Immobilization and characterization of a new regioselective and enantioselective lipase obtained from a metagenomic library

Article abstract of DOI:10.1371/journal.pone.0114945

In previous work, a new lipase and its cognate foldase were identified and isolated from a metagenomic library constructed from soil samples contaminated with fat. This new lipase, called LipG9, is a true lipase that shows specific activities that are comparable to those of well-known industrially-used lipases with high activity against long-chain triglycerides. In the present work, LipG9 was co-expressed and co-immobilized with its foldase, on an inert hydrophobic support (Accurel MP1000). We studied the performance of this immobilized LipG9 (Im-LipG9) in organic media, in order to evaluate its potential for use in biocatalysis. Im-LipG9 showed good stability, maintaining a residual activity of more than 70% at 50°C after incubation in n-heptane (log P 4.0) for 8 h. It was also stable in polar organic solvents such as ethanol (log P -0.23) and acetone (log P -0.31), maintaining more than 80% of its original activity after 8 h incubation at 30°C. The synthesis of ethyl esters was tested with fatty acids of different chain lengths in n-heptane at 30°C. The best conversions (90% in 3 h) were obtained for medium and long chain saturated fatty acids (C8, C14 and C16), with the maximum specific activity, 29 U per gram of immobilized preparation, being obtained with palmitic acid (C16). Im-LipG9 was sn-1,3-specific. In the transesterification of the alcohol (R,S)-1-phenylethanol with vinyl acetate and the hydrolysis of the analogous ester, (R, S)-1-phenylethyl acetate, Im-LipG9 showed excellent enantioselectivity for the R-isomer of both substrates (E> 200), giving an enantiomeric excess (ee) of higher than 95% for the products at 49% conversion. The results obtained in this work provide the basis for the development of applications of LipG9 in biocatalysis.

Full text of DOI:10.1371/journal.pone.0114945

RESEARCH ARTICLE
Immobilization and Characterization of a
New Regioselective and Enantioselective
Lipase Obtained from a Metagenomic Library
1
2
3
Robson Carlos Alnoch , Viviane Paula Martini , Arnaldo Glogauer , Allen Carolina dos
4
4
1
1
Santos Costa , Leandro Piovan , Marcelo Muller-Santos , Emanuel Maltempi de Souza ,
Fábio de Oliveira Pedrosa , David Alexander Mitchell , Nadia Krieger *
1
1
4
1
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046 Centro
Politécnico, Curitiba 81531–980, Paraná, Brazil, 2 Instituto Federal do Paraná–Campus Irati, Irati 84500–
0
8
00, Paraná, Brazil, 3 Agência Tecpar de Inovação, Instituto de Tecnologia do Paraná—Tecpar, Curitiba
1350–010, Paraná, Brazil, 4 Departamento de Química, Universidade Federal do Paraná, Cx. P. 19081
Centro Politécnico, Curitiba 81531–980, Paraná, Brazil
*
nkrieger@ufpr.br
Abstract
OPEN ACCESS
In previous work, a new lipase and its cognate foldase were identified and isolated from a
metagenomic library constructed from soil samples contaminated with fat. This new lipase,
called LipG9, is a true lipase that shows specific activities that are comparable to those of
well-known industrially-used lipases with high activity against long-chain triglycerides. In
the present work, LipG9 was co-expressed and co-immobilized with its foldase, on an inert
hydrophobic support (Accurel MP1000). We studied the performance of this immobilized
LipG9 (Im-LipG9) in organic media, in order to evaluate its potential for use in biocatalysis.
Im-LipG9 showed good stability, maintaining a residual activity of more than 70% at 50°C
after incubation in n-heptane (log P 4.0) for 8 h. It was also stable in polar organic solvents
such as ethanol (log P -0.23) and acetone (log P -0.31), maintaining more than 80% of its
original activity after 8 h incubation at 30°C. The synthesis of ethyl esters was tested with
fatty acids of different chain lengths in n-heptane at 30 °C. The best conversions (90% in 3
h) were obtained for medium and long chain saturated fatty acids (C8, C14 and C16), with
the maximum specific activity, 29 U per gram of immobilized preparation, being obtained
with palmitic acid (C16). Im-LipG9 was sn-1,3-specific. In the transesterification of the alco-
hol (R,S)-1-phenylethanol with vinyl acetate and the hydrolysis of the analogous ester, (R,
S)-1-phenylethyl acetate, Im-LipG9 showed excellent enantioselectivity for the R-isomer of
both substrates (E> 200), giving an enantiomeric excess (ee) of higher than 95% for the
products at 49% conversion. The results obtained in this work provide the basis for the de-
velopment of applications of LipG9 in biocatalysis.
Citation: Alnoch RC, Martini VP, Glogauer A, Costa
ACdS, Piovan L, Muller-Santos M, et al. (2015)
Immobilization and Characterization of a New
Regioselective and Enantioselective Lipase Obtained
from a Metagenomic Library. PLoS ONE 10(2):
e0114945. doi:10.1371/journal.pone.0114945
Academic Editor: Willem J.H. van Berkel,
Wageningen University, NETHERLANDS
Received: September 10, 2014
Accepted: November 16, 2014
Published: February 23, 2015
Copyright: © 2015 Alnoch et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The work was supported by the following:
Brazilian National Council for Scientific and
Technological Development (CNPq) http://www.cnpq.
br/; funding and research scholarships: NK EMS FOP
DAM VPM AG MMS; Pronex/Fundação Araucária
(
Funding)(www.fappr.pr.gov.br/) EMS FOP; and
Coordination for the Development of Higher
Education Personnel (CAPES): research
scholaships: RCA ACS. The funders had no role in
PLOS ONE | DOI:10.1371/journal.pone.0114945 February 23, 2015
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Immobilization of a Lipase Obtained from a Metagenomic Library
study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Introduction
Lipases (glycerol ester hydrolases EC 3.1.1.3) are carboxylesterases that normally catalyze the
hydrolysis of long and short-chain acyl esters in aqueous media [1]. However, when used in
water-restricted media, they can catalyze esterification and transesterification reactions, with
high regio- and enantioselectivity [1,2]. Due to these properties, lipases have been used in or-
ganic media for the synthesis of biodiesel [3,4] and of chiral compounds that have applications
in the chemical, pharmaceutical and cosmetic industries [5,6].
