Purification and partial characterization of Lactobacillus species SK007 lactate dehydrogenase (LDH) catalyzing phenylpyruvic acid (PPA) conversion into phenyllactic acid (PLA)

Article abstract of DOI:10.1021/jf0731503

Phenyllactic acid (PLA) is a novel antimicrobial compound synthesized by lactic acid bacteria (LAB), and its production from phenylpyruvic acid (PPA) is an effective approach. In this work, a lactate dehydrogenase (LDH), which catalyzes the reduction of PPA to PLA, has been purified to homogeneity from a cell-free extract of Lactobacillus sp. SK007 by precipitation with ammonium sulfate, ion exchange, and gel filtration chromatography. The purified enzyme had a dimeric form with a molecular mass of 78 kDa (size exclusion chromatography) or 39 kDa (SDS-PAGE). The ratio of enzyme activity with PPA to that with pyruvate being almost invariable at every purification step indicated that, in Lactobacillus sp. SK007, LDH is responsible for the conversion of PPA into PLA. HPLC profiles of PPA transformation into PLA by growing cells, cell-free extract, and purified LDH of Lactobacillus sp. SK007 were also investigated. Results showed that the presence of NADH was found to be necessary for the enzymatic production of PLA from PPA. The purified LDH displayed optimal activity for PPA at pH 6.0 and 40°C. The K_m values of the enzyme for PPA and pyruvate were 1.69 and 0.32 mM, respectively. Moreover, because other screened LAB strains exhibiting relatively high LDH activity toward PPA produced also considerable amounts of PLA, LDH activity for PPA could be therefore used as a screening marker for PLA-producing LAB.

Full text of DOI:10.1021/jf0731503

2392 J. Agric. Food Chem. 2008, 56, 2392–2399
Purification and Partial Characterization of
Lactobacillus Species SK007 Lactate Dehydrogenase
(LDH) Catalyzing Phenylpyruvic Acid (PPA)
Conversion into Phenyllactic Acid (PLA)
†
XINGFENG LI,† BO JIANG,*,† BEILEI PAN,‡ WANMENG MU,† AND TAO ZHANG
State Key Laboratory of Food Science and Technology (Jiangnan University),
Wuxi 214122, People’s Republic of China, and Chinese Institute of Food Science and Technology,
Beijing 100833, People’s Republic of China
Phenyllactic acid (PLA) is a novel antimicrobial compound synthesized by lactic acid bacteria (LAB),
and its production from phenylpyruvic acid (PPA) is an effective approach. In this work, a lactate
dehydrogenase (LDH), which catalyzes the reduction of PPA to PLA, has been purified to homogeneity
from a cell-free extract of Lactobacillus sp. SK007 by precipitation with ammonium sulfate, ion
exchange, and gel filtration chromatography. The purified enzyme had a dimeric form with a molecular
mass of 78 kDa (size exclusion chromatography) or 39 kDa (SDS-PAGE). The ratio of enzyme activity
with PPA to that with pyruvate being almost invariable at every purification step indicated that, in
Lactobacillus sp. SK007, LDH is responsible for the conversion of PPA into PLA. HPLC profiles of
PPA transformation into PLA by growing cells, cell-free extract, and purified LDH of Lactobacillus sp.
SK007 were also investigated. Results showed that the presence of NADH was found to be necessary
for the enzymatic production of PLA from PPA. The purified LDH displayed optimal activity for PPA
at pH 6.0 and 40 °C. The Km values of the enzyme for PPA and pyruvate were 1.69 and 0.32 mM,
respectively. Moreover, because other screened LAB strains exhibiting relatively high LDH activity
toward PPA produced also considerable amounts of PLA, LDH activity for PPA could be therefore
used as a screening marker for PLA-producing LAB.
KEYWORDS: Phenyllactic acid; phenylpyruvic acid; lactic acid bacteria; Lactobacillus; lactate dehydro-
genase; purification
INTRODUCTION
reus, and Escherichia coli O157:H7 (5–7). Due to its broad
inhibitory activity against a variety of foodborne microorgan-
isms, PLA has interesting potential for practical application as
an antimicrobial agent in the food industry (1, 4).
Foodborne fungi, both yeasts and molds, cause serious
spoilage of stored foods. Molds may also produce health-
damaging mycotoxins, that is, aflatoxins, trichothecenes, fumo-
nisin, ochratoxin A, and patulin (1). Consumer demands for
reduced use of chemical preservatives have stimulated research
on natural antifungal compounds as biopreservatives. Phenyl-
lactic acid (PLA) is a novel antimicrobial compound (2), which
has been recently isolated from lactic acid bacteria (LAB) (3).
