Mol Biotechnol (2010) 44:242–249

DOI 10.1007/s12033-009-9232-2

RESEARCH

Cloning, Expression, Purification, and Characterization of a Novel

Esterase from Lactobacillus plantarum
Fábio Cristiano Angonesi Brod • Javier Vernal •

Jean Borges Bertoldo • Hernán Terenzi •

Ana Carolina Maisonnave Arisi

Published online: 22 December 2009

Ó Springer Science+Business Media, LLC 2009

Abstract Lactobacillus plantarum is an important lactic Introduction

acid bacterium, usually found as natural inhabitant of food,
such as fermented vegetables and meat products. However, Esterases belongs to the carboxylic ester hydrolases group
little information about lactic acid bacteria, especially (EC 3.1.1), lipases being defined as carboxylesterases that
concerning L. plantarum, as a source of useful enzymes has catalyze hydrolysis and synthesis of water-insoluble long-
been reported. The aim of this study was to clone, express chain acylglycerols (acyl chain [10 carbons), whereas
in Escherichia coli, purify, and characterize an esterase esterases catalyze hydrolysis of water-soluble glycerolest-
from L. plantarum ATCC 8014. The esterase gene ers with short-chain lengths (acyl chain \10 carbons) [1].
(1014 bp) was amplified and cloned in pET14b expres- Despite being widely spread in several species, microbial
sion vector to express a His6-tagged protein in E. coli. esterases and lipases present high industrial potential and
Recombinant L. plantarum esterase was purified by are often more useful than the enzymes from other sources
Ni-NTA resin, presenting an apparent molecular mass of due to their substrate specificity, region, and enantiose-
about 38 kDa. It presented highest activity at pH 6.0 and lectivity, ability to remain active in organic solvents, high
40°C. Also, it presented preference for p-nitrophenyl yields, and ease of genetic manipulation [2].
butyrate, but hydrolyzed more efficiently p-nitrophenyl Esterases show the characteristic a/b-hydrolase fold and
acetate. Besides, this study shows, for the first time, CD present the consensus sequence Gly-X-Ser-X-Gly around
data about secondary structure of an esterase from an active residue of Ser, which is part of the catalytic triad
L. plantarum. containing also an Asp/Glu and a His residues [3]. The
pentapeptide motif is usually located between a b-strand
Keywords L. plantarum
Esterase
E. coli expression
and the a-helix, forming an extremely sharp turn called
Biochemical characterization
Circular dichroism ‘‘nucleophile elbow’’ [1].
Although several articles describing the cloning and
expression of microbial esterases and lipases have been
published [4–10], little information about lactic acid bac-
teria as a source of useful enzymes has been reported.
Electronic supplementary material The online version of this
article (doi:10.1007/s12033-009-9232-2) contains supplementary Despite the fact that the role of lactobacilli in the pro-
material, which is available to authorized users. duction of fermented food products is well documented and
studied, scarce information is available concerning ester-
F. C. A. Brod
A. C. M. Arisi (&)
ases from Lactobacillus spp., probably as a consequence of
Department of Food Science and Technology, Universidade
Federal de Santa Catarina, Rod. Admar Gonzaga, 1346, the lower production of such enzymes by these microor-
88034-001 Florianópolis, Brazil ganisms, mainly when compared with Staphylococcus spp.
e-mail: arisi@cca.ufsc.br [5]. Lactobacillus plantarum has a long history of natural
occurrence and safe use in a variety of fermented food
J. Vernal
J. B. Bertoldo
H. Terenzi
Departmento de Bioquı́mica, Universidade Federal de Santa products, being involved in the development of flavor
Catarina, 88040-900 Florianópolis, Brazil and aroma in such kind of products. Besides, lipolytic
Mol Biotechnol (2010) 44:242–249 243

capability of a L. plantarum strain was demonstrated by Construction of pET14b-Est_Lpl Expression Vector

