Báo cáo khoa học: Characterization of recombinant prolidase from Lactococcus lactis – changes in substrate specificity by metal cations, and allosteric behavior of the peptidase pdf

Characterization of recombinant prolidase from
Lactococcus lactis – changes in substrate specificity by
metal cations, and allosteric behavior of the peptidase
Soo I. Yang and Takuji Tanaka
Department of Food and Bioproduct Sciences, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, Canada
Fermented foods have significant nutritional value and
are receiving growing attention from health-conscious
consumers. During fermentation, microbial activity
changes the chemical, physical and nutritional attri-
butes of the food materials. One of the main changes
during fermentation is the production of peptides and
amino acids via proteolysis. These compounds are
major factors contributing to the flavor of fermented
foods. Of these amino acids and peptides, hydrophobic
peptides exhibit undesirable bitterness in the fermented
foods [1]. For example, in Cheddar cheese, hydropho-
bic peptides ranging from 2–23 residues were found to
be responsible for bitterness [2].
Hydrophobic peptides produced during fermenta-
tion undergo further hydrolysis through general pepti-
dase reactions, which result in reduced bitterness.
However, peptides that contain proline behave differ-
ently from other peptides during general proteolysis.
As proline is structurally and chemically unique
among the 20 naturally occurring amino acids due to
its imine structure, proline-containing peptides are
much less susceptible to further enzymatic hydrolysis
[3,4]. Thus, hydrolysis of peptides during fermentation
can ultimately produce proline-containing dipeptides,
such as Xaa-Pro and Pro-Xaa. Ishibashi et al. [5]
reported two interesting observations regarding pro-

line-containing dipeptides: most of these dipeptides
are bitter, and Xaa-Pro is generally more bitter than
Pro-Xaa. Proline-containing dipeptides tend to be
accumulated as a result of the low susceptibility to
enzymatic hydrolysis, and these dipeptides are bitter;
therefore, peptidases specific for proline-containing
dipeptides could control the bitterness of fermented
foods.
Keywords
bitterness; metallopeptidase;
overexpression; PepQ; proline
Correspondence
T. Tanaka, Department of Food and
Bioproduct Sciences, College of Agriculture
and Bioresources, University of
Saskatchewan, 51 Campus Drive,
Saskatoon, Saskatchewan S7N 5A8, Canada
Fax: +1 306 966 8898
Tel: +1 306 966 1697
E-mail:
(Received 1 August 2007, revised 15
October 2007, accepted 16 November
2007)
doi:10.1111/j.1742-4658.2007.06197.x
The Lactococcus lactis NRRL B-1821 prolidase gene was cloned and over-
expressed in Escherichia coli. Under suboptimum growth conditions,
recombinant soluble and active prolidase was produced; in contrast, inclu-
sion bodies were formed under conditions preferred for cell growth.
Recombinant prolidase retained more than half its full activity between 30
and 60 °C, and was completely inactivated after 30 min at 70 °C. CD anal-

ysis confirmed that prolidase was inactivated at 67 °C. The enzyme was
active under weak alkali to weak acidic conditions, and showed maximum
activity at pH 7.0. Although these characteristics are similar to those for
other reported prolidases, this prolidase was distinctive for two kinetic
characteristics. Firstly, different substrate specificity was observed for its
two preferred metal cations, zinc and manganese: Leu-Pro was preferred
with zinc, whereas Arg-Pro was preferred with manganese. Secondly, the
enzyme showed an allosteric response to changes in substrate concentra-
tions, with Hill constants of 1.53 for Leu-Pro and 1.57 for Arg-Pro. Mole-
cular modeling of this prolidase suggests that these unique characteristics
may be attributed to a loop structure near the active site.
Abbreviations
IPTG, isopropyl thio-b-
D-galactoside; LAB, lactic acid bacteria.
FEBS Journal 275 (2008) 271–280 ª 2007 The Authors Journal compilation ª 2007 FEBS 271
Lactic acid bacteria (LAB) are widely used to pro-
duce fermented foods. LAB are nutritionally fastidious
and require amino acids as exogenous nutrients [6,7].
Required amino acids are assimilated in the form of
peptides that are produced from proteins by LAB
extracellular proteinase [8]. The assimilated peptides
are further hydrolyzed by peptidases in LAB in order
to supply free amino acids for metabolism. LAB may
have as many as 18 peptidases for efficient hydrolysis
of the imported peptides [9]. Of these peptidases, four
are proline-specific: proline iminopeptidase, prolinase,
X-prolyl dipeptidyl aminopeptidase and prolidase [9].
Prolidase (EC 3.4.13.9) is specific to Xaa-Pro dipep-
tides, which can only be hydrolyzed by this peptidase
[8]. As mentioned above, Xaa-Pro dipeptides are bitter.

