., 1996). Our new approach using PAGE with SDS-BigCHAP facilitates the purification of autolysins. Electrophoresis in a polyacrylamide gel with only BigCHAP cannot take place because BigCHAP is a nonionic detergent. The use a combination of the two detergents in a ratio of 33% SDS to 67% BigCHAP facilitates migration during the separation of proteins by electrophoresis while maintaining the protection of enzyme activity. In general, the pH, the temperature, the composition of the suspension medium and other factors have a great influence on the activity of the hydrolases of lactic acid bacteria and consequently on the autolysis (Dolinger et al., 1988; Lemee et al., 1995). Thus, Mou et al. (1976), in the study of the autolysis of Streptococcus reported that the autolytic activity was weaker at both highly acidic and basic pH values. Tipper (1969) suggests as well that this would be due to the denaturation of autolysins by these extreme pH values. Using the extraction and purification protocol presented here we characterised the main autolysin of L. lactis subsp. cremoris, PA49.5, as having an optimal pH of 7.5 and an optimal temperature of 45 °C. The high level of lysis observed (80% at 45 °C and 65% at 37 °C) clearly shows the lytic activity of purified PA49.5. Some lysis does occur in the autoclaved cell walls incubated without the enzyme (control) at both temperatures. This activity could be attributed to the fragile state of the glucosidic and/or peptidic bonds within the peptidoglycan structure induced by the heat treatment, the autoclaving and subsequent treatments with SDS and Triton X-100 and not to endogenous enzyme activity which would otherwise be denatured. At 45 °C PA49.5 therefore increases lysis by 55% over the control at the same temperature. The presence of fatty acids in the precipitate (Table 2) from which we purified PA49.5 suggests that this protein has a strong affinity to these

Table 4 Fatty acid content of cell extracts as determined by GC Fatty acid chains

ACE (Crude extract of Mc5)

PCE (ACE precipitate)

SCE (ACE supernatant)

Average: (%Wt)

Average: (%Mol)

Average: (%Wt)

Average: (%Mol)

Average: (%Wt)

Average: (%Mol)

C4:0 C6:0 C8:0 C10:0 C12:0 C13:0 C14:0 C14:1 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3 C20:0 Total:

0.00 0.00 3.81 2.90 1.04 0.00 6.79 0.00 0.00 0.00 0.00 0.00 19.01 0.00 57.51 8.94 0.00 100.00

0.00 0.00 6.65 4.30 1.33 0.00 7.74 0.00 0.00 0.00 0.00 0.00 17.59 0.00 53.94 8.45 0.00 100.00

0.00 0.00 9.39 6.27 4.14 0.00 14.46 0.00 0.00 0.00 0.00 0.00 47.40 0.00 18.35 0.00 0.00 100.00

0.00 0.00 15.09 8.56 4.92 0.00 15.17 0.00 0.00 0.00 0.00 0.00 40.40 0.00 15.85 0.00 0.00 100.00

0.00 0.00 8.55 6.25 2.62 0.00 7.94 0.00 0.00 0.00 0.00 0.00 30.36 0.00 44.28 0.00 0.00 100.00

0.00 0.00 14.05 8.72 3.18 0.00 8.52 0.00 0.00 0.00 0.00 0.00 26.45 0.00 39.09 0.00 0.00 100.00

Fatty acid chains

ACE

Short (C4-8) Medium (10. 12. 13) Long (C14-20) Unsaturated Saturated

%Wt

PCE %Mol

%Wt

SCE %Mol

%Wt

%Mol

3.81 3.94

6.65 5.64

9.39 10.42

15.09 13.48

8.55 8.87

14.05 11.90

92.25 66.45 33.55

87.72 62.38 37.62

80.20 18.35 81.65

71.43 15.85 84.15

82.58 44.28 55.72

74.06 39.09 60.91

Fatty acids were identified by their retention times.

