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Applied and Environmental Microbiology, August 1999, p. 3360-3363, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Kinetics, Stereospecificity, and Expression of
the Malolactic Enzyme
Ceri E.
Arthurs* and
David
Lloyd
Microbiology Group (Cardiff School of
Biosciences), Cardiff University, Cardiff CF1 3TL, Wales, United
Kingdom
Received 2 November 1998/Accepted 6 May 1999
 |
ABSTRACT |
Mass spectrometric measurement of carbon dioxide production was
used to study malolactic fermentation (MLF) in Lactobacillus collinoides isolated from cider. The kinetics and
stereospecificity of the malolactic enzyme (MLE) were studied, and the
stoichiometry of the reaction sequence was investigated. The optimum pH
for activity of the MLE was 4.9. MLF was more rapid (in both intact cells and cell extracts) when L-malic acid was used than
when D-malic acid or the racemic mixture was added. The
enzyme was found to be constitutively present in L. collinoides. Addition of L-malic acid (37 mM) to the
growth medium resulted in increased MLE activity; addition of the
D isomer alone or the racemic mixture resulted in lower
activities. Addition of the main sugars in apple juice (fructose,
sucrose, and glucose) to the growth medium in the presence of malic
acid repressed production of MLE to similar extents in all three cases;
in the absence of malic acid, instead of inhibiting MLF, addition of
sugars to the growth medium somewhat increased the residual MLE activity.
| |
INTRODUCTION |
Two distinct processes are involved
in cider making (9, 11). The initial step is alcoholic
fermentation performed by yeast (Saccharomyces sp.) (6,
12, 19, 22). This is followed by malolactic fermentation (MLF)
(18) performed by lactic acid bacteria (LAB) (3, 8,
21). The overall result of the secondary fermentation is
production of the characteristic flavor and aroma of cider.
Approximately 8% of the fresh weight of an apple is accounted for by a
mixture of sugars (10), and apple juice contains principally
fructose, glucose, and sucrose (9). Fructose is present at
the highest concentration in apples and accounts for about 70% of the
total sugar (10). Sucrose and glucose are much less abundant
and account for 10 to 20% of the total sugar (3). Following
alcoholic fermentation all of the fermentable sugar should be converted
to alcohol. Any residual sugar may influence the secondary
fermentation. Hence, we decided to investigate the effects of residual
sugars on the MLF, both in the presence and in the absence of malic
acid. We selected concentrations of sugars which reflected the low
residual levels which may occur. Sugar (fructose and glucose)
catabolism is inhibited in the highly acidic environment that is
characteristic of cider fermentation; the low pH values maximize the
rate of malate utilization and inhibit the rate of glucose consumption
by some LAB (7).
The malolactic enzyme (MLE) which is responsible for the MLF is present
in many species belonging to the genera Lactobacillus, Leuconostoc, Oenococcus, and Pediococcus and has been
purified (4, 20). This enzyme decarboxylates
L-malic acid (the main acid found in apples
[1]) to form L-lactic acid directly (to date no intermediate products have been detected), which results in an
overall decline in acidity (7, 14). For each malate molecule
metabolized, equimolar amounts of lactate and carbon dioxide are
produced; i.e., the reaction is stoichiometrically equivalent. The
level of recovery of lactic acid from malic acid is about 90%
(17). The rate of the MLF can therefore be accurately determined by measuring the rate of pH change, CO2
formation, or lactic acid formation, although it should be noted that
other naturally occurring organic acids can also be metabolised by LAB (e.g., quinic acid is converted into dihydroshikimic acid)
(1).
In this paper we describe the use of a membrane inlet quadrupole mass
spectrometer to study (i) the kinetics, (ii) the stoichiometry, (iii)
the stereospecificity, and (iv), the conditions which favor expression
of the enzyme involved in MLF.
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MATERIALS AND METHODS |
Cell growth.
An LAB isolate obtained from cider fermentation
was provided by H. P. Bulmer Ltd. (The Cider Mills, Hereford,
United Kingdom). This organism was tentatively identified (by using the
API system [API] bioMérieux, Vercieu, France]) as
Lactobacillus collinoides. Cultures were routinely grown in
250 ml of modified Lactobacillus MRS medium (5)
(Merck Ltd., Dorset, United Kingdom) containing 37 mM DL-,
D, or L-malic acid in stationary 250-ml
Erlenmeyer flasks without baffles sealed with rubber bungs and
incubated at 25°C (standard conditions). The LAB was also grown in
MRS medium supplemented with fructose, sucrose, or glucose both in the
presence and in the absence of L-malic acid under
conditions identical to those described above. The final pH was
adjusted by using 1 M NaOH and 1 M HCl.