Therefore, reduction of the dipeptides via prolidase
may lead to the reduction of bitterness in fermented
food products.
Prolidases have been reported from some microbial
sources, such as Lactobacillus delbrueckii subsp. bulgar-
icus CNRZ 397 [4,10], Lactobacillus casei subsp. casei
IFPL 731 [11], Pyrococcus furiosus [12] and Lb. helveti-
cus [13]. These prolidases have preferential activity on
Xaa-Pro dipeptides that have a hydrophobic amino
acid as the N-terminal residue. The prolidases do not
hydrolyze Pro-Pro or Gly-Pro, and have little activity
on hydrophilic Xaa-Pro peptides. Previous research
has shown that, in the absence of prolidase, LAB
growth is retarded by 13% [14]. Most previous
research has concentrated on kinetic characterization
of LAB physiological activity. As a consequence, there
is little information on the expression of recombinant
prolidases, the functionality of each residue or protein
engineering of this enzyme.
In the present study, the prolidase-coding gene, pepQ,
was isolated from Lactococcus lactis NRRL B-1821 and
cloned. Characterization of the recombinant protein
revealed some interesting characteristics of this proli-
dase. Moreover, this research provides the means to
investigate the structure–function relationships of proli-
dase, hence providing a greater understanding of the
characteristics of this peptidase, which would be of
industrial use in the debittering of fermented foods.
Results and Discussion
Cloning and expression of Lc. lactis prolidase

The prolidase gene of Lc. lactis NRRL B-1821 was
successfully isolated using PCR. The DNA sequence of
the isolated gene (GenBank accession number
EU216565) was virtually identical to that reported
for Lc. lactis Il1403 (GenBank accession numbers
NC_002662 and AE006395). The sequence of the iso-
lated gene has a base difference compared with Lc. lac-
tis Il1403, and this substitution results in an amino
acid change from Tyr67 to His67 in the putative amino
acid sequence. Attempts were made to express the gene
using the tac promoter on pKK223-3, and the expres-
sion system produced a large amount of recombinant
protein under the preferred growth conditions (37 °C
in LB broth) of the Escherichia coli TOP 10 F’ hosts.
The amount of recombinant protein reached about
50% of total cell extracts, as determined from SDS–
PAGE gel densitometry (data not shown). However,
the growth under these conditions yielded recombinant
prolidase as inclusion bodies. In theory, inclusion
bodies can be refolded into an active form; however, it
is uncertain whether the refolded proteins have an
identical fold to that of the native proteins. Therefore,
the above expression system was refined in order to
optimize the conditions to produce soluble protein
without inclusion body formation.
Optimization of expression and purification
of recombinant Lc. lactis prolidase
The overexpression of recombinant proteins can dis-
turb host cell metabolism through their activities. To
avoid these negative effects, host cells often produce

recombinant proteins as inclusion bodies [15]. As pro-
lidases are highly specific for Xaa-Pro, which does not
have any specific roles in metabolism of the host cells,
activity of prolidases would not have harmful effects
on the host cells. Therefore, the rapid and ample
expression itself would have caused sufficient stress to
the host metabolism. Growth conditions were exam-
ined in order to decrease host stress by altering the
conditions for expression. Two possibilities for
decreasing the stress were postulated: (a) that unfavor-
able conditions for E. coli growth (resulting in slower
growth) would retard expression of the recombinant
prolidase, and (b) that conditions allowing the host
more resources for themselves, i.e. rich media or weak
induction of expression, could compensate for the con-
sumption of energy and substances diverted to produce
the recombinant prolidase. Various culture conditions
were examined using a pKK223-3–prolidase clone as
described in Experimental procedures, and it was
found that low temperatures with vigorous aeration
yielded recombinant prolidase as a soluble protein.
Optimum results were achieved using a low concentra-
tion of chloramphenicol (1 lgÆmL
)1
), induction with
1mm isopropyl thio-b-d-galactoside (IPTG) at
A
600
= 0.5, and vigorous aeration (200 r.p.m. with a
low volume of medium in a large vessel) at 16 °C.

Recombinant Lactococcus lactis prolidase S. I. Yang and T. Tanaka
272 FEBS Journal 275 (2008) 271–280 ª 2007 The Authors Journal compilation ª 2007 FEBS
Under these conditions, 40 h cultures produced 20–
40% of soluble proteins as recombinant prolidase, as
determined by densitometry of the SDS–PAGE gel.
Extension of the culture beyond 40 h did not increase
prolidase production.
Purification of recombinant prolidase was achieved
using a two-step process: ammonium sulfate precipita-
tion and anion-exchange column chromatography
(Fig. 1). Crude extracts were found to contain a sub-
stantial amount of recombinant prolidase (40 kDa;
lane C), compared with non-induced cell extracts (lane
B). Ammonium sulfate precipitation removed most of
the contaminating proteins from the crude extracts
(lane D). A final purification step using a DEAE–
Sephacel column resulted in a single prolidase band, as
evidenced by SDS–PAGE (lane E). A 900 mL culture
yielded 18.2 mg of purified prolidase, with a purifica-
tion factor of 11.8 and 89% recovery of activity from
the crude extracts (Table 1). The purified prolidase
had 197.2 unitsÆmg
)1
(where one unit is as defined in
Experimental procedures) of specific activity using
2mm Leu-Pro dipeptide as the substrate in 20 mm
sodium citrate buffer (pH 6.5) ⁄ 1mm ZnCl
2
at 50 °C.
Characterization of recombinant Lc. lactis

