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Applied and Environmental Microbiology, January 2000, p. 262-267, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Increased Production of Hydrogen Peroxide by
Lactobacillus delbrueckii subsp. bulgaricus upon
Aeration: Involvement of an NADH Oxidase in Oxidative
Stress
C.
Marty-Teysset,
F.
de la Torre, and
J.-R.
Garel*
Laboratoire d'Enzymologie et de Biochimie
Structurales du CNRS, 91198 Gif-sur-Yvette, France
Received 21 June 1999/Accepted 20 October 1999
 |
ABSTRACT |
The growth of Lactobacillus delbrueckii subsp.
bulgaricus (L. delbrueckii subsp.
bulgaricus) on lactose was altered upon aerating the
cultures by agitation. Aeration caused the bacteria to enter early into
stationary phase, thus reducing markedly the biomass production but
without modifying the maximum growth rate. The early entry into
stationary phase of aerated cultures was probably related to the
accumulation of hydrogen peroxide in the medium. Indeed, the
concentration of hydrogen peroxide in aerated cultures was two to three
times higher than in unaerated ones. Also, a similar shift from
exponential to stationary phase could be induced in unaerated cultures
by adding increasing concentrations of hydrogen peroxide. A significant
fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the
reduction of molecular oxygen by NADH catalyzed by an
NADH:H2O2 oxidase. The specific activity of
this NADH oxidase was the same in aerated and unaerated cultures,
suggesting that the amount of this enzyme was not directly regulated by
oxygen. Aeration did not change the homolactic character of lactose
fermentation by L. delbrueckii subsp.
bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no
(or a very small) energetic role and could be involved in eliminating oxygen.
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INTRODUCTION |
Lactobacillus delbrueckii
subsp. bulgaricus (L. delbrueckii subsp.
bulgaricus) is an important species of lactic acid bacteria currently used in the industrial production of fermented milk products.
L. delbrueckii subsp. bulgaricus is an
aerotolerant anaerobe that obtains most of its energy from homolactic
fermentation (12). It does not require strict anaerobic
growth conditions and tolerates the concentration of O2 in
air. Even though L. delbrueckii subsp. bulgaricus
does not use O2 in its energetic metabolism, it is likely
that the presence of oxygen in its environment can influence its
physiology. Indeed, some lactic acid bacteria possess oxidases that
utilize molecular oxygen to oxidize substrates such as pyruvate
(22) or NADH (2, 5, 8, 16, 23-25). As these
oxidation reactions cannot occur under anaerobic conditions, metabolism
in the presence of oxygen cannot be identical to that in the absence of
oxygen. Also, the activities of these oxidases can produce partially
reduced oxygen species such as the superoxide radical
(O2.
), hydrogen peroxide
(H2O2), the hydroxyl radical (HO.),
and other peroxyl radicals or peroxides that will cause an oxidative
stress in the cell. It is therefore expected that the presence of
oxygen will induce a specific cellular response to such oxidative
stress. In this work, a difference in the growth of L. delbrueckii subsp. bulgaricus in the presence and
absence of oxygen was observed. It was also found that L. delbrueckii subsp. bulgaricus could reduce oxygen into
hydrogen peroxide with an NADH oxidase, probably to eliminate the
oxygen present. However, this detoxification of oxygen led to an
overproduction of hydrogen peroxide that caused oxidative stress and
triggered an early entry of the cells into stationary phase.
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MATERIALS AND METHODS |
Chemicals.
NADH and NAD+ were purchased from
Boehringer Mannheim, sodium dithionite was purchased from Merck, and
thiamine pyrophosphate, flavin adenine dinucleotide (FAD), and hydrogen
peroxide were purchased from Sigma. Peroxidase (EC 1.11.1.7), pyruvate
oxidase (EC 1.2.3.3), catalase (EC 1.11.1.6), and superoxide dismutase (EC 1.15.1.1) were obtained from Boehringer Mannheim.
Bacterial strains and growth conditions.
L.
delbrueckii subsp. bulgaricus B107 was obtained from
the Centre International de Recherche Daniel Carasso, Danone group. Bacteria were grown at 42°C in modified MRS medium (6)
without manganese, supplemented with 2% lactose, initial pH 6.5. Unaerated and aerated cultures were grown under the same conditions of
medium and temperature, except that permanent aeration was provided to aerated cultures by vigorous shaking, whereas no shaking was used for
unaerated cultures.