Competing Interests: The authors have declared
that no competing interests exist.
A major barrier to the industrialization of lipase-catalyzed processes is their low productivity
when compared to chemical routes of synthesis. Additional disadvantages are the high costs of
the enzymes and their low stability in water-restricted media. In fact, denaturation of the lipase is
frequently a serious problem in the case of biodiesel synthesis, where one of the substrates is a
short-chain alcohol, such as methanol or ethanol [7,8]. It is therefore necessary to obtain new li-
pases that have a high and stable activity in organic solvents, including short-chain alcohols.
Over the years, many researchers have tried to obtain new lipases either by modifying cur-
rent lipases using techniques of molecular biology or by screening samples obtained from natu-
ral or polluted environments [9–11]. One promising approach is the screening for new
enzymes in metagenomic libraries [12]. These libraries contain genetic material that has been
extracted from environmental samples and cloned into hosts such as Escherichia coli. This
technique is particularly promising for the isolation of new lipases: since it does not involve iso-
lation and cultivation of whole microorganisms, it enables lipases to be obtained from so-called
“
non-culturable” microorganisms [12,13].
Over the last decade, several lipases and esterases have been obtained from metagenomic li-
braries derived from soil or sediments collected from mangroves [14], Atlantic Forest [15], a com-
post heap [16,17], an oil-contaminated area [18] and a hot spring [19]. However, relatively little
work has been done to characterize the potential of these enzymes for catalyzing reactions in or-
ganic media [20] and the work that has been undertaken has involved the use of free enzymes.
This is in spite of the fact that, in commercial processes, lipases are typically used in immobilized
form, since this makes it easy to recover the enzyme from the reaction medium and reuse it.
In previous work, we identified a new lipase, LipG9, in a metagenomic library constructed
from soil samples contaminated with animal fat [18]. LipG9 was overexpressed and prelimi-
nary studies were performed with the purified enzyme [21]. It was shown that LipG9 was active
only when co-expressed with its foldase (LifG9) and that after the purification process, LipG9
and LifG9 were co-eluted and remained complexed. The specific activities of LipG9 in the hy-
drolysis of long-chain triglycerides were comparable to those of several well-known commer-
cial lipases, such as the lipases from Thermomyces lanuginosus, Rhizopus oryzae and
Rhizomucor miehei [21]. These features suggested that LipG9 may have potential for use in bio-
catalysis. However, more studies are needed to confirm this potential.
In the present work, we immobilize LipG9, co-expressed with its foldase, on the polypropylene
support Accurel MP1000 and study its behavior in water-restricted media. This immobilized
complex (hereafter referred to as Im-LipG9) has remarkable stability in polar and non-polar or-
ganic solvents, catalyzes ester synthesis with various saturated and unsaturated fatty acids and is
regioselective (being sn-1,3-specific) and enantioselective. These properties of LipG9 justify future
efforts towards the development of its applications in biocatalysis.
Materials and Methods
Reagents and Materials
E. coli BL21 (DE3), pET28a (+) (Novagen, WI, USA), pT7–7 (USB, OH, USA) and IPTG (Iso-
propyl β-D thiogalactopyranoside) (Invitrogen Life Technologies, CA, USA) were used in the
PLOS ONE | DOI:10.1371/journal.pone.0114945 February 23, 2015
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Immobilization of a Lipase Obtained from a Metagenomic Library
recombinant protein expression system. The HiTrap Chelating Sepharose HP column
(
HiTrap) was purchased from GE Healthcare (Uppsala, Sweden). Activity assays involved the
substrates triolein (C18, purities of 65% and 99%), tricaprylin (C8, 99%), caprylic acid (C8:0,
9%), lauric acid (C12:0, 99%), myristic acid (C14:0, 99%), palmitic acid (C16:0, 99%), stearic
9
acid (C18:0, 99%), oleic acid (C18:1, 90%) and linoleic acid (C18:2, 99%) (Sigma-Aldrich,
St. Louis, MO, USA). For the immobilization, polypropylene beads (Accurel MP 1000, Mem-
brane GmbH, BY, Germany) were used, with the following characteristics: surface area of
2
-1
-3
5
5.985 m g , particle density of 1.993 g cm and particle diameter <1500 mm. All other re-
agents and substrates used were of analytical grade.
Overexpression and purification of LipG9
Both the overexpression and the purification of the complex Lip-LifG9 were performed accord-
ing to Martini et al. [21]. E. coli BL21(DE3) cells carrying the plasmids pET28a-lipG9 and pT7–
7
-lifG9 were grown in 200 mL of LB medium at 37°C until an OD600 of 0.5 and induced by the
addition of IPTG to a final concentration of 0.5 mM. The induced culture was incubated for a
further 16 h at 20°C before harvesting of the cells by centrifugation (3000×g for 5 min) at 4°C.
