Discovery and characterization of a thermostable D-lactate dehydrogenase from Lactobacillus jensenii through genome mining

Article abstract of DOI:10.1016/j.procbio.2012.11.013

The demand on thermostable D-lactate dehydrogenases (d-LDH) has been increased for d-lactic acid production but thermostable d-DLHs with industrially applicable activity were not much explored. To identify a thermostable d-LDH, three d-LDHs from different Lactobacillus jensenii strains were screened by genome mining and then expressed in Escherichia coli. One of the three d-LDHs (d-LDH3) exhibited higher optimal reaction temperature (50 °C) than the others. The T₅₀¹⁰ value of this thermostable d-LDH3 was 48.3 °C, much higher than the T₅₀¹⁰ values of the others (42.7 and 42.9 °C) and that of a commercial D-lactate dehydrogenase (41.2 °C). The T_m values were 48.6, 45.7 and 55.7 °C for the three d-LDHs, respectively. In addition, kinetic parameter (k _cat/K_m) of d-LDH3 for pyruvate reduction was estimated to be almost 150 times higher than that for lactate oxidation at pH 8.0 and 25 °C, implying that D-lactate production from pyruvate is highly favored. These superior thermal and kinetic features would make the d-LDH3 characterized in this study a good candidate for the microbial production of D-lactate at high temperature from glucose if it is genetically introduced to lactate producing microbial.

Full text of DOI:10.1016/j.procbio.2012.11.013

Process Biochemistry 48 (2013) 109–117
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Discovery and characterization of a thermostable d-lactate dehydrogenase from
Lactobacillus jensenii through genome mining
Chanha Juna,1, Young Seung Saa,1, Sol-A Gua, Jeong Chan Jooa, Seil Kimb,
Kyung-Jin Kimc, Yong Hwan Kima,∗
a Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea
b Korea Institute of Science and Technology (KIST), Seoul 136-791, Republic of Korea
c School of Life Sciences and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 June 2012
Received in revised form 7 October 2012
Accepted 13 November 2012
Available online 21 November 2012
The demand on thermostable d-lactate dehydrogenases (d-LDH) has been increased for d-lactic acid
production but thermostable d-DLHs with industrially applicable activity were not much explored. To
identify a thermostable d-LDH, three d-LDHs from different Lactobacillus jensenii strains were screened
by genome mining and then expressed in Escherichia coli. One of the three d-LDHs (d-LDH3) exhibited
higher optimal reaction temperature (50 ◦C) than the others. The T5010 value of this thermostable d-LDH3
was 48.3 ◦C, much higher than the T50 values of the others (42.7 and 42.9 ◦C) and that of a commer-
10
Keywords:
d-Lactate dehydrogenase
d-Lactic acid
Lactobacillus jensenii
Thermostability
cial d-lactate dehydrogenase (41.2 ◦C). The Tm values were 48.6, 45.7 and 55.7 ◦C for the three d-LDHs,
respectively. In addition, kinetic parameter (kcat/Km) of d-LDH3 for pyruvate reduction was estimated to
be almost 150 times higher than that for lactate oxidation at pH 8.0 and 25 ◦C, implying that d-lactate
production from pyruvate is highly favored. These superior thermal and kinetic features would make the
d-LDH3 characterized in this study a good candidate for the microbial production of d-lactate at high
temperature from glucose if it is genetically introduced to lactate producing microbial.
Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction
fermentation at high yields and titers at temperatures between
30 and 40 ◦C [12,13]. However, despite the fact that several lac-
Lactic acid, a monomer required for the production of polylactic
acid (PLA), is the major end product of carbohydrate fermentation
by industrially important homofermentative lactic acid bacteria.
Two isomers of lactic acid, the dextrorotatory (d-) and levoro-
tatory (l-) isomers, can be produced by lactate dehydrogenases
(LDHs). Optically pure isomers can be produced as separate prod-
ucts using chiral-specific D- or L-lactate dehydrogenase (d- or
l-LDH) enzymes [1]. d-LDHs catalyze the NAD-dependent conver-
sion of pyruvate to d-lactic acid and the reverse reaction [2].
