Characterization of an acid phosphatase from Lactobacillus pentosus: regulation and biochemical properties

Authors


Y. Sanz, Instituto de Agroquímica y Tecnología de Alimentos (C.S.I.C), PO Box 73, 46100 Burjasot (Valencia), Spain (e-mail: yolsanz@iata.csic.es).

Abstract

Aims:  To screen for phosphatase and phytase activities in Lactobacillus isolated from diverse ecosystems and to determine the biochemical properties and the factors that regulate the synthesis of the enzyme responsible for these activities in the selected strain, Lactobacillus pentosus CECT 4023.

Methods and Results:  These activities were determined spectrophotometrically by using p-nitrophenyl phosphate and sodium phytate as substrates. They were maximal at the onset of the stationary phase of growth and repressed in the presence of high glucose concentration and inorganic phosphate. The enzyme responsible for these activities was an acid phosphatase (E.C.3.1.3.2.), with a molecular mass of 69 kDa. The activity was optimum at pH 5·0 and 50°C. It hydrolysed mono-phosphorylated substrates and phytate, albeit at lower rates. It was inhibited by iodoacetic acid, phenyl-methylsulphonyl fluoride, di-sodium pyrophosphate and Ca+2 while activated by Co+2 and low concentrations of l-ascorbic acid and EDTA.

Conclusions: Lactobacillus pentosus CECT 4023 produces a nonspecific acid phosphatase that hydrolyses a number of mono-phosphorylated substrates and phytate.

Significance and Impact of the Study:  The results suggest that the phosphatase from L. pentosus CECT 4023 could partly contribute to reduce the phosphorylation degree of phytate and its derivatives and, thereby, their anti-nutrient properties during fermentation processes.

Introduction

Lactic acid bacteria (LAB) are important in the fermentation processes of an array of foods, particularly in dairy products, fermented meats and sourdough bread. The overall activity of this bacterial group enhances the shelf-life and nutritional value of the final products and contributes to their unique organoleptic properties. LAB utilize carbohydrates by lactic acid fermentation, which constitutes their main metabolic trait. This bacterial group is also generally involved in peptide and amino acid metabolism by means of a complex set of peptidases and amino acid converting enzymes (Sanz et al. 1999; De Angelis et al. 2002; Sanz and Toldrá 2002; Vogel et al. 2002). The species best adapted to specific ecosystems show, however, subtle differences in their metabolic properties that seem to be triggered by ecological factors. These biochemical characteristics could explain their prevalence and contribution to the improvement of particular fermentation processes (Vogel et al. 1996). In this context, studies on (inducible) enzymes that face up to the nutritional situation in specific microenvironments, as in the case of phytate as a source of phosphate in cereal fermentations, are necessary for the understanding of particular traits of these bacteria that are relevant for their rational exploitation as starters.

Phytate (myo-inositol hexaphosphate) is the major source of phosphorus accumulated in plant seeds during their maturation. It is regarded as an anti-nutritional compound, as it is a chelating agent of proteins, amino acids and mineral cations, which reduces their dietary bioavailability (Skoglund et al. 1997; Zamudio et al. 2001; Lopez et al. 2002; Reddy 2002; Sandberg and Andlid 2002). In humans, retention of calcium, iron and zinc can be significantly decreased by high phytate diets, which is specially important for infants, children, elderly and people in clinical situations (Mendoza 2002). The phosphorylation degree of myo-inositol phosphates determines in which proportion the mineral absorption is inhibited, enhanced or not affected (Reddy 2002). The lower inositol phosphates (IP1−4) and myo-inositol are, on the other hand, recognized as beneficial through different biological roles (Shamsuddin 2002).

