The hormone-sensitive lipase from Psychrobacter sp. TA144: New insight in the structural/functional characterization

Article abstract of DOI:10.1016/j.biochi.2010.04.001

Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 °C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C₂ to C₁₂ and the preferred substrate was pNP-pentanoate showing a k_cat = 26.2 ± 0.1 s^-1, K_M = 0.122 ± 0.006 mM and a k_cat/K_M = 215 ± 11 mM^-1 s^-1. The enzyme was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Mn²⁺ ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C₅₀ values of 1.98 M and 0.92 M, respectively. The region surrounding PsyHSL catalytic site showed an unexpected homology with the human HSL. Further, both enzymes are characterised by the presence of an extra N-terminal domain, which role in the human protein has been related to regulative function. To clarify the function of PsyHSL N-terminal domain, a 97 amino acids deleted version of the enzyme was produced in E. coli in soluble form, purified and characterised. This mutant was inactive towards all tested substrates, indicating the involvement of this region in the catalytic process.

Full text of DOI:10.1016/j.biochi.2010.04.001

Biochimie 92 (2010) 949e957
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Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
The hormone-sensitive lipase from Psychrobacter sp. TA144: New insight
in the structural/functional characterization
,
Concetta De Santi a, Maria Luisa Tutino b c, Luigi Mandrich a, Maria Giuliani b,
,
,
*
Ermenegilda Parrilli b c, Pompea Del Vecchio d, Donatella de Pascale a
a Institute of Protein Biochemistry, CNR, Via Pietro Castellino 111, I-80131 Naples, Italy
b Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Complesso Universitario Monte Sant’Angelo, Via Cinthia 4, I-80126 Naples, Italy
c School of Biotechnological Sciences, University of Naples Federico II, Complesso Universitario Monte Sant’Angelo, Via Cinthia 4, I-80126, Naples, Italy
d Department of Chemistry “Paolo Corradini”, University of Naples Federico II, Complesso Universitario Monte Sant’Angelo, Via Cinthia 4, I-80126 Naples, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine
environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from
Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-
expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from
which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature
dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 ꢀC at
pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C2 to C12 and
the preferred substrate was pNP-pentanoate showing a kcat ¼ 26.2 ꢁ 0.1 sꢂ1, KM ¼ 0.122 ꢁ 0.006 mM and
a kcat/KM ¼ 215 ꢁ 11 mMꢂ1 sꢂ1. The enzyme was strongly inhibited by Hg2þ, Zn2þ, Cu2þ, Fe3þ, Mn2þ ions and it
resulted to be activated in presence of methanol and acetonitrile, with calculated C50 values of 1.98 M and
0.92 M, respectively.
Received 23 December 2009
Accepted 2 April 2010
Available online 9 April 2010
Keywords:
Psychrophilic micro-organisms
Cold-active enzymes
a/b hydrolase fold
Ester synthesis
Esterases
Lipases
The region surrounding PsyHSL catalytic site showed an unexpected homology with the human HSL.
Further, both enzymes are characterised by the presence of an extra N-terminal domain, which role in
the human protein has been related to regulative function. To clarify the function of PsyHSL N-terminal
domain, a 97 amino acids deleted version of the enzyme was produced in E. coli in soluble form, purified
and characterised. This mutant was inactive towards all tested substrates, indicating the involvement of
this region in the catalytic process.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
(the nucleophile), a histidine, and an acidic residue (aspartic acid or
glutamic acid) which follows the same secondary structure
Cold-adapted enzymes have been often observed to catalyse
reactions at rates up to one order of magnitude higher than
those observed for mesophilic counterparts at low and even
moderate temperatures [1]. Low temperature activity has
generally been associated with low conformational stability as
seen by the shift of the apparent temperature optima to lower
temperatures and low stability versus chemical denaturants.
The super family of carboxyl ester hydrolases (which comprises
lipases and esterases) includes ubiquitous enzymes of great phys-
iological function with a wide range of substrate specificities; they
element in the a/b fold architecture. These proteins have been
classified into four separate groups (named group C, L, H and X
respectively), based on their sequence similarity. The C group
includes cholinesterases and fungal lipases; the L group contains
lipoprotein lipases and bacterial lipases; the H group, named after
the mammalian hormone-sensitive lipase (HSL), gathers together
proteins exhibiting sequence similarity to hormone-sensitive
lipase; and the X group includes a wide variety of proteins that
contain the a/b hydrolase motif and do not belong to any of the
other groups [2e4].
are member of the
a ‘catalytic triad’ of residues that is usually composed by a serine
a
/
b
hydrolase family. Enzymatic catalysis utilises
Among these classes, the hormone-sensitive lipase group
comprises enzymes expressed in multiple tissues and that play
a crucial role in lipid metabolism, especially in hibernating animals
[5,6]. This family corresponds to the Pfam entry Abhydrolase_3 and,
for bacterial enzymes, to the family IV of the classification of
Arpigny and Jaeger [7]. Two other families have been included
* Corresponding author. Tel.: þ390816132314; fax: þ390816132248.
E-mail address: d.depascale@ibp.cnr.it (D. de Pascale).