l-LDHs have been thoroughly studied because l-lactic acid is
widely used in food, cosmetics and medicine [3,4]. In contrast, there
are few applications of d-lactic acid [5,6], and detailed research on
d-LDHs has been comparatively neglected [7,8]. Increasing inter-
est in stereocomplex PLA, comprising PDLA and PLLA, which has
better properties than racemic PLA – such as increased melting
point and impact strength – has boosted the production of optically
pure d-lactic acid, which is required for the preparation of PDLA
[9–11]. l-Lactic acid can be produced commercially by microbial
tic acid bacteria, such as Lactobacillus delbrueckii and Lactobacillus
coryniformis, produce d-lactic acid rather than l-lactic acid [14–16],
the productivity of d-lactic acid fermentation using Lactobacillus
was relatively lower than that of l-lactic acid fermentation at
40 ◦C incubation temperature, which may imply that thermosta-
bilty of d-LDHs can be weaker than that of l-LDHs. Compared
with thermostable l-LDHs [17], thermostable d-LDHs have not
been well characterized at genetic level, and d-LDHs have lower
thermostabilities than the l-LDHs in the same hosts [18,19]. Hyper-
thermostable d-LDHs have been found in thermophiles such as
Sulfolobus tokodaii, but these enzymes cannot be used in the pro-
duction of d-lactic acid because thermophilic enzymes are not
highly active at normal culture temperatures (∼40 ◦C) [20]. Wang
et al. successfully produced d-lactate by microbial fermentation at
50 ◦C using a thermostable d-LDH [12]. However, this enzyme had
evolved from a glycerol dehydrogenase, and it had dual activities
toward glycerol and d-lactate. Therefore, the discovery or engi-
neering of thermostable d-LDHs, which should be active at culture
temperatures above 40 ◦C, is necessary for the economical produc-
tion of d-lactic acid.
This work attempted to search for thermostable d-LDHs that
could convert pyruvate into d-lactic acid. Even though the d-LDHs
in supernatant of Lactobacillus jensenii culture broth were reported
∗
Corresponding author. Tel.: +82 2 940 5675; fax: +82 2 941 1785.
E-mail address: metalkim@kw.ac.kr (Y.H. Kim).
These authors contributed equally to this work.
1
1359-5113/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2012.11.013

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C. Jun et al. / Process Biochemistry 48 (2013) 109–117
active at up to 50 ◦C [21], however there has been no available
information for these d-LDHs including gene and protein sequence.
We have tried the functional expression of genes encoding three
L. jensenii d-LDHs in E. coli to find and characterize thermostable
d-LDHs up to 50 ◦C.
2.4. d-LDH activity assays
To determine the enzymes’ activities, the absorbance at 340 nm was continu-
ously recorded used to monitor the NADH concentration during the redox reactions
catalyzed by the d-LDHs for 1 min. One unit of activity was defined as the amount
of enzyme that catalyzed the oxidation of 1 ␮mol NADH per minute under standard
conditions (25 ◦C, pH 7.0). The determination of enzymes’ activities for the reduction
of pyruvate was performed using 50 mM Tris–HCl (pH 7.0) buffer solution containing
10 mM sodium pyruvate, 0.2 mM NADH, 0.1 ␮g of d-LDH1, 2 and 1 ␮g of d-LDH3.
To determine the enzymes’ activities for the oxidation of sodium d-lactate, assay
mixtures containing 50 mM buffer (Tris–HCl (pH 8.0) for d-LDH1, 2 and sodium
bicarbonate–NaOH (pH 10.0) for d-LDH3), 100 mM sodium d-lactate, 2 mM NAD+
and 10 ␮g d-LDHs were used. The reactions were started by the addition of the
enzyme solutions.
2. Materials and methods
2.1. Phylogenetic analysis
Genes encoding d-LDHs were identified in these all Lactobacillus types which
are L. jensenii 1153 (ABWG00000000), L. jensenii JV-V16 (ACGQ00000000), L. jensenii
27-2-CHN (ACOF00000000), L. jensenii 269-3 (ACOY00000000), L. jensenii SJ-7A-US
(ACQD00000000), L. jensenii 115-3-CHN (ACQN00000000) and L. jensenii 208-
1 (ADEX00000000) and their relatives in the GenBank database. The retrieved
sequences were aligned, and the phylogenetic tree for d-LDH was generated using
the maximum-likelihood method. The tree was evaluated using the bootstrap
method with 1000 resamplings [22]. The alignment and the phylogenetic analysis
were carried out using MEGA5 [23]. The analysis involved 27 amino acid sequences.
All positions containing gaps and missing data were eliminated. The final data set
comprised 328 positions. Three genes were selected as representative d-LDH genes
from L. jensenii: ZP05866095 (d-LDH1 from L. jensenii SJ-7A-US), ZP05557096 (d-
LDH2 from L. jensenii 27-2 CHN) and ZP04645201 (d-LDH3 from L. jensenii 269-3).
2.5. Substrate specificity of d-LDHs
Substrate specificities of d-LDHs were examined using 50 mM sodium phos-
phate buffer (pH 8.0) containing 10 mM various substrates (sodium pyruvate,
2-ketobutyric acid, oxaloacetic acid, sodium phenylpyruvate) for reduction reac-
tion and 10 mM various substrates (sodium d-lactate, (R)-2-hydroxybutyric acid,
d-(+)-malic acid, d-(+)-3-phenyllactic acid) for oxidation reaction.
