FEMS Microbiology Letters 180 (1999) 55^60

Tyrosine decarboxylase activity of Lactobacillus brevis IOEB 9809

isolated from wine and L. brevis ATCC 367
V. Moreno-Arribas, A. Lonvaud-Funel *
Laboratoire de Biotechnologie et Microbiologie Appliquëe. Facultë d'Oenologie, Unitë Associëe INRA-Universitë Victor Segalen Bordeaux 2,
351, cours de la Libëration, 33405 Talence cedex, France

Received 13 July 1999; received in revised form 8 September 1999; accepted 10 September 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-5P-phosphate (PLP)-dependent and it was stabilized by the
substrate and coenzyme. In contrast, glycerol and L-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. ß 1999 Federation of European Microbiological Societies. Published
by Elsevier Science B.V. All rights reserved.

Keywords : Tyrosine decarboxylase ; Tyramine; Wine; Lactic acid bacterium; Lactobacillus brevis

1. Introduction becoming an economic problem directly linked to the

in£uence of these compounds on health. In addition
In fermented foods such as cheese, wine, sausages to their own physiological e¡ects, their toxicity may
and sauerkraut, the production of biogenic amines be potentiated by the other amines and ethanol [4,5].
mainly results from the presence of lactic acid bac- The case most studied is histidine decarboxylase
teria (LAB) that can produce biodegradative en- (HDC) which catalyzes the formation of histamine.
zymes decarboxylating the corresponding amino Since HDC is speci¢c for histidine [6], the other
acids [1^3]. amines are produced by the other enzymes: i.e. tyr-
The occurrence and hazard levels of biogenic amine by tyrosine decarboxylase (TDC). The struc-
amines, such as histamine and tyramine, in foods is tural and functional properties of HDC have been
extensively studied in a variety of prokaryotic organ-
isms, Lactobacillus 30a [7], Lactobacillus buchneri,
Clostridium perfringens [8], Micrococcus sp. [9], Pho-
* Corresponding author. Tel.: +33 5 56 846466; Fax: +33 5 tobacterium histaminum [10] and Oenococcus oeni
56 846468; E-mail: [email protected] [11]. With respect to TDC in bacteria, investigations

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 6 0 - 7

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56 V. Moreno-Arribas, A. Lonvaud-Funel / FEMS Microbiology Letters 180 (1999) 55^60

to date have been limited to the TDC from Strepto- in 0.2 M sodium acetate bu¡er at pH 5.0 and sus-
coccus faecalis [12^14]. pended in 20 ml of the same bu¡er. One milliliter of
In a recent study in our laboratory (unpublished the bacterial suspension at OD 0.6 was equivalent to
results), a tyramine-producing strain identi¢ed as approximately 1U108 cfu.
Lactobacillus brevis (IOEB 9809) was isolated from
a wine with high concentrations of tyramine. In this 2.4. Cell-free extract preparation
work, we report the main characteristics of its TDC
activity and compare it to the enzyme of L. brevis The bacterial suspension was homogenized and
ATCC 367, also a tyramine producer. The condi- the cells were disrupted with an ultrasonic disintegra-
tions for enzyme stability and the in£uence of several tor (MSE Scienti¢c Instruments, Crawley, UK) at
e¡ectors common in wines were also determined. Ex- 150 W, 10U30 s with 30 s of pause, supplied with
periments were carried out on cells and crude ex- an thermostatic bath (4³C). The cell-free extract was
tracts. separated from the bacterial debris by centrifuging at
14 000Ug for 20 min at 4³C.