0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2010.04.001

950
C. De Santi et al. / Biochimie 92 (2010) 949e957
recently in the “H” block, namely HSL-like-1 and plant carbox-
ylesterases. Although more than 1000 genes have been ascribed to
this block, only a restricted number of proteins has been charac-
terised and only 31 crystal structures have been solved (December
2009 ESTHER Database: http://bioweb.ensam.inra.fr/ESTHER/
general?what¼index).
were engineered to have a 50 SalI restriction site and 30 NotI
restriction sites. PCR reactions were carried out using a MyCycler
(Bio-Rad). The reaction conditions used were: 1 cycle (98 ꢀC for
5 min), 25 cycles (98 ꢀC 30 s, 59 ꢀC 1.5 min, and 72 ꢀC 1 min), and
a final cycle of 72 ꢀC 10 min. The amplified PCR products were
cloned into pET-22b expression vector previously digested by NdeI
and NotI restriction enzymes. The expression cassette includes an
in-frame C-terminal fusion purification 6xHis-Tag. The DNA
sequence of the resulting construct (pET-22b-lip2) was verified by
bidirectional DNA sequencing.
Deleted PsyHSL, after called PsyHSLdel, was obtained by gene
synthesis distributor (MrGene, Nuernberg De). It is the result of
a deletion of the gene region encoding the first 97 of PsyHSL; the
new gene products starts from Gly98. The fragment was cloned into
pET-28b, and the resulting recombinant construct was named pET-
28b-lip2del.
Members of the HSL-like family present the conserved catalytic
triad Ser-His-Asp; the catalytic Ser is embedded in the conserved
sequence Gly-X-Ser-X-Gly (the nucleophilic elbow), as all the other
members of the C and L groups [8]. Moreover, the HSL-like family
contains the highly conserved sequence His-Gly-Gly-Gly ahead of
the catalytic serine, which is only partially conserved in members
of the other blocks [9]. Among the HSL studied so far, the human
HSL (named hHSL) is an intracellular neutral lipase that is capable
of hydrolyzing triacylglycerols, diacylglycerols, monoacylglycerols,
and cholesteryl esters, as well as other lipid and water-soluble
substrates [10]. It is regarded as an adipose tissue-specific enzyme
that plays an important role in the catalysis of hormone-stimulated
fat cell lipolysis in mammals [11,12]. Feller and co-workers, in the
1990, cloned, sequenced and expressed in Escherichia coli three
lipase encoding genes of the Antarctic bacterium Psychrobacter sp.
TA144 (previously classified as Moraxella TA144), lip1, lip2 and lip3,
but they never purified the encoded products [13]. The region
surrounding the putative catalytic site of the lip2 gene product was
reported to be homologous to the corresponding region of hHSL [6].
In the present paper we report the lip2 gene cloning and expression
in E. coli; the recombinant protein (after called PsyHSL) was purified
to homogeneity and deeply characterised. Furthermore, a 98 amino
acids deletion mutant of PsyHSL was constructed, recombinantly
produced and characterised with the aim of understanding the role
of this extra N-terminal region, with respect to the extra N-terminal
domain of hHSL, which has been demonstrated to be a regulative
domain [6].
2.3. PsyHSL and PsyHSLdel recombinant productions in E. coli cells
E. coli BL21(DE3) carrying pET-22b-lip2 vector was grown in
shake flask containing LB medium supplemented with 100 mg/mL
ampicillin for 16 h at 37 ꢀC. Growing culture was diluted to a cell
density of about 0.08 OD600 in 3 L of LB medium supplemented
with 100 mg/mL ampicillin using a 7.5 L Techfors S fermentor (Infors
HT Switzerland). The temperature of the medium was controlled at
25 ꢁ 1 ꢀC and the pH at 7.0 ꢁ 0.5. Throughout the fermentation, the
dissolved oxygen tension was maintained always above 30% of
saturation by acting on air supply at constant agitation (250 rpm).
PsyHSL induction was performed when culture density reached
0.5e0.6 at OD600 by addition of filter-sterilized IPTG to a final
concentration of 1 mM.
E. coli BL21(DE3) carrying pET-28b-lip2del vector was grown in
shake flask containing LB medium supplemented with 20 mg/mL
kanamycin for 16 h at 37 ꢀC. The obtained culture was diluted to
a cell density of about 0.08 OD600 in 250 mL of LB medium sup-
2. Materials and methods
plemented with 20 m
g/mL kanamycin at 30 ꢀC. PsyHSLdel induction
2.1. Bacterial strains, plasmids and growth conditions
was performed when culture density reached 0.5e0.6 at OD600 by
addition of filter-sterilized IPTG to a final concentration of 1 mM.
In both the cases, the cultures were grown for approximately 3 h
post-induction. Cells were harvested by centrifugation at 3200 ꢃ g
at 4 ꢀC for 20 min, divided into 0.50 g aliquots and frozen at ꢂ80 ꢀC.
The strain Psychrobacter sp. TA144 was collected in the sea water
near the Antarctic station of Dumont d’Urville and was previously
classified as Moraxella TA144 [13]. E. coli BL21(DE3) Fꢂ (ompT hsdSB
(rBꢂ mBꢂ) gal dcm (DE3)); and E. coli TOP10 (supE44,
D
lacU169 (F80
lacZ
D
M15), hsdR17, recA1, endA1, gyrA96, thiꢂ1 relA1) were used as
2.4. Isolation and solubilization of PsyHSL inclusion bodies
host strains for transformation. The vector used for cloning and
expression was pET-22b (Novagen).
The bacterial pellet (0.50 g) was frozen and thawed twice,
resuspended in 10 mL of Buffer TN: 100 mM TriseHCl, pH 8.0,
containing 150 mM NaCl and subjected to sonication. Lysozyme
Psychrobacter sp. TA144 was grown in TYP rich medium (16 g/L
Bacto-Triptone, 16 g/L Yeast Extract, 10 g/L NaCl pH 7.5) at 4 ꢀC in
shake flask. E. coli cells were routinely grown in LB medium [14] at
(200 mg), RNase (200 mg), DNase (50 mg) and 30 mM MgCl2 were
37 ꢀC and ampicillin at 100
mg/mL was added to select transformants.
added to the suspension and incubated for 30 min at 37 ꢀC.