prolidase
The molecular mass of recombinant prolidase was esti-
mated using mass spectrometry and gel filtration. Based
on the gene sequence, an estimated molecular mass for
the prolidase monomer of 40 kDa (39 970 Da) was
determined. The estimated molecular mass (40 164 Da)
determined by mass spectroscopy of purified prolidase
confirmed this value. This molecular mass may not
reflect the native state as mass spectrometers dissociate
the protein molecules during the analysis process. The
molecular mass of prolidase in the native state (active
form) was roughly estimated using four size-exclusion
columns. Prolidase appeared in the void volume fraction
of the Bio-Gel P-60 column (exclusion limit of 60 kDa)
and in later fractions using other columns: Sephadex
G-100 (100 kDa) and G-150 (150 kDa), and Bio-Gel
P-200 (200 kDa). These results indicate that prolidase is
larger than 60 kDa, but smaller than 100 kDa. Only a
dimeric structure can have a molecular mass within this
range, as the monomer of this prolidase is 40 kDa as
shown by mass spectrometry, SDS–PAGE and gene
sequence. We therefore propose that Lc. lactis prolidase
forms a homodimeric structure with a molecular mass
of approximately 80 kDa. This proposed dimeric struc-
ture is in agreement with the X-ray crystal structures of
P. furiosus (Protein Data Bank accession number 1PV9
[16]) and Pyrococcus horikoshii OT3 (Protein Data
Bank accession number 1WY2) prolidases.
Recombinant Lc. lactis prolidase exhibited activity
over a broad range of temperatures, showing similar

activities between 35 and 55 °C (Fig. 2). The reaction
rate dropped to 67% at 60 °C, and no activity was
observed above 70 °C. This range is broader than that
for Lb. delbrueckii prolidase, which has its highest
activities between 40 and 50 °C [4]. Figure 2 also
shows the temperature stability. The enzyme retained
more than 60% of its activity after 30 min incubation
below 50 °C; however, incubation at 60 °C decreased
Fig. 1. SDS–PAGE gel showing the final purification of recombinant
Lactococcus lactis prolidase. Samples from each step of purification
process were compared by SDS–PAGE. Lane B, whole cell extracts
of non-induced culture; lane C, crude extracts of induced culture;
lane D, after 60% ammonium sulfate precipitation; lane E, after
DEAE–Sephacel chromatography purification. The arrow indicates a
molecular mass of 40 kDa. Lane A shows the molecular mass
markers.
Table 1. Purification of Lactococcus lactis recombinant prolidase.
Purification process
Total protein
(mg)
Total activity
(units
a
)
Specific activity
(unitsÆmg
)1
)
Yield
(% activity)

Purification
(fold)
Cell extract 272 4542 16.7 100 1
Ammonium sulfate precipitation 41 3665 89.4 81 5.4
DEAE–Sephacel 18.2 3589 197.2 79 11.8
a
One unit of prolidase activity is defined as hydrolysis of 1 lmol of peptide in 1 min.
S. I. Yang and T. Tanaka Recombinant Lactococcus lactis prolidase
FEBS Journal 275 (2008) 271–280 ª 2007 The Authors Journal compilation ª 2007 FEBS 273
the residual activity to 22%. Little activity was
observed after incubation at 70 °C. The thermal
stability is comparable to that of Lb. casei prolidase
[11] and higher than that of Lb. delbrueckii proli-
dase [4].
This loss of activity in the recombinant Lc. lactic
prolidase was most likely due to denaturation between
60 and 70 °C. In order to confirm this speculation, CD
analysis was employed. The CD signal started to
decline at 60 °C and reached a minimum at 71 °C
(Fig. 3), with the denaturation temperature estimated
as 67 °C. This observation indicated that the enzyme
began to lose structure at 60 °C and was completely
denatured at around 70 °C, thereby supporting the
speculation that the loss of prolidase activity results
from denaturation of the enzyme.
Enzyme activity was measured between pH 4 and 10
(Fig. 4). Activity was detected between pH 6.0 and 8.0
and reached a maximum at pH 7 for both Leu-Pro
and Arg-Pro. The optimum pH was consistent with
values reported for Lb. delbrueckii (pH 6.0) [4],