Lactose metabolism by growing cells of L. delbrueckii
subsp. bulgaricus.
The concentrations of residual lactose
and accumulated D-lactate in the medium were determined at
different times with commercially available kits (Boehringer Mannheim),
after eliminating the cells by centrifugation for 4 min at 18,000 × g.
Determination of H2O2 production.
H2O2 concentrations were measured after
eliminating the cells by centrifugation for 4 min at 18,000 × g, as described by Green and Hill (9), from the
amount of quinoneimine formed by the oxidation of 4-aminoantipyrine and
phenol by hydrogen peroxide. The assay mixture contained 0.4 mM
phosphate buffer (pH 6.9), 2% H2O-saturated phenol, 0.4 mg
of 4-aminoantipyrine (Sigma) per ml, and 0.04 U of peroxidase per ml,
and the change in the absorbance was measured at 505 nm
(A505) with an extinction coefficient of 
= 6,400 M1 cm1 for the quinoneimine.
Preparation of crude extracts.
Cells in their early
stationary phase were harvested by centrifugation, washed with 100 mM
phosphate buffer (pH 6.9), and broken with alumina (ca. 2 g of
alumina per g of wet cells) in the same buffer. The crude extract was
obtained by centrifugation for 30 min at 18,000 × g at
4°C, and its protein concentration was determined according to
Bradford (4), with immunoglobulin G as a standard.
Enzyme assays.
NADH oxidase activity was determined
spectrophotometrically by measuring the initial rate of NADH oxidation
at 25°C with = 6,220 M1 cm1 as the
extinction coefficient of NADH at 340 nm. The assay mixture contained
100 to 250 µM NADH and 20 µM FAD in 50 mM phosphate buffer at pH 6. Pyruvate oxidase was assayed by the method of Risse et al.
(18) in a reaction mixture composed of 5 mM pyruvate, 2 mM
4-aminoantipyrine, 7 mM 2-hydroxy-3,5 dichlorobenzene sulfonate, and
0.1 U of peroxidase per ml in 50 mM phosphate buffer (pH 6.5) and 10%
glycerol. D-Lactate dehydrogenase (LDH) was assayed
according to Le Bras and Garel (14) in 400 µM NADH and 7.5 mM pyruvate in 0.4 M potassium acetate (pH 5.5) by the changes at
A340 with an optical path of 0.5 cm. For all enzymes
assayed, 1 U of activity was defined as the amount that converted 1 µmol of substrate per min at 25°C.
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RESULTS AND DISCUSSION |
Influence of aeration on the growth of L. delbrueckii
subsp. bulgaricus.
In the following discussion, the
unaerated cultures of L. delbrueckii subsp.
bulgaricus grown without shaking will be called "still
cultures," as opposed to those grown with vigorous shaking, which
will be called "aerated cultures." In order to eliminate any
influence of aeration on the lag phase and to monitor only the
influence of aeration on growth, a common inoculum in the same medium
was split into two halves that were both grown without shaking until
they reached A600 = 0.2. At this point, one half was
shifted to aerated conditions and grown with strong shaking, while the
other half was left still and further grown as an unaerated culture
without shaking. The still and aerated cultures showed the same maximal
growth rate of 0.8 ± 0.1 h1, but the aerated
culture entered into its stationary phase earlier than the still
culture (Fig. 1). The result of this
earlier entry into stationary phase upon aeration was a reduction of
about 40% in the biomass produced (Fig. 1A). The absorbance of the
aerated cultures at A600 remained constant for about 3 h, suggesting that no bacterial lysis occurred during stationary phase.
The early entry into stationary phase was associated with a stop in
lactose consumption. The interruption in the growth of the aerated
cultures was not, however, due to a lack of energy or carbon source,
since more than half of the initial lactose was still present in the medium. The still cultures did not stop growing because of exhaustion of their lactose supply, as they utilized less than two-thirds of the
initial lactose before settling into stationary phase.
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FIG. 1.
Effect of aeration on the growth of L. delbrueckii subsp. bulgaricus (A), the lactate
concentration (B), and the pH of the culture medium (C). Open circles,
still cultures; solid circles, aerated cultures. The arrow shows the
time at which aeration was started. MRS medium with 2% lactose was
used. Growth was measured through changes in
A600. The lactate concentration and pH of the
medium of the supernatant were determined after rapidly centrifuging
the cells.