The suspension was then diluted in 100 ml of 100 mM TriseHCl, pH
8.0, 150 mM NaCl and gently shaken for 1 h at 4 ꢀC. After 30 min
centrifugation at 7000 ꢃ g, the pellet containing the inclusion
bodies was resuspended in 100 ml of 100 mM TriseHCl, pH 8.0, and
2.2. DNA manipulation and gene cloning
Genomic DNA from Psychrobacter sp. TA144 was purified by
using ChargeSwitch gDNA Mini Bacteria Kit (Invitrogen). General
DNA methods were carried out according to Sambrook and Russell
[14]. PsyHSL gene (lip2) was amplified from Psychrobacter sp.
TA144 genomic DNA in two fragments in order to facilitate cloning
of PCR products. Restriction sites NdeI and SalI were inserted in the
583 bp fragment corresponding to the region 1e583 of lip2 gene.
The forward and reverse primers for the amplification of the first
fragment were: 50-ATCTCATATGCCTATTCTACCAGTACC- 30, 50-
GATAGTCGACACTTACCACCGCCCAG- 30. The second fragment of
719 bp corresponding to the region 583e1302, was amplified using
the Lip2fw and Lip2rev primers (50-AAGTGTCGACTATCGC
ATGGCACCTG-30, 50-TTATGCGGCCGCTGCTCTGAGATTTG-30) which
50 mM b-mercaptoethanol. The suspension was gently shaken for
1 h at 4 ꢀC and then centrifuged at 7000 ꢃ g for 30 min. The pellet
was solubilized in 10 ml of 6 M urea, 100 mM TriseHCl, pH 8.0,
1 mM glycine, 1 mM EDTA, 50 mM
a gently shaking for 12 h.
b
-mercaptoethanol at 4 ꢀC with
2.5. PsyHSL refolding and purification
The solubilized material was centrifuged at 45,000 ꢃ g for 2 h
using a 60Ti rotor at 4 ꢀC. The supernatant was then centrifuged at
49,000 ꢃ g for 2 h using the same rotor at 16 ꢀC. Renaturation of the
supernatant containing the PsyHSL was achieved by a 200-fold

C. De Santi et al. / Biochimie 92 (2010) 949e957
951
dilution of the denaturant in 100 mM TriseHCl buffer, pH 8.0, and
the solution was concentrated again to about 50 ml using an
Amicon ultrafiltration cell (Millipore) equipped with a 10 kDa cut-
off membrane. Starting from 0.50 g of cell pellet, about 10 mg of
pure protein was obtained by this procedure and PsyHSL was stored
at ꢂ20 ꢀC in presence of 20% glycerol.
acetonitrile. Samples of identical composition as the assay mixture,
omitting the enzyme, provided suitable blanks. Assays were done in
duplicate and results were mean of two independent experiments.
One unit of enzymatic activity was defined as the amount of the
protein releasing
1 mM of p-nitrophenoxide/min from pNP-
hexanoate at indicated temperature. The absorption coefficient
used for p-nitrophenoxide was 19,000 Mꢂ1 cmꢂ1 at 35 ꢀC and
pH 8.0.
2.6. PsyHSLdel purification
The bacterial pellet (0.50 g) was frozen and thawed twice,
resuspended in 10 mL of Buffer TN: 100 mM TriseHCl, pH 8.0,
containing 150 mM NaCl and subjected to sonication. The solubi-
lized material was centrifuged at 4000 ꢃ g for 25 min using an SS34
2.10.2. Kinetic measurements and analysis
Initial velocity versus substrate concentration data were fitted to
the LineweavereBurk transformation of the MichaeliseMenten
equation, by weighted linear least-squares analysis with a personal
computer and the GRAFIT program 5.1; pNP-acetate and pNP-
butanoate ranged from 0.04 to 1.6 mM, pNP-hexanoate from 5 to
rotor at 4 ꢀC. More than 500
mg of E. coli homogenate was loaded on
HIS Select spin column (purchased from Sigma) equilibrated with
600 l of 50 mM TriseHCl pH 8.0, containing 500 mM NaCl. After
extensive washing, the purified protein was eluted in 500 l at
m
180 mM. Assays were done in duplicate or triplicate and results for
m
kinetic data were mean of two independent experiments. Kinetic
parameters were calculated using acetonitrile or methanol as
solvent (dissolving pNP-pentanoate in pure acetonitrile or meth-
anol), and also without solvent, dissolving pNP-pentanoate in 0.1 M
TriseHCl pH 8.0 at final concentration of 1 mM.
250 mM imidazole. The eluted solution was dialysed over night
against 10 mM TriseHCl, pH 8.0 at 4 ꢀC, and stored at ꢂ20 ꢀC.
2.7. Electrophoretic analysis
Electrophoretic runs were performed with a Bio-Rad Mini-
Protean II cell unit, at room temperature. 12.5% SDS-PAGE was
performed essentially as described by Laemmli. ‘‘Page Ruler Pre-
stained Protein Ladder’’(Fermentas) was used as molecular weight
standard [15].
