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Tyrosine decarboxylase activity of Lactobacillus brevis IOEB 9809 isolated from wine and L. brevis ATCC 367

V. Moreno-Arribas, A. Lonvaud-Funel
DOI: http://dx.doi.org/10.1111/j.1574-6968.1999.tb08777.x 55-60 First published online: 1 November 1999

Abstract

Tyramine, a frequent amine in wines, is produced from tyrosine by the tyrosine decarboxylase (TDC) activity of bacteria. The tyramine-producing strain Lactobacillus brevis IOEB 9809 isolated from wine and the reference strain L. brevis ATCC 367 were studied. At the optimum pH, 5.0, Km values of IOEB 9809 and ATCC 367 crude extracts for l-tyrosine were 0.58 mM and 0.67 mM, and Vmax was higher for the wine strain (115 U) than the ATCC 367 (66 U). TDC exhibited a preference for l-tyrosine over l-DOPA as substrate. Enzyme activity was pyridoxal-5′-phosphate (PLP)-dependent and it was stabilized by the substrate and coenzyme. In contrast, glycerol and β-mercaptoethanol strongly inhibited TDC. Tyramine competitively inhibited TDC for both strains. Citric acid, lactic acid and ethanol had an inhibitory effect on cells and crude extracts, but none could inhibit TDC at the usual concentrations in wines.

Keywords
  • Tyrosine decarboxylase
  • Tyramine
  • Wine
  • Lactic acid bacterium
  • Lactobacillus brevis

1 Introduction

In fermented foods such as cheese, wine, sausages and sauerkraut, the production of biogenic amines mainly results from the presence of lactic acid bacteria (LAB) that can produce biodegradative enzymes decarboxylating the corresponding amino acids [13].

The occurrence and hazard levels of biogenic amines, such as histamine and tyramine, in foods is becoming an economic problem directly linked to the influence of these compounds on health. In addition to their own physiological effects, their toxicity may be potentiated by the other amines and ethanol [4,5]. The case most studied is histidine decarboxylase (HDC) which catalyzes the formation of histamine. Since HDC is specific for histidine [6], the other amines are produced by the other enzymes: i.e. tyramine by tyrosine decarboxylase (TDC). The structural and functional properties of HDC have been extensively studied in a variety of prokaryotic organisms, Lactobacillus 30a [7], Lactobacillus buchneri, Clostridium perfringens [8], Micrococcus sp. [9], Photobacterium histaminum [10] and Oenococcus oeni [11]. With respect to TDC in bacteria, investigations to date have been limited to the TDC from Streptococcus faecalis [1214].

In a recent study in our laboratory (unpublished results), a tyramine-producing strain identified as Lactobacillus brevis (IOEB 9809) was isolated from a wine with high concentrations of tyramine. In this work, we report the main characteristics of its TDC activity and compare it to the enzyme of L. brevis ATCC 367, also a tyramine producer. The conditions for enzyme stability and the influence of several effectors common in wines were also determined. Experiments were carried out on cells and crude extracts.

2 Materials and methods

2.1 Bacteria

The tyramine-producing strain from wine, L. brevis IOEB 9809, belonged to the bacteria collection of the ‘Faculté d'Oenologie de Bordeaux’ (IOEB). The reference strain from the ATCC (American Type Culture Collection), L. brevis ATCC 367, was also investigated.

2.2 Medium and culture conditions

The basal medium was MRS [15] containing per l: yeast extract, 8 g; beef extract, 8 g; bactopeptone, 10 g; sodium acetate, 5 g; Tris sodium citrate, 2 g; K2HPO4, 2 g; MgSO4·7H2O, 0.2 g; MnSO4·H2O, 0.1 g; Tween 80, 1 ml. It was supplemented with l-tyrosine (1 g l−1) and glucose (1 g l−1) and adjusted at pH 5.0 with HCl. After sterilization by autoclaving for 20 min at 120°C, 1 l of medium was inoculated with 10 ml of a preculture in the exponential growth phase and incubated overnight at 25°C.