PLA had a broad inhibitory activity against a wide range of
fungal species isolated from bakery products, flour, and cereals,
including some mycotoxigenic species, namely, Aspergillus
ochraceus, Penicillium roqueforti, and Penicillium citrinum (4).
Furthermore, the inhibitory properties of PLA have been
demonstrated against both Gram-positive and Gram-negative
bacteria, such as Listeria monocytogenes, Staphylococcus au-
PLA may be produced by food-related microorganisms,
including the fungus Geotrichum candidum (2), propionibacteria
(8), and LAB (1, 3, 7, 9–20). Lavermicocca and co-workers
(3) reported the production of PLA by L. plantarum 21B used
as a starter in sourdough bread, which was the first report
showing the production of PLA by LAB (Table 1). PLA has
also been identified from culture supernatants of several LAB
strains, such as L. plantarum MiLAB 393 (9), L. coryniformis
Si3, and L. sakei (10). However, later studies revealed that from
the 12 tested species 9 of them included strains that are widely
used in the production of fermented foods and able to produce
PLA in the range of 0.17–0.57 mM (11). Moreover, some
commercial probiotic lactobacilli were investigated for their
capacity to produce PLA: L. johnsonii La1 and L. acidophilus
IBB 801 produced 0.25 and 0.15 mM PLA, respectively (15).
In a previous study, 70 of the 112 LAB strains isolated from
Chinese traditional pickles could produce relevant amounts of
* Author to whom correspondence should be addressed (telephone/
fax 0086-510-85919161; e-mail bjiang@jiangnan.edu.cn).
† State Key Laboratory of Food Science and Technology (Jiangnan
University).
‡ Chinese Institute of Food Science and Technology.
10.1021/jf0731503 CCC: $40.75  2008 American Chemical Society
Published on Web 03/12/2008

LDH-Catalyzed Conversion of PPA into PLA
Table 1. Review of Some PLA-Producing LAB Strains
strain
J. Agric. Food Chem., Vol. 56, No. 7, 2008 2393
source
year
PLA (mM)
ref
L. plantarum 21Ba
L. plantarum MiLAB393
L. coryniformis Si3
L. sakei
Enterococcus faecalis
L. plantarum ITM21A
sourdough
grass silage
grass silage
grass silage
tempeh
sourdough
human origin
fish products
sourdough
sourdough
sourdough
human origin
sourdough
cheese
olive phylloplane
sourdough
grass silage
ATCC
Technical University of Munich-Weihenstephan
DIPROVAL collection
LC1, Nestlé
2000
2002
2003
2003
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2004
2005
2006
2006
2006
2006
2006
2006
2006
2006
2006
2007
2007
2007
0.34
nrd
nr
nr
nr
3
9
10
10
7
0.35
0.23
0.37
0.08
0.35
0.20
0.46
0.06
0.09
0.57
0.43
nr
0.12
0.50
0.24
0.25
0.05
0.10
0.05
0.15
0.05
nr
11
11
11
11
11
11
11
11
11
11
11
1
13
13
14
15
15
15
15
15
15
16
17
18
L. rhamnosus ATCC53103
L. alimentarius ATCC29643
L. fermentum ITM18B
L. sanfranciscensis IDMC57
L. acidophilus IDMA2
L. brevis ATCC14869
Weissella confusa ITM14A
Enterococcus faecium ATCC882
Leuconostoc mesenteroides subsp. mesenteroides ITMY30b
L. citreum ITM22A
L. plantarum MiLAB 14
L. sanfranciscensis DSM20451T
L. plantarum TMW1.468
L. plantarum VLT01
L. johnsonii La1
L. casei Shirota
L. rhamnosus GG
Yakult, Yakult Honsha
Gefilus, Valio
corn steep liquor
dairy product
Greek Xynotyri cheese
malted barley
L. amylovorus DCE 471
L. acidophilus IBB 801
L. plantarum ACA-DC 287
L. plantarum FST 1.7
Lactobacillus sp. SK007c
L. plantarum VLT01
Chinese traditional pickles
salami
0.55
0.28
a L. plantarum ITM21B is the first LAB strain reported to produce PLA. b The highest level of PLA (0.57 mM) was produced by L. mesenteroides subsp. mesenteroides
ITMY30 grown in MRS broth. c PLA production by Lactobacillus sp. SK007 used in the present investigation was found to be closer to that of L. mesenteroides subsp.
mesenteroides ITMY30. d Not reported.
Figure 1. Pathway for PLA production from Phe or PPA by LAB: (1) transamination of Phe to PPA by aminotransferase; (2) reduction of PPA to PLA
by dehydrogenase. Transamination (1) is the rate-limited step in PLA production. Modified after refs 4 and 13.