2.10.3. Esterase activity on triacylglycerols
Enzyme activity towards triacylglycerols with acyl chain length
from 4 to 8 carbon atoms by pH titration, using a pH-stat controller
apparatus (Radiometer, Copenhagen). Reaction mix was 5 mM
TriseHCl pH 8.0, containing 3% (v/v) acetonitrile and triacylglycerols
ranged from 50 to 300 mM. Stock solutions of triacylglycerols were
2.8. PsyHSL determination of pH and temperature optima
prepared by dissolving substrates in pure acetonitrile. The assays
were done at 35 ꢀC in duplicate or triplicate and results are the mean
The dependence of the initial velocity on pH was monitored at
348 nm (the pH-independent isosbestic point of p-nitrophenol and
the p-nitrophenoxide ion) with a molar absorption coefficient of
5000 Mꢂ1 cmꢂ1 at 35 ꢀC. The buffers used were 0.1 M MES over the
range 5.0e6.0, 0.1 M Na-phosphate over the range 6.0e7.5, and
0.1 M TriseHCl over the range 7.5e9.0. The assays were carried out
in duplicate or triplicate and the results were the means of two
independent experiments. The measurements of esterase activity
of two independent experiments. The activity was measured as meq
of NaOH used to neutralise the free acid developed from the enzy-
matic reaction in 1 min by 1 mg of enzyme [16]. Enzyme activity was
expressed as units. One enzyme unit corresponds to the release of
1 mmol of acid per minute. The value of the catalytic rate constant
kcat (secꢂ1) was calculated from the specific activity and the
molecular mass [17].
by using pNP-pentanoate at a final concentration of 100
mM in
2.10.4. Thioesterase activity using DTNB-assay
a reaction mixture 0.1 M TriseHCl pH 8.0, containing 3% acetoni-
trile, over the range 5.0e9.0. The dependence of esterase activity
on temperature was studied over the range 5e90 ꢀC, with pNP-
Enzyme activity towards thioester was analysed by the DTNB-
assay [18]. Initial rates of formation of free-thiol groups from
S-methyl thiobutanoate were quantified by measuring absorbance
at 410 nm of TNB, in 1-cm path-length cells with a Cary 100
spectrophotometer (Varian, Australia). Initial rates were calculated
by linear least-square analysis of time courses comprising less than
10% of the total substrate turnover. The kinetic parameters were
calculated in a mixture containing 0.1 M TriseHCl pH 8.0, 1 mM
DTNB, 3% acetonitrile and S-methyl thiobutanoate in a range from
0.1 to 2.5 mM, at 35 ꢀC. A stock solution of S-methyl thiobutanoate
was prepared by dissolving the substrate in pure acetonitrile. One
unit of enzymatic activity was defined as the amount of the protein
pentanoate (100
mM) as substrate, in a reaction mixture 0.1 M
TriseHCl pH 8.0, containing 3% acetonitrile.
2.9. PsyHSL thermostability
The thermal stability of PsyHSL was studied over the range
1e40 ꢀC. Pure enzymes (0.2 mg/ml), in a 0.1 M TriseHCl pH 8.0,
were incubated in sealed glass tubes at the different temperatures.
Aliquots were withdrawn at 20, 40, 60, 80, 120 min and assayed at
35 ꢀC in standard condition described above.
releasing 1 mmole of TNB per min from DTNB. Assays were carried
out in duplicate or triplicate and results were mean of two inde-
2.10. Biochemical characterization
pendent experiments. The absorption coefficient used for DTNB
was 19,000 Mꢂ1 cmꢂ1
.
2.10.1. PsyHSL esterase activity
The time course of the esterase-catalysed hydrolysis of pNP-
esters was followed by monitoring of p-nitrophenoxide production
at 405 nm, in 1-cm path-length cells with a Cary 100 spectropho-
tometer (Varian, Australia). Initial rates were calculated by linear
least-squares analysis of time courses comprising less than 10% of
the total substrate turnover. Assays were performed at 35 ꢀC in
a mixture of 0.1 M TriseHCl pH 8.0, 3% acetonitrile at the indicated
2.10.5. Effect on enzymatic activity by organic solvents,
detergents and metals
Enzyme activity was evaluated in the standard assay (0.1 M
TriseHCl pH 8.0, 3% acetonitrile, 35 ꢀC) using pNP-pentanoate as
substrate.
2.10.5.1. Organic solvents. The activity was measured without
organic solvents (acetonitrile or methanol) and using an increasing
concentration of the solvents from 0 to 15% (v/v) in the assay
pH, containing pNP-esters (100
mM). Stock solutions of pNP-esters
from C2 to C12 were prepared by dissolving substrates in pure

952
C. De Santi et al. / Biochimie 92 (2010) 949e957
mixture. Results were reported as relative activity with respect to
the value measured without solvents.
rate of 5 nm minꢂ1, were signal-averaged over at least five scans,
and baseline corrected by subtracting a buffer spectrum, at 5 ꢀC.
2.10.5.2. Detergents and metals. The effect of detergents on enzy-
matic activity of PsyHSL was evaluated incubating 0.2 mg/ml of
pure protein in presence of 5 mM SDS, Triton X-100, Tween-20 and
Tween-80 at 4 ꢀC for 1 h. The residual enzymatic activity was
measured in the standard condition as described above.
3. Results
3.1. Purification and physical properties of PsyHSL and PsyHSLdel
IPTG induction of recombinant E. coli BL21(DE3)/pET-22b-lip2
cells resulted in the accumulation of recombinant PsyHSL as
inclusion bodies (IB). The IB were purified, solubilized with 6 M
urea, and the refolding was achieved by rapid dilution in 0.1 M
TriseHCl pH 8.0 as described in “Experimental Procedures”. Ten
milligrams of pure enzyme were obtained starting from 0.5 g of
E. coli cell pellet. Recombinant PsyHSL identification was carried out
by N-terminal sequencing of the electroblotted protein band. The
obtained sequence was M-P-I-L-P-V.
The mutant PsyHSLdel was purified to homogeneity by a single
purification step using HIS Spin columns loaded with the soluble
fraction of the E. coli BL21(DE3)/pET-28b-lip2del recombinant cells.