2.3 Cell suspension preparation

When the optical density at 600 nm (932 Uvikon Spectrophotometer, Kontron) reached 0.6, the LAB of 1 l of culture were harvested by centrifugation (10 000×g for 15 min at 4°C). The pellet was washed in 0.2 M sodium acetate buffer at pH 5.0 and suspended in 20 ml of the same buffer. One milliliter of the bacterial suspension at OD 0.6 was equivalent to approximately 1×108 cfu.

2.4 Cell-free extract preparation

The bacterial suspension was homogenized and the cells were disrupted with an ultrasonic disintegrator (MSE Scientific Instruments, Crawley, UK) at 150 W, 10×30 s with 30 s of pause, supplied with an thermostatic bath (4°C). The cell-free extract was separated from the bacterial debris by centrifuging at 14 000×g for 20 min at 4°C.

2.5 Determination of protein concentration

The proteins of the crude extract were determined spectrophotometrically by the reaction with bicinchoninic acid (BCA Protein Assay, Pierce) [16] using bovine serum albumin (BSA) standards for the calibration curve.

2.6 Measurement of enzymatic activity

The TDC activity was determined by measuring the CO2 released from tyrosine with a specific CO2 electrode (Eischweiler and Co., Kiel, Germany) by the method of Lonvaud and Ribéreau-Gayon [17]. The reaction mixture contained tyrosine (3.6 mM) and pyridoxal-5′-phosphate (PLP) (400 µM) in 2.0 ml of 0.2 M sodium acetate buffer (pH 5.0). The experiments were conducted at 25°C and the reaction was started by adding the bacterial suspension or the cell-free extract containing the enzyme. Release of CO2 was monitored for 20 min and activity was expressed as nmol of CO2 released per min for 1×108 cfu for resting cells and as nmol of CO2 released per min per mg of protein for cell-free extracts. All the results are the means of two determinations.

For the determination of optimal pH, the following buffers (0.2 M) were used: citrate-phosphate (pH 2.0–6.0); sodium acetate (pH 3.6–5.6); sodium phosphate (pH 5.6–8.0) and Tris (pH 7.2–9.0); pH values were adjusted with HCl or NaOH.

3 Results and discussion

3.1 Effect of pH and kinetics of TDC reaction

The influence of pH on the decarboxylation of l-tyrosine was investigated from pH 2.0 to 9.0 at a constant concentration of substrate (3.6 mM). TDC was active in the pH range 3.0–7.0 with an optimal activity at pH 5.0 for both cell suspension and cell-free extracts (Fig. 1), but strain L. brevis from wine (IOEB 9809) showed higher activity (354 nmol CO2 min−1 for 1×108 cfu and 541 nmol CO2 min−1 mg−1) than the reference L. brevis ATCC 367 strain (95 nmol CO2 min−1 for 1×108 cfu and 190 nmol CO2 min−1 mg−1), respectively for cell suspension and cell-free extract. Kinetic parameters of the crude extract were determined at optimal pH, 5.0. TDC exhibited a simple Michaelis-Menten kinetic (not shown) and the apparent Km values of L. brevis ATCC 367 (0.58 mM) and IOEB 9809 (0.67 mM) were very similar. These values are close to the Km values described for S. faecalis, 0.6 mM [12] and 0.35 mM [18]. The pH range for TDC from S. faecalis was 2.5–6.0, with a maximum at pH 5.5 [13]. The apparent Vmax for the conversion of tyrosine to tyramine was two-fold greater for L. brevis IOEB 9809 from wine (115 nmol min−1 mg−1 protein) than for the ATCC 367 strain (66 nmol min−1 mg−1 protein).

Figure 1

Effect of pH on TDC activity of cell suspension (◯) (nmol min−1 for 1×108 cfu) and cell-free extracts (•) (nmol min−1 mg−1 protein).

3.2 Substrate specificity

The substrate specificity of TDC from L. brevis strains was tested with different amino acids (histidine, lysine, phenylalanine, tryptophan and ornithine; final concentration 5 mM in 0.2 M sodium acetate buffer, pH 5.0) potential precursors of the other biogenic amines in wine via decarboxylation. Under these conditions, none of these compounds was decarboxylated by TDC.