2.6. Thermostability and thermal inactivation of d-LDHs
For the study of thermal inactivation of d-LDHs at fixed constant temperature,
the d-LDHs containing solutions were incubated at 45 ◦C for different time intervals
from 0 to 90 min, and then cooled on ice for 10 min. Thermal inactivation constants
of d-LDHs were determined by estimating values of half-life (t1/2). All data analyses
were performed by using linear regression fitting.
The kinetic stabilities of the d-LDHs were determined by measuring the residual
activity after incubation at different temperatures. Enzyme solutions were incu-
bated at 25–55 ◦C for 10 min and cooled for 10 min on ice before measuring the
residual enzyme activities. Resistance to heat inactivation (T5010) is defined as the
temperature at which half of an enzyme’s activity remains after 10 min incubation
relative to that after 10 min incubation at 25 ◦C.
2.2. Construction of the expression plasmid
The genes for d-LDHs tagged with hexa-histidine at the C-terminus were chem-
ically synthesized by GenScript (USA) in the pUC57 vector. The N-terminus was
designed to contain an NdeI restriction site, and the C-terminus contained an
EcoRI restriction site. The pUC57 vector containing the genes encoding d-LDHs was
amplified using E. coli DH5␣ cells (RBC Bioscience, Taiwan) and isolated by using
LaboPassTM Plasmid Mini Purification Kit (Cosmo Genetech, Korea).
The d-LDH genes were digested with the corresponding restriction enzymes
and ligated with the NdeI-EcoRI-digested pET-22b(+) vector (Novagen, USA). The
resulting plasmid containing the inserts was transformed into E. coli DH5␣ cells. The
constructed pET-22b(+)-d-LDH1–3 vector was also transformed into an expression
host, E. coli BL21 (␭DE3) (RBC Bioscience, Taiwan).
Differential scanning fluorimetry (DSF) using
a real-time quantitative PCR
thermal cycler (LightCycler 480, Roche, USA) was utilized to determine the confor-
mational stabilities of the enzymes by measuring the temperatures of their melting
transitions (Tm). SYPRO Orange dye (Invitrogen, USA) was used to monitor enzyme
unfolding. The hydrophobic dye could successfully detect the exposure of hydropho-
bic residues in the core during thermal unfolding [25]. A 100× stock solution was
prepared by adding 20 ␮L of SYPRO Orange (5000× stock in DMSO) to 980 ␮L of
phosphate buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Enzyme–dye solution
(50 ␮L) containing 20 ␮L of purified d-LDH (10–50 ␮M), 20 ␮L of phosphate buffer
and 10 ␮L of 100× SYPRO Orange stock (20× in the final solution) was placed in
96-well qPCR microplates and heated from 20 to 80 ◦C.
2.3. Expression and purification of the d-LDHs
For small-scale experiments, E. coli transformants were picked from the plate
and inoculated overnight in 5 mL of Luria–Bertani (LB) medium (10 g tryptone, 5 g
yeast extract, 10 g sodium chloride per liter) supplemented with 100 ␮g/mL ampi-
cillin for cells harboring pET-22b(+)-d-LDH1-3 Fresh LB medium (150 mL) with
100 ␮g/mL ampicillin was inoculated and incubated at 37 ◦C and 200 rpm. When
the OD600 reached 0.6–0.8, the expression of the d-LDHs was induced for 4 h at
25 ◦C and 200 rpm by adding 1.0 mM isopropyl-␤-d-thiogalactopyranoside (IPTG).
Cells were harvested by centrifugation (13,000 × g, 30 min and 4 ◦C), the super-
natant was decanted, and the pellet was resuspended in BugBuster Master Mix
(Novagen, USA) according to the manufacturer’s guidelines. The resulting suspen-
sion was incubated at 25 ◦C for 30 min with gently shaking. The suspension was
separated into soluble and insoluble fractions by centrifugation at 13,000 × g for
30 min at 4 ◦C.
Because d-LDH genes were fused with a hexa-histidine affinity purification motif
(6× His-tag) at their C-termini, affinity chromatography (Ni-NTA column, Qiagen,
USA) was used to purify the recombinant d-LDHs. Supernatants containing the solu-
ble recombinant d-LDHs were incubated with 1 mL of Ni-NTA agarose bead (Qiagen,
USA) at 4 ◦C for 60 min with gently shaking. The Ni-NTA agarose bead was loaded
onto column after incubation, which were washed with 100 ml of washing buffer
(50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole (pH 8.0)). The recombinant d-
LDHs were finally eluted with 2 mL of elution buffer (50 mM NaH2PO4, 300 mM
NaCl and 250 mM imidazole (pH 8.0)).