PLA (17). In particular, Lactobacillus sp. SK007 produced 0.55
mM PLA, and this amount is similar to that of L. mesenteroides
subsp. mesenteroides ITMY30. Screening of high-PLA-produc-
ing LAB strains from different environments has been actively
pursued (18–20).
bottleneck could be overcome using PPA instead of Phe as
substrate (Figure 1), with a 14-fold increase of PLA content
(17). Traditionally, PPA has been used to produce L-Phe, which
is in high demand for the production of the artificial sweetener
“aspartame” (21). Therefore, compared with Phe, PPA can be
obtained easily at a lower price, and PLA production from PPA
can be an effective approach. Hence, it is necessary to
investigate the enzyme’s catalyzing the reduction of PPA to
PLA from Lactobacillus sp. SK007.
Although PLA can be produced by a wide range of Lacto-
bacillus species, its production is rather low. PLA was formed
by LAB growing in de Man, Rogosa, and Sharpe (MRS) broth
in levels up to 0.57 mM (11, 13). It has been shown that PLA
is a product of phenylalanine (Phe) metabolism (a schematic
overview is given in Figure 1); in particular, Phe can be
transaminated to phenylpyruvic acid (PPA), which is further
metabolized into PLA by reduction (4). Previously, all studies
on PLA production by lactobacilli have employed amino acids
as substrates. PLA production by L. plantarum ITM21B was
improved using increased concentrations of Phe (11). Neverthe-
less, amino acid metabolism by LAB is limited by the
availability of amino acceptors in the transamination reaction.
Vermeulen and co-workers (13) reported that Phe transamination
is a limiting factor in PLA production by L. sanfranciscensis
DSM20451T and L. plantarum TMW1.468. They found also
that PLA yields increased from 5 to >30% upon the addition
of R-ketoglutarate in L. plantarum TMW1.468 culture. In
previous research, the rate-limiting step in PLA production by
Lactobacillus sp. SK007 was also found to be Phe, and this
Lactate dehydrogenase (LDH, EC 1.1.1.27) is widely dis-
tributed in a variety of sources including LAB, and its
biochemical properties have been characterized. It is well-known
that LDH catalyzes the reduction of pyruvate to lactate.
However, LDH also catalyzes the conversion of PPA into PLA
due to its broad substrate specificity. Meister (22) first showed
the evidence that crystalline beef heart LDH also catalyzes the
reduction of PPA to PLA in the presence of NADH. Hummel
and co-workers (23) described LDH from L. confusus, which
also converts PPA into PLA for the production of various
pharmaceuticals. Although LDH from LAB has been extensively
studied for a long time, there have been few reports on the
enzyme characterization for the production of PLA. Therefore,
the aim of this study was to purify LDH converting PPA into
PLA from Lactobacillus sp. SK007 and to determine some of

2394 J. Agric. Food Chem., Vol. 56, No. 7, 2008
Li et al.
60 min in different buffers at the indicated pH values.
its properties. LDH in some LAB strains and their abilities to
produce PLA were also investigated.
Effects of Temperature on Enzyme Activity. For determining the
optimum temperature, enzyme activity was measured at different
temperatures ranging from 20 to 70 °C. To examine the thermal
stability, the enzyme was preincubated at temperatures ranging from
20 to 70 °C for different periods of time before assay at 30 °C as
described previously.
MATERIALS AND METHODS
Materials. PPA, PLA, and NADH were purchased from Sigma
Chemical Co. (St. Louis, MO). All chromatography columns were
obtained from Pharmacia Co. (Amersham Bioscience, Uppsala, Swe-
den), and electrophoresis reagents were imported from Bio-Rad
Laboratories Inc. (Nanjing, China). Other chemicals for enzyme assay
and characterization were provided by Sinopharm Chemical Reagent
Co., Ltd. (Shanghai, China).
Strains and Culture Conditions. Lactobacillus sp. SK007 (Gen-
Bank accession no. DQ534529), isolated from Chinese traditional
pickles, was used in the present investigation. The strain was grown in
1.0 L of MRS broth at 30 °C for 24 h without shaking. The culture
was centrifuged at 10000g (4 °C) for 20 min, and the cells were
collected for further purification. Another nine lactobacilli also isolated
from Chinese traditional pickles and maintained in the laboratory were
also investigated for their ability to produce PLA.
Enzyme Extraction and Purification. Unless otherwise specified,
all purification steps were carried out at 4 °C. Cells harvested were
washed twice with sterile 50 mM Tris-HCl buffer (pH 7.1), resuspended
in the same buffer, and disrupted by sonication (300 W, pulse on, 1 s;
pulse off, 3 s) for 40 min. Cell debris was removed by centrifugation
at 10000g for 20 min, and the supernatant was used as crude
extract.