The decarboxylase activity of L. brevis TDC toward l-DOPA (3.6 mM) as substrate was also determined at pH 5.0. At this pH, l-DOPA was decarboxylated with a relative activity of 18 (IOEB 9809) and 22% (ATCC 367) when compared with l-tyrosine (100%). In further experiments, both strains were inoculated in MRS broth (Section 2) enriched with DOPA (1 g l−1) instead of tyrosine and the tyramine formed was determined by HPLC (unpublished results) 5 days after. The TDC activity was also quantified after this incubation time. Results are compared to those obtained in the tyrosine supplemented MRS medium. Since tyramine concentrations were around 20 times lower in DOPA-supplemented medium, it suggests that the amine was produced from the tyrosine of the MRS components (yeast extract, beef extract, etc.). Moreover, TDC proved to be 50-fold more active toward tyrosine than toward DOPA. No dopamine was detected in the DOPA-added MRS. Investigations of TDC in plants [19,20] have demonstrated that the enzyme shows similar affinities for tyrosine and DOPA. The TDC from S. faecalis has also been reported to exhibit no distinct preference for either substrate [14]. However, l-DOPA was not a suitable substrate for L. brevis TDC activity.

3.3 Effect of PLP on TDC activity

The enzymatic activity TDC from L. brevis was enhanced at pH 5.0 with PLP (not shown), as for TDC from S. faecalis [12]. Although a high level of activity was detected even without addition of PLP (around 200 U for IOEB 9809 and 80 U for ATCC 367), in its presence the activity of the cell extracts increased about three-fold for L. brevis IOEB 9809, showing a high stimulation for a concentration range over 0–100 µM of PLP. For L. brevis ATCC 367, it was enhanced about two-fold, but reached an activity plateau at 100 µM of cofactor.

3.4 Influence of tyramine and several wine effectors on TDC activity

The effect of tyramine, the product of decarboxylation of tyrosine, was assayed over a concentration range from 1 to 100 mM (Table 1). The TDC activity decreased in the presence of the amine, but it was more affected for the cell suspension than for the cell-free extract in both strains. Histamine showed the same effect on HDC activity and the inhibition of the antiport histidine/histamine of the cell membrane has been proposed to explain this [11]. In the present work, the transport of tyrosine by the cell membrane might also be involved, but, we have not found any references concerning the mechanism of this transport. The enzymatic activity TDC of L. brevis ATCC 367 was more strongly inhibited by tyramine than that of the other strain. The mode of inhibition was determined in the presence of 1, 5, 10 and 50 mM of the amine. Tyramine acted as a competitive inhibitor of TDC with an apparent inhibition constant (Ki) of 8 mM for strain ATCC 367 and of 13 mM for IOEB 9809.

View this table:
Table 1

Influence of tyramine, citric acid, l-lactic acid and ethanol on TDC activity

L. brevis ATCC 367L. brevis IOEB 9809
Cell suspensionCell-free extractCell suspensionCell-free extract
Tyramine (mM)0100100100100
188979396
579858189
1049605777
2532464565
5020383560
10014313058
Citric acid (mM)0100100100100
1100100100100
2879584100
440485565
l-lactic acid (mM)0100100100100
10100100100100
2096969398
8053656972
Ethanol (%)0100100100100
8100100100100
128210089100
2080968098
  • Activity is expressed as a percentage of control without effector

We also studied the influence on TDC activity of several compounds naturally present in wine (citric acid, lactic acid and ethanol) (Table 1). Citric acid and lactic acid inhibited TDC activity, but to a lesser extent for cell-free extract. Again, a lower inhibition was caused on the TDC from strain IOEB 9809. In wine, citric acid is metabolized during MLF. At the maximal concentrations encountered in wines (2 mM), citric acid had practically no effect on cell extract and it only slightly decreased TDC activity on whole cells. However, lactic acid is the main product resulting from MLF; around to 2 g l−1 (20 mM) are formed after this transformation. At this concentration, lactic acid did not significantly inhibit the decarboxylation of tyrosine either for cell suspension or crude extract. Results obtained with ethanol over a range of 0 to 10% (v/v) showed that TDC activity was unaffected. The addition of 12% ethanol (the average concentration in wine) resulted in a slight inhibition of the activity of cell suspension, while there was no variation in cell-free extract. This could be due to the role of ethanol on the cell membrane, as has been previously reported for inhibition of HDC by ethanol [11]. These results provide evidence that, even at the highest levels of these compounds in wine, tyramine formation may not be prevented.