Pure protein was eluted by using 0.1 M TriseHCl pH 8.0, 250 mM
imidazole.
Several metal ions, such as Hg 2þ, Mg2þ, Ca2þ, Cu2þ, Co2þ, Mn2þ
,
Zn2þ, Fe3þ were added to 0.2 mg/ml of pure protein at final
concentration of 5 mM and incubated at 4 ꢀC for 1 h. The residual
enzymatic activity was measured in the standard condition as
described above.
2.10.5.3. Inhibition by paraoxon of esterase activity. Inhibition by
paraoxon was evaluated by assay the activity for the enzyme at
35 ꢀC and 0.1 M TriseHCl pH 8.5 containing 3% acetonitrile, using
pNP-pentanoate as substrate at the concentration of 100
the KM value) and paraoxon at two different concentrations 5 and
50 M. The activity was expressed as residual activity with respect
mM (about
m
to the activity without inhibitor.
Recombinant pure protein was identified by N-terminal
sequencing of the electroblotted protein band, which gave the
following sequence at the N-terminal: M-G-T-D-A-V.
2.11. Multiple sequence alignment
Multiple sequence alignments were carried out with the
program T-COFFE Regular Version 7.81 using the following lipase
sequences belonging to the HSL family: EstEI from metagenomic
source (PDB ID 2c7b); HerE heroin esterase from Rhodococcus sp.
(PDB ID 1lzl); EST2, carboxylesterase from Alicyclobacillus acid-
ocaldarius (PDB ID 1evq); and AFEST, carboxylesterase from
Archaeoglobus fulgidus (PDB ID 1jji). The alignments featuring the
highest score was obtained using the BLOSUM 62 matrix [19] and
standard Multalin parameters.
Purity of the proteins preparation were also evaluated by SDS-
PAGE analysis. As shown in Fig. 1, two single bands were observed
with an apparent molecular mass of about 48,000 Da and
37,500 Da, for the wild type protein PsyHSL and the mutant Psy-
HSLdel, respectively.
Both PsyHSL and PsyHSLdel adopt a folded conformation in
solution as indicated by the far-UV CD spectra recorded at 5 ꢀC in
20 mM sodium phosphate buffer, 1 mM DTT at pH 7.5 and reported
in Fig. 2. In fact, the analysis of CD spectra by means of Dicroweb
[23], has provided values of 31%
PsyHSL and 14% -helix, 29% -strand content for the mutant Psy-
HSLdel. These results suggest that the deleted N-terminal domain
has a full -helix conformation, in agreement with results coming
from a secondary structure prediction of the same region (data not
shown).
a-helix, 33% b-strand content for
a
b
2.12. Homology model
a
The three dimensional model of PsyHSL was obtained using
heroin esterase from Rhodococcus strain H1 [20] (1lzl PDB entry) as
template. The atomic coordinates of HerE from Rhodococcus in its
open conformation were obtained from the Brookhaven Protein
Data Bank. The template was selected in according with position of
the catalytic triad (Ser, Asp, His). To generate 3D-model the
sequence alignment was submitted to the comparative structural
modeling program MODELLER 8v2 [21] which uses sequence
alignment to extract a large number of spatial restraints for
homology modeling of the target protein. These spatial restraints
are then expressed as 50 PDBs, which are combined into an
objective function. The model was selected on the basis of the
lowest value of the MODELLER objective function. Hydrogen atoms
were added to the model and the strain has to be removed by 100
steps of energy minimization of the structure using GROMACS
package [22].
2.13. Circular dichroism
CD spectra were recorded with
a
Jasco J-715 spec-
tropolarimeter equipped with a Peltier type temperature control
system (Model PTC-348WI). Molar ellipticity per mean residue,
(
(
q
) in deg cm2 dmolꢂ1, was calculated from the equation:
q
) ¼ (
q)obs$mrw/10 l C, where (q)obs is the ellipticity measured in
degrees, mrw is the mean residue molecular weight, C is the
protein concentration in g mLꢂ1 and l is the optical path length of
the cell in cm. CD spectra were recorded by using a 0.1 cm path-
length cell, a time constant of 4 s, a 2 nm band width, and a scan
Fig. 1. SDS-PAGE (12% acrylammide) of PsyHSL and PsyHSLdel at the end of denatur-
ation-refolding and purification procedure respectively. Lane 1, PsyHSL purified at
homogeneity; lane 2, PsyHSLdel purified at homogeneity; lane 3, molecular weight
markers.

C. De Santi et al. / Biochimie 92 (2010) 949e957
953
10
5
0.8
0.6
0
0.4
PsyHSLdel
PsyHSL
-5
0.2
-10
0
0
20
40
60
80
100
190
200
210
220
230
240
250
Temperature (°C)
wavelength (nm)
Fig. 4. Effect of temperature on the lipase activity.
Fig. 2. Far-UV CD spectra of PsyHSL (open circles) and PsyHSLdel (open squares)
recorded at 5 ꢀC in 20 mM sodium phosphate, 1 mM DTT, pH 8.0 buffer.
3.3. Kinetic studies
3.2. PsyHSL pH optimum, thermophilicity and thermostability
As for others enzymes belonging to the same family, we inves-
tigated the activity of PsyHSL versus different types of substrates. In
particular we determined the kinetic parameters on pNP-esters
with acyl chain length from 2 to 12 carbon atoms, triacyl esters of
glycerol from 4 to 8 carbon atoms and on S-methyl thiobutanoate.