The expression and purity of the recombinant d-LDHs were monitored by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native
PAGE. The SDS-PAGE was performed by the method of Laemmli using 5% stacking
gel and 10% separating gel [24]. The same system was used for native PAGE except
the SDS. 5% stacking gel and 10% separating gel was used as the resolving gel. After
SDS-PAGE and native PAGE, the gels were stained by 0.1% Coomasie brilliant blue R-
250 (Sigma–Aldrich, USA) in mixture of methanol/acetic acid/water, 45:10:45 (v/v),
for 30 min and then destained in mixture of methanol/acetic acid/water, 10:10:80
(v/v), for overnight. The protein concentrations were determined using the Bradford
reagent (Sigma–Aldrich, USA) according to the manufacturer’s instructions.
The multimeric state of d-LDH3 was determined by measuring the apparent
molecular weight using a SuperdexTM 75 column and fast protein liquid chro-
matography (GE Healthcare, USA). The apparent molecular weight of d-LDH3 was
calculated using molecular markers (Sigma–Aldrich, USA) including cytochrome
c (12.4 kDa), carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehydroge-
nase (150 kDa) and amylase (200 kDa) in running buffer (50 mM sodium phosphate
buffer, pH 8.0, 300 mM NaCl).
2.7. Determination of optimal temperature and pH for d-LDHs
The temperature and pH dependencies of d-LDHs were determined by using the
standard activity assays for reduction and oxidation as described above. All param-
eters were kept constant except the temperature or pH. Assay solution without
enzymes was preincubated at the fixed temperature from 10 to 60 ◦C for 60 min
and reaction was initiated by the addition of enzyme to determine the optimal
temperature. Relative activities were expressed respective to maximum activity.
The optimal pH for d-LDH activity was assessed by varying the pH using standard
potassium–citrate activity buffer (pH 5.0–6.0), Tris–HCl (pH 7.0–9.0) and sodium
bicarbonate–NaOH (pH 10.0–11.0).
2.8. Kinetic parameters of d-LDHs
A kinetic study of d-LDHs was performed by measuring the oxidation and reduc-
tion rate for NADH and NAD+ at pH 8.0 and 25 ◦C. Various concentrations of NADH
(0.02–0.50 mM) were tested with pyruvate at a constant concentration of 50 mM.
NAD+ was tested at 0.02–2.00 mM with d-lactate at a constant concentration of
100 mM. The kinetic parameters (kcat and Km) were determined by Lineweaver–Burk
plots. All experimental data in this study were the average values of triplicate mea-
surements.
3. Results and discussion
3.1. Extraction of the d-LDH genes from the genome of L. jensenii
and their amino acid compositions
The d-LDH genes of L. jensenii were clustered in two mono-
phyletic groups in the phylogenetic tree (Fig. 1). Cluster I formed
a monophyletic group with genes from L. gasseri, L. johnsonii and

C. Jun et al. / Process Biochemistry 48 (2013) 109–117
111
Fig. 1. The phylogenetic tree of d-LDH inferred using the maximum-likelihood method. The percentages at the nodes are the levels of bootstrap support based on maximum-
likelihood analyses of 1000 resampled data sets. Only values over 50% are shown. The d-LDH gene of Vibrio cholerae V52 (ZP01680838) was used as an outgroup. Scale bar,
0.2 amino acid substitution per position. 1d-LDH1; 2d-LDH2, 3d-LDH3.
L. acidophilus, and cluster II formed a monophyletic group with
genes from L. delbrueckii, which can grow at 45 ◦C [26]. The sim-
ilarity between d-LDH2 and d-LDH3 was only 47%, indicating that
the genes in cluster I are paralogs of the genes in cluster II. Both the
phylogenetic analysis and the levels of similarity between the d-
LDH genes suggested that d-LDH3 likely has unique characteristics
compared with d-LDH1 and d-LDH2.
3.2. Expression and purification of the d-LDHs
Specific activities of purified enzymes were calculated to be
3100–3150 U/mg for d-LDH1, 2 and 380 U/mg for d-LDH3 in the
reduction of sodium pyruvate with NADH as a cofactor at 25 ◦C.
And the purified d-LDH1 and d-LDH2 showed specific activities
of 88–97 U/mg and the purified d-LDH3 showed specific activities
of 42 U/mg in the oxidation of sodium d-lactate with NAD+ as a
cofactor at 25 ◦C.