Phosphatases are enzymes ubiquitous in nature, which catalyse the hydrolysis of the C-O-P linkage of a wide variety of phosphate esters (Abdallah et al. 1999a,b). They are usually classified as acid or alkaline phosphatases (Greiner et al. 1993; Konietzny et al. 1995) depending on their optimum pH, but they can also be subdivided as alkaline phosphatases, high- , low-molecular weight acid phosphatases and protein phosphatases (Wyss et al. 1999). Phytases are a particular subgroup of phosphatases that hydrolyse phytates to myo-inositol and phosphoric acid in a stepwise manner forming myo-inositol phosphate intermediates (Vohra and Satyanarayana 2003). In cereal products, the use of micro-organisms with phosphatase and phytase activities or their enzymes improves the bioavailability of minerals and amino acids (Phillippy 1998). LAB are predominant components of the sourdough microflora and other food fermentations. However, these activities have been scarcely studied in this bacterial group. Overall, phosphatases from LAB are thought to be involved in the dephosphorylation of phospho-peptides and -proteins during dairy fermentations and in the hydrolysis of phytate and their derivatives during cereal fermentations (Akuzawa and Fox 1998; Abdallah et al. 1999a,b; Zamudio et al. 2001; De Angelis et al. 2003). In recent years, several general phosphatases have been purified and characterized from strains isolated from both fermented products while the identification of phytases is not well documented (Sreeramulu et al. 1996; Akuzawa and Fox 1998; Abdallah et al. 1999a,b; Zamudio et al. 2001; Fredikson et al. 2002; De Angelis et al. 2003). Currently, these activities have been assessed for few LAB and the studies on their biochemistry, physiology and technological functions are limited.

The first aim of this study was to screen a wide number of lactobacilli strains isolated from different ecosystems (most of them previously unexplored) in order to find phytase and phosphatase activities. Then, the environmental factors regulating the synthesis as well as the biochemical properties of the acid phosphatase responsible for these activities were determined for the selected strain Lactobacillus pentosus CECT 4023. The potential physiological and technological roles of the characterized enzyme during cereal fermentations are discussed.

Material and methods

Bacterial strains and culture conditions

The lactobacilli strains included in this study were wheat sourdough isolates (SD-1 to SD-10), meat isolates (Lactobacillus sakei 23K and L. sakei L115; Berthier et al. 1996; Sanz et al. 1998) and strains provided by the Colección Española de Cultivos Tipo (CECT, Spain; Lactobacillus plantarum CECT 220, Lactobacillus brevis CECT 216, Lactobacillus reuteri CECT 925, Lactobacillus gasseri CECT 4479, L. pentosus CECT 4023, Lactobacillus hilgardii CECT 4786, Lactobacillus agilis CECT 4131 and Lactobacillus graminis CECT 4017).

For activity assays and purification purposes, lactobacilli were grown in modified MRS broth (MRS-MOPS) in which inorganic phosphate was replaced by 0·65 g l−1 sodium phytate and 0·1 m 3-[N-morpholino] propanesulphonic acid (MOPS, Sigma-Aldrich, St Louis, MO, USA), and the contents of glucose, yeast extract and meat extract were reduced to 10, 2 and 4 g l−1, respectively, to reduce the final phosphate content in the culture medium and promote the enzyme synthesis. The medium was inoculated (1%) with an overnight culture and incubated at 30°C till cells reached the stationary phase of growth. Cells were harvested by centrifugation (6 000 g, 10 min, 4°C) and washed with 50 mm Tris–HCl, pH 6·5. The culture supernatant was kept for activity assays of enzymes possibly released into the extracellular medium. The cell pellets were suspended in 50 mm sodium acetate-acetic acid, pH 5·0, and also used for activity assays of possible intracellular or cell-envelope-associated enzymes.

The effect of the composition of the growth medium on enzyme activities was studied in a complex defined medium (CDM-L) and in the MRS-MOPS-based medium. The CDM-L was prepared according to Ledesma et al. (1977), but increasing the concentrations of glutamic acid, arginine, lysine, proline and glycine to 1 g l−1, including 10 g l−1 glucose and replacing inorganic phosphate by MOPS and sodium phytate. The MRS-MOPS broth was also used for this purpose but adding different concentrations of glucose (5, 10 or 20 g l−1) or different sources of phosphorus (K2HPO4 or sodium phytate).