The cell pellet was resuspended in 35 mL of lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM
NaCl, 10 mM imidazole, 10 mM β-mercaptoethanol, 10% (v/v) glycerol, 0.25% (w/v) Nonidet
P-40) and disrupted by ultrasonication in an ice bath (10 cycles of 60-s pulses, 90 W, with 30-s
intervals), using a SONICATOR XL 2020 (Heat systems-Ultrasonics Inc., NY, USA). The
crude extract was then centrifuged at 30000×g for 30 min at 4°C to pellet the cell debris. The su-
pernatant containing the His-tagged LipG9 complexed with its foldase LifG9 was loaded onto a
2+
HiTrap column, previously loaded with Ni and equilibrated with lysis buffer, using an ÄKTA
basic (Uppsala, Sweden) chromatography system. The column was washed with 5 volumes of
the lysis buffer and then with 5 volumes of elution buffer (50 mM Tris-HCl pH 8.0, 500 mM
NaCl, 10 mM imidazole, 10% (v/v) glycerol). The complex Lip-LifG9 was eluted with an in-
creasing gradient of imidazole (up to 1 M) in elution buffer. The elution of the complex was
monitored at 280 nm and the protein fractions were analyzed by SDS-PAGE, pooled, dialyzed
(
50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM CaCl , 20% (v/v) glycerol) and stored at 4°C
2
until use. Hereafter, Lip-LifG9 is referred to simply as LipG9.
Gel electrophoresis and protein determination
Electrophoresis of protein samples (SDS-PAGE) was done with 12% (w/v) gel [22]. The gel was
stained with Coomassie Brilliant Blue R-250 and destained with methanol/acetic-acid/water
(
5/1/4 v/v/v). Protein content was determined by the method of Smith [23], using the Pierce
BCA Protein Assay Kit (Pierce Biotechnology, IL, USA) with bovine serum albumin as
the standard.
Lipase activity assay
The hydrolysis activity in aqueous solution was determined by titration using an automatic ti-
trator pHStat (Metrohm 718 Stat Titrino) [24]. The substrate emulsions consisted of 67 mM
triacylglycerol, 3% (w/v) gum arabic, 2 mM CaCl , 2.5 mM Tris-HCl pH 8.0 and 150 mM
2
NaCl, dispersed in distilled water [25]. The enzyme was added to 20 mL of the emulsion under
magnetic stirring (300 rpm) at 30°C and the reaction was followed for 5 min. One unit of hy-
drolytic activity in aqueous medium corresponds to the release of 1 μmol of fatty acid per min-
ute, under the assay conditions.
The hydrolytic activity in organic medium was determined by adding 5 mL of a reaction
medium containing 4.9 mL of n-heptane, 70 mM triolein and 2% (v/v) distilled water to a
PLOS ONE | DOI:10.1371/journal.pone.0114945 February 23, 2015
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Immobilization of a Lipase Obtained from a Metagenomic Library
2
5-mL Erlenmeyer flask. The reaction was started by the addition of either free (Fr-LipG9) or
immobilized LipG9 (Im-LipG9). The mixture was then incubated in an orbital shaker at 200
rpm and 40°C. At fixed intervals, 100-μL samples of the mixture were collected and analyzed
for residual free fatty acids by the Lowry-Tinsley method [26]. One unit of hydrolytic activity
in organic medium corresponds to the release of 1 μmol of fatty acid per minute, under the
assay conditions.
Immobilization of LipG9
LipG9 was immobilized by physical adsorption on Accurel MP 1000 according to Al-Duri and
Yong [27]. The support was wetted with ethanol solution (50% (v/v) in water) for 30 min, then
washed with distilled water and filtered. To study the effect of the protein loading (i.e. mg of
protein offered for immobilization per g of support), 0.1 g of the support and 10 mL of enzyme
solution with the stated protein concentration were added to a 25-mL Erlenmeyer flask and
incubated in an orbital shaker at 150 rpm and 4°C. The time course of immobilization was
evaluated by determining the residual activity and protein concentration in aliquots of the su-
pernatant removed over time (0–48 h). Finally, the immobilized enzyme was removed from
the mixture by filtration with qualitative filter paper (Whatman n° 1), dried in a vacuum desic-
cator for 16 h and stored at 4°C.
The immobilization efficiency (IE, %) was calculated as:
A ꢀ A
i
f
IE ¼
ꢁ 100%
ð1Þ
A
i
where A is the hydrolytic activity (U) of the enzyme solution before immobilization and A is
i
f
the hydrolytic activity (U) remaining in the supernatant at the end of the immobilization pro-
cedure [28].
The retention of activity (R, %) was calculated as:
AO
R ¼ AT ꢁ 100%
ð2Þ
where A is the observed hydrolytic activity in organic medium of the immobilized preparation
O
-
1
-1
(
U g of support) and A is the theoretical activity of the immobilized preparation (U g of
T
support), calculated based on the amount of activity removed from the supernatant during the
immobilization procedure [28].
In order to confirm the immobilization of LipG9 in Accurel MP 1000, proteins were de-
sorbed from the support by incubating Im-LipG9 in a solution of SDS 10% (w/v) at 180 rpm
for 2 h, at 40°C.
Thermal stability of LipG9
The thermal stabilities of Fr-LipG9 and Im-LipG9 were assessed by incubation in a water bath
for 8 h at temperatures from 30 to 60°C. Fr-LipG9 (0.045 mg) was incubated in 300 μL buffer
(
50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM CaCl ) and its residual activity was evaluated
2
in aqueous medium by the titrimetric method (pHStat). Im-LipG9 (20 mg) was incubated in
mL of n-heptane (to prevent desorption of the enzyme from the support) and its residual ac-
1
tivity was evaluated by hydrolysis of triolein in organic medium. The residual activities were
calculated in relation to controls that were treated identically, but without incubation.