Harvesting.
Growth was monitored by measuring absorbance at
660 nm compared with medium blanks. Once the late exponential growth
phase had been reached, the cells were harvested by centrifugation with a Sorvall model RC-5B centrifuge fitted with a type GSA rotor (6,000 × g, 6 min, 25°C). Usually, they were then
washed and resuspended in 10 ml of 5 mM
2-(N-morpholino)ethanesulfonic acid (MES) (Sigma, Dorset,
United Kingdom) (pH 4.5) before they were stored briefly (1 to 4 h) on ice prior to use. Preliminary experiments indicated that storage
resulted in no significant changes in activity. On other occasions (as
indicated below) the procedure was repeated, and the harvested cells
were resuspended in 5 mM MES adjusted to pH 3.5, 4.1, 4.3, 4.5, 4.9, and 5.5.
Preparation of cell extracts.
A model Soniprep 150 sonicator
(amplitude, 22 µm; five 30-s pulses over a 5-min period) was used to
break the walls of cells that had been resuspended at pH 4.5. The cell
extract was then obtained by centrifugation with a Sorvall model RC-5B
centrifuge equipped with a type SS34 rotor (6,000 × g,
6 min, 25°C) and collection of the supernatant.
MLF. Resuspended intact cells or cell extracts were added to
a specially constructed Lucite reaction vessel (total volume, 4 ml)
that was surrounded by a water jacket (kept at 25°C) and was fitted
with a sealed cap with a 0.5-mm-diameter injection port. A pH probe
attached to an analogue pH meter (model PW9418; Philips Analytical,
Cambridge, United Kingdom) and a mass spectrometer probe were inserted
and cemented in place. The reaction mixture was magnetically stirred at
200 rpm. A quadrupole mass spectrometer (Hiden Analytica, Ltd.,
Warrington, United Kingdom) in electron ionization mode measuring at
m/z 44 was equipped with a membrane inlet probe. A tubular
silicone membrane (inside diameter, 0.5 mm; outside diameter, 1.0 mm;
Vygon) was used with the 1.6-mm stainless steel probe (2,
13).
The MLF was initiated by adding
D-,
L-, or
DL-malic acid in 5 mM MES (pH 4.5) to the vessel containing
washed intact cells
or cell extracts (pH 4.5) which had been grown in
medium supplemented
with 37 mM
DL-malic acid. Both the
amounts of CO
2 produced and
pH changes were
recorded.
The procedure was then repeated by adding
DL-malic acid
preparations with the pH adjusted to 3.5, 4.1, 4.3, 4.5, 4.9, and
5.5 to the vessel containing washed whole cells (previously grown
in MRS
medium [pH 4.5] containing 37 mM
DL-malic acid) at pH
3.5,
4.1, 4.3, 4.9, and 5.5, respectively. The reactions were monitored
by measuring CO
2 production by mass
spectrometry.
DL-Malic acid (37 mM) in 5 mM MES (pH 4.5) was used to
initiate the MLF in washed whole cells that had been grown in media
supplemented with sugar in the presence and in the absence of
different
isomers of malic acid. The reaction rates were again
determined by
measuring CO
2 evolution by mass
spectrometry.
NaHCO
3 dissociates in the presence of HCl to produce
CO
2, the concentration of which depends on the amount of
NaHCO
3 added.
Hence, adding 10 mM NaHCO
3 to
excess HCl was used to calibrate
the mass spectrometer for
CO
2 at
m/
z 44.
Protein analysis.
The protein contents of intact cell
suspensions and cell extracts were determined by using a modification
of the method described by Lowry et al. (15); bovine serum
albumin was used as the standard. Protein was extracted by boiling
preparations (for 5 min with 1 M NaOH in closed tubes) before the assay.
Reproducibility of results.
The experiments in which intact
organisms and cell extracts were used were all performed in triplicate,
and the standard errors of the means were found to be less than 0.2%.
| |
RESULTS AND DISCUSSION |
MLF investigated at different pH values by measuring
CO2 production.