Determination of Kinetic Constant of LDH with PPA and
Pyruvate. To determine the apparent Michaelis–Menten constant (Km),
enzyme activities using PPA and pyruvate were measured by varying
the concentrations of each substrate under optimum conditions of pH
and temperature. The Km value of LDH at each substrate concentration
was calculated according to the Lineweaver–Burk method by plotting
1/V against 1/[S].
PLA Production from PPA by Growing Cells, Cell-Free Extract,
and Purified LDH from Lactobacillus Spcies SK007. The production
of PLA from PPA by fermentation was carried out according to the
methodof Li et al. (17). For enzymatic production of PLA, 10 mM
PPA and 6 mM NADH were dissolved in 100 mM potassium phosphate
buffer (pH 6.5). The reaction was initiated by adding either crude extract
or purified enzyme (2 units of activity with PPA). The reaction mixture
was maintained at 40 °C for 1 h and then terminated by the addition of
1 M HCl.
Activities of LDH from 10 Isolated LAB Strains and Their
Abilities To Produce PLA. The activities of LDH produced by 10
lactobacilli were measured using pyruvate and PPA as substrates in
cell-free extract according to the enzyme assay procedure described
previously. PLA production by the 10 LAB strains was carried out
according to the method of Li et al. (17), and PLA content was measured
after growth of the strains in MRS broth containing 30.12 mM PPA at
30 °C for 24 h.
HPLC Analysis of PLA and PPA. The product obtained from PPA
reduction by growing cells, cell-free extract, and purified LDH was
determined using reverse phase HPLC equipped with an Agilent Zorbax
SB-C18 column (4.6 mm × 150 mm, 5 µm). Elution was performed
with methanol/0.05% TFA (solvent A) and water/0.05% TFA (solvent
B) at 1 mL/min and A/B ratios of 10:90, 100:0, 100:0, and 10:90,
with run times of 0, 20, 23, and 25 min, respectively. PLA and PPA
were analyzed with DAD (Agilent 1100 series) at 210 nm according
to the procedure described by Valerio et al. (11) with minor
modifications.
Purification of LDH catalyzing PPA conversion into PLA was
accomplished from cell-free extract by three successive steps. The cell-
free extract was subjected to ammonium sulfate precipitation,
but the precipitate obtained after saturation up to 60% had no activity
for PPA or pyruvate. Hence, the resulting supernatant was saturated
up to 100%, and the precipitate was collected by centrifugation at
10000g for 20 min, dissolved in a small amount of 50 mM Tris-HCl
buffer (pH 7.1), and dialyzed in the same buffer for 24 h with three
changes of the buffer during dialysis. The dialyzed enzyme was
loaded on a DEAE-Sepharose Fast Flow column (2.6 cm × 30 cm)
and equilibrated with 20 mM Tris-HCl buffer (pH 7.1). The absorbed
proteins were eluted (1.5 mL/min) with a linear gradient of 0–1.0 M
NaCl in the same buffer. Fractions exhibiting activity toward PPA or
pyruvate were concentrated and desalted by ultrafiltration. Further
purification was carried out using a Sephacryl S-200 HiPrep 16/60
column (1.6 cm × 60 cm, AKTA purifier) previously equilibrated with
20 mM phosphate buffer (pH 6.6) containing 0.15 M NaCl. Proteins
were eluted with equilibrating buffer at a flow rate of 0.5 mL/min.
The active fraction was pooled and concentrated by ultrafiltration and
used as the purified enzyme for further analyses. The purity of enzyme
and molecular mass were analyzed by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) and visualized with
Coomassie brilliant blue (24). Protein concentrations were determined
according to the Lowry method (25) using bovine serum albumin (BSA)
as standard. LDH purity was checked using high-performance liquid
chromatography (HPLC) by injecting the enzyme on a Zorbax 300SB
RPC18 column (4.6 × 250 mm), via elution with a linear gradient for
20 min using the elution A (100% H2O, 0.05% TFA) and B (80%
acetonitrile, 0.05% TFA) at a flow rate of 1 mL/min.
Enzyme Assay. The activity of LDH toward PPA and pyruvate as
substrates was determined by measuring the rate of disappearance of
NADH at 340 nm (22, 23). The assay mixture contained in 100 mM
potassium phosphate buffer (pH 6.5) 0.6 µmol of NADH, 19.6 µmol
of PPA or 2.27 µmol of pyruvate, and relevant amounts of enzyme in
a total volume of 3.0 mL. The presence of NADH was omitted from
the control, and PPA solution was freshly prepared before each use.
The enzyme assay was performed at 30 °C, and 1 unit of enzyme
activity was defined as the amount of enzyme that catalyzes the
degradation of 1 µmol of NADH per minute.