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The entry into stationary phase of aerated cultures was accompanied by
a stop in the production of lactate. The final lactate
concentration in
the medium of aerated cultures was around 30
mM, significantly lower
than that for still cultures, of close
to 72 mM (Fig.
1B). The aerated
and still cultures showed the
same ratio of 1.9 ± 0.1 millimoles
of lactate produced per millimoles
of lactose consumed, indicating that
aeration did not significantly
alter the homolactic character of
lactose fermentation by
L. delbrueckii subsp.
bulgaricus. Because aeration lowered the production of
lactate, the final pH reached by aerated cultures was about 0.7
pH
units higher than that of still cultures (Fig.
1C), suggesting
that the
early entry into stationary phase of aerated cultures
was not caused by
excessive acid
stress.
Another difference between aerated and still cultures was that aerated
cultures showed little, if any, production of lactate
and decrease in
pH during their stationary phase, whereas still
cultures maintained a
significant consumption of lactose (not
shown), production of lactate
(Fig.
1B), and lowering of pH (Fig.
1C) after they stopped growing.
This indicated that the physiological
states of stationary cells were
different in aerated and still
cultures, and that the residual
maintenance metabolism was almost
quiet in aerated cells but still
active in unaerated cells. This
difference suggested that what caused
the early interruption of
growth with aeration was absent in still
cultures and could possibly
be related to oxidative
stress.
Production of H2O2 by L. delbrueckii subsp. bulgaricus culture and its effects
on bacterial growth.
Oxidative stress results from an overexposure
to reactive oxygen derivatives, such as superoxide anion
(O2) and hydrogen peroxide
(H2O2). Although superoxide is a charged species, it could diffuse sufficiently across the cell membrane that a
significant fraction of intracellular superoxide would be eliminated in
the presence of external superoxide dismutase. The addition of
superoxide dismutase to the medium had no effect on the growth curves
of aerated cultures, indicating that early entrance into stationary
phase was not directly related to an excessive concentration of
external superoxide.
The presence of catalase in the medium of aerated cultures completely
prevented the early entry of the bacteria into stationary
phase.
Indeed, aerated cultures in the presence of catalase showed
the same
growth curves (Fig.
2), lactate
production, final pH,
and lactose/lactate ratio (data not shown) as
those of still cultures
(Fig.
2), strongly suggesting that hydrogen
peroxide was somehow
involved in the early interruption of growth. The
simultaneous
presence of catalase and superoxide dismutase had the same
influence
on the growth curves as that of catalase alone (data not
shown).
In order to confirm that the early interruption of growth in
L. delbrueckii subsp.
bulgaricus with aeration
was due to the presence
of hydrogen peroxide, we measured
H
2O
2 concentrations in the media
of still and
aerated cultures. One component of MRS medium, beef
extract, interfered
with the spectrophotometric measurement of
H
2O
2
concentration, and we had to grow
L. delbrueckii subsp.
bulgaricus in an MRS medium without beef extract. The use of
this incomplete
MRS medium led to a lower biomass production, but a
comparison
of still and aerated cultures gave the same results as those
observed
for cultures grown in complete MRS medium: aeration did not
change
the maximal growth rate but markedly reduced both biomass (Fig.
1A and
3A) and lactate production. The
final lactate concentration
in the medium was indeed lowered from 24 mM
for still cultures
to 14 mM for aerated cultures. The concentration of
hydrogen peroxide
in the medium was largely increased by aeration (Fig.
3B). In
still cultures, hydrogen peroxide concentration rapidly
increased
to 130 µM at the beginning of the exponential phase and
then increased
slowly to 160 µM during the rest of the exponential
and the stationary
phases (Fig.
3). In aerated cultures, hydrogen
peroxide concentration
increased throughout the exponential phase to
reach 360 µM and
remained at this level during the stationary phase.
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FIG. 2.
Growth of L. delbrueckii subsp.
bulgaricus under aerated ( ) and nonaerated ( )
conditions and in aerated conditions in the presence of 4 U of catalase
per ml in the growth medium ( ). The arrow shows the time at which
aeration was started.
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FIG. 3.