Enzyme activities

Acid phosphatase activity was determined using p-nitrophenyl-phosphate (Fluka Chemie, Buchs, Spain) as substrate. The reaction mixture consisted of 62·5 μl of 50 mm sodium acetate-acetic acid (pH 5·0) containing 6 mm substrate and 62·5 μl of enzyme sample. After incubation for 15 min at 50°C, the reaction was stopped by addition of 125 μl of 1 m NaOH. The p-nitrophenol released was determined by measuring the absorbance at 405 nm in a multi-well plate spectrophotometer (SPECTRAmax 190, Microplate Spectrophotometer; Molecular Devices, Sunnyvale, CA, USA). Samples in which the enzyme was added after the addition of NaOH solution were used as controls. One unit of phosphatase activity (U) was defined as the amount of enzyme that produces 1 μmol of p-nitrophenol per hour at 50°C.

Phytase activity was determined using sodium phytate as substrate (Sigma-Aldrich). The reaction mixture consisted of 100 μl of 50 mm sodium acetate-acetic acid (pH 5·0) containing 1·2 mm substrate and 50 μl of enzyme sample. After 90 min of incubation at 45°C, the reaction was stopped by addition of 25 μl of 20% (w/v) trichloroacetic acid (TCA) solution. Samples in which the enzyme was added after the addition of 20% (w/v) TCA solution were used as controls. The inorganic phosphorous released was quantified by the ammonium molybdovanadate method at 405 nm (Tanner and Barnett 1986; Haros et al. 2001a,b). One unit of phytase activity (U) was defined as the amount of enzyme that produces 1 μmol of inorganic phosphorous per hour at 45°C.

Purification

  • iPreparation of cell-free extracts. Cells from 1·5-l batch cultures were harvested by centrifugation (6000 g, 10 min, 4°C), washed with 50 mm Tris–HCl (pH 6·5), and suspended in 50 mm sodium acetate-acetic acid (pH 5·0). Two volumes of glass beads (0·1 mm diameter; Sigma) were added per each volume of cell suspension. Cell disruption was carried out in a Bead Beater (Biospec Products, Washington, DC, USA) by 10 shakings for 30 s each with 5-min intervals on ice. Glass beads, unbroken cells and debris were removed by centrifugation (12 000 g, 10 min, 4°C) and the supernatant constituted the cell-free extract used for purification.
  • iiAnion-exchange chromatography. The cell-free extract was filtered through a 0·22 μm membrane filter and applied to a TSK gel DEAE-5PW (75 × 7·5 cm; TosoHaas, Barcelona, Spain) column, previously equilibrated with 20 mm Tris–HCl (pH 7·2). Proteins were eluted at 1 ml min−1 by applying a linear gradient from 200 to 300 mm NaCl in the same buffer. Twenty-five fractions of 1 ml were collected and assayed for phosphatase and phytase activities.
  • iiiGel filtration chromatography. The active fractions obtained from the previous chromatography step were pooled and 250 μl samples were applied to a TSKgel G3000 PWXL column (78 × 30 cm, TosoHaas). The column was equilibrated with 20 mm Tris–HCl (pH 7·5) containing 0·1 m NaCl. Proteins were eluted at 0·4 ml min−1 and 60 fractions of 0·4 ml were collected and assayed for enzyme activity.

Both chromatographic separations were carried out on an HPLC system (HP1090 Liquid Chromatograph, Hewlett Packard, Waldbronn, Germany) equipped with a 1040 HPLC- Detection System (Hewlett Packard).

Determination of molecular mass

The molecular mass of the enzyme was estimated by gel filtration chromatography, as previously described. The following proteins were used as standards (in kDa): α-lactoalbumin (14·4), trypsin inhibitor (20), carbonic anhydrase (30), ovoalbumin (43), bovine albumin (67), B phosphorylase (94), alcohol dehydrogenase (150) and β-amylase (200).

Determination of protein concentration

The protein concentration was measured by the method of Bradford (1976), using bovine serum albumin as standard. Protein fractions eluting from the chromatographic separations were monitored at 280 nm.