Lopes et al. [11], and two lipases were purified from
L. plantarum cultures [5, 12]. The aim of this study was to DNA fragments obtained from digestion of pGEM-Est_Lpl
perform the cloning, expression, purification, and charac- with BamHI were ligated into pET14b vector previously
terization of a new esterase from L. plantarum. digested with the same enzyme and dephosphorilated.
Insertion of the lipase gene was confirmed by restriction
analysis with HindIII and sequencing. Recombinant plas-
Materials and Methods mid was named pET14b-Est_Lpl. Expressed protein carries
N-terminal His6-Tag encoded by the expression vector.
Bacterial Strains, Plasmids, Media, and Chemicals GenBank accession number for recombinant esterase
sequence described in this study is GQ497844.
Escherichia coli strain DH5a was used as host for gene
cloning and plasmid propagation, and strain BL21 (DE3) Expression and Purification of Recombinant Esterase
pLysS for protein expression. Plasmid pGEM-T Easy
Vector (Promega) was used for sub-cloning, while pET- Escherichia coli BL21 (DE3) pLysS harboring the
14b (Novagen) was used as cloning and expression vector. pET14b-Est_Lpl vector were inoculated in 10 ml LB broth
BamHI and HindIII were purchased from Promega and supplemented with 100 lg/ml ampicillin and 50 lg/ml
Ni-NTA resin from QIAGEN. The p-nitrophenyl esters and chloramphenicol. Overnight cultures were transferred to
isopropyl-L-thio-b-D-galactopyranoside (IPTG), as well as 250 ml of the same medium and cultivated at 37°C until
phenylmethylsulfonyl fluoride (PMSF), diethyl pyrocar- OD600 of 0.8. IPTG was added to a final concentration of
bonate (DEPC), phenylarsine oxide (PAO), and phenyl- 1 mM, and the following conditions were applied for
glyoxal hydrate (PGO) were purchased from Sigma. protein expression: 15, 30, and 37°C for 5 and 18 h. After
L. plantarum ATCC 8014 strain was obtained from the induction, cells were harvested by centrifugation (30009g,
Collection André Tosello Foundation (Brazil). 15 min, 4°C), and the pellet was washed once with 50 mM
NaH2PO4, pH 8.0. Cells were resuspended in buffer A
Construction of pGEM-Est_Lpl (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH
8.0), and 40 lg/ml PMSF and disrupted by gentle sonica-
Gene of lipase/esterase from L. plantarum was amplified tion (7 cycles, 20 s) on ice. Cell debris was separated from
with primers LipLPF (50 -GGA TCC GAT GCC AAC AAT supernatant by centrifugation (100009g, 20 min, 4°C) to
TAA TTC G-30 ) and LipLPR (50 -GGA TCC CTA AAT obtain the crude extract. Crude extract containing the
TAA CGC GGC CGC-30 ) containing a restriction site for recombinant esterase was purified under native conditions
BamHI (restriction sites are underlined). Primers were by Ni-NTA (QIAGEN) resin previously equilibrated with
designed from the sequence of the putative lipase/esterase buffer A according to manufacturer’s recommendations.
gene published [13] (GenBank accession number lp_0973; Recombinant protein was eluted with 250 mM imidazole.
GenBank accession number for L. plantarum complete Eluted protein was dialyzed twice against 1 l 50 mM
genome AL935263). Amplification reactions were per- NaH2PO4 buffer containing 300 mM NaCl (pH 8.0).
formed in a final volume of 25 ll containing 19 PCR Purity and apparent molecular mass were determined by
buffer (20 mM Tris–HCl, pH 8.4, 50 mM KCl), 2 mM SDS polyacrylamide gel electrophoresis. SDS-PAGE was
MgCl2, 0.2 mM of each dNTP, 0.2 lL of each primer, 1 carried out in gels containing 10% (w/v) polyacrylamide
unit of Taq DNA polymerase, and 2 ll of DNA. Amplifi- according to standard protocols using Bio-Rad Mini-
cations were carried out in a MinicyclerTM (MJ Research PROTEANÒ equipment. Gels were stained with Coo-
Inc., Watertown, MA) with the following program: dena- massie Brilliant Blue R-250 and destained with methanol/
turation at 95°C for 5 min; followed by 40 cycles of 94°C acetic-acid/water (5/1/4 v/v/v). Protein concentration was
for 1 min, 54°C for 1 min, and 72°C for 1.5 min; final determined using a Bio-Rad protein assay kit with BSA as
extension at 72°C for 7 min. DNA fragment obtained standard.
(1014 bp) was excised from the gel, purified with WizardÒ
SV Gel and PCR Clean-Up System (Promega), and ligated Enzyme Activity Assay
into pGEM-T Easy Vector (Promega). Recombinant plas-
mid pGEM-Est_Lpl was used for transformation of E. coli Enzyme activity was determined by monitoring the release
DH5a competent cells. Positive transformants were ana- of p-nitrophenol from p-nitrophenyl butyrate (pNPC4) at
lyzed by restriction analysis with BamHI, and those pre- 348 nm in a spectrophotometer (HITACHI U2910) for
senting the lipase gene were propagated in E. coli DH5a 5 min at 40°C [6]. In order to carry out the reaction, a stock
and used for construction of the expression vector. solution of 100 mM of pNPC4 was prepared in isopropanol
244 Mol Biotechnol (2010) 44:242–249