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Immobilization of a Lipase Obtained from a Metagenomic Library
Stability of immobilized LipG9 in organic solvents
The effect of organic solvents on the stability of Im-LipG9 was determined using the following
solvents (with their log P values): n-heptane (4), hexane (3.5), cyclohexane (3.2), toluene (2.5),
tert-butanol (1.45), n-butanol (0.8), ethyl acetate (0.68), tetrahydrofuran (0.46), n-propanol
(
0.25), ethanol (-0.23), acetone (-0.31), acetonitrile (-0.33), methanol (-0.76) and dimethylsulf-
oxide (-1.3). Im-LipG9 (20 mg) was incubated in each solvent for 8 h at 30°C, without agita-
tion. The solvent was then removed by filtration through filter paper (Whatman n° 1) and
Im-LipG9 was then dried in a vacuum desiccator (16 h, at 4°C) and its residual activity was
evaluated by the hydrolysis of triolein in organic medium. The residual activities were calculat-
ed relative to a control performed without pre-incubation in the solvents.
Application of immobilized LipG9 in ester synthesis
The performance of Im-LipG9 in the synthesis of ethyl-oleate in organic medium was compared
to that of Fr-LipG9. Given that lyophilized Fr-LipG9 did not have activity when added directly to
the organic medium, a purified aqueous extract containing the enzyme was added instead. The
reaction medium (5 mL) contained 4.9 mL of n-heptane, 70 mM oleic acid and 210 mM ethanol,
in a 25-mL Erlenmeyer flask. The reaction was started by the addition of either Fr-LipG9 or
Im-LipG9, followed by incubation in an orbital shaker at 200 rpm and 40°C. At fixed intervals,
100-μL samples of the mixture were collected and analyzed for residual free fatty acids by the
Lowry-Tinsley method [26]. The reaction yield and the activity of esterification of Im-LipG9 were
calculated based on the consumption of free fatty acids. One unit of lipase esterification activity (U)
corresponds to the consumption of 1 μmol of fatty acid per minute, under the assay conditions.
The effects of the chain length and the degree of unsaturation of the fatty acid were evaluat-
ed. The esterification reactions were performed as described above, except that various fatty
acids were used: caprylic (C8:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), stearic
(
C18:0) and linoleic (C18:2).
Evaluation of the regioselectivity of LipG9
In order to determine the regioselectivity of Fr-LipG9 and Im-LipG9, an assay was carried out,
in organic medium, using triolein (99% purity, Sigma-Aldrich, MO, USA) as the substrate, and
the reaction products were examined by thin-layer chromatography (TLC), according to Sugi-
hara et al. [29]. The reaction medium was composed of 20 mM triolein, 0.1 mL distilled water
(
2%, v/v) and 4.9 mL of n-heptane. The reaction was started by addition of 30 mg of Im-LipG9
or 50 μL (containing 0.07 mg protein) of the solution of Fr-LipG9. The mixture was kept in an
orbital shaker for 30 min at 30°C and 180 rpm. Aliquots (2 μL) were applied on a silica gel 60
plate (Merck, Darmstadt, HE, Germany) and developed with a 95:4:1 (v/v) mixture of chloro-
form, acetone and acetic acid. The spots were visualized using a saturated iodine chamber and
their R values were compared with those of the standards 1 (2)-monoolein, oleic acid, 1,2
f
(
2,3)-diolein, 1,3-diolein and triolein (Sigma-Aldrich, MO, USA).
Evaluation of the enantioselectivity of Im-LipG9
The enantioselectivity of Im-LipG9 was evaluated in two reactions: (1) the transesterification
of (R,S)-1-phenyl-1-ethanol [(R,S)-1] with vinyl acetate as the acyl moiety donor (Fig. 1A); and
(
(
[
2) the hydrolysis of (R, S) 1-phenylethyl acetate [(R,S)-1a], with H O as the nucleophile
Fig. 1B). Both reactions were carried out with n-hexane as the solvent. The secondary alcohol
(R,S)-1] and its respective ester [(R,S)-1a], both racemic, were chosen because they have a sig-
2
nificant difference between the sizes of the groups at the stereocenter (i.e. phenyl and methyl
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Immobilization of a Lipase Obtained from a Metagenomic Library
Fig 1. Model reactions for the evaluation of the enantioselectivity of immobilized LipG9 (Im-LipG9) in
(
A) transesterification and (B) hydrolysis reactions.
doi:10.1371/journal.pone.0114945.g001
groups). According to Kazlauskas’s rule, substrates with substituents of significantly different
sizes are better resolved by lipases [30]. Compounds (R,S)-1 and (R,S)-1a were synthesized ac-
cording to methodologies that are already described in the literature and characterized by spec-
troscopic techniques (see S1 Text).
For the transesterification reaction, 100 mg of Im-LipG9 (corresponding to 22 U of triolein-
hydrolyzing activity in organic medium) was added to a solution of (R,S)-1-phenyl-1-ethanol
(
12 mg, 0.1 mmol) and vinyl acetate (35 mg, 4 mmol) in n-hexane (2 mL). The reaction was
carried out at 35°C and 150 rpm. Samples were removed and analyzed by gas chromatography
in a GC-2010 chromatographer (Shimadzu Co., Kyoto, Japan), equipped with a hydrogen
flame ionization detector, and a chiral column CP Chirasil-DEX CB (25 m × 0.25 mm diame-
ter, 0.25 μm film thickness). A sample of 1 μL was injected with a split ratio of 1:50, using N as
2
the carrier gas. The injector and detector were set at 220°C. The oven program was as follows:
-
1
initial value of 110°C, with heating at 1°C min to 120°C. Retention times were: (R)-1 3.63
min, (S)-1 3.93 min, (S)-1a 4.43 min and (R)-1a 4.68 min.
For the hydrolysis reaction, 100 mg of Im-LipG9 (corresponding to 22 U of triolein-hydro-
lyzing activity in organic medium) was added to a reaction medium containing (R,S) 1-pheny-
lethyl acetate (16 mg, 0.1 mmol) and water (2.2 mmol, 4 mg, corresponding to 2%) in n-
hexane (2 mL). The reaction was carried out at 35°C and 150 rpm. Samples were removed and
analyzed by GC as described above for the transesterification reaction.