A single band with an apparent molecular size of 34–43 kDa
appeared, confirming the calculated molecular masses of 39.93,
37.73 and 37.41 kDa derived from the amino acid sequences of
single subunits (Fig. 3a). However, the native PAGE gel (Fig. 3b)
Multiple sequence alignment was used to compare the d-LDH
sequence identities (Fig. 2). The enzymes had conserved cat-
alytic and NAD-binding residues. d-LDH1 and d-LDH2 showed 98%
sequence identity. The sequence of d-LDH1 without first 20 amino
acids at the N-terminus (MLHNKSCIIKISDCNIWRYN) is identical to
that of d-LDH2 except for six amino acids (based on the num-
bering of d-LDH1; Met147 to Leu, Gln183 to Lys, Lys206 to Glu,
Val265 to Ile, Lys291 to Glu and Gln294 to Lys). d-LDH2 and d-LDH3
had 33% sequence identity, and their amino acid compositions are
quite different (Table 1). The comparison of d-LDH2 and d-LDH3
revealed that d-LDH2 contained more charged residues (30.33%;
9.31% Asp, 7.21% Glu, 2.40% His, 7.51% Lys, 3.90% Arg) and that d-
LDH3 contained more hydrophobic residues (40.00%; 11.52% Ala,
7.27% Val, 8.48% Ile, 9.09% Leu, 3.64% Phe). Even though simple
comparison based on the primary amino acid sequences and com-
position cannot the properties of folded enzymes, there are reports
that hydrophobic residues in the core or at the protein–protein
interface could contribute to greater thermostability of enzymes
[27,28]. Moreover, d-LDH3 showed low sequence similarity with
known mesophilic d-LDHs from Lactobacillus helveticus (PDB code:
2dld, 39%) and Lactobacillus bulgaricus (PDB code: 1j49, 35%) for
which X-ray crystal structures were available. On the other hand,
d-LDH1 and 2 are highly similar to mesophilic d-LDHs (80–90%),
indicating that the high thermostability of d-LDH3 may the result
of the sequence differences discussed above. Although no struc-
tural information is available, the amino acid compositions could
be useful in explaining the differences in the thermostabilities of
d-LDH1, 2 and 3.
showed that d-LDH3 could have
a multimeric form. Unlike
other reports in BRENDA (http://www.brenda-enzymes.info/) stat-
ing that most d-LDHs can form homodimers, the size-exclusion
chromatography analysis revealed that d-LDH3 could form a
homotetramer (Fig. 3c). d-LDH1 and d-LDH2 seemed to form a
mixture of oligomers and homodimers, respectively.
The enantiopreference of the d-LDHs was confirmed by the
oxidation of d- and l- lactate. All enzymes isolated in this study
exhibited d-LDH activity and no L-LDH activity (Table 2).
For reduction reaction catalyzed by various d-LDHs, d-LDH1 and
d-LDH2 showed very similar substrate preference, on the while
d-LDH3 showed different preference as shown in Table 3. Only
d-LDH3 catalyzed the reduction of 2-ketobutyric acid consider-
ably (54% of pyruvate reduction activity), however, other d-LDHs
showed higher reduction activity for oxaloacetic acid (around 90%
of pyruvate reduction activity). These results of d-LDH1 and 2
showed very similar substrate specificities of d-LDH from L. bul-
garicus [29,30]. Morever, d-LDH from L. bulgaricus showed high
sequence similarity with d-LDH1 and 2 as mentioned above. Even
though oxidation activity of d-LDH3 showed much lower than that
of reduction activity for pyruvate, d-LDH3 showed relatively high
oxidation rate for (R)-2-hydroxybutyric acid, which implies that

112
C. Jun et al. / Process Biochemistry 48 (2013) 109–117
Fig. 2. Multiple sequence alignment of d-LDH1, 2 and 3. Asterisks and dots indicate identities and similarities, respectively. The solid and dashed arrows indicate conserved
catalytic residues (Arg, Glu and His) and the NAD-binding residue (Asp) of the d-LDH family, respectively.
three dimensional structure of d-LDH3 can be quite different with
those of other d-LDHs.
determined by reduction reactions with NADH and oxidation reac-
tions with NAD+ (Fig. 4). d-LDH1, d-LDH2 and d-LDH3 showed
similar activity–pH profile, and optimal activity was observed at pH
7 at reduction reactions. Maximal activities of oxidation reaction of
d-LDH1 and 2 were obtained at pH 8. However, the optimum pH
of d-LDH3 was shifted to a more basic pH (pH 10) (Fig. 4b). Garvie
et al. reported that the d-LDH of L. jensenii has an optimum pH of
7.8 [33], similar to the values for d-LDH1 and d-LDH2. d-LDH3 had
more alkaline pH optimum. Since d-LDH3 showed basic pH opti-
mum for oxidation reaction compared with other d-LDHs, d-LDH3
can yield higher net reduction rate for pyruvate to produce d-lactate
at neutral pH, which can be a leading point.
3.3. Optimum pH of the d-LDHs
d-LDHs from other hosts, such as L. delbrueckii subsp. bulgari-
cus, L. delbrueckii and Staphylococcus epidermidis, showed optimum
pHs of 7.4–8.5 [30–32]. The optimum pH of the d-LDHs was
Table 1
Amino acid compositions of the investigated d-LDHs.