P. furiosus (pH 7) [12], Lb. casei (pH 6.5–7.5) [11] and
partially purified Lc. lactis subsp. cremoris AM2 pro-
lidases (pH 7.35 and 8.25) [17], although these enzymes
worked in narrower pH ranges for Leu-Pro than that
for Lc. lactis prolidase.
The reported prolidases vary in their metal require-
ments, e.g. Lb. delbrueckii prolidase requires zinc [4],
P. furiosus prolidase prefers cobalt and manganese
[12], and Lb. casei enzyme can utilize magnesium,
manganese and cobalt [11]. The recombinant Lc. lactis
prolidase showed its highest activity for Leu-Pro with
zinc, but the activity with manganese was 21.5% of
that with zinc (Table 2). Activity was not detected with
other divalent cations, i.e. cobalt, magnesium, nickel,
copper and calcium.
Substrate specificity of recombinant Lc. lactis
prolidase
To date, all known prolidases are dipeptide-specific,
and cannot hydrolyze larger peptides. Similar results
were observed for the prolidase examined in this study
(Table 2). Recombinant Lc. lactis prolidase exhibited
activity for Leu-Pro, Val-Pro, Phe-Pro, Arg-Pro and
Lys-Pro. Based on the peptide hydrolysis assay
employed in this study, i.e., quantification of free
0
25
50
75
100
20 30 40 50 60 70

Tem
p
erature (°C)
Relative activity ( : 40°C = 100%)
Residual activity ( : 20°C = 100%)
Fig. 2. Effect of temperature on recombinant Lactococcus lactis
prolidase activity. Open circles represent the observed activity of
fresh prolidase at each temperature. The activities are expressed
as activities relative to the activity at 40 °C. Closed triangles repre-
sent the residual activity of Lc. lactis prolidase after 30 min treat-
ment at each temperature. The residual activities are expressed as
activities relative to the activity after 20 °C incubation.
Tem
p
eratue (°C)
Normalized value (%)
0
20
40
60
80
100
20 30 40 50 60 70 80 90
Fig. 3. Thermal denaturation observed by CD. The observed CD
signal at 222 nm is plotted against temperature. The signal intensity
is expressed relative to the value at 20 °C. The determined dena-
turing temperature, 67 °C, is indicated by a vertical dashed line.
0
20
40

60
80
100
45678910
pH
Relative activity (pH 7 = 100%)
Fig. 4. pH dependency of recombinant Lactococcus lactis proli-
dase. The activity of Lc. lactis prolidase was measured using two
dipeptides over a range of pH values. The observed activities are
expressed as the activity relative to that at pH 7.0. Open squares
and closed circles represent Leu-Pro and Arg-Pro, respectively.
Recombinant Lactococcus lactis prolidase S. I. Yang and T. Tanaka
274 FEBS Journal 275 (2008) 271–280 ª 2007 The Authors Journal compilation ª 2007 FEBS
proline, no hydrolysis was observed for Gly-Pro, Glu-
Pro, Asp-Pro or the two tripeptides Leu-Leu-Pro and
Leu-Val-Pro. Interestingly, substrate specificity was
dependent on the catalytic metal cation. No activity
towards Pro-Pro was seen in the presence of zinc, but
low activity was seen in the presence of manganese.
Moreover, the preference for dipeptide changed from
Leu-Pro to Arg-Pro in the presence of manganese.
A comparison of the crystal structure of P. furiosus
prolidase and the sequence-based model of Lc. lactis
prolidase (Fig. 5) indicated that the S
1
sites are
composed mainly of hydrophobic residues (Phe190,
Leu193 and Ile308 of Lc. lactis prolidase), suggesting
a preference towards hydrophobic residues at the
N-terminus of the dipeptides. In fact, the Xaa-Pro

dipeptides preferred by Lc. lactis prolidase were mostly
hydrophobic peptides, as shown in Table 2. However,
the preference for Arg-Pro, and the metal-dependent
substrate specificity cannot be explained by the nature
of the S
1
site residues described. A notable difference
between P. furiosus and Lc. lactis prolidases in the
active site area is the length of the loop structure that
is contributed by the other subunit and covers the S
1
site (yellow ribbon for P. furiosus and cyan ribbon for
Lc. lactis in Fig. 5). The crystal structure of P. furiosus
suggests that this loop forms part of the S
1
site. The
loop is longer in Lc. lactis by four residues, and the
middle of the loop is composed of charged residues
(Asp36, His38, Glu39 and Arg40), whereas the
Arg295
Ser307
Arg295
Ser307
S
1
′
S
1
′
S