The absolute configurations of the enantiomers, of both the alcohol and the ester, were attribut-
ed indirectly by comparing the retention times of the substrates and products involved in the reac-
tions catalyzed by Im-LipG9 with the retention times of the enantiomers produced in the same
reactions catalyzed by the lipase B of Candida antarctica (CALB, Novozymes, Denmark). The
(R)-enantiopreference of CALB for these compounds is already described in the literature [31,32].
The enantiomeric excesses of substrate (ee , %) and product (ee , %) were determined based
s
p
on the differences in the relative percentages of each enantiomer:
ꢀ
ꢁ
R ꢀ S
R þ S
ee ¼
ꢁ 100%
ð3Þ
where R is the concentration of the appropriate R-enantiomer and S is the concentration of the
appropriate S-enantiomer.
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Immobilization of a Lipase Obtained from a Metagenomic Library
Fig 2. SDS-PAGE analyses of the fractions eluted from the nickel column. The lanes were loaded as
follows: lane M, protein molecular weight standards; lane 1, supernatant of the bacterial cell lysate; lane 2,
crude cell extract (precipitated); lane 3, column wash; lane 4–10, eluted fractions during purification on nickel
2
+
column (Ni ). Proteins were stained with Coomassie brilliant blue R-250.
doi:10.1371/journal.pone.0114945.g002
The conversion (c, %) and enantioselectivity (E) were calculated from the values of ee and
s
ee according to Chen et al. [33]:
p
ees
c ¼
ꢁ 100%
p
ð4Þ
ð5Þ
ðee þ ee Þ
s
ln½ð1 ꢀ cÞð1 ꢀ ee Þꢂ
s
E ¼
ln½ð1 ꢀ cÞð1 þ ee Þꢂ
s
Results
Purification and immobilization of LipG9
Protein bands corresponding to the molecular masses of LipG9 (32 kDa) and its foldase LifG9
24 kDa) were observed after SDS-PAGE of the eluate from the HiTrap column (see lanes 4 to
0 in Fig. 2). The eluted fractions were pooled and dialyzed in buffer containing 20% (v/v) glyc-
(
1
erol with no imidazole. Table 1 summarizes the results of the purification step, showing an ac-
tivity yield of 41% and a purification factor of 12. The specific hydrolytic activity in aqueous
Table 1. Summary of the purification of the lipase LipG9 by affinity chromatography on nickel column.
a
b
-1
Step
Volume
mL)
Total Protein
(mg)
Total Activity
(U)
Specific activity (Umg
protein)
Purification
Factor
Activity Yield
(%)
(
Crude extract
35
9
197.0
6.7
43550
17650
221
1
100
41
Purified
2634
12
extract
a
Protein was determined by the method of Smith et al. [23].
b
A unit of activity (U) represents the production 1 μmol of fatty acid per min using tricaprylin as substrate in the titrimetric method using a pHStat, at 30°C
for 5 min.
doi:10.1371/journal.pone.0114945.t001
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Immobilization of a Lipase Obtained from a Metagenomic Library
Table 2. Principal parameters for immobilization of the lipase LipG9 on Accurel MP 1000.
-
1
a
-1
-1
b
Protein loading (mg g
support)
Efficiency of Immobilization
(%), IE
Lipase activity (U g
support)
Specific activity (U mg
protein)
Retention of activity
(%), R
c
5
68
54
36
219 ± 28
66 ± 9
43 ± 1
46 ± 5
248
164
178
10
5
230 ± 6
1
249 ± 22
a
Calculated as the difference between the initial and final activities in the supernatant after immobilization using the titrimetric method (pHStat) and
tricaprylin as substrate.
b
-1
Retention of activity (%), measured as the ratio between the real activity (U g support) of immobilized LipG9 in organic media, and theoretical activity of
-1
the immobilized LipG9 (U g support), calculated from the difference in the activities measured in the supernatant at the beginning and end of the
immobilization process.
c
SEM: Standard error of the mean.
doi:10.1371/journal.pone.0114945.t002
medium of the purified fraction of LipG9 (complexed with its foldase), used in further experi-
-
1
-1
ments, was 2634 U mg with tricaprylin and 911 U mg with triolein.
-1
Protein loadings of 5, 10 and 15 mg g were evaluated for the immobilization of LipG9 by physi-
cal adsorption on Accurel MP 1000. The best immobilization efficiency (IE 68%) and the highest
specific hydrolytic activity in organic medium (66 U mg of protein) were obtained for the loading
of 5 mg g . For higher protein loadings (10 and 15 mg g ), lower IE values were obtained (54% and
6%, respectively), whereas the specific activity of the immobilized preparation remained almost
constant (43 and 46 U mg , respectively), indicating saturation of the support. For assays done with
loadings varying from 1 to 4 mg g , Im-LipG9 showed low activity, in spite of the fact that all activi-
ty disappeared from the supernatant after the immobilization procedure (IE 100%, data not shown).
The activity retention values (R), calculated in comparison to the specific activity of the
aqueous extract of Fr-LipG9 in organic medium (26 U mg ), were above 100% for all immobi-
lized preparations (Table 2). This shows that LipG9 was activated upon immobilization on
Accurel MP 1000. The highest value of R, 249%, was obtained with the protein loading of
-1
-1
-1
3
-1
-1
-
1
-
1
5
mg g . This loading was used for the characterization experiments.