Amino acids
d-LDHs (number and percentage)
d-LDH1
d-LDH2
d-LDH3
3.4. Optimal temperatures of the d-LDHs
Alanine
Cysteine
30 (8.50%)
2(0.57%)
30(9.01%)
0(0.00%)
38(11.52%)
2(0.61%)
Aspartic acid
Glutamic acid
Phenylalanine
Glycine
Histidine
Isoleucine
Lysine
32(9.07%)
22(6.23%)
13(3.68%)
23(6.52%)
9(2.55%)
21(5.95%)
27(7.65%)
22(6.23%)
13(3.68%)
18(5.10%)
14 (3.97%)
10 (2.83%)
14(3.97%)
9(2.55%)
31(9.31%)
24(7.21%)
13(3.90%)
23(6.91%)
8(2.40%)
18(5.41%)
25(7.51%)
22(6.61%)
11(3.30%)
15(4.50%)
14(4.20%)
8(2.40%)
13(3.90%)
7(2.10%)
17(5.11%)
40(12.01%)
3(0.90%)
23(6.97%)
22(6.67%)
12(3.64%)
17(5.15%)
7(2.12%)
28(8.48%)
27(8.18%)
30 (9.09%)
9(2.73%)
The optimal temperatures of the d-LDHs were determined
by measuring reduction reactions of sodium pyruvate as a sub-
strate and oxidation reactions of sodium d-lactate as a substrate.
Maximal activity of reduction reaction of the d-LDHs was observed
at 50 ◦C. Only d-LDH3 was active above 55 ◦C (Fig. 5a). At 55 ◦C, d-
LDH3 exhibited 80% of its activity at 50 ◦C, but the other d-LDHs
exhibited less than 5% of their activities at 50 ◦C. In the oxida-
tion reaction, only d-LDH3 was active above 40 ◦C (Fig. 5b). At
45 ◦C, d-LDH3 exhibited 85% of its activity at optimal tempera-
ture (40 ◦C), but the other d-LDHs exhibited less than 5% of their
activities at optimal temperature (30 ◦C). Only d-LDH3 was active
over a broad temperature range, and thus this enzyme could be a
suitable d-LDH for the production of d-lactate at high-temperature
cultures. It implies that d-LDH3 may have more rigid folding struc-
ture compared with those of other d-LDHs. Considering most d-LDH
Leucine
Methionine
Asparagine
Proline
Glutamine
Arginine
Serine
Threonine
Valine
16(4.85%)
11(3.33%)
5(1.52%)
9(2.73%)
19(5.76%)
18(5.45%)
24(7.27%)
0 (0.00%)
17(4.82%)
41 (11.61%)
4(1.13%)
Tryptophan
Tyrosine
12(3.40%)
11(3.30%)
13(3.94%)

C. Jun et al. / Process Biochemistry 48 (2013) 109–117
113
Fig. 3. Expression and purification of recombinant d-LDHs. (a) SDS-PAGE analysis: Lane M: molecular mass markers (GenDEPOT, USA), (1): purified d-LDH1 (0.5 ␮g), (2):
purified d-LDH2 (0.5 ␮g), (3): purified d-LDH3 (0.5 ␮g). (b) Native PAGE analysis: Lane M: molecular mass markers (Sigma–Aldrich, USA), (1): purified d-LDH1 (10 ␮g),
(2): purified d-LDH2 (10 ␮g), (3): purified d-LDH3 (10 ␮g). (c) The apparent molecular weight of d-LDH3 determined by the FPLC system. AMY: amylase, ADH: alcohol
dehydrogenase, LDH: d-LDH3, BSA: albumin, CAH: carbonic anhydrase, CytC: cytochrome c.
showed low temperature as optimum, d-LDH3 can attract more
attention since it can be used useful biocatalysts in vitro as well as
in vivo [2].
time (Fig. 6). The time required for the residual activity to be
reduced to half (t1/2) of d-LDHs at 45 ◦C was calculated to be
39.4, 47.6 and 72.2 min, respectively (Table 4). It means that d-
LDH3 showed much longer lifetime to be thermally inactivated at
45 ◦C compared with other d-LDHs. In addition of the half time for
inactivation, other parameters showing kinetic and thermody-
namic stabilities also point out the superior property of d-LDH3
(Table 4).
3.5. Thermostability and thermal inactivation of d-LDHs
The thermal inactivation rate constants (kd) of d-LDHs were
determined from the slope of the logarithmic plot of activity against

114
C. Jun et al. / Process Biochemistry 48 (2013) 109–117
Table 2
Table 3
The enantioselectivity of the three d-LDHs toward D- and L-lactate.
The substrate specificity of the three d-LDHs.