1
S
1
Fig. 5. Active site superposition of Lactococcus lactis and Pyrococcus furiosus prolidases. The residues in the active sites of a Lc. lactis
prolidase subunit are indicated by thick lines. The S
1
site (Phe190, Leu193 and Val302; blue), S
1
‘ site (His292, Tyr329 and Arg337; green),
and substrate size-limiting residues (Pro306, Ser307 and Ile308; orange) and metal-chelating residues (Asp221, Asp232, His296, Glu325 and
Glu339; cyan) are shown. Corresponding residues in P. furiosus are indicated by thin lines. The size-limiting arginine, Arg295, in P. furiosus
prolidase [16], and the corresponding residue, Ser307, in Lc. lactis prolidase are labeled. Ribbon models show the loop contributed from the
other subunit. The Lc. lactis and P. furiosus prolidase loop structures are in cyan and yellow, respectively. Leu37B of P. furiosus prolidase
and Asp36B of Lc. lactis prolidase are shown in the line model on the ribbons. The illustration was generated using the
VMD molecular
modelling program [27].
Table 2. The relative activities of recombinant Lactococcus lactis
prolidase in the presence of zinc or manganese for various peptide
substrates. Activity was measured with 2 m
M peptides in 20 mM
sodium citrate (pH 6.5) and 1 mM metal (zinc or manganese) chlo-
ride; activities are expressed relative to the activity for Leu-Pro in
the zinc reaction mixture.
Substrates Zinc Manganese
Leu-Pro 100.0 ± 0.4 21.5 ± 0.6
Phe-Pro 23.8 ± 0.5 15.2 ± 2.7
Val-Pro 14.4 ± 0.4 14.7 ± 3.8
Arg-Pro 12.0 ± 0.9 42.5 ± 1.2
Lys-Pro 6.6 ± 0.5 2.5 ± 0.4
Pro-Pro < 0.1 0.8 ± 0.1

Glu-Pro < 0.1 < 0.1
Gly-Pro < 0.1 < 0.1
Asp-Pro < 0.1 < 0.1
Leu-Leu-Pro < 0.1 < 0.1
Leu-Val-Pro < 0.1 < 0.1
S. I. Yang and T. Tanaka Recombinant Lactococcus lactis prolidase
FEBS Journal 275 (2008) 271–280 ª 2007 The Authors Journal compilation ª 2007 FEBS 275
P. furiosus loop has two hydrophilic residues (Thr34
and Ser35). It is speculated that this loop structure
contributes to the preference for the Arg-Pro dipep-
tide, i.e. methylene groups (b, c, d–carbons) are
accommodated in the S
1
site, and the amino group of
the side chain is associated with the negatively charged
residues (Asp 36 and Glu39) on the loop structure.
Allosteric behavior of recombinant Lc. lactis
prolidase
The relationships between substrate concentrations and
reaction rates were examined in order to determine
kinetic parameters. Plots of substrate concentration
against observed catalytic rate showed sigmoidal
curves for both Leu-Pro and Arg-Pro (Fig. 6). The
allosteric behavior indicated by the sigmoidal curves
was analyzed using the Hill plot, and Hill coefficients
of 1.53 and 1.57, respectively, were obtained for Leu-
Pro and Arg-Pro (Fig. 6). Although allosteric behavior
is not common among proteinases ⁄ peptidases, it has
been reported in several proteinases, e.g. cathepsin C
[18] and Helicobacter pylori leucyl aminopeptidase [19].

Interestingly, the latter enzymes share characteristics
with Lc. lactis prolidase: they hydrolyze peptides with
leucine at the N-terminus, they are metallopeptidases,
and their 3D structures share similar domain struc-
tures (based on the bovine leucyl aminopeptidase
structure, 1LAM [20]). Their similarities in 3D struc-
ture include (a) two distinctive domains that fold in
a ⁄ b structures, (b) an active site located at the center
of the C-terminal domain, and (c) an active site that
faces another subunit.
The Lineweaver–Burk plot using the Hill coefficient
(1 ⁄ s
H
against 1⁄ v plot) gave a Michaelis constant for
Leu-Pro of 3.7 mm and a rate constant of 247.9 s
)1
.
The constants for known prolidases are: Lb. del-
brueckii, 2.2 mm and 225.9 s
)1
; Lb. casei, 0.2 mm and
55.1 s
)1
[11]; P. furiosus, 3.0 mm and 271 s
)1
[12].
Similar to known prolidases [4,12,17], this Lc. lactis
prolidase exhibited substrate inhibition above 5 mm
Leu-Pro; at 8 mm, the observed activity was 47% of
that at 5 mm.