Effect of aeration on the growth of L. delbrueckii subsp. bulgaricus (A) and the concentration
of hydrogen peroxide concentration in the culture medium (B). Open
circles, unaerated still cultures; closed circles, aerated cultures.
The arrow shows the time at which aeration was started. MRS medium
without beef extract and with 2% lactose was used. Growth was measured
through changes at A600. The hydrogen peroxide
concentration of the supernatant was determined after rapidly
centrifuging the cells.
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The levelling off of hydrogen peroxide concentrations in the medium of
aerated and still cultures was probably due to different
causes. It is
shown below that a large amount of the hydrogen
peroxide was produced
by an NADH:H
2O
2 oxidase that catalyzed the
reduction of molecular oxygen by NADH with a 1/1 stoichiometry.
In
aerated cultures, the cellular energetic metabolism was very
quiet
during stationary phase, as judged from the low lactose
consumption
(data not shown) and low lactate production (Fig.
1B). Therefore,
although oxygen was present, the production of
H
2O
2 could have been limited by the
availability of NADH. In still
cultures, however, cells in late
exponential or stationary phase
had a metabolism actively producing
lactate (Fig.
1B), showing
that NADH was not limiting. Rather, the
factor limiting H
2O
2 production
could have been
the availability of oxygen. Indeed, the final
H
2O
2 concentration of 160 µM in still
cultures corresponded roughly
to the solubility of molecular oxygen at
the growth temperature
of 42°C, suggesting that hydrogen peroxide was
a waste product
of oxygen elimination by the
cells.
The influence of hydrogen peroxide on the growth of
L. delbrueckii subsp.
bulgaricus was studied directly by
adding either
different H
2O
2 concentrations,
from 0.5 to 5 mM, during the exponential
phase of still cultures or the
same H
2O
2 concentration of 0.75
mM at different
times during the exponential phase. It was found
that such additions of
hydrogen peroxide caused still cultures
to enter into stationary phase
(Fig.
4A) independently of the
time of
addition during exponential phase (Fig.
4B). Absorbance
at 600 nm
remained constant for several hours after the addition
of up to 5 mM
H
2O
2, indicating that no bacterial lysis was
occurring.
That millimolar concentrations of hydrogen peroxide were
sufficient
to stop the growth of
L. delbrueckii subsp.
bulgaricus suggested
that the earlier entry into stationary
phase and subsequent lower
biomass production of aerated cultures were
due to the higher
accumulation of H
2O
2 in the
medium.
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FIG. 4.
Influence of hydrogen peroxide on the growth of L. delbrueckii subsp. bulgaricus under nonaerated
conditions. MRS medium with 2% lactose was used. Growth was measured
through changes in A600. (A) Influence of
hydrogen peroxide concentration. Hydrogen peroxide was added at the
time indicated by the arrow to reach final concentrations of 0 (),
0.5 ( ), 1 ( ), and 5 ( ) mM. (B) Influence of the time of
addition. Hydrogen peroxide was added at different times to give the
same final concentration of 0.75 mM in parallel unaerated cultures.
H2O2 was added immediately after inoculation
(X), when A600 = 0.150 (), when
A600 = 0.350 (), when
A600 = 0.600 (+), and when
A600 = 1 (). , growth without
addition of hydrogen peroxide.
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Some strains of
Streptococcus mutans also accumulated high
levels (up to 2 mM) of hydrogen peroxide during growth, because
they
produced H
2O
2 faster than they eliminated it
(
24). Since
little was known of the metabolism (production
and/or elimination)
of hydrogen peroxide in
L. delbrueckii
subsp.
bulgaricus, a characterization
of the enzyme(s) that
could be involved in H
2O
2 formation was
undertaken.
Evidence for the presence of an NADH:H2O2
oxidase in L. delbrueckii subsp. bulgaricus.
L.
delbrueckii subsp. bulgaricus has been reported to lack
a superoxide dismutase that could have produced hydrogen peroxide by
the dismutation of O2 (3).
Therefore, the reactions that were the best candidates for the
formation of H2O2 in L. delbrueckii
subsp. bulgaricus were those catalyzed by oxidases. Three
H2O2-producing oxidases (lactate, pyruvate, and
NADH oxidases) were assayed for activity in crude extracts from
L. delbrueckii subsp. bulgaricus. The lactate and
pyruvate oxidases were assayed by their H2O2
production, and NADH oxidase was assayed by its consumption of NADH.