2. Materials and methods 2.5. Determination of protein concentration

2.1. Bacteria The proteins of the crude extract were determined

spectrophotometrically by the reaction with bicin-
The tyramine-producing strain from wine, L. bre- choninic acid (BCA Protein Assay, Pierce) [16] using
vis IOEB 9809, belonged to the bacteria collection of bovine serum albumin (BSA) standards for the cali-
the `Facultë d'Oenologie de Bordeaux' (IOEB). The bration curve.
reference strain from the ATCC (American Type
Culture Collection), L. brevis ATCC 367, was also 2.6. Measurement of enzymatic activity
investigated.
The TDC activity was determined by measuring
2.2. Medium and culture conditions the CO2 released from tyrosine with a speci¢c CO2
electrode (Eischweiler and Co., Kiel, Germany) by
The basal medium was MRS [15] containing per l: the method of Lonvaud and Ribëreau-Gayon [17].
yeast extract, 8 g; beef extract, 8 g; bactopeptone, 10 The reaction mixture contained tyrosine (3.6 mM)
g; sodium acetate, 5 g; Tris sodium citrate, 2 g; and pyridoxal-5P-phosphate (PLP) (400 WM) in 2.0
K2 HPO4 , 2 g; MgSO4 W7H2 O, 0.2 g; MnSO4 WH2 O, ml of 0.2 M sodium acetate bu¡er (pH 5.0). The
0.1 g; Tween 80, 1 ml. It was supplemented with L- experiments were conducted at 25³C and the reaction
tyrosine (1 g l31 ) and glucose (1 g l31 ) and adjusted was started by adding the bacterial suspension or the
at pH 5.0 with HCl. After sterilization by autoclav- cell-free extract containing the enzyme. Release of
ing for 20 min at 120³C, 1 l of medium was inocu- CO2 was monitored for 20 min and activity was ex-
lated with 10 ml of a preculture in the exponential pressed as nmol of CO2 released per min for 1U108
growth phase and incubated overnight at 25³C. cfu for resting cells and as nmol of CO2 released per
min per mg of protein for cell-free extracts. All the
2.3. Cell suspension preparation results are the means of two determinations.
For the determination of optimal pH, the follow-
When the optical density at 600 nm (932 Uvikon ing bu¡ers (0.2 M) were used: citrate-phosphate (pH
Spectrophotometer, Kontron) reached 0.6, the LAB 2.0^6.0); sodium acetate (pH 3.6^5.6); sodium phos-
of 1 l of culture were harvested by centrifugation phate (pH 5.6^8.0) and Tris (pH 7.2^9.0); pH values
(10 000Ug for 15 min at 4³C). The pellet was washed were adjusted with HCl or NaOH.

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3. Results and discussion

3.1. E¡ect of pH and kinetics of TDC reaction

The in£uence 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
min31 for 1U108 cfu and 541 nmol CO2 min31 Fig. 1. E¡ect of pH on TDC activity of cell suspension (a)
mg31 ) than the reference L. brevis ATCC 367 strain (nmol min31 for 1U108 cfu) and cell-free extracts (b) (nmol
(95 nmol CO2 min31 for 1U108 cfu and 190 nmol min31 mg31 protein).
CO2 min31 mg31 ), respectively for cell suspension
and cell-free extract. Kinetic parameters of the crude apparent Vmax for the conversion of tyrosine to tyr-
extract were determined at optimal pH, 5.0. TDC amine was two-fold greater for L. brevis IOEB 9809
exhibited a simple Michaelis-Menten kinetic (not from wine (115 nmol min31 mg31 protein) than for
shown) and the apparent Km values of L. brevis the ATCC 367 strain (66 nmol min31 mg31 protein).
ATCC 367 (0.58 mM) and IOEB 9809 (0.67 mM)
were very similar. These values are close to the Km 3.2. Substrate speci¢city
values described for S. faecalis, 0.6 mM [12] and 0.35
mM [18]. The pH range for TDC from S. faecalis The substrate speci¢city of TDC from L. brevis
was 2.5^6.0, with a maximum at pH 5.5 [13]. The strains was tested with di¡erent amino acids (histi-

Table 1
In£uence of tyramine, citric acid, L-lactic acid and ethanol on TDC activity
L. brevis ATCC 367 L. brevis IOEB 9809
Cell suspension Cell-free extract Cell suspension Cell-free extract
Tyramine (mM) 0 100 100 100 100
1 88 97 93 96
5 79 85 81 89
10 49 60 57 77
25 32 46 45 65
50 20 38 35 60
100 14 31 30 58
Citric acid (mM) 0 100 100 100 100
1 100 100 100 100
2 87 95 84 100
4 40 48 55 65
L-lactic acid (mM) 0 100 100 100 100
10 100 100 100 100
20 96 96 93 98
80 53 65 69 72
Ethanol (%) 0 100 100 100 100
8 100 100 100 100
12 82 100 89 100
20 80 96 80 98
Activity is expressed as a percentage of control without e¡ector