Effects of pH on Enzyme Activity toward PPA. The effect of pH
on the activity of purified enzyme toward PPA was measured in 100
mM sodium acetate buffer (pH 3.0–5.0), 100 mM potassium phosphate
buffer (pH 6.0–8.0), and 100 mM Tris-HCl buffer (8.0–9.0). The pH
stability of LDH was tested by preincubating the enzyme at 30 °C for
Statistical Analysis. Analysis of variance (ANOVA) was carried
out using SAS software (The SAS System for Windows version 8.1).
Experimental data were expressed as the means ( SD of values obtained
from triplicate measurements.
RESULTS
Purification of LDH from Lactobacillus Species SK007
Catalyzing PPA Conversion into PLA. The cell-free extract
of Lactobacillus sp. SK007 was subjected to ammonium sulfate
precipitation and separated into two fractions: the precipitate
obtained after saturation up to 60% and that corresponding to
saturation up to 100%. No activity with PPA or pyruvate was
detected in the first fraction, whereas >80% of activity was
present in the second. After removal of salts by dialysis, the
sample was concentrated and then loaded onto a DEAE-
Sepharose Fast Flow column. Three different fractions were
eluted from the gel matrix, and among these fractions only one
showing activity with both PPA and pyruvate could be detected.
For further purification, the enzyme was loaded onto a Sephacryl
S-200 HiPrep 16/60 column. The elution profile of gel filtration
chromatography showed a single sharp peak active toward PPA
and pyruvate. Following concentration by ultrafiltration, the
fraction reached 57.98 and 516.42 units/mL of activity with PPA
and pyruvate, respectively. The purified LDH averaged 19.27%
of yield with a 34-fold purification level. The specific activities
of purified enzyme with pyruvate and PPA were 203.3 and 22.48

LDH-Catalyzed Conversion of PPA into PLA
J. Agric. Food Chem., Vol. 56, No. 7, 2008 2395
Table 2. Characteristics of LDH from Lactobacillus Species SK007 at Different Stages of Purification
specific activityb
total activity with
PPAa (units)
total activity with
pyruvateb (units)
step
proteinb (mg)
(units/mg)
foldb
yieldb (%)
relative activityc (%)
cell-free extract
ammonium sulfate
DEAE-Sepharose Fast Flow
AKTA Sephacryl S-200
296.40
237.04
115.96
57.98
2679.95
2133.78
1073.32
516.42
451.83
125.29
17.98
2.54
5.93
17.03
59.70
1
100
11.06
11.11
10.80
11.22
2.87
10.07
34.29
79.62
40.05
19.27
203.30
a Enzyme activity was determined using PPA as substrate. b Enzyme activity was determined using pyruvate as substrate. c Relative activity is the percentage of activity
with PPA to that with pyruvate.
Figure 2. SDS-PAGE of purified LDH by Lactobacillus sp. SK007. SDS-
PAGE analysis of the enzyme was performed in 12% (w/v) polyacrylamide
gel. Lane 1 represents protein makers and lane 2, LDH obtained after
purification with AKTA Sephacryl S-200. Rabbit phosphorylase b (97.4
kDa), BSA (66.2 kDa), rabbit actin (43.0 kDa), bovine carbonic anhydrase
(31.0 kDa), trypsin inhibitor (20.1 kDa), and hen egg white lysozyme (14.4
kDa) were used as protein markers.
units/mg, respectively. A summary of the LDH purification
process is shown in Table 2.
Protein purification was successfully achieved to homogeneity
as evidenced by a single band corresponding to 39 kDa on SDS-
PAGE (Figure 2). The apparent molecular mass of native LDH
was estimated to 78 kDa by size exclusion chromatography,
indicating that LDH produced by Lactobacillus sp. SK007 is a
dimeric enzyme. The purity of concentrated LDH was deter-
mined to be about 98% by HPLC.
Conversion of PPA into PLA by LDH from Lactobacillus
Species SK007. After purification, only one fraction active
toward PPA and pyruvate was isolated from the cell-free extract
of Lactobacillus sp. SK007. The enzyme activity toward either
PPA or pyruvate was determined at each purification step (Table
2). The activity of LDH with PPA was always lower than that
with pyruvate, but the relative activity (the percentage of enzyme
activity with PPA to that with pyruvate) was almost invariable
from the crude extract to the purified LDH. Results from Table
2 show that LDH from Lactobacillus sp. SK007 is responsible
for the conversion of PPA into PLA by reduction. This finding
is in agreement with that of Rijnen (26), who reported that LDH
from Lactobacillus lactis is responsible for PPA reduction to
PLA.
Figure 3. HPLC profiles of PPA conversion into PLA by growing cells,
cell-free extract, and purified LDH from Lactobacillus sp. SK007: (A)
conversion of PPA into PLA by growing cells; (B) conversion of PPA into
PLA by cell-free extract; (C) conversion of PPA into PLA by purified LDH.