Because all the used esters show a low solubility in water, we
evaluated the effect of the presence of acetonitrile in the assay
mixture on PsyHSL activity (see below). No negative effect on
PsyHSL catalytic activity was recorded when acetonitrile was added
up to 3% v/v. Therefore, all the characterization were performed at
35 ꢀC in the presence of 3% acetonitrile. In this condition, the
psychrophilic enzyme displays highest kcat and kcat/KM values on
pNP-pentanoate; for this substrate, kinetic parameters were also
determined in absence of acetonitrile (Table 1). The clear effect of
acetonitrile addition on kcat is not unusual and can be regarded as
the consequence of its positive effect on the substrate solubility in
water assay conditions. As for the kinetic parameters, PsyHSL
The dependence of PsyHSL activity on the substrate pNP-
pentanoate in function of pH was examined at 35 ꢀC. Kinetics were
followed spectrophotometrically at 348 nm, which is the pH-
independent isosbestic wavelength between the p-nitrophenol and
p-nitrophenolate ion. As reported in Fig. 3, by using three different
buffers over the pH range 5.0e10.0, the maximum PsyHSL activity
was recorded at pH 8.0 in 0.1 M TriseHCl.
The relationship between PsyHSL activity and temperature in
the range 5e90 ꢀC was obtained using pNP-pentanoate as substrate
(Fig. 4). The apparent maximal activity was recorded at 35 ꢀC,
a value similar to that recorded for other enzymes from psychro-
philic bacteria, i.e. the cold-active esterase from Psychrobacter sp.
Ant300 [24]. All characterizations were performed at 35 ꢀC.
The PsyHSL thermal stability was evaluated in the range 1e40 ꢀC
(Fig. 5). Enzyme samples were incubated at anygiven temperature for
different lengths of time (up to 2 h) and residual activity was
measured. After 2 h incubation at 4 ꢀC, no changes in activity were
observed, while after 20 min of incubation at 40 ꢀC the enzymatic
activity dramatically drops down. A 10% decrease was observed after
2 h of incubation at 1 ꢀC. At 20 ꢀC, a t1/2 value of 20 min was observed.
4 °C
10 °C
1 °C
2
1,5
1
15 °C
20 °C
0.6
0.4
0.2
25 °C
30 °C
35 °C
0,5
0
40 °C
0
20
40
60
80
100
120
140
0
5
5.5
6.0
6.5
7.0
pH
7.5
8.0
8.5
9.0
Time (min)
Fig. 5. Thermostability of PsyHSL at various temperatures, such as 1 ꢀC (C), 4 ꢀC (B),
10 ꢀC (,), 15 ꢀC (-), 20 ꢀC ( ), 25 ꢀC (:), 30 ꢀC ( ), 35 ꢀC ( ), 40 ꢀC (>). The
Fig. 3. Effect of pH on the lipase activity of PsyHSL. The enzyme was incubated with
0.1 mM pNP-pentanoate and assayed in (B) 0.1 M MES, (C) 0.1 M Na-phosphate and
(,) 0.1 M TriseHCl.
6
7
;
enzyme was incubated in 0.1 M TriseHCl pH 8.0 at the indicated temperatures for the
times indicated. The residual activity was measured at 35 ꢀC.

954
C. De Santi et al. / Biochimie 92 (2010) 949e957
Table 1
for other enzymes [26], in both tested conditions, PsyHSL catalytic
activity was increased up to a critical threshold concentration of the
co-solvent, and further addition leaded to a gradual protein inac-
tivation. We calculated the threshold concentration (C50) for
acetonitrile (0.92 M) and methanol (1.98 M), defined as the
concentration (in molarity) of this organic solvent at which one-
half of the enzyme activity is observed.
PsyHSL was 1 h-incubated in the presence of various denatur-
ants or metal ions, and the residual activity was measured using
pNP-pentanoate as substrate at 35 ꢀC. The resulting values are
summarised in Table 2, from which it can be observed that all tested
compounds have an inhibitory effect on PsyHSL activity, although at
various extents. The strongest inhibitory effect was observed in the
presence of Hg2þ metal ions, supporting the presence of function-
ally important thiol group(s) needed for enzymatic activity. On the
contrary, the presence of Mg2þ ions slightly enhanced the enzyme
activity. The incubation with Ca2þ ions led to a partial inactivation
of the enzymatic activity suggesting the absence of the Ca2þ
binding motif sequence. Treatment with PMSF strongly inhibits the
PsyHSL activity, probably due to the covalently binding to the serine
residue (Ser239) that is essential for the catalytic activity. Contrary
to what observed for other esterase and lipase [27], we found
a decrease of PsyHSL activity in the presence of various detergents
such as Triton X-100, Tween-20 and Tween-80.
Kinetic parameters. All parameters were calculated at 35 ꢀC, in TriseHCl 0.1 M pH
8.0, containing acetonitrile at final concentration of 3%.