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58 V. Moreno-Arribas, A. Lonvaud-Funel / FEMS Microbiology Letters 180 (1999) 55^60

dine, lysine, phenylalanine, tryptophan and orni- 3.3. E¡ect of PLP on TDC activity
thine; ¢nal concentration 5 mM in 0.2 M sodium
acetate bu¡er, pH 5.0) potential precursors of the The enzymatic activity TDC from L. brevis was
other biogenic amines in wine via decarboxylation. enhanced at pH 5.0 with PLP (not shown), as for
Under these conditions, none of these compounds TDC from S. faecalis [12]. Although a high level of
was decarboxylated by TDC. activity was detected even without addition of PLP
The decarboxylase activity of L. brevis TDC to- (around 200 U for IOEB 9809 and 80 U for ATCC
ward L-DOPA (3.6 mM) as substrate was also deter- 367), in its presence the activity of the cell extracts
mined at pH 5.0. At this pH, L-DOPA was decar- increased about three-fold for L. brevis IOEB 9809,
boxylated with a relative activity of 18 (IOEB 9809) showing a high stimulation for a concentration range
and 22% (ATCC 367) when compared with L-tyro- over 0^100 WM of PLP. For L. brevis ATCC 367, it
sine (100%). In further experiments, both strains was enhanced about two-fold, but reached an activ-
were inoculated in MRS broth (Section 2) enriched ity plateau at 100 WM of cofactor.
with DOPA (1 g l31 ) instead of tyrosine and the
tyramine formed was determined by HPLC (unpub- 3.4. In£uence of tyramine and several wine e¡ectors
lished results) 5 days after. The TDC activity was on TDC activity
also quanti¢ed after this incubation time. Results
are compared to those obtained in the tyrosine sup- The e¡ect of tyramine, the product of decarboxy-
plemented MRS medium. Since tyramine concentra- lation of tyrosine, was assayed over a concentration
tions were around 20 times lower in DOPA-supple- range from 1 to 100 mM (Table 1). The TDC activ-
mented medium, it suggests that the amine was ity decreased in the presence of the amine, but it was
produced from the tyrosine of the MRS components more a¡ected for the cell suspension than for the
(yeast extract, beef extract, etc.). Moreover, TDC cell-free extract in both strains. Histamine showed
proved to be 50-fold more active toward tyrosine the same e¡ect on HDC activity and the inhibition
than toward DOPA. No dopamine was detected in of the antiport histidine/histamine of the cell mem-
the DOPA-added MRS. Investigations of TDC in brane has been proposed to explain this [11]. In the
plants [19,20] have demonstrated that the enzyme present work, the transport of tyrosine by the cell
shows similar a¤nities for tyrosine and DOPA. membrane might also be involved, but, we have
The TDC from S. faecalis has also been reported not found any references concerning the mechanism
to exhibit no distinct preference for either substrate of this transport. The enzymatic activity TDC of L.
[14]. However, L-DOPA was not a suitable substrate brevis ATCC 367 was more strongly inhibited by
for L. brevis TDC activity. tyramine than that of the other strain. The mode

Table 2
Relative stability for TDC activity of free-cell extracts stored in several bu¡ers at di¡erent temperatures
Addition agents Storage temperature (³C) Storage time (days)
0 1 3 6 15 30 60 90
a
Bu¡er 25 100 80 70 63 36 14 nd nd
4 100 77 78 69 55 30 18 nd
320 100 100 100 100 73 63 42 20
Bu¡era + glycerol (24%) 25 100 83 64 40 13 4 nd nd
4 100 73 69 60 49 26 nd nd
320 100 96 92 90 60 44 10 nd
Bu¡era + MCE (1 mM) 25 100 79 38 10 4 nd nd nd
4 100 72 56 12 7 nd nd nd
320 100 100 73 27 10 nd nd nd
nd: not detected ; MCE : L-mercaptoethanol
a
0.2 M Sodium acetate bu¡er containing 0.1 mM EDTA, 200 WM PLP and 3.6 mM L-tyrosine