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lipophilic components. In his study about the arrangement and the organization of membrane proteins, Frindlay (1987) has shown that there are several types of association between double layer of phospholipids and the integral membrane proteins. He presents a minimum of 5 types of protein integrations in cell membranes that certainly reflect the nature of their biosynthesis. There are many examples of the attachment of fatty acids to proteins. However, Frindlay (1987) reported that it is easier to prove that fatty acids are associated with isolated membranous proteins than to establish that the association is of the covalent type or to explain the position or the exact nature of this bond. The detection of fatty acids by gas chromatography confirms the presence of these acids but certainly not the type of bond that they have with the isolated proteins. The first and clearest demonstration comes from Hantke and Braun (1973) where they showed that the terminal amines of the constituent lipoprotein of the Gram-negative cell walls are build mainly with palmitic acid (C16). Furthermore, the side-chains and the cysteine N-terminal groups are coupled with diacyl glycerol by a thioether bond. The presence of fatty acids in the crude extract could also be attributed to the dispersive force of LiCl, a chaotropic compound (Thatcher et al., 1996), favouring the almost total disorganization of the bacterial structure. From this point of view, some lipophilic elements that have affinities with most of the denatured proteins are found in the supernatant. The release of hydrolases at the end of the exponential phase of growth effectively points out that these enzymes are implicated in the cell wall synthesis and in cell division. (Tomasz, 1984; Koch, 1991). Their presence in the culture medium can also explain the stationary stage and the decrease in the growth curve. In this case, they would act as growth inhibitors (Dako et al., 2003b). According to the analysis of protein release during growth (Fig. 7) the presence of PA49.5 activity in peak II at the end of the exponential phase of growth partly accounts for the presence of this second peak of protein release. This phenomenon suggests that, at the end of the exponential stage of growth, there is a release of hydrolases characterizing the end of the synthesis of peptidoglycan, an essential constituent of the new wall. According to the studies of Mou et al. (1976) and Dako et al. (2003a) regarding the localization and specificity of autolytic activity during cell division, the autolysins found in the division zone were firmly bound to, or were an integral part of the new wall. Those observations suggest that the autolysins are localized at or in the most recently synthesized wall, or they are involved in the integration of the new wall (Shockman et al., 1967). Moreover, they are responsible for the sensitivity of cells collected during the exponential stage of growth (Shockman et al., 1967; Tomasz, 1984; Lortal et al., 1989; Valence and Lortal, 1995; Dako et al., 1995). Also, Koch (1991) pointed out the secretion of two autolysins (glucosaminidase and amidase) through the cytoplasmic membrane during the growth of B. subtilis. The experiments by Fein and Rogers (1976) showed that autolysins could be transferred from one organism to the other. It could be hypothesised that the use of BigCHAP detergent tends to eliminate the ionic bonds involved in the formation of the fatty acids–protein complexes. Based on the sole criterion of solubility for example, the salt-bridge model could also be rejected. The detergent action itself can also be responsible for the release of the carboxylic acids from, for example, supramolecular inclusion complexes involving this class of compounds (Lehn, 1997). In this respect many lipophilic protein cavities can host the long aliphatic chains of the carboxylic acids, as opposed to the hydrophilic cavities which rather will host the carboxylic group ends of the same acids. After centrifugation, one observes that from the 66.45% of unsaturated fatty acids in the ACE, only 18.35% and 44.28% respectively are found in the precipitate and in the supernatant as witnessed by the presence of linoleic acid (C18:3). However, more long-chained fatty acids, and particularly stearic acid (C18), are found in the precipitate as well as in the supernatant. Thus, one can assume that the presence of the six fatty acids detected in the precipitate is somehow related to the insolubility of

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PA49.5. Even after centrifugation, which would normally eliminate at least part of the fatty acids, some fatty acids were still found in the precipitate and could have developed some interactions with PA49.5 that were difficult to eliminate. A thermic reaction (100 °C/2 min) was necessary to break them off in the presence of 0.1% CHAPS so as to solubilize PA49.5 (Dako et al., 2003b). The use of 95% of ethanol did not the solubilization of more than 5% of insoluble substances (teichoic or lipoteichoic acid) (Brown, 1972). Nevertheless, in this present work, we were able to solubilize 99% of PA49.5 in the presence of 0.1% of the nonionic detergent BigCHAP. 5. Conclusion The micro-purification of PA49.5 from a SDS-BigCHAP gel provides new perspectives concerning the characterization of lytic enzymes of lactic bacteria that are generally insoluble. This easy and quick process will allow an easier access to membranous hydrolases that are difficult to purify. Since most Lactococcus are rich in ripening enzymes, their spontaneous lysis due to the stimulated action of PA49.5 will favour the availability of theses enzymes resulting in, for example, the accelerated ripening of cheese. This study demonstrates clearly that the purified autolysin has more effect on the lactic acid bacteria autolysis than the unpurified. Thus, 80% autolysis is observed after 5 min at 45 °C with the PA49.5 purified extract, compared to more than 48 h under the same conditions for the unpurified extract, as previously reported by Dako et al. (2003a). Likewise, knowing that those lytic enzymes are produced during the end of exponential growth phase represents an important way to ensure their purification. It would definitely be much more beneficial to be use at this stage of the process of the growth. Moreover, the lactic acid bacteria are more fragile when harvested at the exponential phase of growth stage. We have also shown that the PA49.5 does not contain any disulphide bridge nor any free cysteine, since the β-mercaptoethanol did not have any effect on it. This molecular characteristic represents an advantage for the prebiotic PA49.5 purification, isolation and detergent action. PA49.5 will be therefore much less sensitive to the detergents that act on the cysteine–cysteine equilibrium in S-bound proteins or enzymes (disulphide bridge). This study also showed the presence of fatty acids such as C8, C10, C12, C14, C18:0, C18:1, C18:2 and C18:3 in the extracts of L. lactis subsp cremoris. We believe that the presence of these fatty acids was responsible for PA49.5 insolubility in the first three steps of the purification process. In addition, their presence partially rationalises the membrane insolubility of proteins in general and that of PA49.5 in particular. Finally, this new method allowed the purification of PA49.5 by a factor of 3 045 times with a purification yield of 52%. A partial characterisation of the enzyme found that it has an optimal pH of 7.5 and an optimal temperature of 45 °C. Acknowledgements We are indebted to the Laboratory of Biotechnology of School of Food Science, Nutrition and Family Studies, University of Moncton (NB, Canada). This project was also supported by the Université de Moncton, Faculty of Research and Graduate Studies and Laval University, Department of Food Science and Nutrition. We thank Marlene Spence, Nicole Haché, Prisca Antoine and Sheila Antle for assistance and technical help. References Aoshuang, X., Sluszny, C., Yeung, E.S., 2005. 25th International Symposium on Chromatography, 16 September 2005. Journal of Chromatography A 1087 (1–2), 177–182.

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