Substrate
Kinetic parameters
kcat (sꢂ1
)
KM (M ꢃ 10ꢂ3
)
s ¼ kcat/KM
(secꢂ1 Mꢂ1 ꢃ 103)
pNP-acetate
3.5 ꢁ 0.4
3.9 ꢁ 0.3
0.63 ꢁ 0.01
0.40 ꢁ 0.06
0.12 ꢁ 0.02
0.12 ꢁ 0.01
0.11 ꢁ 0.02
0.07 ꢁ 0.01
0.075 ꢁ 0.005
0.022 ꢁ 0.001
0.021 ꢁ 0.002
5.5 ꢁ 1.5
pNP-propionate
pNP-butanoate
pNP-pentanoate
pNP-pentanoate*
pNP-hexanoate
pNP-octanoate
pNP-decanoate
pNP-dodecanoate
9.8 ꢁ 1.6
6.2 ꢁ 0.5
51.3 ꢁ 13.6
215 ꢁ 11
162 ꢁ 35
62.8 ꢁ 10.7
48.1 ꢁ 5.2
51.4 ꢁ 1.0
8.2 ꢁ 0.6
26.2 ꢁ 0.1
17.8 ꢁ 1.5
4.4 ꢁ 0.1
3.6 ꢁ 0.6
1.13 ꢁ 0.02
0.17 ꢁ 0.01
Glyceryl tributanoate
Glyceryl trihexanoate
21.5 ꢁ 2.1
16.7 ꢁ 1.3
0.115 ꢁ 0.030
0.175 ꢁ 0.030
187 ꢁ 50
95.5 ꢁ 20.0
S-methyl thiobutanoate
1.5 ꢁ 0.1
0.6 ꢁ 0.1
2.5 ꢁ 0.3
*Kinetic parameters were determined in water.
displays a significant decrease in activity when substrate acyl chain
length decreases or increases with respect of pNP-
pentanoate (Table 1).
Natural lipase substrates, such as triacylglycerols with acyl chain
length from four to eight carbon atoms were tested as substrate of
PsyHSL. Assays were performed by pH titration in buffered solution
containing 3% acetonitrile and the given triacylglycerol. One
enzyme unit corresponds to the release of one microequivalent acid
per minute. As reported in Table 1, kinetic parameters comparable
to that recorded with the optimal pNP-esters (pNP-pentanoate)
were recorded using glyceryl tributanoate. Glyceryl trihexanoate
was a less efficient substrate, although the reported kinetic
parameters were still better that the other pNP-esters tested. Due to
low solubility of the substrate in the assay conditions, it was
impossible to determine PsyHSL kinetics parameters on glyceryl
trioctanoate, although a clear but low enzymatic activity was
detected (data not shown). These results are suggestive that PsyHSL
natural substrates may be triacylglycerols with short length acyl
chain. Other enzymes belonging to the HSL family, such as EST2,
AFEST, have been endowed with “catalytic promiscuity” because all
of them show activity versus different class of substrates. PsyHSL
has been shown that besides the hydrolysis of different pNP-esters
with acyl chain length from 2 to 12 carbon atoms and glyceryl
triesters with acyl chain length from 4 to 8 carbon atoms. PsyHSL
kinetic parameters were also measured on the thioester substrate,
S-methyl thiobutanoate: the values reported in Table 1 are very low
and suggestive of a low specificity for the thioester substrate. This
result can be interpreted as a result of a PsyHSL promiscuous
enzymatic activity. The term “catalytic promiscuity” [25] is used to
describe the capability of an enzyme to catalyse different chemical
reactions, so-called secondary activities, at the active site respon-
sible for the primary activity. This is a well known characteristic of
3.5. Modelling
In order to model the structure of PsyHSL, its amino acid sequence
was aligned with those of other homologous enzymes with known
3D structure. As shown in the resulting alignment presented in Fig. 6,
sequence similarity extends along all the protein sequence, with the
exceptions of PsyHSL N-terminal region, where the psychrophilic
protein displays an extra domain. The alignment allowed to clearly
identify the conservation of the canonical HGGG motif, the so-called
nucleophilic elbow, and the putative residues involved in the catalytic
triad, i.e. the residues Ser239, Asp361 and His391 (where the numbering
corresponds to PsyHSL sequence). Higher sequence conservation was
observed in the PsyHSL central region, where two extra-loops char-
acterised the psychrophilic enzyme.
The crystal structure (PDB ID Ilzl) [20] of heroin esterase from
Rhodococcus strain H1 (HerE) was selected as template for the
construction of a 3D PsyHSL model, since it shares the highest
sequence identity (about 30%) with the psychrophilic enzyme.
Table 2
Effect of various compounds on PsyHSL activity.
Compound (5 mM)
Residual
activity (%)a
No addition
HgCl2
MgCl2
CaCl2
CuCl2
CoCl2
MnCl2
FeCl3
ZnS04
100
0.15
113.1
22.8
3.6
31.6
8.8
3.2
14.3
39
69
92
2.8
many enzymes within the
a/b-hydrolyse fold and as a consequence
it has been assumed that this scaffold can tolerate large insertions
modifying in the active site architecture so that different substrates
can be accommodated.
2-mercaptoethanol
DTT
EDTA
3.4. Effect of organic solvents, denaturants and metal
ions on PsyHSL activity
SDS
PMSF
56.6
17.1
74.6
28.7
The effect of the presence of water-miscible solvents on PsyHSL
enzymatic activity on pNP-pentanoate at 35 ꢀC in 0.1 M TriseHCl
buffer (pH 8.0) was investigated. The effect of growing concentra-
tion of methanol and acetonitrile were recorded and, as reported
Triton X-100
Tween-20
Tween-80
a
The residual activity was measured with pNP-pentanoate at 35 ꢀC.

C. De Santi et al. / Biochimie 92 (2010) 949e957
955
Fig. 6. Alignment of PsyHSL sequence with those of other homologous enzymes. EstEI, esterase from metagenomic source (PDB ID 2c7b); HerE, heroin esterase from Rhodococcus sp.
(PDB ID 1lzl); EST2, carboxylesterase from Alicyclobacillus acidocaldarius (PDB ID 1evq); and AFEST, carboxylesterase from Archaeoglobus fulgidus (PDB ID 1jji). The conserved
residues at least in three sequences are in grey and bold. The residues in red indicate the HGGG motif (the nucleophilic elbow) and the active site residues. Filled triangle indicate
the PsyHSLdel starting residue. (For the interpretation of the reference to color in this figure legend the reader is referred to the web version of this article.)