and mixed with 50 mM phosphate buffer (pH 6.0) to obtain Effect of Metal Ions and Inhibitors on Esterase Activity
a substrate final concentration of 1 mM. Reaction was ini-
tiated by adding 1 lg of protein. Blanks (NaH2PO4 50 mM, The inhibitory effect of modifying reagents for serine
300 mM NaCl, pH 8.0) were always used instead of enzyme (PMSF), histidine (DEPC), arginine (PGO), and cysteine
solution. Molar extinction coefficient for p-nitrophenol was (PAO), as well as the effect of several metal solutions
experimentally determined as 5487.25 M-1 cm-1. One unit (CaCl2, MgCl2, AlCl3, MnCl2, and ZnSO4), EDTA,
of esterase activity was defined as the amount of enzyme DMSO, and ethanol were determined according to Rhee
that released 1 lmol of p-nitrophenol per minute. et al. [6] with some modifications. Enzyme solution (1 lg
of protein) was incubated in 50 mM phosphate buffer (pH
6.0) containing 1 mM of each modifying reagent, metal
Effect of pH and Temperature on Esterase Activity
solution or 1% of DMSO or ethanol at room temperature
(25°C) for 10 min. Residual activities were measured at
Effect of pH was investigated by assaying esterase activity in
25°C as described above.
a range of pH values from 5.0 to 9.0. Buffers used were
acetate 50 mM (pH 5.0 and 5.5), phosphate 50 mM (pH 6.0,
CD Spectroscopy
6.5 and 7.0), Tris–HCl 50 mM (pH 8.0), and CHES 50 mM
(pH 9.0). Reactions were carried out by mixing the appro-
Protein was submitted to size-exclusion chromatography
priate buffer with a stock solution of 100 mM of pNPC4 to
on a Superdex 200 Prep Grade (GE Healthcare) connected
obtain a substrate final concentration of 1 mM. Reactions
to an ÄKTA HPLC System (GE Healthcare) prior to cir-
were initiated by adding 1 lg of enzyme. In order to inves-
cular dichroism assay. CD spectra were measured at 40°C
tigate temperature effect, reactions were performed in pH 6.0
using a JASCO J-815 spectropolarimeter. The far-UV
at 25, 30, 37, 40, 45, 50, 55, and 60°C as described above.
spectrum of L. plantarum esterase was measured over a
Molar extinction coefficient for p-nitrophenol was experi-
wavelength range from 195 to 260 nm as an average of five
mentally determined for each pH as 5487.25 M-1 cm-1 (pH
spectra with 100 nm/min scan speed and a step resolution
5.0, 5.5, and 6.0); 6010.23 M-1 cm-1 (pH 6.5); 9554.33
of 0.1 nm. All the measurements were carried out in a
M-1 cm-1 (pH 7.0); 18015.43 M-1 cm-1 (pH 8.0); and
0.1-cm path length cuvette using protein at a concentration
18669.31 M-1 cm-1 (pH 9.0).
of 4.5 lM in phosphate buffer pH 6.0. All the spectra were
subtracted from buffer CD signal.
Substrate Specificity