PPA utilization and PLA formation were monitored by HPLC.
HPLC profiles of the transformation of PPA into PLA by
growing cells, cell-free extract, and purified LDH from Lacto-
bacillus sp. SK007 were also investigated (Figure 3). Results
showed that growing cells could convert PPA into PLA, as well
as crude extract and purified LDH. However, both crude extract
and purified LDH required NADH addition as coenzyme to

2396 J. Agric. Food Chem., Vol. 56, No. 7, 2008
Li et al.
Figure 5. Effects of temperature on the activity of LDH from Lactobacillus
sp. SK007 toward PPA: (A) optimal temperature of purified LDH; (B)
thermostability of purified LDH; (9) 20 °C; (0) 30 °C; (2) 40 °C; (b)
50 °C; (]) 60 °C. The optimum temperature for the enzyme activity toward
PPA was assayed at various temperatures from 20 to 70 °C. Thermo-
stability of the enzyme was determined after incubation of the enzyme in
100 mM potassium phosphate buffer (pH 6.0) at various temperatures
for different incubation times. Residual activity was measured, and the
relative activity of preincubated sample at 4 °C was regarded as 100%.
Each point represents the mean (n ) 3) ( standard deviation.
Figure 4. Effects of pH on the activity of LDH from Lactobacillus sp.
SK007 toward PPA: (A) optimal pH of purified LDH; (B) pH stability of
purified LDH. The enzyme was mixed with various buffers: 100 mM sodium
acetate buffer (pH 3.0–5.0), 100 mM potassium phosphate buffer (pH
6.0–8.0), and 100 mM Tris-HCl buffer (8.0–9.0), respectively. The pH
stability of LDH was tested by preincubating the enzyme at 30 °C for 60
min in different buffers of respective pH values. Residual activity was
expressed as a percentage of the maximum enzyme activity under the
assay conditions. Each point represents the mean (n ) 3) ( standard
deviation.
Km values of LDH from other Lactobacillus species with either
PPA or pyruvate have been compared with that of LDH from
Lactobacillus sp. SK007 in Table 3. Regardless of the strain,
LDH affinity for PPA is lower than that for pyruvate. Moreover,
the Km value of LDH toward PPA was found to vary from 0.8
to 20 mM among lactobacilli (23, 27–29). For all strains, LDH
from Lactobacillus sp. SK007 showed a relatively high affinity
for PPA.
initiate the reduction, whereas growing cells did not. Hence,
the presence of NADH was found to be necessary for the
enzymatic production of PLA from PPA.
Properties of Purified LDH Catalyzing the Conversion of
PPA into PLA. Effects of pH on Enzyme ActiVity toward PPA.
LDH from Lactobacillus sp. SK007 showed an optimum activity
at pH 6.0 when PPA was used as substrate (Figure 4A) and
was found to be virtually inactive below pH 3.0 or above pH
9.0. The effect of pH on LDH stability was studied in the pH
range of 3.0–9.0. The enzyme was rather stable from pH 4.0 to
8.0, but a rapid decline in activity was observed below pH 4.0
or above pH 8.0 (Figure 4B).
Effects of Temperature on LDH ActiVity toward PPA. The
temperature profile of purified LDH from Lactobacillus sp.
SK007 was determined from 20 to 70 °C with PPA as substrate.
The purified LDH displayed an optimum temperature at 40 °C;
the enzyme retained 90% of its relative activity at 50 °C and
50% at 60 °C (Figure 5A). The purified LDH lost no activity
following exposure at temperatures up to 40 °C for 2 h, but
only 20% of initial activity could be retained when the enzyme
was exposed at 50 °C for 2 h (Figure 5B).
LDH Activity toward PPA as a Screening Marker for
PLA-Producing LAB. In a previous study, 10 lactobacilli,
including Lactobacillus sp. SK007, isolated from Chinese
traditional pickles could produce up to 0.55 mM PLA from Phe
in MRS broth (17). In this study, activities of LDH from the
10 isolated strains and their abilities to produce PLA were
investigated. As shown in Table 4, LDH from all tested strains
showed activity toward pyruvate and PPA, and the relative
activity (the percentage of enzyme activity with PPA to that
with pyruvate) varied from 1.08 to 12.51%. With regard to PLA
production, strains SK005 and SK008 showed the highest LDH
activities toward pyruvate but did not provide also the highest
amounts of PLA. In contrast, strains SK006 and SK007 having
low LDH activities toward pyruvate displayed the highest yields
of PLA. Obviously, strains SK002 and SK004 exhibiting the
lowest LDH activities toward pyruvate produced also the lowest
quantities of PLA. The correlation between LDH activity with
Kinetic Parameters. Using PPA and pyruvate as substrates,
the kinetic parameters of purified LDH from Lactobacillus sp.