3.5 Enzyme stability

Concerning TDC stability, a progressive loss of activity was observed when crude extracts were stored at 4°C. PLP-dependent amino acid decarboxylases, and particularly TDC, are characterized by this apparent lability [14,20]. Therefore, experimental conditions for improving TDC stability were established using L. brevis IOEB 9809. Preliminary assays with addition of PLP (200 µM) or l-tyrosine (3.6 mM) to the storage buffer (0.2 M sodium acetate, pH 5.0) did not affect the initial enzymatic activity of cell extract, but maintained it for several months at −20°C (Fig. 2). In contrast, for preparations in acetate buffer alone, the loss of activity was 85% and 60% after 24 h respectively at 4°C or −20°C. When both PLP and tyrosine were added to the storage buffer, the enzyme showed a half-life of around 6 d stored at 25°C and of around 15 d at 4°C (Table 2). Furthermore, it was stable for at least two months at −20°C. This suggests that PLP stimulates decarboxylation of tyrosine and besides protects the TDC enzyme against inactivation during storage. Attempts to stabilize TDC further by the addition of glycerol and β-mercaptoethanol (MCE) were not successful (Table 2). On the contrary, the enzyme lost activity more rapidly in the presence of these agents. In the most favorable conditions (−20°C), after 15 d, 60% of the relative activity remained in the presence of glycerol and only 10% with MCE, while 73% of the decarboxylase activity was maintained in the absence of both of them. For the enzyme of S. faecalis [12,14], PLP, l-tyrosine, glycerol and MCE act as protective agents. For L. brevis TDC, the addition of PLP and l-tyrosine to the storage buffer is essential, yet a considerable inhibition of the enzyme due to glycerol and particularly MCE was observed.

Figure 2

Stability of TDC of cell-free extracts from L. brevis IOEB 9809 as a function of storage time in the absence (•) and presence of 200 µM PLP (▪) or 3.6 mM l-tyrosine (▲), at −20°C. The storage buffer was 0.2 M sodium acetate, pH 5.0, containing 0.1 mM EDTA.

View this table:
Table 2

Relative stability for TDC activity of free-cell extracts stored in several buffers at different temperatures

Addition agentsStorage temperature (°C)Storage time (days)
013615306090
Buffer251008070633614ndnd
4100777869553018nd
−2010010010010073634220
Buffer+ glycerol (24%)25100836440134ndnd
41007369604926ndnd
−20100969290604410nd
Buffer+ MCE (1 mM)251007938104ndndnd
41007256127ndndnd
−20100100732710ndndnd
  • nd: not detected; MCE: β-mercaptoethanol

  • 0.2 M Sodium acetate buffer containing 0.1 mM EDTA, 200 µM PLP and 3.6 mM l-tyrosine

Further research is being carried out in our laboratory in order to obtain more information about the structure and functional properties of the enzyme. For this, a procedure for the purification of TDC from L. brevis IOEB 9809 is being developed.

Acknowledgements

V. Moreno-Arribas is funded by the Agriculture and Fisheries program (FAIR-98-5018; Grant Proposal 97-1292) of the European Commission.

References

  1. [1].
  2. [2].
  3. [3].
  4. [4].
  5. [5].
  6. [6].
  7. [7].
  8. [8].
  9. [9].
  10. [10].
  11. [11].
  12. [12].
  13. [13].
  14. [14].
  15. [15].
  16. [16].
  17. [17].
  18. [18].
  19. [19].
  20. [20].
View Abstract