Substrate specificity was assessed using the following p- Results and Discussion
nitrophenyl esters: acetate (pNPC2), butyrate (pNPC4),
caprylate (pNPC8), caprato (pNPC10), laurate (pNPC12), Cloning and Sequence Analysis of the Esterase
myristate (pNPC14), and palmitate (pNPC16), as substrates
according to Maia et al. [14] with minor modifications. A Genomic DNA extracted from L. plantarum strain ATCC
stock solution of each p-nitrophenyl ester was prepared in 8014 [15] was amplified with primers LipLPF and LipLPR
isopropanol. Substrates were emulsified to a final concen- and a fragment with approximately 1014 bp was obtained.
tration of 1 mM in 50 mM phosphate buffer, pH 6.0, As this fragment consists of the complete putative lipase/
containing 1.1 mg/ml Arabic gum and 4.4 mg/ml Triton esterase gene from L. plantarum WCFS1 [13], it was
X-100. Reaction mix consisted of 998 ll of emulsified directly cloned in pGEM-T Easy vector. The obtained
substrate and 2 ll of enzyme solution (1 lg of protein). recombinant plasmid named pGEM-Est_Lpl was propa-
Reactions were carried out at 40°C in a spectrophotometer gated in E. coli DH5a and sequenced. After the gene
(HITACHI U2910) as described above. sequence was confirmed by sequencing, pGEM-Est_Lpl
recombinant plasmid was digested with BamHI and the
Kinetic Parameters DNA fragment ligated in pET14b vector, resulting in the
recombinant pET14b-Est_Lpl. Presence of the insert in
Esterase activity was measured as a function of different the recombinant plasmid was verified by digestion with
concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 1, and 3 mM) of HindIII and identity and orientation was confirmed by
pNPC4 and pNPC2. Michaelis–Menten substrate affinity sequencing, which showed that esterase gene was suc-
constant (Km), maximum velocity (Vmax), and the turnover cessfully inserted in frame with the following sequence
number (kcat) were calculated using GraphPad Prism 5.01 encoding the His6-tag: MGSSHHHHHHSSGLVPAGSH
software. Catalytic efficiency (kcat/Km) was also determined. MLG.
Mol Biotechnol (2010) 44:242–249 245

Fig. 1 Alignment of the deduced amino acid sequence of L.planta- Lactobacillus brevis ATCC 367; ZP_03666814.1 lipase from Listeria
rum esterase (Est_Lpl) with other proteins showing a oxyanion hole monocytogenes Finland 1988; YP_001135818.1 alpha/beta hydrolase
loop, b pentapeptide containing active serine, and c probable aspartate domain-containing protein from Mycobacterium gilvum PYR-GCK;
and histidine residues forming active site. Filled inverted triangle YP_001879185.1 acetyl esterase from Shigella boydii CDC 3083-94;
putative catalytic triad. Est_Lpl esterase from Lactobacillus planta- pdb|1JJI carboxylesterase from Archaeoglobus fulgidus; pdb|2C7B
rum ATCC 8014; NP_784685.1 lipase/esterase from Lactobacillus from Uncultured archaeon estE1 carboxylesterase; pdb|1EQV carb-
plantarum WCFS1; EEI39690.1 esterase/lipase from Gordonia oxylesterase from Alicyclobacillus acidocaldarius
bronchialis DSM 43247; YP_796253.1 esterase/lipase from