Modelling of Lc. lactis prolidase
Molecular modeling provides insight regarding the
allosteric nature of this enzyme. A molecular model of
Lc. lactis prolidase was successfully constructed and
used to evaluate the structure–function relationship of
this prolidase. The Lc. lactis model was superposed on
the P. furiosus model by comparison of their a-car-
bons, yielding a root mean square deviation of 1.59 A
˚
.
Figure 5 shows models of P. furiosus (1PV9) and
Lc. lactis prolidases around the active site zinc ions.
The P. furiosus enzyme, which did not exhibit alloste-
ric behavior, had a smaller loop structure over the
active site (shown as a yellow ribbon in Fig. 5). Maher
et al. [16] discussed the contribution of this loop struc-
ture as part of the substrate binding site, and it was
suggested that Leu37B (B indicates the contribution
from the other subunit) formed the hydrophobic S
1
site in cooperation with Phe178, Ile181 and Ile 290.
The comparable residues in the Lc. lactis prolidase are
Asp36B, Phe190, Leu193 and Ile308, respectively. The
positions and characteristics of the latter three residues
are comparable to those of P. furiosus prolidase. How-
ever, unlike Leu37B of P. furiosus prolidase, Asp36B is
a hydrophilic charged residue, and is located on the
longer loop structure (cyan ribbon in Fig. 5) that was
discussed in the substrate specificity section. In some
enzymes, the loop structures have been shown to con-

tribute to the activity of enzymes by changing their
shape [21,22]. This suggests that the structure of this
loop could take a different shape in the event of sub-
strate binding, thus the residue comparable to Leu37B
of P. furiosus might not be Asp36B but instead
another residue on the longer loop. This flexibility may
mediate changes in the overall structure of the enzyme
via subunit–subunit interaction. Such changes may
trigger the allosteric behavior of Lc. lactis prolidase.
Another possibility is that Ser307 works as a key resi-
due in the allosteric behaviour of this enzyme. This
residue is located close to the substrate-binding site,
and is replaced by Arg295 in P. furiosus (Fig. 5).
Maher et al. [16] suggested that this residue limited the
substrate to dipeptides. We suggest that this residue
can cooperate with the loop and contribute allosteric
behavior to the peptidase. These suggestions, i.e. the
0
0.001
0.002
0.003
0.004
0.005
0.006
01234
Substrate concentration (mM)
Observed rate (µmole·min
–1
)
–4

–2
0
2
–3 –2 –1 0 1 2
Ln (substrate)
Ln [v/(V
max
–v
)]
Fig. 6. Allosteric behavior of Lactococcus lactis prolidase. The plots
show the relationship between the observed activity and the con-
centration of Leu-Pro (open squares) and Arg-Pro (closed circles).
The inset shows the Hill plot of the assay with Leu-Pro.
Recombinant Lactococcus lactis prolidase S. I. Yang and T. Tanaka
276 FEBS Journal 275 (2008) 271–280 ª 2007 The Authors Journal compilation ª 2007 FEBS
contributions of the loop and Ser307, are being exam-
ined by our group.
Conclusion
In this study, we have produced recombinant prolidase
in a soluble, active form. The techniques use to achieve
solubilization could be used for other difficult-
to-express proteins. Recombinant Lc. lactis prolidase
exhibited characteristics similar to other prolidases,
but possessed distinctive properties of allosteric behav-
ior and metal-dependent substrate specificity. Further
structure–function relationship studies will provide
insights into the behaviour of prolidase, thus contrib-
uting to applications of prolidase in fermented food
processing.
Experimental procedures

Enzymes and chemicals
Enzymes for genetic engineering were purchased from Fer-
mentas (Burlington, Canada) and Invitrogen (Burlington,
Canada). All chemicals used in this study were commer-
cially available ACS grade, and were purchased from VWR
International (Edmonton, Canada).
Cultivation of Lactococcus lactis and genomic
DNA isolation
Lactococcus lactis NRRL B-1821 (Agricultural Research
Service culture collection, Peoria, IL, USA) was cultivated
in 100 mL of Lactobacillus MRS medium (BD-Difco,
Franklin Lakes, NJ, USA) for 24 h at 37 °C without shak-
ing. The culture was harvested by centrifugation at 4000 g
for 5 min at 4 °C. Harvested cells were treated with pro-
teinase K (1 mgÆmL
)1
in 50 mm Tris–HCl pH 8.0, 50 mm
EDTA, 100 mm NaCl, 0.5% SDS; Roche Diagnostics,
Montreal, Canada) at 50 °C for 1 h, then disrupted using
phenol. Extracted nucleic acids were collected, and RNA
was removed by RNase (Fermentas) treatment. Genomic
DNA was purified by ethanol precipitation from the reac-
tion mixture.
Isolation and cloning of the gene
A pair of primers (5¢-GGAGAATTCATGAGCAAAA
TTGAACGTATT-3¢;5¢-ATT
CTGCAGTTAGAAAATT
AATAAGTCATG-3¢) for PCR was designed based on the
sequence of the Lc. lactis spp. Il1403 prolidase coding gene
(GenBank accession numbers NC_002662 and AE006395)