These assays showed that no lactate oxidase could be detected, and that
some pyruvate oxidase was present. Pyruvate oxidase activity was
nondialyzable and heat labile, suggesting that it was associated with a
protein. It also had properties similar to those of the pyruvate
oxidase from Lactobacillus plantarum (18), in
that it required FAD and was largely enhanced by adding 50 µM
thiamine pyrophosphate and 1 mM Mn2+ ion to the assay. It
is thus likely that a part of the H2O2 produced by L. delbrueckii subsp. bulgaricus was due to
pyruvate oxidase.
It was also found that extracts of
L. delbrueckii subsp.
bulgaricus contained a nondialyzable and heat-labile
activity that
could rapidly reoxidize NADH into NAD
+. NADH
reoxidation was not dependent on the addition of any electron
acceptor
and apparently needed only dissolved molecular oxygen,
as indicated by
the marked reduction of the rate of reoxidation
upon degassing the
assay buffer and flushing it with nitrogen.
The involvement of oxygen
in this reoxidation of NADH was confirmed
by the complete disappearance
of this activity in the presence
of the oxygen scavenger sodium
dithionite in the assay. The rate
of NADH reoxidation was strongly
increased by the addition of
FAD to the assay mixture, suggesting that
the NADH oxidase from
L. delbrueckii subsp.
bulgaricus was a flavoprotein, as are NADH
oxidases from
other gram-positive bacteria (
1,
13,
20,
21). Other evidence
for NADH oxidase being a flavoprotein was
the inhibition caused by
diphenyleneiodonium chloride, a reagent
that binds covalently to
reduced flavins (S. Arnould and J.-M.
Camadro, personal
communication).
The reoxidation of NADH by NADH oxidase could produce either
H
2O or H
2O
2, depending on the
number of electrons used to reduce
molecular oxygen. Two types of NADH
oxidases have indeed been
described, the first one catalyzing the
four-electron reduction
of O
2 into 2 H
2O
(
13,
17,
19,
21) and the second the two-electron
reduction
of O
2 to H
2O
2 (
2,
10,
11,
24). These enzymes
are present in several lactic acid bacteria
that possess either
a NADH-H
2O
2 or a
NADH-H
2O oxidase and sometimes both (
5,
7).
The
NADH oxidase from
L. delbrueckii subsp.
bulgaricus showed
a ratio of 1 ± 0.05 between the
amounts of NADH reoxidized and
that of H
2O
2
liberated, indicating a stoichiometric reaction.
This indicated not
only the involvement of a NADH:H
2O
2 oxidase
in
the production of H
2O
2, but also the absence of
any NADH:H
2O
oxidase. The NADH:H
2O
2
activity in extracts of
L. delbrueckii subsp.
bulgaricus decreased very slowly over time during storage
at
4°C, indicating that the NADH oxidase was not a fragile enzyme.
The
optimum pH for its activity was around pH 5.5.
This NADH oxidase contributed much more strongly to the production of
hydrogen peroxide than did pyruvate oxidase. Indeed,
under the same
conditions, the specific activity of NADH oxidase
was 25-fold higher
than that of pyruvate oxidase, 0.65 and 0.025
µmol/min/mg of protein,
respectively. This NADH oxidase thus appeared
as an important enzyme in
H
2O
2 production by
L. delbrueckii
subsp.
bulgaricus.
The specific activity of NADH oxidase was determined throughout the
growth of
L. delbrueckii subsp.
bulgaricus with
crude
extracts taken from still and aerated cultures to check whether
or not the presence of oxygen and/or the growth phase had any
influence
on the cellular amount of this enzyme. The same specific
activity of
0.65 ± 0.2 µmol/min/mg of protein was measured for
NADH oxidase
with and without aeration, and it remained constant
during the
exponential and stationary phases (Table
1). Therefore,
the expression of the NADH
oxidase gene was apparently not regulated
by its substrate, oxygen, or
its product, hydrogen peroxide. Also,
no regulation of the amount of
NADH oxidase was associated with
a shift from exponential to stationary
phase. The higher production
of H
2O
2 by aerated
cultures of
L. delbrueckii subsp.
bulgaricus was
thus not due to a higher amount of NADH oxidase but probably
to a
higher concentration of oxygen. This was consistent with
the above
hypothesis that H
2O
2 production by still
cultures was
limited by oxygen availability, and that dissolved oxygen
could
be eliminated efficiently from the medium by this NADH oxidase.