Deduced amino acid sequence of the recombinant protein Shigella boydii CDC 3083-94 (42%). Alignment with
presented 337 residues with a theoretical molecular mass of esterases of known three dimensional structures (pdb 1JJI,
39.075 kDa (for the full His6-tagged protein) and showed an pdb 2C7B, and pdb 1EQV, Fig. 1) suggests that Ser174,
identity of 100% with the putative lipase/esterase of Asp283, and His313 could be the putative catalytic triad,
L. plantarum WCFS1 (NP_784685.1). Also, both sequences because such residues are highly conserved in lipases and
(GQ497844 from ATCC 8014 strain, and NP_784685.1 esterases [1, 16].
from WCFS1 strain) presented 99% identity at nucleotide
level. L. plantarum esterase amino acid sequence presented Expression and Purification of the Recombinant
two regions that are commonly associated with esterases/ Esterase
lipases, the Gly-X-Ser-X-Gly pentapeptide, covering the
residues 172–176, which contains the putative active serine The pET14b-Est_Lpl recombinant plasmid was trans-
(Ser174) and the oxyanion hole loop, covering residues formed in BL21 (DE3) pLysS E. coli strains to express the
105–108 (Fig. 1). Deduced amino acid sequence was used His6-tagged recombinant esterase. Different induction
to perform a BLAST search, and the highest similarity periods (5 and 18 h) and temperatures (15, 30, and 37°C)
was found with L. plantarum esterase and other esterases/ were applied for expression of recombinant esterase.
lipases, including an esterase/lipase (YP_796253.1) from Recombinant esterase was expressed after 18 h of induc-
L. brevis ATCC 367 (57%), an esterase/lipase (EEI39690.1) tion at 15°C and purified to homogeneity from cell lysates
from Gordonia bronchialis DSM 43247 (50%), a lipase by metal-affinity chromatography on nickel nitriloacetate
(ZP_03666814.1) from Listeria monocytogenes Finland resin. His6-tagged protein was eluted with 250 mM imid-
1988 (45%), an a/b-hydrolase domain-containing protein azole, which migrated as a single band with an apparent
(YP_001135818.1) from Mycobacterium gilvum PYR-GCK molecular mass of about 38 kDa in SDS-PAGE (Fig. 2).
(45%), and an acetyl esterase (YP_001879185.1) from Such electrophoretic mobility was consistent with the
246 Mol Biotechnol (2010) 44:242–249

1 2 3

50 kDa

37 kDa

25 kDa

Fig. 2 SDS-PAGE of recombinant L. plantarum esterase purification

by Ni-NTA resin. Lane 1 molecular marker, lane 2 total soluble
proteins of recombinant of E. coli BL21(DE3) pLysS after 18 h
induction at 15°C, lane 3 purified esterase

theoretical molecular mass value (39.07 kDa) predicted for

the full-length recombinant protein.

Esterase Activity Fig. 3 Effect of pH (a) and temperature (b) on L. plantarum

recombinant esterase activity. For pH effect measurements, enzyme
activity was measured with pNPC4 using different buffers: 50 mM
Remaining imidazole, an interferent for the enzymatic assay,
acetate (pHs 5.0 and 5.5), 50 mM phosphate (pHs 6.0, 6.5, and 7.0),
was removed from the purified enzyme solution by dialysis 50 mM Tris–HCl (pH 8.0), and 50 mM CHES (pH 9.0). For
as described in ‘‘Materials and Methods’’ section. Esterase temperature effect measurements, enzyme activity was measured
activity was determined spectrophotometrically at 348 nm using pNPC4 at pH 6.0. Results are mean ± SE of three independent
experiments
for 5 min using pNPC4 as substrate. Purified His6-tagged
esterase presented a specific activity of 17.2 ± 3.12 U/mg.
activity at pH 5.5–6.0 and 30°C. A lipase [12] and a tri-
Effect of pH and Temperature on Esterase Activity butyrin esterase [18] from L. plantarum 2739 presented
highest activities at pH 7.5 and 7.0, respectively, at 35°C.
Several pH values (5.0, 5.5, 6.0, 6.5, 7.0, 8.0, and 9.0) and On the contrary, an arylesterase from L. helveticus
temperatures (25, 30, 37, 40, 45, 50, 55, and 60°C) were CNRZ32 presented an optimum pH of 8.0 and temperature
applied to determine pH and temperature effect on activity range of 35–40°C [4]. An esterase cloned and purified from
of recombinant esterase. The pNPC4 was used instead of a metagenome library [6] presented the highest activity at
pNPC2 because pNPC4 was more stable than pNPC2 in pH 6.0 and maintained more than 80% in the pH range of
aqueous solution, mainly at high pH values and elevated 5.5–7.5. The observed pH optimum of 6.0 presented by the
temperatures. As shown in Fig. 3a, this esterase is more recombinant esterase is quite normal, since L. plantarum is
active in pH ranging from 5.0 to 7.0. Relative activity a lactic acid bacteria.
increased from 50.3% at pH 5.0 to a maximum (100%) at
pH 6.0, then decreased to 43.9% at pH 8.0 and, with drastic Substrate Specificity
reduction, maintained only 2.6% of total activity at pH 9.0.
With regard to temperature, recombinant esterase presented Substrate specificity was assessed by testing enzymatic
highest activity (100%) at 40°C (Fig. 3b), but presented activity against several p-nitrophenyl esters of different chain
high stability in temperatures ranging from 30 to 50°C, lengths (pNPC2, pNPC4, pNPC8, pNPC10, pNPC12, pNPC14,
with relative activities from 73.3 to 78%, respectively. The and pNPC16) at pH 6.0 and 40°C. Recombinant esterase pre-
behavior presented by this esterase is consistent with sented the highest activity for pNPC2 (128.9 U/mg), followed
findings of other esterases from Lactobacilli. An intracel- by pNPC4 (15.7 U/mg). No activity was detected for the other
lular esterase from L. casei LILA [17] presented the highest p-nitrophenol esters in the tested conditions. Similar substrate
Mol Biotechnol (2010) 44:242–249 247