and custom-synthesized (Integrated DNA Technologies
Inc., Coralville, IA, USA). The primers possessed EcoRI
(N-terminus) and PstI (C-terminus) restriction enzyme sites
(indicated by underlining) that flanked the ends of the open
reading frame. The PCR reaction mixture contained geno-
mic DNA (20 lg), primers (20 pmol each), dNTPs (40 lm
each) and Pfu DNA polymerase (0.5 units; Fermentas) in
100 lL of the buffer recommended by the manufacturer.
Each PCR reaction cycle consisted of 94 °C for 1 min,
55 °C for 1 min, and 68 °C for 3 min (5 s was added to the
68 °C step for each cycle), and was repeated 30 times. The
amplified PCR fragments were hydrolyzed using EcoRI and
PstI, and were then introduced into EcoRI–PstI-digested
pUC18 plasmids. The recombinant DNA was transformed
into E. coli TOP10F’, and positive clones were verified by
DNA sequencing. First, the sequenced gene was isolated
using EcoRI–PstI restriction enzymes, and subcloned into
the same sites of the pKK223-3 vector [23]. Then, the con-
structed recombinant DNA was transformed into E. coli
TOP10F’. The expression was examined under a variety of
conditions in order to obtain recombinant prolidase in a
soluble form. The various conditions studied were: (a) cul-
ture medium (LB or 2YT medium), (b) the concentration
of the inducing agent for the tac promoter (0.1, 1 or 10 mm
IPTG), (c) protein synthesis inhibition using sublethal con-
centrations of chloramphenicol (0.1 or 1 lgÆmL
)1
), (d)
media with higher osmotic pressures (0.5 or 2% w ⁄ v NaCl),
(e) schedules of the induction (induced at A

600
=0.4, 0.5,
0.8 or 1.2), (f) the pH of the medium (pH 5.5 or 7.5), (g)
the aeration conditions (100 or 200 r.p.m.), (h) the culture
temperature (16, 18, 22, 29, 30, 33 or 37 °C), and (i) the
duration of the culture (16, 40, 72 or 96 h). These condi-
tions were tried individually or concurrently.
Purification of the recombinant Lc. lactis
prolidase
The recombinant E. coli was cultured in LB broth (pH 5.5)
at 16 °C. The culture was carried out in 18 500 mL flasks
with 50 mL medium in each (total 900 mL). Expression
was induced by addition of 1 mm IPTG and chlorampheni-
col (1 lgÆmL
)1
) when the A
600
reached 0.5. The culture was
vigorously shaken at 200 r.p.m. for 40 h before harvesting.
The harvested cells were resuspended in a lysis buffer solu-
tion (20 mm sodium citrate buffer, pH 6.0, 1 mm zinc
sulfate, 100 mm sodium chloride, 8 lgÆmL
)1
RNase and
0.2 mgÆmL
)1
lysozyme), and disrupted using ultrasonica-
tion. After removal of some proteins from the crude
extracts by 40% saturated ammonium sulfate precipitation,
the prolidase fraction was recovered using 60% saturated

ammonium sulfate precipitation. The recovered prolidase in
the precipitate was dissolved in 20 mm sodium citrate
(pH 6.0) ⁄ 1mm zinc sulfate and dialyzed against 2 L of the
same buffer twice. The dialyzed sample was applied to a
DEAE–Sephacel anion exchange column (3 diameter ·
15 cm; GE Healthcare, Chalfont St Giles, Buckingham-
shire, UK), and prolidase was eluted using a 600 mL linear
gradient from 0 to 0.5 m NaCl in 20 mm sodium citrate
S. I. Yang and T. Tanaka Recombinant Lactococcus lactis prolidase
FEBS Journal 275 (2008) 271–280 ª 2007 The Authors Journal compilation ª 2007 FEBS 277
(pH 6.0) ⁄ 1mm zinc sulfate. The prolidase fractions were
concentrated and desalted using an YM30 Amicon Ultracell
filtration system (Millipore, Billerica, MA, USA). The pur-
ity of the prolidase was densitometrically estimated by
SDS–PAGE using Coomassie Brilliant Blue G250 and NIH
image software (developed at the US National Institutes of
Health and available at />The purified sample in 20 mm sodium citrate
(pH 6.0) ⁄ 1mm zinc sulfate was mixed with the same vol-
ume of glycerol and stored at )20 °C until use.
Enzyme activity assay
The amount of proline liberated from the peptide substrates
was determined using the ninhydrin method [24]. Dipep-
tides were hydrolyzed in 20 mm sodium citrate buffer
(pH 6.5) ⁄ 1mm zinc chloride. The reaction was initiated by
the addition of enzyme solution. At 1 min intervals, an
aliquot (20 lL) was withdrawn and mixed with 50 lLof
glacial acetic acid and 50 lL of ninhydrin reagent (3% w ⁄ v
ninhydrin, 60% v ⁄ v glacial acetic acid, 40% v ⁄ v phosphoric
acid). The mixture was boiled for 10 min to develop the
color, and then cooled on ice. The resulting chromophore