Effects of temperature and pH

The effect of temperature on phosphatase activity was measured in the range from 37 to 60°C according to the standard assay. The effect of pH on phosphatase activity was determined at pH 0·5 intervals in the range from 4 to 6 by incubation at optimum temperature according to the usual procedure. The buffers used were the following: sodium citrate-NaOH, pH 3·0–5·5; 50 mm sodium acetate-acetic acid, pH 4·5–5·5, and 50 mm MES-NaOH, pH 6·0.

Effect of potential activator or inhibitors

The effects of potential activators or inhibitors on phosphatase activity were determined by addition of each chemical compound at 1 and 10 mm concentrations to the reaction mixture. The activity was measured as described above and expressed as a percentage of the activity obtained in the absence of any added compound.

Substrate specificity

The relative activities of the purified phosphatase against different phosphate esters at 6 mm was determined by measuring the released inorganic phosphorous as previously described for the phytase assay. The activities were expressed as a percentage of the activity obtained against the substrate hydrolysed at the highest rate.

Kinetic parameters

Kinetic parameters of the purified phosphatase were estimated for p-nitrophenyl-phosphate by using concentrations ranging from 1 to 6 mm. Activities were determined by the standard procedure and kinetic parameters were calculated from Lineweaver-Burk plots.

Results

Screening for phosphatase and phytase activities in lactobacilli strains

The phosphatase and phytase activities of several lactobacilli strains belonging to different species and isolated from various ecosystems were tested in whole-cell suspensions (Table 1). The studied strains showed remarkable differences in their specific activity levels especially against p-nitrophenyl-phosphate. The tested strains of the species L. pentosus CECT 4023, L. reuteri CECT 925 and L. agilis CECT 4131 showed the highest phosphatase activities while those of the remaining strains, including the sourdough isolates, were lower than 15% of the value of the most active strain. The strains of L. pentosus CECT 4023, L. gasseri CECT 4479 and the sourdough isolate SD-4 displayed the highest activities against sodium phytate. In general, low levels of activity against p-nitrophenyl-phosphate were correlated with low levels of activity against sodium phytate except for L. agilis CECT 4131, only endowed with significant phosphatase activity and the sourdough isolate SD-4, which showed the opposite trend. Phosphatase and phytase activities were not detected in the extracellular medium of any of the studied lactobacilli strains. Amongst all tested strains, L. pentosus CECT 4023 was selected for further studies on the basis of its highest activity against the specific substrates for both type of enzymes.

Table 1.  Phosphatase and phytase activities of several lactobacilli strains
StrainsOriginPhosphatasePhytase
Specific activity (U/mg prot.)* Activity (U ml−1)Specific activity (U/mg prot.)* Activity (U ml−1)
  1. *Specific activities (μmol h−1/mg prot.) were expressed as a percentage of those obtained with L. pentosus CECT 4023, which were given a value of 100%.

SD-1Sourdough7·53·30·40·2
SD-2Sourdough3·91·50·60·2
SD-3Sourdough2·91·60·20·1
SD-4Sourdough5·02·41·40·7
SD-5Sourdough3·81·70·60·3
SD-6Sourdough6·53·10·50·2
SD-7Sourdough6·12·60·50·2
SD-8Sourdough7·93·60·50·2
SD-9Sourdough5·82·60·60·3
SD-10Sourdough4·92·20·60·3
L. plantarum CECT 220Maize silage4·52·30·70·3
L. brevis CECT 216Beer5·02·60·80·4
L. reuteri CECT 925Intestine55·245·50·90·7
L. gasseri CECT 4479Human gut11·15·61·20·6
L. pentosus CECT 4023Maize silage84·749·41·60·9
L. sakei L 115Meat3·61·50·40·2
L. sakei 23KMeat3·21·30·00·0
L. hilgardii CECT 4786Wine4·92·70·80·4
L. agilis CECT 4131Sewage52·927·70·10·0
L. graminis CECT 4017Grass silage4·12·10·20·1

Effect of growth phase and medium composition on phosphatase and phytase activities