specificity was found in an esterase from Xanthomonas oryzae Table 2 Effect of metal ions, amino acid modifying reagents, ethanol
[19], which hydrolyzed only pNPC2 and pNPC4, with the and DMSO on recombinant L. plantarum esterase activity
highest activity for pNPC2. An arylesterase from L. plantarum Treatment Relative activity (%)
[4] presented very high activities for pNPC4 and pNPC2, and
Control 100 ± 11.6
also for pNPC12. An esterase from L. casei CL96 [1] hydro-
lyzed pNPC2 to pNPC12, with higher activity for the latter. AlCl3 123.9 ± 13.7
This substrate hydrolysis profile is characteristic of an ester- CaCl2 98.3 ± 7.9
ase, which is an enzyme usually active only in short-chain MgCl2 101.2 ± 8.5
fatty acid esters, i.e., in water-soluble substrates. MnCl2 106.1 ± 8.4
ZnSO4 112.7 ± 14.7
Kinetic Parameters EDTA 105.2 ± 6.6
DEPC 23.8 ± 11.8
Kinetic parameters were determined by spectrophotometric PAO 105.4 ± 5.1
assay using pNPC2 and pNPC4 as substrates. Recombinant PGO 105.9 ± 9.2
PMSF 90.4 ± 6.5
Etanol (1%) 84.0 ± 5.6
DMSO (1%) 88.6 ± 7.8
Residual enzyme activities were determined spectrophotometrically
using pNPC4 1 mM as substrate at 25°C in 50 mM phosphate buffer
(pH 6.0) and metal ions, amino acid modifying reagents at 1 mM.
Enzyme (1 lg) was incubated for 10 min in each substance solution
before readings were carried out. Results are mean ± SD from three
independent experiments. DEPC (diethyl pyrocarbonate), PAO
(phenylarsine oxide), PGO (phenylglyoxal hydrate), PMSF (phenyl-
methylsulfonyl fluoride), DMSO (dimethyl sulfoxide)

esterase exhibited a simple hyperbolic Michaelis–Menten

kinetics (Fig. 4) for these two substrates. Vmax, Km, kcat,
and kcat/Km were calculated and are presented in Table 1. It
can be seen that recombinant esterase is more selective for
pNPC4, presenting a Km value of 0.1185, despite presenting
a lower Vmax for this substrate than that for pNPC2.
However, kcat and kcat/Km values showed that recombinant
esterase is more efficient in hydrolyzing pNPC2 than
pNPC4.

Effect of Metal Ions and Inhibitors on Esterase Activity

Recombinant esterase was incubated for 10 min at room

temperature in the presence of different metal ions and
inhibitors before determining residual activity (Table 2). In
Fig. 4 Michaelis–Menten plots of recombinant esterase from L.
plantarum. Kinetic data were measured spectrophotometrically using
general, metal ions did not produce significant effect on
a pNPC2 and b pNPC4 as substrates. Inset graphs show the double recombinant esterase activity, even calcium being known
reciprocal Lineweaver–Burk plot of the transformed data. Assays to stabilize lipolytic enzymes [20]. However, the presence
were performed in 50 mM phosphate buffer pH 6.0 at 40°C of Al3? seemed to produce a small increase in recombinant