The time course of LipG9 immobilization was followed in order to determine the best im-
mobilization time (Fig. 3). There was no loss of activity in a control in which the enzyme
Fig 3. Kinetics of adsorption of LipG9 on Accurel MP1000. Key: (▲) Residual activity in the supernatant,
determined with tricaprylin as the substrate, by the titrimetric method using a pHStat, at 30°C for 5 min; (●)
Residual protein in the supernatant, determined by method of Smith et al. [23]. Adsorption was performed
-
1
with an initial protein loading (mg protein per g support) of 5 mg g , at 4°C, 150 rpm.
doi:10.1371/journal.pone.0114945.g003
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Immobilization of a Lipase Obtained from a Metagenomic Library
solution was incubated for 48 h under the same conditions but without the support. The immo-
bilization efficiency was higher (IE 88%) after 48 h as compared to 24 h (IE 68%). However, the
-
1
specific activity and the retention of activity at 48 h were lower (52 U mg , R 152%) than at
-
1
2
4 h (66 U mg , R 249%) (Table 2). This result suggests that, from 24 h on, the surface of the
support might already be saturated with protein and that at longer immobilization times the
protein is absorbed in thicker layers that restrict activity of some of the enzyme due to mass
transfer limitations. Based on this result, the time of 24 h was chosen for the immobilization of
LipG9.
After 2 h incubation of the immobilized preparation in an SDS solution, SDS-PAGE of the
supernatant gave bands at 32 kDa and 23 kDa (see lane 4 of Fig. 4). These bands correspond to
LipG9 and LifG9, respectively. This result shows that the lipase was immobilized on the sup-
port with its foldase.
Thermal stability of LipG9
When incubated for 8 h, Im-LipG9 retained 100% of its activity at 30°C, more than 70% at
4
0°C and 50°C and around 50% at 60°C (Fig. 5). Under the same incubation conditions, the re-
sidual activity of Fr-LipG9 was approximately 50% at 50°C, but only 20% at 60°C. The thermal
stability of LipG9 is therefore enhanced upon immobilization, as has been shown for other
Fig 4. SDS-PAGE analyses of the fractions desorbed from the support Accurel MP 1000 after
immobilization of LipG9. The lanes were loaded as follows: lane M, protein molecular weight standards;
lane 1, purified LipG9; lane 2, supernatant at time 0 h, lane 3, supernatant after 48 h of immobilization; lane 4,
fractions desorbed from Accurel MP 1000 after being washed with 10% (w/v) SDS solution. Proteins were
stained with Coomassie brilliant blue R-250.
doi:10.1371/journal.pone.0114945.g004
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Immobilization of a Lipase Obtained from a Metagenomic Library
Fig 5. Thermal stability of free and immobilized LipG9. Key: (ꢃ) Residual activity of free enzyme (Fr-
LipG9); (●) Residual activity of immobilized enzyme (Im-LipG9). Fr-LipG9 was incubated in 1 mL of buffer for
8
h. The residual activity of Fr-LipG9 was determined by the titrimetric method using a pHStat. Im-LipG9 was
incubated in 1 mL of n-heptane for 8 h and its residual activity was measured by the hydrolysis of triolein in
organic media. The error bars represent the standard error of the mean.
doi:10.1371/journal.pone.0114945.g005
lipases [34–37]. This improved stability is due to interactions between the enzyme and the sup-
port [34], which confer a more rigid structure on the enzyme [38,39]. The higher thermal sta-
bility of Im-LipG9 could also be due, in part, to the fact that it was incubated in a hydrophobic
organic solvent in the absence of free water: this can also make the enzyme more rigid and
therefore less prone to denaturation [40].
Stability of Im-LipG9 in organic solvents
We investigated the stability of Im-LipG9 in polar and non-polar solvents, with log P values
ranging from -1.30 to 4.0. Im-LipG9 retained over 80% of its activity after 8 h incubation in n-
heptane, n-hexane, toluene, tetrahydrofuran, acetone and ethanol (Table 3). Low residual activ-
ity (30%) was observed in methanol and DMSO completely denatured the enzyme. Normally,
lipases are more active and stable in non-polar solvents with log P>2.5 [41,42]. Polar solvents,
such as acetone and alcohols, tend to lead to greater denaturation rates, probably because they
remove the solvating water layer from the surface of the enzyme, thereby destabilizing it
[40,41]. However, in this work, there was no clear correlation between Im-LipG9 stability and
log P values, with Im-LipG9 showing significant stability (>80% residual activity) in polar sol-
vents such as ethanol and acetone (Table 3). The lack of correlation becomes clearer when the
residual activities of Im-LipG9 are compared for two polar solvents with similar log P values,
tetrahydrofuran (log P 0.48) and ethyl acetate (log P 0.68): in these solvents the residual activi-
ties were quite different, with values of 82% and 43%, respectively. Similar results have been re-
ported for some other lipases when treated with organic solvents [43–46], so it appears that the
specific functional groups and molecular constitution of the solvents are important [45,47].
Regioselectivity of LipG9
Lipases can be classified into two groups based on their positional specificity for the hydrolysis
of the ester bond in triglycerides: sn-1,3-specific and non-specific [29]. In order to determine
the positional specificity of Im-LipG9, hydrolysis assays were performed in n-heptane using
pure triolein (99%) as the substrate, with 2% (v/v) of water. After 30 min of reaction, the main
products present in the medium were oleic acid and 1,2 (2,3)-diolein (Fig. 6). The same profile
was observed for the products of the reaction catalyzed by Fr-LipG9 (lane 7, Fig. 6), indicating
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Immobilization of a Lipase Obtained from a Metagenomic Library
Table 3. Residual activity of immobilized LipG9 (Im-LipG9) after incubation in different organic
solvents.
a
c
d
Organic solvents
log P
Residual Activity (% ± SEM )
b
Control
***
100
n-Heptane
n-Hexane
4.0
89 ± 2
79 ± 2
76 ± 2
86 ± 1
71 ± 1
76 ± 1
43 ± 2
82 ± 3
63 ± 3
85 ± 1
83 ± 3
72 ± 3
30 ± 1
0
3.50
3.2
Cyclohexane
Toluene
2.50
1.45
0.80
0.68
0.46
0.25
- 0.23
-0.31
-0.33
-0.76
-1.30
tert-Butanol
n-Butanol
Ethyl acetate
Tetrahydrofuran
n-Propanol
Acetone
Ethanol
Acetonitrile
Methanol
Dimethyl sulfoxide
a
Im-LipG9 was incubated in 1 mL of solvent for 8 h at 30°C.