Our results indicated that the pH at
which the mass spectrometry analysis was carried out was very
influential in determining the MLE activity. Figure
1 shows that L. collinoides
produced very little CO2 from malate at pH 3.5 and 5.5 but
exhibited a high level of activity at pH 4.9 (Km, 100.2 mM; Vmax 1,410 nmol/min/mg of protein). The optimum pH for L-malate
transport in Lactobacillus plantarum has previously been
found to be 4.5 (16).
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FIG. 1.
MLF rates in samples from L. collinoides
cultures growing under standard conditions and analyzed at different pH
values.
|
|
Although malate-driven changes in pH were monitored by using samples of
washed whole-cell suspensions taken at intervals during
the MLF, the
results obtained were not as reliable as the results
obtained when
carbon dioxide production was measured by mass spectrometry.
This may
be attributed to the less sensitive method for measuring
pH in the
presence of a buffer (5 mM MES) which counteracted pH
changes.
MLF in intact cells in which different isomers of malic acid were
used as substrates for the MLE.
The MLF rate when a racemic
mixture of malic acid was used as the substrate was greater than the
MLF rate when equimolar amount of either isomer alone was used. The
results obtained for L. collinoides are shown in Table
1. Although the racemic mixture was
inferior as a substrate compared with the L isomer, this
mixture was more effective than the D isomer. The
L isomer induced L-malate transport in L. plantarum to a greater degree (100%) than either the racemic mixture (82%) or the D isomer (66%) (16).
The
Vmax values obtained reflect either the
transport of malic acid into the cell or, alternatively, the metabolism
of malic
acid. There are a number of possible explanations for our
findings.
The results which we obtained may be attributed to a concerted
transport mechanism, in which one isomer reduces the uptake
and,
therefore, the metabolism of the other. This proposal supports
the
hypothesis that malic acid cannot readily diffuse across the
cytoplasmic membrane (
16). On the basis of the
Vmax values obtained
(Table
1), we deduced that
it is uptake and metabolism of the
L isomer that are
reduced in the presence of
D-malate. It has
been reported
previously that the
L-malate carrier is stereospecific
and
is not significantly inhibited by
D-malate (
16).
Alternatively, racemization (intracellular or extracellular) may be the
rate-limiting factor. If this is so and intracellular
racemization
occurs, both isomers of malate would be transported
into the cell at
similar rates. Once inside the cell, the
D isomer
would
then have to be transformed into the
L isomer before it
was
accepted as a substrate for the MLE in order to undergo the
decarboxylation reaction that results in production of lactic
acid.
A third possibility is that extracellular racemization is the
rate-limiting step. If this were the case, the
L isomer
would
be expected to exhibit the highest
Vmax
value, followed by the
racemic mixture and finally the
D
isomer. The results obtained
in this study clearly show that this was
the case (Table
1).
The final possibility is that MLF rates (as calculated in this study)
are determined primarily by the ability of the MLE to
convert
L-malic acid to lactic acid. In this process the
D isomer
might interact with the enzyme to alter its
affinity for
L-malate.
In order to distinguish between the hypotheses described above and thus
determine whether the measured
Vmax values are
the
Vmax values for the malic acid transporter
or are
Vmax values
for the MLE itself, MLF was
studied in cell extracts. With a cell
extract if identical
Vmax were obtained with all three substrates
(the
D isomer, the
L isomer, and the racemic
mixture), then the
substrate affinity-determining step would be assumed
to be the
transport mechanism. However if different
Vmax values were observed,
the results could be
explained by either (i) the enzyme having
a different affinity for each
of the isomers or (ii) the
D isomer
requiring racemization
before it could be utilized as a substrate
by the
MLE.
MLF in cell extracts in which different isomers of malic acid were
used as substrates for the MLE.
The MLF was investigated with cell
extracts in order to determine whether the Vmax
values obtained with intact cells represented the rate of transport of
malic acid into the cell or were a measure of the rate of conversion of
malic acid into lactic acid (Table 1).
The cell extract
Vmax values were lower than the
Vmax values for intact cells, indicating that
the transport system for malic
acid favors the
L isomer
over the
D isomer (Table
1); i.e.,
L-malic
acid
is transported into the cell by a higher-affinity system
than the
system used for
D-malic acid. We concluded, therefore,
that
the presence of
D-malic acid significantly reduces the
uptake
of
L-malic acid into the
cell.