Substrate
Specific d-LDHs activity [U/mg]
Substrate
Enzyme activity [U/mg] (%)*
d-LDH1
d-LDH2
d-LDH3
d-LDH1
d-LDH2
d-LDH3
l-Lactate
d-Lactate
<0.01
96.88
<0.01
88.71
<0.01
41.91
Sodium pyruvate
2-Ketobutyric acid
Oxaloacetic acid
3838 (100)
68(0.27)
3151(82.12)
7 (0.18)
2997 (100)
41 (1.37)
2877(96.00)
9 (0.31)
348 (100)
189(54.21)
18(5.05)
Sodium phenylpyruvate
28(8.07)
Resistance to heat inactivation (T5010) was determined to
compare the kinetic stabilities of the d-LDHs. Lactobacillus mesen-
teroides D-LDH (Megazyme Ireland Ltd, Ireland) was also tested to
allow comparison with a commercially available d-LDH. d-LDH3
was the most thermostable of the four tested d-LDHs (Fig. 7a).
After 10 min incubation at 45 ◦C, d-LDH1 and 2 and the commer-
cial D-LDH showed less than 20% residual activity, whereas d-LDH3
Sodium d-lactate
(R)-2-Hydroxybutyric acid
D-(+)-Malic acid
17.82 (100)
0.05 (0.27)
0.00 (0.00)
0.00 (0.00)
14.97 (100)
0.05 (0.32)
0.00 (0.00)
0.00 (0.00)
9.74 (100)
10.24 (105)
0.11 (1.09)
3.70 (38.0)
D-(+)-3-Phenyllactic acid
*
Values in parenthesis mean the relative values for sodium pyruvate and sodium
d-lactate, respectively.
10
retained 70% of its activity. The T50 values of d-LDH1 and 2 and
thed-LDHofL. jensenii62Glost25%ofitsactivityafter5 min incuba-
tion at 50 ◦C. According to this study, this loss of activity at 50 ◦C also
seems to occur for the d-LDHs of L. jensenii 62G that are homologous
to d-LDHs 1 or 2 but not for the d-LDH homologous to d-LDH3, a
result that is supported by similar optimum pHs of d-LDHs 1 and 2.
the commercialenzyme were calculatedto be 42.7, 42.9 and 41.2 ◦C,
respectively. The T5010 value of d-LDH3 was calculated to be 48.3 ◦C.
The NAD-dependent d-LDHs from other bacteria, such as L. plan-
tarum, L. acidophilus, L. fermentum, L. leichmannii and Staphylococcus
haemolyticus [31,33], were inactivated completely at 50 ◦C, indi-
cating d-LDH3 is more thermostable than the previously reported
NAD-dependent LDHs. In previous study, Gasser et al. reported that
Fig. 5. The activity–temperature profile of the d-LDHs to determine optimal temper-
ature. (a) The assay solutions without enzyme were pre-incubated at the indicated
temperature for 1 h, and the activity was then measured at pH 7.0 using sodium
pyruvate as a substrate for 1 min. (b) The enzymes and assay solutions were pre-
incubated at the indicated temperature for 1 h, and the activity was then measured
at pH 8.0 (Tris–HCl) for d-LDH1, 2 and at pH 10.0 (sodium bicarbonate–NaOH) for
d-LDH3 using sodium d-lactate as a substrate for 1 min. Relative activities were
expressed respective to maximum activity (ꢀ) d-LDH1, (᭹) d-LDH2, (ꢁ) d-LDH3.
Fig. 4. The activity–pH profile of the d-LDHs measured at room temperature to
determine optimal pH. (ꢀ) d-LDH1, (᭹) d-LDH2, (ꢁ) d-LDH3. Relative activities
were expressed respective to maximum activity. (a) The reduction reaction using
sodium pyruvate as a substrate. (b) The oxidation reaction using sodium d-lactate
as a substrate.

C. Jun et al. / Process Biochemistry 48 (2013) 109–117
115
Table 4
Thermostabilities and thermal inactivation constants of d-LDHs.
10
T50
Tm
[◦C]
Kd
[min−1
t1/2
[min]
[◦C]
]
d-LDH1
d-LDH2
d-LDH3
42.7
42.9
48.6
48.6
45.7
55.7
17.60 × 10−3
14.57 × 10−3
9.60 × 10−3
39.4
47.6
72.2
The thermodynamic stabilities (Tm) of the d-LDHs were deter-
mined by the DSF method (Fig. 7c). d-LDH3 showed the highest
thermodynamic stability among the recombinant d-LDHs. The cal-
culated Tm values of d-LDHs 1, 2 and 3 were 48.59, 45.66 and
55.72 ◦C, respectively. Interestingly, d-LDHs 1 and 2 showed only
single transition peaks during their thermal unfolding, but d-LDH3
had two transition peaks (51.45 and 55.72 ◦C, data not shown).