Comparison between the fluxes of NADH reoxidized by NADH oxidase
and lactate dehydrogenase.
In the classical paradigm of lactic
fermentation, NADH is reoxidized by LDH. The amount of LDH, like that
of NADH oxidase, did not seem to be regulated by aeration or the growth
phase (Table 1). The two enzymes, LDH and NADH oxidase, compete for
NADH, and their relative contributions to NADH reoxidation could be estimated from the ratio between the concentrations of their respective products, lactate and H2O2, liberated in the
medium, assuming that L. delbrueckii subsp.
bulgaricus did not rapidly degrade H2O2 (see above). The ratio between lactate and
H2O2 was the lowest at the end of aerated
cultures, when the concentration of H2O2 reached 360 µM (Fig. 3B) and that of lactate was only 14 mM. The lowest value of the lactate/H2O2 ratio was thus
close to 40-fold, indicating that about 97 to 98% of NADH had been
reoxidized by LDH and only 2 to 3% by NADH oxidase. Comparing the
specific activities of LDH and NADH oxidase in extracts of L. delbrueckii subsp. bulgaricus confirmed that a much
higher flux of NADH could be reoxidized with pyruvate than with oxygen.
Indeed, during the exponential and stationary phases of still and
aerated cultures, the specific activity of LDH was about 15 times
higher than that of NADH oxidase (Table 1). Therefore, only a small
percentage (at most) of NADH could be reoxidized by oxygen, which
suggested that NADH oxidase had little (if any) significant role in
energy production in L. delbrueckii subsp.
bulgaricus, in agreement with the fermentation remaining
homolactic, with the same lactate/lactose ratio of 1.9 ± 0.1 (see
above), with and without aeration. Aeration of wild-type L. delbrueckii subsp. bulgaricus was thus not sufficient
to induce a visible shift from homolactic to mixed-acid fermentation.
However, a very large increase in NADH oxidase activity could result in a significant fraction of NADH being reoxidized with oxygen, so that
the pyruvate that would not be reduced into lactate could be
metabolized further into acetyl coenzyme A, acetate, acetoin, diacetyl,
and other fermentation end products. Indeed, it has been recently shown
that a 100-fold overexpression of a foreign NADH oxidase gene in
Lactococcus lactis caused a shift from homolactic to
mixed-acid fermentation during growth on glucose in the presence of
oxygen (15). A similar metabolic shift should occur in
L. delbrueckii subsp. bulgaricus if the level of
its NADH oxidase activity could be raised by 10- to 100-fold or if the
activity of its LDH could be lowered by 10- to 100-fold.
Conclusion.
The present results have shown that aeration
provoked an early entry into stationary phase by L. delbrueckii subsp. bulgaricus growing on lactose,
probably because of the oxidative stress caused by an excessive
liberation of hydrogen peroxide in the medium. This
H2O2 production was largely due to NADH
oxidase, an enzyme that seemed to be involved in the elimination of
dissolved oxygen. This NADH:H2O2 oxidase from
L. delbrueckii subsp. bulgaricus could be a
flavoprotein, similar to that isolated from S. mutans
(11). This NADH oxidase had apparently no crucial energetic
role in wild-type L. delbrueckii subsp.
bulgaricus, since its activity was much lower than that of
LDH. This enzyme could, however, be a valuable target for enzyme
engineering, either for increasing the biomass production in presence
of oxygen or for shifting lactose fermentation from homolactic to mixed acid.
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ACKNOWLEDGMENTS |
We are grateful to S. Arnould and J.-M. Camadro for communication
of their results prior to publication and to M.-C. de Givry for her
help in obtaining the L. bulgaricus stocks.
This work was supported by grants from CNRS (UPR9063), Université
Pierre-et-Marie Curie (9270300), and the Danone group.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire
d'Enzymologie et de Biochimie Structurales du CNRS, 91198 Gif-sur-Yvette, France. Phone: 33 1 69 82 34 75. Fax: 33 1 69 82 31 29. E-mail: garel{at}lebs.cnrs-gif.fr.
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Applied and Environmental Microbiology, January 2000, p. 262-267, Vol. 66, No. 1
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Abstract
Introduction
