Dr T. Requena, Department of Dairy Science and Technology, Instituto del Frío (CSIC), Ciudad Universitaria, 28040 Madrid, Spain.
An intracellular esterase from Lactobacillus casei subsp. casei IFPL731 was purified 1000-fold by ion exchange chromatography and gel filtration chromatography. The relative molecular mass of the native enzyme was 105 kDa, while the subunit molecular mass was estimated to be 38 kDa. The esterase hydrolysed tributyrin and had a preference for esters of short-chain fatty acids (butyrate, caproate and caprylate), while it did not hydrolyse palmitate and sterate esters. The apparent Michaelis-Menten constant of the enzyme on p-nitrophenyl butyrate was 0·3 mmol l−1 while on p-nitrophenyl caprylate, it was 0·04 mmol l−1. The esterase was active over a broad range of pH and temperature values, and retained about 50% of maximal activity at pH 5·0 and 12 °C. Activity was strongly inhibited by 5 mmol l−1 phenylmethylsulphonyl fluoride, β-mercaptoethanol and N-ethylmaleimide, and was stimulated by Zn2+ at 1 mmol l−1.
Lipolysis occurs to a small degree in most cheese varieties and is only especially marked in mould-ripened varieties, due mainly to very active extracellular lipases produced by Penicillium roqueforti or P. camemberti, and in hard Italian cheeses manufactured with rennet paste containing pregastric lipase and esterases (Fox & Stepaniak 1993). Lactic acid bacteria are considered to be weakly lipolytic compared with the activities of psychrotrophs, micrococci or brevibacteria (Kalogridou-Vassiliadou 1984; Bhowmik & Marth 1990; Lambrechts et al. 1995), and little is known about their contribution to cheese lipolysis and flavour development.
Addition of selected strains of lactobacilli as adjuncts to starter cultures for semi-hard cheese manufacture has been shown to increase proteolysis and improve cheese flavour (Broome et al. 1991; Trépanier et al. 1991; Lynch et al. 1996) and in some studies, it has corresponded to an increase in free fatty acid content (Requena et al. 1992; Rodríguez et al. 1997). In preliminary assays, esterolytic and lipolytic activity of several strains of lactobacilli and enterococci were compared and Lact. casei subsp. casei IFPL731 was found to be the most esterolytic strain (unpublished data). In the present work, the isolation and characterization of an intracellular esterase from Lact. casei subsp. casei IFPL731 is described. The possible contribution of the esterase to the release of fatty acids in cheese ripening conditions is also discussed.
Materials and methods
Micro-organism and growth conditions
Lactobacillus casei subsp. casei IFPL731 from the Culture Collection of the Instituto del Frío (CSIC), Spain, was sub-cultured twice overnight in MRS broth (Oxoid) at 30 °C and then grown in the same medium (7 l) from an inoculum of 2% until late exponential growth stage (approximately 109 cfu ml−1). Cells were harvested by centrifugation (7500 g, 15 min, at 4 °C), washed twice and resuspended in 80 ml 0·05 mol l−1 Tris-HCl buffer, pH 7·5. The cell suspension was mixed (1 : 1, w/v) with glass beads (150–212 μm; Sigma) and shaken in an Osterizer blender (Sunbeam-Oster, Miami, USA) (four times for 4 min each at 4 °C). Glass beads, unbroken cells and cell debris were removed by centrifugation (14 000 g, 20 min, 4 °C). The clear supernatant fluid, which constituted the cell-free extract, was treated with DNase (5 μg ml−1) and RNase (2·5 μg ml−1) (Boehringer Mannheim) for 30 min at room temperature.
All chromatographic steps during the enzyme purification were carried out at 4 °C using an FPLC system (Pharmacia, Uppsala, Sweden).
First anion-exchange chromatography
The cell-free extract was filtered through 0·22 μm filters (Millipore, Bedford, MA, USA) and applied to a DEAE-Sepharose Fast Flow column (Pharmacia) (15 cm × 5 cm) equilibrated with 50 mmol l−1 Tris-HCl buffer, pH 7·5. Bound proteins were eluted with a linear gradient of 0–1 mol l−1 NaCl in the same buffer, at a flow rate of 10 ml min−1. Fractions (10 ml) were assayed for esterase activity.
Active fractions pooled from anion-exchange chromatography were concentrated in an ultrafiltration cell (Amicon Danvers, Beverly, MA, USA) using a 10 kDa Omega membrane (Filtron Technology, Northborough, MA, USA). Portions (200 μl) were loaded onto a Superose 12 HR 10/30 column (Pharmacia) previously equilibrated with 50 mmol l−1 sodium phosphate buffer, pH 7·0, containing 0·15 mol l−1 NaCl. Proteins were eluted with equilibrating buffer at a flow rate of 0·3 ml min−1.