The activities of L. pentosus CECT 4023 against p-nitrophenyl-phosphate and sodium phytate were growth phase-dependent (Fig. 1). These activities were maximal at the onset of the stationary phase of growth and, then, progressively decreased with time. The composition of the growth medium also had an important effect on L. pentosus CECT 4023 activities (Fig. 2). In MRS-MOPS containing sodium phytate, both activities were maximal at the lowest glucose concentration (0·5%). In the same medium (MRS-based medium), the presence of inorganic phosphate (Pi) in the form of dipotassium phosphate instead of MOPS and sodium phytate markedly reduced the activities against both substrates. This inhibitory effect was partially reversed by the simultaneous addition of sodium phytate, suggesting that the substrate could act as inducer. The phosphatase activity from L. pentosus CECT 4023 was maximal in CDM-L plus sodium phytate, whose residual concentration of inorganic phosphate was negligible (nondetected by the standard procedure) in comparison with that contained in MRS-MOPS (66 μg Pi ml−1). However, the growth yields of L. pentosus CECT 4023 in CDM-L as well as in MRS-MOPS containing only 0·5% glucose were quite low when compared with that obtained in last medium containing 1·0% glucose. For this reason, the last one was selected to grow L. pentosus CECT 4023 to obtain a high cell density for enzyme purification.

Figure 1.

Phosphatase and phytase activities of Lactobacillus pentosus CECT 4023 during different phases of growth in MRS-MOPS containing sodium phytate. The growth curve was determined by measuring absorbance at 660 nm (•) and the activities were assayed against p-nitrophenyl phosphate (bsl00001) and sodium phytate (bsl00066), respectively, according to the standard procedures (see Material and methods for details)

Figure 2.

Effect of the composition of the medium on phosphatase (a) and phytase activities (b) from Lactobacillus pentosus CECT 4023. Growth medium: (1–3), MRS-MOPS plus sodium phytate and 0·5, 1·0 and 2·0% glucose, respectively; (4) MRS plus K2HPO4; (5) MRS plus K2HPO4 and sodium phytate; (6) CDM-L-MOPS plus sodium phytate (see Material and methods for details)

Enzyme purification

A phosphatase from L. pentosus CECT 4023 was partially purified using a two-step chromatographic procedure (Table 2). From the first purification step on an anion-exchange column, a unique active peak against p-nitrophenyl phosphate was detected, eluting at 240 mm NaCl which was also slightly active against sodium phytate. This separation resulted in a 1·3-fold increase in specific activity with a recovery yield of 71·5%. From the gel filtration column also a unique peak with p-nitrophenyl phosphate hydrolysing activity was detected. The active fractions were pooled, concentrated by ultrafiltration through a 10-kDa cutoff membrane (Millipore, Bedford, MA, USA) and further used for biochemical characterization. The whole procedure resulted in a 16·1-fold increase in specific activity with a recovery yield of 2·4%.

Table 2.  Purification of an acid phosphatase from L. pentosus CECT 4023
Purification stepTotal protein (mg)Total activity (U)Specific activity (U/mg prot.)Purification (fold)Yield (%)
Cell extract13·57678·1649·971·0100·0
Anion exchange7·61485·0163·711·371·5
Gel filtration0·0317·8593·3311·92·6
Concentration by ultrafiltration0·0216·10805·0016·12·4

Molecular mass determination

The molecular mass of the native enzyme was estimated to be 69 kDa by gel filtration chromatography.

Effects of pH and temperature on enzyme activity

The enzyme was active over a pH range from 3·5 to 6·0, showing a maximum at pH 5·0. The enzyme retained above 50% of its optimal activity at pH 4·5 and 5·5 and around 20% at pH 4·0 and 6·0. The effect of the buffer composition on the activity was determined in the pH interval from 4·5 to 5·5 by using 50 mm sodium acetate-acetic acid and 50 mm sodium citrate-NaOH buffers. The acid phosphatase activity was fivefold higher in acetate buffer than in citrate buffer (data not shown). The enzyme activity was optimal at 50°C, when measured at pH 5·0. The isolated enzyme retained above 40% of its optimal activity in the temperature ranged from 37 to 60°C.