Control without pre-incubation.
b
c
Residual activity was measured by the hydrolysis of triolein in organic media. Conditions: 20 mg of Im-
LipG9, 70 mM triolein in n-heptane, 30°C, 200 rpm.
d
SEM: Standard error of the mean. Assays were carried out in triplicate.
doi:10.1371/journal.pone.0114945.t003
that the enzyme did not change its positional specificity upon immobilization. Since 1,3-diolein
was not detected, LipG9 does not hydrolyze ester bonds at the sn-2 position of triacylglycerols
and therefore can be classified as a sn-1,3-specific lipase [29,48].
Enantioselectivity of LipG9
The enantioselectivity of Im-LipG9 was evaluated during transesterification of (R, S)-1-phenyl-
1
-ethanol and the hydrolysis of its corresponding ester (R, S)-1-phenylethyl acetate, both in n-
hexane. For the resolution of (R,S)-1-phenyl-1-ethanol, Im-LipG9 achieved 49% conversion in
0 h, with high enantiomeric excess of (R)-1-phenylethyl acetate (ee > 95%), and an enantio-
3
p
meric ratio (E) greater than 200 (Table 4). For the resolution of (R, S)-1-phenylethyl acetate,
Im-LipG9 gave 16% conversion in 60 h, with high enantiomeric excess for (R)-1-phenyl-1-eth-
anol (ee > 95%) and an enantiomeric ratio (E) greater than 200 (Table 4). The chro-
p
matographic profiles observed in reactions catalyzed by Im-LipG9 were the same as those
observed in the reactions catalyzed by the lipase CALB (Table 4), confirming the enantioprefer-
ence of Im-LipG9 for the R-isomer.
Reaction conditions: Transesterification reaction: (R,S)-1 (0.10 mmol), Im-LipG9 (100 mg)
or CALB (20 mg), vinyl acetate (0.2 mL), n-hexane (2 mL) and 35°C. Hydrolysis reaction: (R,
S)-1a (0.10 mmol), Im-LipG9 (100 mg) or 20 mg (CALB), water (0.2 mL), n-hexane (2 mL)
and 35°C.
Conversions (c) and enantioselectivity (E) were calculated from the excesses of substrate (%
ee ) and product (% ee ) according to Chen et al. [33].
s
p
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Immobilization of a Lipase Obtained from a Metagenomic Library
Fig 6. Thin layer chromatography profile of the reagents and products of the hydrolysis of triolein catalyzed by LipG9 in organic medium. Lane 1, 1
(2)-monoolein (1(2)-MO); lane 2, oleic acid; lane 3, 1,2(2,3)-diolein (1,2(2,3)-DO); lane 4, 1,3-diolein (1,3-DO); lane 5, triolein (TO); lane 6, the beginning of
the reaction; lane 7, products of triolein hydrolysis catalyzed by Fr-LipG9; lane 8, products of triolein hydrolysis catalyzed by Im-LipG9.
doi:10.1371/journal.pone.0114945.g006
Application of LipG9 in ester synthesis
The ability of Fr-LipG9 and Im-LipG9 to catalyze the synthesis of ethyl-oleate in n-heptane
was evaluated using 22 U of total hydrolytic activity in the reaction medium for both prepara-
-
1
tions. For Im-LipG9, the esterification activity was 16 U per gram of support (U g ) and a
yield of 95% was obtained in 7 h, whereas lyophilized Fr-LipG9 was inactive (Fig. 7). These
Table 4. Kinetic resolution parameters from reactions catalyzed by immobilized LipG9 (Im-LipG9) and Candida antarctica Lipase B (CALB).
Enzyme
Compound
Time (h)
c (%)
ee
s
(%)
ee
p
(%)
E
Im-LipG9
(R,S)-1-phenyl-1-ethanol
30
60
2
49
16
50
50
> 95
18
> 95
> 95
99
> 200
> 200
> 200
> 200
(
R, S) 1-phenylethyl acetate
(R,S)-1-phenyl-1-ethanol
R, S) 1-phenylethyl acetate
doi:10.1371/journal.pone.0114945.t004
CALB
99
(
2
99
99
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Immobilization of a Lipase Obtained from a Metagenomic Library
Fig 7. Kinetics of the synthesis of ethyl-oleate in n-heptane, catalyzed by different preparations of
LipG9. Key: (●) Im-LipG9; (■) lyophilized Fr-LipG9. In all assays, the total activity in the medium was 22 U (as
determined by the hydrolysis of triolein in organic media). Conditions: 5 mL n-heptane, 70 mM oleic acid, 210
mM ethanol. The mixture was incubated at 37°C and 200 rpm. The error bars represent the standard error of
the mean.
doi:10.1371/journal.pone.0114945.g007
results are in agreement with several studies reporting higher yields in the production of esters
using immobilized lipases in relation to free enzymes [49–52].
Further experiments were performed in order to determine the effect of chain length on the
esterification activity of Im-LipG9. The best conversions (> 95% in 3 h) were obtained for satu-
rated fatty acids of medium and long chain lengths (C8, C14 and C16), with the maximum activi-
-
1
ty (29 U g ) being obtained for palmitic acid (C16) (Fig. 8). For long-chain and unsaturated fatty
acids (C18, C18:1 and C18:2), conversions of 90% were obtained after 7 h of reaction. Indeed, the
esterification activity of Im-LipG9 was not directly related to the length of the acyl chain.