Due to the presence of PsyHSL N-terminal extra domain, the model
was limited to its central region, between amino acids Gln63 and
Gln419. Fig. 7(A) shows the ribbon representations of the resulting
PsyHSL model, from which it can be appreciated the occurrence of
the built model in a spatial configuration compatible with the
catalytic action (Fig. 7(B)).
4. Discussion
the classical a/b fold, typical of this class of enzymes. Furthermore,
the residues likely involved in the catalytic activity, and previously
identified by multiple sequence alignment, resulted to be located in
The aim of this work is the recombinant production, purification
and biochemical characterization of PsyHSL and its mutant PsyHSLdel

956
C. De Santi et al. / Biochimie 92 (2010) 949e957
Therefore, homogeneous PsyHSL was obtained by inclusion bodies
purification followed by in vitro refolding. Three different methods
were tested to recover the protein from inclusion bodies: N-laur-
oylsarcosine, guanidine hydrochloride (data not shown) and urea.
The refolding procedure starting from the urea solubilized protein
sample gave the best yields either in terms of protein amounts or
catalytic activity. Applying this procedure, the protein was recov-
ered in quite homogeneous form (about 90e95% pure) and no
additional purification steps were needed.
Several criteria were evaluated to assess the quality of the
refolded enzyme. The recombinant pure protein was analysed by
CD spectroscopy, exhibiting high percentages of
a and b structures,
as estimated from the spectra analyses. The CD data are in agree-
ment with those referring to properly folded forms of esterase
belonging to the same family, including EST2, the carboxylesterase
from A. acidocaldarius [29] and AFEST from Archeoglobus fulgidus
[30], suggesting that PsyHSL was obtained in a properly folded
form.
Furthermore, the purified recombinant PsyHSL resulted to be
also active, efficiently converting several substrates into products.
The recombinant psychrophilic enzyme exhibited optimal apparent
temperature at 35 ꢀC towards pNP-pentanoate under the optimal
pH 8.0.
Stability studies demonstrated that the enzyme behave as a clas-
sical psychrophilic one, since it was characterised by a pronounced
thermal-stability. Indeed, it retained almost 100% of activity after 2 h
of incubation at 4 ꢀC, while a gradual loss of activity was observed
when the enzyme was incubated at temperature above 30 ꢀC, with
practically no remaining activity after only 20 min incubation at 40 ꢀC
(Fig. 5). Interestingly, when PsyHSL was incubated at 1 ꢀC, we
observed a partial loss of activity. This observation suggests that the
protein may be subjected to a cold-denaturation. This finding is in
agreement with current hypothesis that cold-adapted enzymes are
both heat- and cold-labile, contrary to what is intuitively expected.
Psychrophilic enzymes have a fragile molecule edifice that is
uniformly unstable and is stabilised by a fewer number of weak
interactions with respect to those occurring in heat-stable
proteins [1].
In view of the experimentally determined value for Kcat, KM and
s obtained using several substrates, some interesting comments can
be done. As for the KM values are concerned, the affinity constants
decreased when substrate aliphatic chain length increased. These
results suggested that the enzyme active site seems to interact
more efficiently with longer aliphatic chain substrates. The kcat
values displayed a different trend: they increased as the substrate
aliphatic chain length increased up to C5, and they decreased
beyond C5. As a consequence, also the kcat/KM values displayed
a trend similar to kcat values, pointing towards pNP-pentanoate (C5)
as the most preferable substrate for the psychrophilic enzyme.
Because lipases prefer substrates with relatively long aliphatic
chains, these results suggested that PsyHSL exhibits low lipase
activity. Our results elucidated the putative physiological role of
PsyHSL as esterase because it was able to hydrolyse more effectively
substrate with short acyl chain length.
HSLs are often implicated in a variety of metabolic processes, and
they have been reported to cleave several substrates, including tri-
acylglycerols, cholesterol esters and steroid esters [10]. Therefore,
we also tested PsyHSL activity on triacylglycerols; high enzymatic
activity was obtained using glyceryl tributanoate (comparable value
with pNP-pentanoate, Table 1) while low activity was observed using
glyceryl trioctanoate.
Fig. 7. (A) A model representation of overall structure of PsyHSL that shows a typical
a/
b
fold of esterase. (B) A molecular model of the predicted catalytic triad composed of
Ser239, Asp361 and His391. These active site residues are shown by sticks.
(D97mut) from Psychrobacter sp. TA144, a Gram-negative bacterium
isolated from Antarctic sea water. PsyHSL encoding gene (lip2) was
first isolated by Feller and co-workers in the 1991 [28] by screening
a Psychrobacter sp TA144 expression library in E. coli by a classical
approach of lipolytic activity assay on plates. The authors never
purified the encoded product, and its preliminary biochemical
characterization was carried out on the E. coli recombinant cell
lysate [28].
To study PsyHSL in further details, we amplified the lip2 gene
from the bacterial genomic DNA, and cloned it into pET-22b
expression vector allowing us to over-express the psychrophilic
enzyme in E. coli BL21(DE3) strain.
Sequence alignment and enzyme modeling presented in this
paper further supported the inclusion of PsyHSL in the hormone-
sensitive lipase family, mainly due to the conservation of the
nucleophilic elbow and the active site residues (Fig. 6), which
The amount of the enzyme in the whole-cell extract was very
high, but the protein was poorly represented in the soluble fraction,
because PsyHSL largely accumulates as insoluble aggregates.

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in which the first 97 amino acids were removed. The mutant version
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Acknowledgements
This work was supported by P.N.R.A. (Programma Nazionale
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