Table 1 Kinetic parameters for recombinant L. plantarum esterase

Substrate Vmax (lmol/min/mg) Km (mM) kcat (s-1) kcat/Km (s-1 mM-1)

pNPC2 176.7 ± 7.7 0.401 ± 0.100 114.9 ± 5.0 286.4 ± 12.5

pNPC4 17.0 ± 1.1 0.119 ± 0.032 11.1 ± 0.7 93.5 ± 6.1
Enzyme activities were determined at 40°C in 50 mM phosphate buffer (pH 6.0). Results are mean ± SD from three independent experiments
248 Mol Biotechnol (2010) 44:242–249

esterase activity. It was also observed that EDTA had no CD Spectroscopy

detectable influence on enzyme activity, suggesting that
this esterase may be a non-metalloenzyme. The tributyrin The secondary structure of the recombinant esterase was
esterase purified from L. plantarum [18] was moderately investigated by circular dichroism measurements. The far-
stimulated by Ca2? and Mg2?, while the lipase purified UV CD spectrum displayed a minimal peak at 218 nm
from L. plantarum [12] was not affected by 1 mM of these (Fig. 5), indicating a predominance of antiparalell b-sheet
two ions. Besides, both enzymes were negatively affected structure, different from other esterases, which display
by 1 mM EDTA. unordered structures [21] and a-helical structures [22].
With regard to amino acid residues modifying reagents, Analysis of the spectrum [23] showed the presence of
this esterase was strongly inhibited (23.8% relative activ- 38.6% of b-sheet and 23.4% of a-helices. Thus, the pres-
ity) by diethyl pyrocarbonate (DEPC), a histidine modifier, ence of such mixed helical and sheet content resolved by
and slightly inhibited (90.4% relative activity) by phe- CDSSTR algorithm [23] indicates that this esterase may be
nylmethylsulphonyl fluoride (PMSF), a serine modifier, included on a/b-fold, once esterases are primarily charac-
suggesting the dependence of recombinant enzyme on the terized by this fold type, predominantly parallel b-sheet
classical model Ser-Asp/Glu-His catalytic triad. It was and flanked by a-helical connections [24].
suggested that the only Cys residue of an arylesterase from
L. helveticus [4] and an esterase from L. casei [1] were also
important for their activities. However, esterase activity Conclusions
was not affected by the cysteine modifier (PAO), seeming
that the only Cys residue (Cys130) does not play a role in A gene coding for L. plantarum esterase was successfully
esterase activity. Also, the arginine modifier (PGO) did not cloned and expressed. Deduced amino acid sequence
influence the activity of esterase. The tributyrin esterase obtained in this study presented 100% similarity with the
purified from L. plantarum [18] was strongly affected by lipase/esterase from L. plantarum WCFS1 strain. This is
PMSF, while the L. plantarum lipase [12] was slightly the first report to show the cloning, expression, and puri-
affected. Besides, esterases from L. helveticus CNRZ32 fication of an esterase from L. plantarum. Also, biochem-
[4], L. casei LILA [17], L. casei CL96 [1], and from a ical and biophysical characterization were carried out. The
metagenomic library [6] were also affected by PMSF, the His6-tagged protein presented the highest specific activity
last two being also strongly affected by DEPC. at pH 6.0 and 40°C for p-nitrophenyl acetate, followed by
This esterase showed stability toward DMSO and etha- p-nitrophenyl butyrate. It was strongly affected by DEPC
nol, keeping more than 80% of total activity. Stability in and to a lesser extent by PMSF, suggesting the catalytic
the presence of organic solvents is an important and activity depends on serine and histidine residues. Further
valuable feature for biotechnological applications [8]. structural and biochemical characterization are necessary
to achieve a full understanding of this enzyme and its
reaction mechanism, and this will be the subject of future
communications.

Acknowledgments This study was financially supported by CNPq

processes 476285/2007-0 and 552508/2007-1, and Rede Proteoma de
Santa Catarina (FAPESC/FINEP/MCT). FCAB was recipient of a fel-
lowship from CAPES, Ministry of Education, Brazil. JBB was recipient of
a fellowship from CNPq, Ministry of Science and Technology, Brazil.

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