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3.5. Enzyme stability

Concerning TDC stability, a progressive loss of

activity was observed when crude extracts were
stored at 4³C. PLP-dependent amino acid decarbox-
ylases, and particularly TDC, are characterized by
this apparent lability [14,20]. Therefore, experimental
conditions for improving TDC stability were estab-
lished using L. brevis IOEB 9809. Preliminary assays
with addition of PLP (200 WM) or L-tyrosine (3.6
Fig. 2. Stability of TDC of cell-free extracts from L. brevis IOEB mM) to the storage bu¡er (0.2 M sodium acetate,
9809 as a function of storage time in the absence (b) and pres- pH 5.0) did not a¡ect the initial enzymatic activity
ence of 200 WM PLP (F) or 3.6 mM L-tyrosine (R), at 320³C. of cell extract, but maintained it for several months
The storage bu¡er was 0.2 M sodium acetate, pH 5.0, containing
0.1 mM EDTA.
at 320³C (Fig. 2). In contrast, for preparations in
acetate bu¡er alone, the loss of activity was 85% and
60% after 24 h respectively at 4³C or 320³C. When
of inhibition was determined in the presence of 1, 5, both PLP and tyrosine were added to the storage
10 and 50 mM of the amine. Tyramine acted as a bu¡er, the enzyme showed a half-life of around 6 d
competitive inhibitor of TDC with an apparent in- stored at 25³C and of around 15 d at 4³C (Table 2).
hibition constant (Ki ) of 8 mM for strain ATCC 367 Furthermore, it was stable for at least two months at
and of 13 mM for IOEB 9809. 320³C. This suggests that PLP stimulates decarbox-
We also studied the in£uence on TDC activity of ylation of tyrosine and besides protects the TDC
several compounds naturally present in wine (citric enzyme against inactivation during storage. At-
acid, lactic acid and ethanol) (Table 1). Citric acid tempts to stabilize TDC further by the addition of
and lactic acid inhibited TDC activity, but to a lesser glycerol and L-mercaptoethanol (MCE) were not
extent for cell-free extract. Again, a lower inhibition successful (Table 2). On the contrary, the enzyme
was caused on the TDC from strain IOEB 9809. In lost activity more rapidly in the presence of these
wine, citric acid is metabolized during MLF. At the agents. In the most favorable conditions (320³C),
maximal concentrations encountered in wines (2 after 15 d, 60% of the relative activity remained in
mM), citric acid had practically no e¡ect on cell ex- the presence of glycerol and only 10% with MCE,
tract and it only slightly decreased TDC activity on while 73% of the decarboxylase activity was main-
whole cells. However, lactic acid is the main product tained in the absence of both of them. For the en-
resulting from MLF; around to 2 g l31 (20 mM) are zyme of S. faecalis [12,14], PLP, L-tyrosine, glycerol
formed after this transformation. At this concentra- and MCE act as protective agents. For L. brevis
tion, lactic acid did not signi¢cantly inhibit the de- TDC, the addition of PLP and L-tyrosine to the
carboxylation of tyrosine either for cell suspension storage bu¡er is essential, yet a considerable inhi-
or crude extract. Results obtained with ethanol bition of the enzyme due to glycerol and particularly
over a range of 0 to 10% (v/v) showed that TDC MCE was observed.
activity was una¡ected. The addition of 12% ethanol Further research is being carried out in our labo-
(the average concentration in wine) resulted in a ratory in order to obtain more information about the
slight inhibition of the activity of cell suspension, structure and functional properties of the enzyme.
while there was no variation in cell-free extract. For this, a procedure for the puri¢cation of TDC
This could be due to the role of ethanol on the cell from L. brevis IOEB 9809 is being developed.
membrane, as has been previously reported for in-
hibition of HDC by ethanol [11]. These results pro-
vide evidence that, even at the highest levels of these Acknowledgements
compounds in wine, tyramine formation may not be
prevented. V. Moreno-Arribas is funded by the Agriculture

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60 V. Moreno-Arribas, A. Lonvaud-Funel / FEMS Microbiology Letters 180 (1999) 55^60

and Fisheries program (FAIR-98-5018; Grant Pro- [10] Okozumi, M., Hiraishi, A., Kobayashi, T. and Fuji, T. (1994)
Photobacterium histaminum sp. nov., a histamine-producing
posal 97-1292) of the European Commission.
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tidine decarboxylase activity of Leuconostoc oenos 9204. Food
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