Second anion-exchange chromatography
Active fractions (0·9 ml) from gel filtration were diluted threefold with 20 mmol l−1 Bis-Tris propane (Sigma) buffer, pH 6·5, and applied to a Mono Q HR 5/5 column (Pharmacia) equilibrated with the same buffer. Bound proteins eluted at a flow rate of 0·5 ml min−1 with a linear NaCl gradient, 0–1 mol l−1, in the equilibrating buffer. The active fraction was re-chromatographed on the anion-exchange Mono Q column at a flow rate of 0·3 ml min−1. Esterase activity was eluted at 0·37 mol l−1 NaCl and stored at – 80 °C for further studies.
Esterase activity was routinely tested spectrophotometrically using p-nitrophenyl butyrate (Sigma) as substrate. The reaction mixture contained 800 μl 50 mmol l−1 Tris-HCl buffer, pH 7·5, 100 μl substrate (8·1 mmol l−1 in acetone) and 100 μl enzyme solution. Enzyme activity was measured continuously for 10 min at 37 °C by monitoring the release of p-nitrophenol at 346 nm. One unit of enzymatic activity was defined as the amount of enzyme that releases one nanomole of p-nitrophenol (E346 = 4800 l mol−1 cm−1) per minute.
The substrate specificity of the enzyme was determined on β-naphthyl esters of C4–C18 fatty acids (Table 2). The reaction mixture (200 μl) consisted of 50 mmol l−1 Tris-HCl (pH 7·0), 100 μl enzyme solution and 7–20 μl β-naphthyl substrate (15 mmol l−1 C14–C18 in acetone and 5 mmol l−1 C4–C12 in methanol). After incubation for 4 h at 37 °C, 360 μl Fast Garnet GBC (Sigma) preparation (5 mg ml−1 in 10% SDS) were added to terminate the reaction, and the mixture was centrifuged at 11 000 g for 5 min. Absorbance was measured at 560 nm and, using a standard curve, the activity was expressed as μmol β-naphthol released min−1.
Table 2. Relative activity of the esterase from Lactobacillus casei subsp. casei IFPL731 on β-naphthyl esters of fatty acids
Tributyrin esterase activity was assayed following the method described by Calvo et al. (1996). The reaction mixture (10 ml) contained a final concentration of 68 mmol l−1 tributyrin, 0·1 mol l−1 NaCl, 0·1 mol l−1 CaCl2, 1 mmol l−1 Tris HCl, pH 7·2, and 3% acetonitrile as solubilizing agent. Reaction was started by the addition of 10–20 μl enzyme solution and was measured continuously at 37 °C by titration with 20 mmol l−1 NaOH using a pH-stat (Radiometer Copenhagen, Bagsvaerd, Denmark). One unit of enzyme activity was defined as the amount of enzyme that liberates one micromole of fatty acid per minute.
Protein concentration was determined according to the method of Lowry et al. (1951) using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as standard.
Polyacrylamide gel electrophoresis (PAGE)
SDS-PAGE under reducing conditions was performed using the Phast electrophoresis system (Pharmacia) according to the instruction manual. Electrophoresis was performed using 12·5% polyacrylamide gels and SDS buffer strips (Pharmacia). Non-denaturing PAGE was carried out with 12% polyacrylamide gels as described by Benoist & Schwencke (1990). Proteins were silver-stained and the molecular mass of the enzyme was estimated by reference to the migration of marker proteins LMW and HMW calibration kit standards (Pharmacia) for denaturing and native conditions, respectively.
Determination of pH and temperature optima and temperature stability
The effect of pH on the enzyme was measured at 37 °C in the pH range 3–10, using a universal buffer composed of boric acid (57 mmol l−1), citric acid (33 mmol l−1), NaH2PO4 (33 mmol l−1), NaOH (1 mol l−1), and varying amounts of 0·1 mol l−1 HCl. For determination of the temperature optimum, enzyme activity was assayed in the range 5–55 °C using 50 mmol l−1 Tris-HCl buffer, pH 7·0.
Heat stability was determined with aliquots (50 μl) of the enzyme diluted in 50 mmol l−1 phosphate buffer, pH 7·0, and heated in sealed capillary glass tubes at different temperatures (65–75 °C) for different periods of time. After heating, samples were cooled on ice and the remaining activity was measured at 37 °C.
Effect of metal ions and chemical reagents
A mixture (900 μl) containing 25 μl of the purified enzyme solution and the indicated concentration of each reagent or cation (see Table 3) in 50 mmol l−1 Tris-HCl buffer, pH 7·5, was pre-incubated for 30 min at 25 °C. Residual activity was assayed at 37 °C by adding 100 μl substrate solution (8·1 mmol l−1p-nitrophenyl butyrate in acetone), as described previously.