Effect of potential inhibitors and activators

The effects of various chemical compounds on the acid phosphatase activity are shown in Table 3. The sulphydryl-blocking reagent iodoacetic acid exerted an inhibitory effect while l-ascorbic acid (1 mm), which may act as reducing agent, enhanced the activity suggesting that sulphydryl groups could be important for the catalysis. Phenyl-methylsulphonyl fluoride (PMSF), which is a serine protease inhibitor, markedly reduced the enzyme activity especially at the highest concentration. The effects of chelating agents, EDTA and o-phenanthroline, depended on the concentrations. Both diminished the activity at 10 mm although EDTA caused about 30% activation at 1 mm. The effect of di-sodium pyrophosphate, previously described as competitive inhibitor of phosphatases, was also remarkable at both concentrations. The divalent cation Co+2 was a strong activator (336–1531%) while Ca+2 reduced the activity to 53–17% of the optimum.

Table 3.  Effect of chemical compounds on the acid phosphatase activity from L. pentosus CECT 4023
CompoundRelative activity (%)*
1 mm10 mm
MeanSDM†MeanSDM
  1. *Activities were expressed as a percentage of the activity obtained against p-nitrophenyl phosphate in the absence of any chemical compound, which was given a value of 100%.

  2. †The mean of two independent determinations and the standard deviation of the mean are given.

None100·00·00100·00·00
β-Mercaptoethanol110·03·1393·55·21
Di-sodium pyrophosphate65·78·938·41·29
Iodoacetic acid62·59·2373·16·72
PMSF72·25·609·20·57
o-Phenanthroline89·83·974·82·26
CoCl21530·59·21335·76·19
CaCl253·17·6817·31·13
l-Ascorbic acid125·93·0519·72·26
EDTA129·86·0014·51·70

Substrate specificity

The purified acid phosphatase showed broad specificity, hydrolysing a wide variety of monophosphorylated substrates and phytate (Table 4). The substrates hydrolysed at the highest rates were p-nitrophenyl phosphate and d-fructose-6-phosphate followed by d-glucose-6-phosphate. The enzyme showed less preference for other monophosphorylated substrates such as acetyl-phosphate and O-phospho-dl-serine. Sodium phytate (myo-inositol hexaphosphate) and its derivative d-myo-inositol-1-monophosphate were also hydrolysed to some extent (6–15%). Thus, a unique acid phosphatase in L. pentosus CECT 4023 could be responsible for the hydrolysing activities found initially in cell suspensions and cell-free extracts against both p-nitrophenyl phosphate and sodium phytate.

Table 4.  Substrate specificity of the acid phosphatase from L. pentosus CECT 4023
SubstrateRelative activity (%)*
MeanSDM†
  1. *Activities were expressed as a percentage of the activity obtained against p-nitrophenyl phosphate, which was given a value of 100%.

  2. †The mean of two independent determinations and the standard deviation of the mean are given.

p-Nitrophenyl phosphate100·000·00
Acetyl-phosphate10·941·63
d-Fructose-6-phosphate96·602·05
d-Glucose-6-phosphate58·113·04
O-phospho-dl-serine8·300·99
Sodium phytate6·040·64
d-myo-inositol 1-monoP15·090·35

Kinetic parameters

The Km and Vmax values obtained for p-nitrophenyl phosphate were of 2·44 μmol l−1 and 820·0 μmol h−1 per mg protein, respectively.

Discussion

Phosphatases are ubiquitous enzymes of broad specificity that have been recently identified in LAB, while phytases are a particular subgroup of phosphatases, with preference for phytate, that do not seem to be common in this bacterial group (Zamudio et al. 2001; Fredikson et al. 2002). Nevertheless, the specificity of both types of enzymes can partly overlap and the phosphatases produced by micro-organisms also have phytase activity (Simon and Igbasen 2002). In fact, the active sites of phytases show high homology to the active site residues of the members of the acid phosphatase group termed ‘histidine phosphatase’ (Vohra and Satyanarayana 2003). So far, the ability of LAB to degrade phytate and its derived products has been scarcely studied. This property has often been detected in strains from plant origin but not in those from dairy environments, although the number of species and strains tested is still rather limited (Sreeramulu et al. 1996; De Angelis et al. 2003).