During denaturation, the homo-tetrameric d-LDH3 first dissociated
into monomers at 51 ◦C before each of the monomers underwent
unfolding at above 55 ◦C. This structural analysis is consistent
with the kinetic stability results of d-LDH3, showing that this
enzyme has 40% residual active after 10 min incubation at 50 ◦C
but is completely inactivated after 10 min incubation at 55 ◦C
(Fig. 7a). Oligomerization can contribute to the thermostabiliza-
tion of proteins, and hydrophobic interactions at protein–protein
interfaces could aid oligomerization [34,35]. The homo-tetrameric
state of d-LDH3 was an important structural feature contributing
to its high thermostability, with its higher portion of hydropho-
bic residues possibly contributing to the multimerization. The
reversibility of the heat-induced unfolding of the d-LDHs was inves-
tigated by cooling the denatured d-LDHs at 1.0 ◦C/min. All d-LDHs
formed aggregates and exhibited no fluorescence changes (data not
shown).
3.6. Determination of kinetic parameters
The kinetic parameters of the d-LDHs were determined using
NADH and NAD+ (Table 5). d-LDHs 1 and 2 had Km values of 0.7
and 2.83 mM with NADH and 2.75 and 2.58 mM with NAD+, respec-
tively. d-LDH3 had Km values of 0.036 mM with NADH and 0.41 mM
with NAD+. These affinities for cofactors were similar to those of
other d-LDHs [8,36]. The catalytic efficiencies were much higher in
the direction of sodium pyruvate reduction than in the direction of
sodium d-lactate oxidation, suggesting that the enzymes catalyze
sodium pyruvate reduction rather than sodium d-lactate oxidation.
Fig. 7. The thermostability of the d-LDHs. (a) Kinetic stability. The enzymes were
pre-incubated at the indicated temperature for 10 min and cooled for 10 min on
ice. Residual activity was measured at 25 ◦C, pH 7.0, using sodium pyruvate as a
substrate. (b) Kinetic stability. The enzymes were pre-incubated at the indicated
temperature for 10 min and cooled for 10 min on ice. Residual activity was measured
at 25 ◦C in Tris–HCl (pH 8.0) for d-LDH1, 2 and sodium bicarbonate–NaOH (pH 10.0)
for d-LDH3, using sodium d-lactate as a substrate. (ꢀ) d-LDH1, (᭹) d-LDH2, (ꢁ) d-
LDH3, (ꢂ) d-LDH from Megazyme. (c) Thermal unfolding curves of d-LDHs measured
by the DSF method. (ꢀ) d-LDH1, (᭹) d-LDH2, (ꢁ) d-LDH3.
Fig. 6. The thermal inactivation of the d-LDHs. Residual activity was measured at
25 ◦C, pH 7.0, using sodium pyruvate as a substrate. (ꢀ) d-LDH1, (᭹) d-LDH2, (ꢁ)
d-LDH3.

116
C. Jun et al. / Process Biochemistry 48 (2013) 109–117
Table 5
Kinetic parameters for reduction and oxidation.
Enzyme
Cofactor
NADH
NAD+
Km
[mM]
kcat
[s−1
kcat/Km
Km
[mM]
kcat
[s−1
kcat/Km
]
[mM−1 s−1
]
]
[mM−1 s−1
]
d-LDH1
d-LDH2
d-LDH3
0.70
2.83
0.04
144.36
516.42
8.28
206.29
182.57
232.63
2.75
2.58
0.41
4.42
3.65
0.64
1.61
1.41
1.56
It is interesting that d-LDH3 had a lower turnover number than d-
LDH1 and 2, which could be compensated for by a higher cofactor
binding affinity. Therefore, d-LDH3 showed a higher catalytic effi-
ciency in d-lactate production than d-LDH1 and 2. In addition, the
relative catalytic efficiency of pyruvate reduction to d-lactate oxi-
dation ([kcat/Km,reduction]/[kcat/Km,oxidation]) of d-LDH3 (149.12) was
higher than that of d-LDH1 (128.13) and d-LDH2 (129.48), imply-
ing that d-LDH3 has the potential to efficiently produce d-lactate
in other transformed hosts.
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4. Conclusions
Three genes for d-LDHs were retrieved from the genome of
L. jensenii and then successfully expressed in E. coli. The three
enzymes, originating from the same species, were clustered into
two different groups and showed different characteristics, sug-
gesting that d-LDH genes with distinct activities can be found
through genome-data mining, including the genomes of uncul-
turable organisms or environmental metagenomes. The searching
allowed the identification and subsequent characterization of a
thermostable d-LDH, which could be used in the production of d-
lactate monomer for the synthesis of PDLA and stereocomplex PLA
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Acknowledgments
This research work was supported by the R&D program of
MKE/KEIT (10031717) and Kwangwoon University Grant for Pro-
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