SK007 were investigated. The apparent Km values of enzyme
with PPA and pyruvate were 1.69 and 0.32 mM, respectively.

LDH-Catalyzed Conversion of PPA into PLA
J. Agric. Food Chem., Vol. 56, No. 7, 2008 2397
lactobacilli possessing high LDH activities toward PPA are able
to produce also high amounts of PLA. However, as shown in
Figure 6A, Lactobacillus species with high LDH activities
toward pyruvate are not necessarily high PLA producers (R2 )
0.1639). Lactobacillus sp. SK007, the LDH activity of which
with pyruvate and relative activity were 2679.95 units and
11.06%, respectively, produced 14.04 mM PLA in MRS broth
containing 30.12 mM PPA. To our knowledge, this is the highest
PLA production by LAB strains reported so far. Therefore, LDH
activity may be used as a screening marker for PLA-producing
LAB.
DISCUSSION
In this study, LDH from Lactobacillus sp. SK007, which
catalyzes the reduction of PPA to PLA, was purified and
characterized. Results from both SDS-PAGE and HPLC indi-
cated that the enzyme has been purified to homogeneity. The
native enzyme was found to have a molecular mass of 78 kDa
and to consist of two identical subunits of 39 kDa. In general,
monomeric molecular mass for LDH produced by LAB species
ranges from 32 to 40 kDa (30), and the LDH molecule is
supposed to be either a tetramer or a dimer. The molecular mass
and the dimeric structure of LDH from Lactobacillus sp. SK007
agree with previous findings (31).
In Lactobacillus sp. SK007, LDH is responsible for the
synthesis of PLA from PPA, and this is in agreement with a
recent study on L. lactis (26). Previously, in addition to LDH,
Schütte and co-workers (32) isolated the so-called L-2-hydroxy-
isocaproate dehydrogenase (L-HicDH) from L. confuses, which
catalyzes the stereospecific reduction of straight- and branched-
chain aliphatic 2-keto-carboxylic acids including PPA to the
corresponding L-2-hydroxycarboxylic acids. Furthermore, the
authors found that L-HicDH activity amounted to only 2% of
LDH activity when PPA was used as a substrate for both
enzymes in L. confuses crude extract. L-HicDH activity was
measured using the method described by Schütte et al. (32).
However, in the present study, L-HicDH activity could not be
detected in the cell-free extract of Lactobacillus sp. SK007 (data
not shown). This may be due to the fact that the enzyme is
strain-dependent or depends on the strain’s nature. Considering
the predominance of LDH in LAB strains, it is evident that LDH
is mainly responsible for the production of PLA, despite the
presence of L-HicDH.
Figure 6. Correlation between LDH activities with PPA or pyruvate and
PLA production: (A) correlation between LDH activity with pyruvate and
PLA production; (B) correlation between LDH activity with PPA and PLA
production. The activities of LDH produced by 10 isolated lactobacilli were
measured using pyruvate or PPA as substrates in cell-free extract. PLA
content was measured after the strains had been grown in MRS broth
containing 30.12 mM PPA at 30 °C for 24 h.
Table 3. LDH Produced by Some LAB Species: Apparent Km with PPA or
Pyruvate
strain
PPA (mM)
pyruvate (mM)
ref
Lactobacillus sp. SK007
L. pentosus
L. pentosus
L. plantarum
L. confusus
1.69
0.8
15
20
3.0
0.32
0.12
1.8
1.2
0.68
this work
27
28
29
23
LDH is a NADH-dependent oxidoreductase and requires
pyridine nucleotide cofactors for catalysis. In this work, it was
shown that NADH is essential for the enzymatic production of
PLA from PPA. The high cost of pyridine cofactors, however,
necessitates in situ cofactor regeneration for preparative ap-
plications. At present, the most profitable methods are the use
PPA and PLA production is represented by a linear regression
(R2 ) 0.9606) in Figure 6B. These results demonstrated that
Table 4. Activities of LDH Produced by 10 Isolated LAB Strains and Their Abilities To Produce PLA from PPA
activity with
pyruvatea (units)
activity with
PPAa (units)
relative
activityb (%)
LAB strain
species
PLAa (mM)
SK007
SK001
SK002
SK003
SK004
SK005
SK006
SK008
SK009
SK010
Lactobacillus sp.