The present study includes strains of Lactobacillus spp. typically present in different ecological niches, and most of them were explored for the first time. The strains isolated from ecosystems containing phytate, such as the intestinal- and plant-related isolates, showed the highest phytase and phosphatase activities. They are L. pentosus CECT 4023 isolated from silage, L. reuteri CECT 925 and L. gasseri CECT4479 isolated from human intestine and SD-4 isolated from whole wheat sourdoughs. Despite the fact that phosphatase seems to be a frequent enzyme in LAB, remarkable differences were found in the activity levels depending on the strain, whose physiological meaning remained to be determined. Bacterial phosphatases and phytases are either periplasmic or cell-associated enzymes, with the exception of the phytases described in Bacillus subtilis, Lactobacillus amylovorus and Enterobacter sp.4 which are extracellular (Vohra and Satyanarayana 2003). In all tested lactobacilli, the activities against specific substrates for phosphatases and phytases were not detected in the extracellular medium but associated to whole-cell suspensions. Accordingly, the acid phosphatase from Lactococcus lactis was confirmed to be a cell-membrane-associated enzyme (Akuzawa and Fox 1998). Overall, the levels of both activities seemed to be related suggesting that a unique enzyme could be responsible for the hydrolysis of both substrates, as anticipated (Zamudio et al. 2001).

The physiological and environmental factors affecting the production of these activities in Lactobacillus pentosus CECT 4023 were determined. The activities were maximal at the onset of the stationary phase as described for the phytase of L. amylovorus and Lactobacillus sanfranciscensis (Sreeramulu et al. 1996; De Angelis et al. 2003). The acid phosphatases from Gram-negative bacteria were also induced when cultures enter the stationary phase (Dassa et al. 1982). The carbon source present in the culture medium greatly influences the phytase production, glucose being normally the preferred substrate (Sreeramulu et al. 1996; Vohra and Satyanarayana 2003). In L. pentosus CECT 4023 the effect of various glucose concentrations was studied. The specific activities were maximal at the lowest glucose concentration (0·5%), suggesting that the synthesis of the enzyme(s) can respond to limiting concentrations of carbon source. Moreover, the biomass was reduced in the presence of 0·5% glucose and, therefore, the total activity recovered was higher when adding 1·0% glucose in the culture medium. This glucose concentration was also optimal for total production of phytase in L. amylovorus and Enterobacter sp.4, although values of specific activities were not reported (Sreeramulu et al. 1996; Vohra and Satyanarayana 2003). In general, the starvation for inorganic phosphate stimulates the production of these enzymes in yeast, moulds and bacteria (Sreeramulu et al. 1996; Thaller et al. 1997; Pandey et al. 2001; Quan et al. 2001; Vohra and Satyanarayana 2003). In L. pentosus CECT 4023, apart from this evidence, the inhibitory effect caused by the presence of inorganic phosphate in the growth medium could be partially restored by the simultaneous addition of sodium phytate, indicating that the substrate could act as an inducer. On the other hand, sodium phytate did not exert a stimulating effect on the enzyme production in yeast (Segueilha et al. 1993). In L. pentosus CECT 4023 the activities against specific substrates for phosphatases and phytases were regulated in a similar manner what indicate that a sole enzyme could be responsible for both. In fact, a unique peak of p-nitrophenyl phosphate hydrolysing activity, which was also active against sodium phytate, was detected after two consecutive chromatographic separations. However, the possibility that different enzymes eluted from the two chromatographic columns in the same fractions cannot be completely disregarded. The partial purified enzyme was characterized as an acid phosphatase.