Discussion
In the current work, we immobilized LipG9, complexed with its specific foldase (LifG9), on the
hydrophobic support Accurel by physical adsorption and studied its potential in several model re-
actions in water-restricted media. Not only is this the first study of the immobilization of a lipase-
foldase complex obtained from metagenomics, but also there are only few studies involving lipases
complexed with their specific foldases and they are normally devoted to investigation of strategies
for co-expression in vitro and in vivo in order to achieve expression of active enzymes [53–55].
The only previous study of immobilization of a lipase-foldase complex was that of Peng
et al. [56], for the lipase-foldase complex from Pseudomonas aeruginosa CS2 immobilized on
Celite-54. Although the systems are quite different, our immobilized preparation gave better
esterification results than did theirs: Our best conversion, of close to 100% in 3 h for the syn-
thesis of ethyl palmitate in n-heptane (Fig. 8) was obtained much faster than their best result, a
conversion of 98% after 10 h for the synthesis of butyl-acetate in n-heptane.
In the particular case of the use of lipases from metagenomics to catalyze reactions in organ-
ic media, there are only relatively few studies and Im-LipG9 compares quite favorably. The
first metagenome-derived lipase that was shown to have a significant esterification activity in
-
1
solvent-free medium (0.12 U mg for the synthesis of 1-propyl laurate) was LipS, isolated by
Chow et al. [20]. More recently, Brault et al. [57] demonstrated the potential of using whole-
cell biocatalyst with a recombinant metagenomic lipase (LipIAF5–2) for the synthesis of short-
chain flavor esters. In our case, Im-LipG9 catalyzes the synthesis of ethyl esters of fatty acids of
various chain lengths and degrees of unsaturation (C8, C12, C14,C16, C18, C18:1, C18:2) in n-
heptane, in all cases giving yields above 85% after 7 h of reaction.
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Immobilization of a Lipase Obtained from a Metagenomic Library
Fig 8. Effect of fatty acid chain length in the yields of esterification reactions catalyzed by
immobilized LipG9. Key: (●) caprylic acid (C8); (
^
) lauric acid (C12); (ꢃ) myristic acid (C14); (▲) palmitic acid
(
C16); (Δ) stearic acid (C18); (□) oleic acid (C18:1); and (■) linoleic acid (C18:2). In all assays, the total
activity in the medium was 22 U (as determined by the hydrolysis of triolein in organic media). Conditions: 5
mL n-heptane, 70 mM of fatty acids, 210 mM ethanol. The mixture was incubated at 37°C and 200 rpm. The
error bars represent the standard error of the mean.
doi:10.1371/journal.pone.0114945.g008
In the current work, Im-LipG9 showed good stability, with over 80% residual activity after 8
h incubation in various pure organic solvents, among them acetone and ethanol. No other li-
pase obtained through metagenomics exhibits satisfactory stability when incubated for 8 h in
1
00% acetone or ethanol: Two previously isolated metagenomic lipases, LipIAF1–6 [58] and
LipC12 [18], only showed good stability in acetone and ethanol at solvent concentrations up to
0% (v/v). In fact, the good solvent stability of Im-LipG9 may, in part be due to the fact that it
3
was co-immobilized with its foldase, which may help it to maintain its tertiary structure in con-
ditions that might otherwise denature it.
The high triolein-hydrolyzing activity in organic medium of Im-LipG9 in comparison to the
free enzyme (Table 2) corresponds to retention of activity values (R) well above 150% for all pro-
tein loadings. The suggested explanation for this phenomenon is that the lipase is adsorbed in its
open structural conformation, in other words, the lipase simultaneously binds to the support and
undergoes interfacial activation [49]. In our work, this activation was facilitated by the hydropho-
bicity of Accurel and by the fact that LipG9 has a hydrophobic lid sub-domain [21].
Im-LipG9 appears to have good potential for application in media containing organic sol-
vents. Its enantioselectivity is comparable to those of commercial lipases from Candida antarc-
tica B (CALB), Pseudomonas cepacia and P. fluorescens, which show enantiomeric ratios
greater than 200 in the resolution of 1-phenyl-1-ethanol [59]. Of particular interest is the sta-
bility presented by Im-LipG9 in hydrophilic solvents such as ethanol and acetone, since such
solvents are used in many organic synthesis reactions and most lipases, including commercial
formulations such as Novozym 435, have poor stability in these solvents [60].
In conclusion, we have reported the most thorough characterization to date of the potential
for the application of a metagenomic lipase in organic solvents. The combined characteristics
of Im-LipG9, namely its good activity and stability in organic solvents, along with its regios-
electivity and enantioselectivity, make Im-LipG9 an interesting prospect for use in biocatalysis.
Nucleotide sequence accession number
The lipG9 and lifG9 nucleotide sequences reported here are available in the GenBank database
under the accession numbers GenBank: KM023399 and GenBank: KM023400, respectively.
PLOS ONE | DOI:10.1371/journal.pone.0114945 February 23, 2015
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Immobilization of a Lipase Obtained from a Metagenomic Library
Supporting Information
S1 Text. Synthesis and characterization of racemates used to determine the enantioselectiv-
ity of LipG9.
(
DOCX)
Acknowledgments
We thank Roseli Prado and Valter A. de Baura for technical support.
Author Contributions
Conceived and designed the experiments: NK EMS MMS LP AG RCA. Performed the experi-
ments: RCA VPM ACS. Analyzed the data: NK DAM EMS LP FOP AG RCA MMS. Contribut-
ed reagents/materials/analysis tools: NK FOP EMS LP MMS. Wrote the paper: RCA NK DAM
AG EMS MMS LP.
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