L. plantarum
L. plantarum
L. plantarum
L. plantarum
L. plantarum
L. pentosus
L. pentosus
L. pentosus
L. pentosus
2679.95 ( 67.23
3406.80 ( 80.92
620.05 ( 7.29
1344.00 ( 15.12
367.71 ( 14.78
8061.31 ( 127.62
3336.78 ( 94.90
7100.82 ( 128.03
5938.89 ( 147.69
1864.56 ( 76.46
296.40 ( 4.80
170.34 ( 3.37
53.20 ( 0.81
143.54 ( 1.63
46.00 ( 1.85
260.38 ( 5.40
287.63 ( 7.12
173.97 ( 3.67
64.14 ( 1.47
183.10 ( 7.38
11.06
5.00
8.58
10.68
12.51
3.23
8.62
2.45
1.08
9.82
14.04 ( 0.42
8.86 ( 0.33
2.78 ( 0.19
6.33 ( 0.35
1.87 ( 0.09
10.97 ( 0.53
13.06 ( 0.47
9.82 ( 0.26
3.55 ( 0.32
7.59 ( 0.48
a Data were expressed as the mean ( SD from triplicate experiments. b Relative activity is the percentage of enzyme activity with PPA to that with pyruvate.

2398 J. Agric. Food Chem., Vol. 56, No. 7, 2008
Li et al.
of formate dehydrogenase for NADH regeneration (33). Re-
cently, PLA production using resting cells was reported to be
effective with glucose addition as cosubstrate (17). Glucose and
glucose dehydrogenase may be applied for NADH regeneration
in PLA production by enzyme. Further studies are needed to
develop NADH regeneration methods suitable for the enzymatic
production of PLA.
PLA is an effective marker of LAB antifungal action, and it
is therefore becoming an important selection criterion for
LAB (11, 14). Previously, very few LAB strains had been shown
to produce PLA, but recently, 22 of the 29 tested strains were
found to be involved in the production of such metabolites that
contribute to food quality preservation along with the sensorial
characteristics of fermented products (11). PLA production by
many LAB strains may be attributed to LDH catalyzing the
reduction of pyruvate to lactate, which could convert PPA to
PLA, although only a few amounts of PPA are generated from
Phe after transamination (Figure 1).
Although LDH is widely distributed in LAB, PLA-producing
ability was found to vary among the LAB strains tested in this
study. In fact, LAB strains producing LDH with high activity
toward pyruvate are not necessarily high PLA producers. After
transamination, PPA reduction was found to be the second
bottleneck in PLA production. LDH has long been recognized
in LAB, but its activity toward PPA and pyruvate is different
depending on the strain. LDH from Lactobacillus helVeticus
CNRZ 32 had higher activity with pyruvate, but no activity was
detected with PPA (30). In contrast, LDH from L. plantarum
had a high activity with PPA (29). In this study, a positive
correlation between LDH activity toward PPA in 10 isolated
LAB strains and their abilities to produce PLA from PPA were
demonstrated. In fact, strains possessing high LDH activities
toward PPA produced also high amounts of PLA. Therefore,
high production of PLA may be achieved by screening a relevant
number of LAB strains displaying high LDH activity with PPA.
PLA, being a novel antimicrobial compound produced by
LAB, represents a promising natural substance for controlling
contaminants in food systems (1, 4). On the other hand, the
use of functional starter cultures in the food fermentation
industry is being explored (34). Specifically, LAB strains, being
regarded as functional starter cultures, are able to produce
antimicrobial substances, sugar polymers, sweeteners, aromatic
compounds, useful enzymes, or nutraceuticals (35). From this
point of view, PLA-producing LAB may be applied as functional
starter cultures for food preservation due to their broad inhibitory
activity against a variety of foodborne microorganisms (35, 36).
For instance, L. plantarum ITM21B, which produced 0.34 mM
PLA in MRS medium, when used as a sourdough starter, has
been shown to delay the growth of Aspergillus nigerand
Pennicillium roqueforti for up to 7 days and to significantly
prolong the shelf life of bread (3). Lactobacillus sp. SK007,
which was isolated from Chinese traditional pickles, showed
99% similarity with L. plantarum by 16S rDNA sequence (17)
and produced 14.04 mM PLA in MRS broth containing PPA.
These properties provide the possibility of utilizing Lactobacillus
sp. SK007 as a functional starter culture in the production of
some fermented foods. However, further studies are needed to
ascertain the contribution of this strain to food quality and
preservation.
sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
BSA, bovine serum albumin; HPLC, high-performance liquid
chromatography.
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ABBREVIATIONS USED
PLA, phenyllactic acid; LAB, lactic acid bacteria; PPA,
phenylpyruvic acid; Phe, phenylalanine; MRS, de Man,
Rogosa, and Sharpe; LDH, lactate dehydrogenase; SDS-PAGE,

LDH-Catalyzed Conversion of PPA into PLA
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Received for review October 27, 2007. Revised manuscript received
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