Table 3. Effect of metal ions (1 mmol l−1) and chemical reagents (5 mmol l−1) on the esterase activity of Lactobacillus casei subsp. casei IFPL731
Activity on p-nitrophenyl butyrate in absence of metal ion or chemical reagent was taken as 100%.
Phenylmethylsulphonyl fluoride (PMSF)
Enzyme solution was incubated at 37 °C with various concentrations of p-nitrophenyl butyrate or p-nitrophenyl caprylate ranging from 0·5 to 2 mmol l−1, and activity was measured continuously as described previously. A Lineweaver-Burk plot was constructed to calculate the apparent Michaelis-Menten constant (Km) and the maximum velocity of hydrolysis (Vmax).
Purification of the enzyme
A summary of the purification procedure is shown in Table 1. Application of the cell-free extract to the first anion-exchange chromatography (DEAE Sepharose) resulted in a single peak with activity on p-nitrophenyl butyrate eluting at 0·5 mol l−1 NaCl (results not shown). Elution of esterase activity on gel filtration chromatography corresponded to a single fraction, but it was included in the same peak as most of the proteins loaded onto the column. Nevertheless, a 26-fold increase in specific activity was attained. Final purification of the enzyme was obtained after a second anion-exchange chromatography in a Mono-Q column in a peak eluting at 0·37 mmol l−1 NaCl, and after re-chromatography (Fig. 1). The specific activity after the second Mono-Q chromatography was increased about 1000-fold compared with the cell-free extract fraction, with a final recovery of 4% (Table 1).
Table 1. Purification procedure for the esterase from Lactobacillus casei subsp. casei IFPL731
*Expressed as nmol p-nitrophenol released from p-nitrophenyl butyrate min−1.
†Activity expressed as nmol p-nitrophenol released min−1 and mg−1 protein.
Molecular mass after the last chromatographic step was estimated to be about 105 kDa by non-denaturing PAGE (Fig. 2). SDS-PAGE of the different fractions obtained during the purification of the esterase of Lact. casei subsp. casei IFPL731 revealed a band corresponding to an apparent molecular mass of 38 kDa (results not shown). These results suggested that the esterase may be a trimer in the native configuration.
Table 2 shows the relative activity of the esterase of Lact. casei subsp. casei IFPL731 on different β-naphthyl substrates. The esterase was most active on β-naphthyl caprylate; this activity was taken as 100%. The enzyme hydrolysed the C4 and C6 with a relatively high activity whereas it hydrolysed C12 and C14 with a low specificity. The enzyme did not hydrolyse palmitate and sterate esters (Table 2). The purified enzyme showed a notable activity on tributyrin which released 47 μmol butyrate min−1 and mg−1 protein.
Effects of pH and temperature
The enzyme was active over a broad range of pH and temperature values (Fig. 3). It retained more than 70% of maximum activity from pH 6–8·5; the pH optimum was at pH 7·5. At pH 7·0, the optimum temperature was in the range 25– 30 °C; between 12 and 37 °C, the relative activity of the esterase was higher than 60%. At pH 5 and 12 °C, the enzyme retained about 50% of maximal activity.
At pH 7·0, the enzyme was relatively stable, retaining about 45% of its original activity after heating at 60 °C for 1 min. The enzyme was completely inactivated by heating at 65°C for 30 s.
Effect of activators and inhibitors
The effects of divalent cations and chemical reagents on esterase activity are summarized in Table 3. Among the metal ions studied, enzyme activity was only enhanced by Zn2+, whereas Mg2+, Mn2+ and Co2+ caused a moderate inhibitory effect (all at 1 mmol l−1). Activity was strongly inhibited by the serine proteinase inhibitor, phenylmethylsulphonyl fluoride (PMSF), the reducing agent β-mercaptoethanol, and the sulphhydryl modifying agent N-ethylmaleimide (all at 5 mmol l−1). Moderate inhibition of the esterase was observed in the presence of 5 mmol l−1 of the metal-chelating agents EDTA and o-phenantroline (Table 3).
The apparent Michaelis-Menten constant (Km) and maximum velocity (Vmax) for the enzyme, using different concentrations of p-nitrophenyl butyrate and calculated from a Lineweaver-Burk plot, were 0·328 mmol l−1 and 75 μmol min−1 mg−1, respectively. The corresponding values using p-nitrophenyl caprylate as substrate were 0·038 mmol l−1 and 20 μmol min−1 mg−1, respectively.