was quantified using 515 nm absorption. All measurements
were carried out at least in triplicate. One unit of prolidase
activity is defined as hydrolysis of 1 lmol of peptide in
1 min.
Measurement of substrate specificity
The peptide substrates examined were Leu-Pro, Val-Pro,
Phe-Pro, Gly-Pro, Arg-Pro, Lys-Pro, Pro-Pro, Asp-Pro,
Glu-Pro, Leu-Val-Pro and Leu-Leu-Pro. The peptides
(2 mm) were hydrolyzed in 20 mm sodium citrate
(pH 6.5) ⁄ 1mm zinc chloride or manganese chloride at
50 °C.
pH dependency
The pH dependency of prolidase was examined using the
following buffer solutions in place of the sodium citrate
buffer in the method described above: 20 mm sodium cit-
rate (pH 4–5.5), 20 mm MES (pH 6.0–7.0), 20 mm Tris–
HCl (pH 7.5–9) and 20 mm sodium borate (pH 10). The
activity was analyzed using 2 mm Leu-Pro or Arg-Pro and
1mm manganese chloride at 50 °C.
Thermal stability and dependency
Recombinant prolidase was incubated in 20 mm sodium cit-
rate buffer (pH 6.5) ⁄ 1mm zinc chloride at the designated
temperature (20, 30, 40, 50, 60 or 70 °C) for 30 min. The
residual activity was determined in order to evaluate the
stability of prolidase. The temperature dependency was
separately examined in reactions using fresh enzyme at vari-
ous temperatures (20, 30, 35, 40, 45, 50, 55, 60 and 70 °C). In
both experiments, 2 mm Leu-Pro was used as the substrate.
Metallic ion dependency
A variety of metal cations were tested for their effects on

the prolidase activities. Metal salts of zinc chloride, nickel
chloride, cobalt nitrate, copper sulfate, manganese chloride,
magnesium chloride and calcium chloride were used. The
activities were measured in 20 mm sodium citrate
(pH 6.5) ⁄ 1mm solutions of each metal salt with 2 mm Leu-
Pro at pH 6.5.
Thermal denaturation temperature measurement
for recombinant prolidase
The CD spectrum of purified prolidase was analyzed in
20 mm sodium phosphate buffer (pH 6.0) using a PiStar-
180 spectroscope (Applied Photophysics Ltd, Leatherhead,
Surrey, UK) at the Saskatchewan Structural Sciences Cen-
tre (University of Saskatchewan, Saskatoon, Canada). The
thermal denaturation temperature was determined from the
change in the CD spectrum at 222 nm over a temperature
range from 25 to 90 °C. The denaturation temperature was
determined as the temperature at which the rate of CD
spectrum change reached its maximum [15].
Mass spectrometry
The molecular mass of the recombinant prolidase molecule
was determined using a mass spectrometer (API Q-star XL
hybrid MS system; Applied Biosystems, Foster City, CA,
USA) using the electrospray ionization method at the Sas-
katchewan Structural Sciences Centre; measurements were
carried out on the desalted sample in deionized water.
Molecular mass estimation using gel filtration
The purified prolidase protein (0.8 lgin10lL) was loaded
onto gel filtration columns: Sephadex G-100 and G-150
(GE Healthcare) and Bio-Gel P-60 and P-200 (Bio-Rad
Laboratories, Hercules, CA, USA). The size of the columns

was 0.5 diameter · 10 cm, and 20 mm sodium citrate buffer
(pH 6.5) ⁄ 1mm ZnCl
2
was used as the eluant. The eluate
was fractionated, and the activities of the fractions were
qualitatively checked using Leu-Pro in order to determine
the prolidase fractions.
Computational molecular modelling
The protein sequence, deduced from the DNA sequence of
the prolidase gene, was submitted to the 3d-jigsaw server
Recombinant Lactococcus lactis prolidase S. I. Yang and T. Tanaka
278 FEBS Journal 275 (2008) 271–280 ª 2007 The Authors Journal compilation ª 2007 FEBS
( [25] in order to
create an initial molecular model of Lc. lactis prolidase. The
initial model was derived based on the crystal structure of
P. furiosus (Protein Data Bank accession number 1PV9 [16])
using the default parameters of the server. This model was
then energy-minimized using the namd molecular modelling
program [26]. The calculation used topology force field data
provided with the program, and was carried out in a water-
filled box. The cut-off distance was set to 15 A
˚
and the calcu-
lation run for 5000 iterations. The minimized model was
compared with the P. furiosus model (Protein Data Bank
accession number 1PV9 [16]) using the vmd software package
[27] on a Macintosh G4 computer.
Acknowledgements
This research was supported by a grant from the Natu-
ral Sciences and Engineering Research Council of

Canada. The authors appreciate the assistance of Lili
Liu and Guodong Zhang with the laboratory work.
George Khachatourians, Rickey Yada (Guelph,
Canada), Nicholas Low, Michael Nickerson and Sylvia
Yada (Guelph, Canada) are acknowledged for their
helpful suggestions in the preparation of the manu-
script.
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