The molecular mass determined for the native enzyme of L. pentosus CECT 4023 differs from the ones reported for other bacteria. Most of the characterized bacterial phosphatases such as those from L. plantarum DPC 2739, Lactobacillus curvatus and enteric bacteria have a molecular mass of 100–110 kDa (Thaller et al. 1997; Abdallah et al. 1999a,b). All are tetrahomodimers with a subunit molecular mass of about 25–27 kDa. However, lower as well as higher molecular masses than 100–110 kDa have been reported for the corresponding enzymes of other strain of L. plantarum and for L. sanfranciscensis and Lactococcus lactis, which appeared to be monomers (Akuzawa and Fox 1998; Zamudio et al. 2001; De Angelis et al. 2003).

Most bacterial acid phosphatases have an optimum pH in the range from 4·0 to 5·5 including those characterized from LAB (Akuzawa and Fox 1998; Abdallah et al. 1999a,b; Zamudio et al. 2001; De Angelis et al. 2003), according to what found for L. pentosus CECT 4023 enzyme. Even amongst the limited number of phosphatases characterized from LAB, major differences have been found in relation to its optimal temperature that range from 40 to 70°C (Abdallah et al. 1999a,b).

The response of L. pentosus CECT 4023 enzyme to possible inhibitors was quite similar to what it was found for the acid phosphatase of Lactococcus lactis (Akuzawa and Fox 1998). The opposite effects of iodoacetic acid, which is a sulphydryl-blocking reagent, and ascorbic acid, which is a reducing agent, suggested the involvement of sulphydryl groups in the catalysis. The effect of the chelating agents (EDTA and o-phenanthroline) varies greatly depending on the concentrations. These compounds were inhibitors of phosphatases from enteric bacteria, but stimulated or had no effect on the phosphatase activities of LAB (Thaller et al. 1997; Akuzawa and Fox 1998; Abdallah et al. 1999a,b; De Angelis et al. 2003). The stimulating effect of Co+2 as well as the inhibitory effect of Ca+2 exerted on the L. pentosus CECT 4023 enzyme has not been reported for acid phosphatases from other LAB, as most divalent cations did not exert any effect. Only Zn+2 was a strong inhibitor of a L. curvatus phosphatase and Hg+2 and Fe+2 of a L. sanfranciscensis enzyme (Abdallah et al. 1999a; De Angelis et al. 2003).

The purified enzyme from L. pentosus CECT 4023 shows preference for p-nitrophenyl phosphate as well as for simple phosphorylated carbohydrates. The regulation of the enzyme production responded to carbon and phosphate limitation. Therefore, a physiological role of this enzyme in the hydrolysis of organic phosphomonoesters related to energy metabolism cannot be disregarded if we take into consideration its specificity as well as the factors that control its biosynthesis. On the other hand, the hydrolysis (6–15%) of sodium phytate and its derivative, d-myo-inositol-1-phosphate, were notably higher to the relative hydrolysis found in enzymes from other LAB (Zamudio et al. 2001). The only exception is the recently characterized enzyme from L. sanfranciscensis (De Angelis et al. 2003). During cereal fermentations, phytases of broad specificity can readily liberate all five equatorial phosphate groups of phytic acid but myo-inositol monophosphates are accumulated (Wyss et al. 1999). Thus, a partial function of lactobacilli acid phosphatases in the degradation chain leading to the generation of myo-inositol and in the improvement of mineral solubility should be considered (Lopez et al. 2000; Zamudio et al. 2001), particularly, in the case of the L. pentosus CECT 4023 enzyme, whose synthesis is induced in the presence of phytate.

The evaluation of the role of the acid phosphatase from L. pentosus CECT 4023 in the hydrolysis of myo-inositol phosphates as well as the meaning of its biochemical and regulatory properties during cereal fermentations should be carried out; this can be a valuable trait provided by strains of this bacterial group when used as starters for the improvement of the nutritional value of cereal-based products.

Acknowledgements

This work was supported by Consejo Superior de Investigaciones Científicas (CSIC), Spain, AGL2002–04093-C03–02 ALI. The scholarship from C.S.I.C. to M.C. Palacios is acknowledged.

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