Isolation of esterase activity from Lact. casei subsp. casei IFPL731 throughout all chromatographic steps gave no indication that more than one esterase was present, in contrast to other reports which have claimed that lactobacilli possess more than one esterolytic enzyme (Lee & Lee 1990; Gobbetti et al. 1997a). Addition of ammonium sulphate to pre-purified enzyme solutions and application to hydrophobic interaction columns (TSK-butyl or phenyl-Sepharose) led to a very poor recovery of activity. Therefore, this chromatographic step was omitted for the enzyme purification procedure. Strong reduction of esterase activity by ammonium sulphate has also been reported for lactococci by Holland & Coolbear (1996) and Chich et al. (1997).
The esterase was highly active over a broad range of pH (6–8·5) and temperature (12–37 °C) values. Other authors have also found pH and temperature optima for esterases from bacteria to be relatively broad (Chich et al. 1997; Gobbetti et al. 1997b; Rattray & Fox 1997). The enzyme retained about 50% of maximal activity at pH 5·0 and 12 °C and therefore, Lact. casei subsp. casei IFPL731 could contribute to cheese lipolysis if intracellular esterase is released during ripening. In contrast, 4% NaCl exerted a high inactivation effect on the enzyme (results not shown).
The esterase of Lact. casei subsp. casei IFPL731 exhibited preference for β-naphthyl esters of short-chain fatty acids (C4–C8) and remarkable activiy on tributyrin. Activity declined with medium- and long-chain fatty acid substrates. Kinetic studies of the enzyme at pH 7·0 and 37 °C have shown high affinity for the substrates p-nitrophenyl butyrate and p-nitrophenyl caprylate (Km 0·3 mmol l–1 and 0·04 mmol l–1, respectively). On a molar basis, butyric acid (C4) is present at more than 35% on the sn-3 position of the bovine milk fat triglyceride, which also contains, in this position, a variety of other short-chain fatty acids (Christie & Clapperton 1982). The preference for short-chain fatty acids described for esterases from lactic acid bacteria (Lee & Lee 1990; Østdal et al. 1996; Gobbetti et al. 1997a) seems to be similar to the highly sterospecific activity of pregastric esterases by the sn-3 position of milk fat triglycerides (Kwak et al. 1989). This means that the activity of the enzyme towards cheese fat would release short-chain fatty acids which, even at low concentrations, are important as flavour components during cheese ripening (Urbach 1993).
Inactivation of the esterase by PMSF suggests that a serine residue might be involved in the catalytic mechanism of the enzyme. It has been recognized that most of the proteins in the family of esterases and lipases have a Ser-Asp-His catalytic triad, similar to that observed in serine proteinases (Drablos & Petersen 1997). Inhibition by sulphhydryl modifying reagents such as p-hidroxymercuribenzoate, N-ethylmaleimide and iodoacetate, and by the reducing agent β-mercaptoethanol, was observed, indicating that sulphhydryl groups and disulphide bridges are important in maintaining an active conformation in the enzyme. Comparable behaviour has been described for the esterase purified from B. linens ATCC 9174 (Rattray & Fox 1997) and the lipase from Lact. plantarum 2739 (Gobbetti et al. 1997b). Moderate inhibition of the esterase from Lact. casei subsp. casei IFPL731 was observed in the presence of EDTA and o-phenantroline. A similar inhibitory effect by metal-chelating agents was found on the esterases purified from Lact. plantarum 2739 and Lact. fermentum DT41 (Gobbetti et al. 1997a,b). However, in these cases, Mg2+ and Ca2+ stimulated enzyme activity, while the esterase from Lact. casei subsp. casei IFPL731 showed a significant inhibition of activity in the presence of Mg2+, being only stimulated by Zn2+.
The esterase of Lact. casei subsp. casei IFPL731 may contribute positively to cheese flavour development as it shows marked activity on tributyrin and the β-naphthyl esters of short-chain fatty acids, and retains a high percentage of activity at the temperature and pH required for cheese ripening, although its sensitivity to NaCl would markedly reduce the activity. The intracellular location of this enzyme, together with the great variety of intracellular peptidases described for Lact. casei subsp. casei IFPL731 (Fernández de Palencia et al. 1997a,b; Fernández-Esplá & Martín-Hernández. 1997; Fernández-Espláet al. 1997a,b), makes this micro-organism a valuable adjunct to cheese starters, providing the medium, on autolysis of the cells, with a collection of enzymes capable of enhancing cheese proteolysis and lipolysis. Different mechanisms for inducing early autolysis of Lact. casei subsp. casei IFPL731 during cheese ripening are currently being investigated.
The authors acknowledge the financial support for this study from the Comisión Interministerial de Ciencia y Tecnología (Project ALI 97–0737) and the Comunidad Autónoma de Madrid (Project 06G/049/96). I. Castillo is the recipient of a scholarship from the Spanish Ministry of Education. The authors thank M.V. Calvo for performing the tributyrin esterase activity analysis.