The
Km values described in this paper (Table
1)
correlate well with previously reported values; e.g., the purified
preparation
of MLE obtained from
L. plantarum was found to
have a
Km for malate
of 9.5 mM by Caspritz
(
4).
The
Vmax values obtained for cell extracts
indicate that the MLE has the greatest affinity for
L-malic
acid (
Vmax 3,600 nmol/min/mg
of protein). When
cell extracts are used, it is the rate of conversion
of
L-malic acid to
L-lactic acid which is the
rate-limiting
step.
Interestingly, the racemic mixture and the
D isomer (when
used individually as substrates for the MLE) had very similar
Vmax values (1,490 and 1,470 nmol/min/mg of
protein, respectively).
Therefore, we propose that the
D
isomer reduces the affinity of
the MLE for
L-malic acid and
also must be converted into
L-malic
acid extracellularly
before it is accepted as a suitable substrate
by the
MLE.
In summary, we propose that the
D isomer has two effects on
MLF in LAB; not only does it reduce the uptake of the
L
isomer,
but it also reduces the affinity of the enzyme for
L-malic acid.
In addition,
D-malic acid
requires extracellular racemization
in order to form
L-malic acid before it can be used as a substrate
for the
MLE. This proposal adequately explains the results obtained
for both
cell extracts and intact cells described in this
paper.
Expression of the MLE with different isomers of malic acid.
LAB were grown in the presence and absence of 37 mM D-,
L-, or DM-malic acid, and the activity of the
MLE was determined. When grown in the absence of malic acid, L. collinoides was able to perform the MLF, which indicated that the
enzyme was constitutively present. L-malate has previously
been reported to be responsible for inducing elevated levels of the
enzyme (14). Thus, as expected, addition of malic acid to
the growth medium also increased the level of expression of the enzyme
in L. collinoides. L-malic acid induced the MLE
to a greater extent than either D-malic acid or the racemic
mixture induced this enzyme (Fig. 2).
Both the D and L isomers of malic acid (when
used individually or in combination) enhanced expression of the MLE,
and it was clear that L-malic acid resulted in greater
expression than the D isomer (Fig. 2).
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FIG. 2.
MLF rates in samples from L. collinoides
cultures growing with various concentrations of D-,
L-, or DL-malic acid. The standard medium was
supplemented with D-malic acid ( ), L-malic
acid ( ), or DL-malic acid ( ).
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|
Effect that sugars in the growth medium have on induction of the
MLE.
When MRS medium containing 37 mM DL-malic acid
(with no added sugars) was used as the growth medium for L. collinoides, the highest level of MLF activity was observed.
Addition of 15 mM sucrose, addition of 28 mM glucose, or addition of 28 mM fructose to this medium inhibited expression of the MLE to similar
extents (Fig. 3). The lowest level of MLF
activity occurred when L. collinoides was grown in MRS
medium (pH 4.5) which had not been supplemented with either malic acid
or carbohydrates.
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FIG. 3.
MLF rates in samples from L. collinoides
cultures growing with sugar supplements. (a) Standard growth medium
containing 37 mM malic acid. (b through d) Medium supplemented with 28 mM fructose ( and  ). (b) 28 mM glucose ( and  ) (c), and 15 mM sucrose ( and  ) (d) in the presence of 37 mM malic acid (solid
symbols) and in the absence of malic acid (open symbols). The rates in
the absence of both malic acid and sugars are also indicated ( ).
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|
When
L. collinoides was grown in the absence of malic acid,
additions of sugars to the growth medium increased MLE activity
instead
of inhibiting MLF. The enhancement of MLF activity was
greater with
either glucose or sucrose than with fructose (Fig.
3). Maximum
expression of the MLE was observed when the growth
medium was
supplemented with 37 mM
L-malic acid
alone.
| |
ACKNOWLEDGMENTS |
This research was funded by a Ministry of Agriculture, Fisheries,
and Food postgraduate agricultural and food studentship.
We thank Martin Forster for advice and materials.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Microbiology
Group (Cardiff School of Biosciences), Cardiff University, P.O. Box
915, Cardiff CF1 3TL, Wales, United Kingdom. Phone: 44 0 1222 874000. Fax: 44 0 1222 874305. E-mail: sabca1{at}cf.ac.uk.
| |
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Applied and Environmental Microbiology, August 1999, p. 3360-3363, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
