Appl Microbiol Biotechnol (2010) 85:597–604
DOI 10.1007/s00253-009-2131-4
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Co-expression of the lipase and foldase of Pseudomonas
aeruginosa to a functional lipase in Escherichia coli
Bhawna Madan & Prashant Mishra
Received: 29 February 2008 / Revised: 6 July 2009 / Accepted: 6 July 2009 / Published online: 21 July 2009
# Springer-Verlag 2009
Abstract The lipA gene, a structural gene encoding for
protein of molecular mass 48 kDa, and lipB gene, encoding
for a lipase-specific chaperone with molecular mass of
35 kDa, of Pseudomonas aeruginosa B2264 were co-
expressed in heterologous host Escherichia coli BL21
(DE3) to obtain in vivo expression of functional lipase.
The recombinant lipase was expressed with histidine tag at
its N terminus and was purified to homogeneity using nickel
affinity chromatography. The amino acid sequence of LipA
and LipB of P. aeruginosa B2264 was 99–100% identical
with the corresponding sequence of LipA and LipB of P.
aeruginosa LST-03 and P. aeruginosa PA01, but it has less
identity with Pseudomonas cepacia (Burkholderia cepacia)
as it showed only 37.6% and 23.3% identity with the B.
cepacia LipA and LipB sequence, respectively. The molec-
ular mass of the recombinant lipase was found to be 48 kDa.
The recombinant lipase exhibited optimal activity at pH8.0
and 37°C, though it was active between pH5.0 and pH9.0
and up to 45°C. Km and Vmax values for recombinant P.
aeruginosa lipase were found to be 151.5±29µM and 217±
22.5µmol min−1 mg−1 protein, respectively.
Keyword Lipase . Co-expression . Foldase . P. aeruginosa .
E. coli
Introduction
Lipases (triacylglycerol ester hydrolases, E.C. 3.1.1.3) cata-
lyse the hydrolysis of long-chain triacylglycerols in aqueous
B. Madan : P. Mishra (*)
Department of Biochemical Engineering and Biotechnology,
Indian Institute of Technology Delhi,
Hauz Khas,
New Delhi 110016, India
e-mail: pmishra@dbeb.iitd.ac.in
media, whereas in non-aqueous media, they catalyse the
esterification and transesterification reactions (Zaks and
Klibanov 1988; Klibanov 2001). Owing to the properties
like wide substrate specificity, enantio- and regioselectivity,
they have applications in organic synthesis, in detergent
formulations and in the food and pharmaceutical industries
(Reetz 2002; Jaeger and Eggert 2002; Chand and Mishra
2003; Madan and Mishra 2009). Whilst lactonising lipase of
Pseudomonas sp. has been used in the synthesis of macrolide
antibiotics (Ihara et al. 1991), Pseudomonas aeruginosa
lipase has exhibited amide hydrolysing activity (Fujii et al.
2005) and high enantioselectivity towards hydrolysis of
optically active trifluoroethylated (3′-indolyl) thiacarboxylic
esters, novel plant growth regulators (Kato et al. 1999) and
chiral model substrate 2-methyldecanoic acid p-nitrophenyl
ester (Liebeton et al. 2000).
In spite of various potential applications of Pseudomonas
lipases, overexpression of functional lipase has been a lim-
iting factor, as lipases from Pseudomonas species require an
assistant protein, a lipase-specific chaperone to fold into an
active conformation (Arpigny and Jaeger 1999; Rosenau et
al. 2004). The foldase for lipase (chaperone) is encoded in
the same operon along with the structural gene of lipase
(lipA). Lipase-specific chaperone is either required during or
after translation. In Pseudomonas sp. Strain KWI-56
activator (chaperone) is required at the time of lipase
synthesis (Yang et al. 2000). The functional expression of
Pseudomonas lipase has been achieved by in vitro refolding
of lipase using various approaches (Jorgensen et al. 1991;
Oshima-Hirayama et al. 1993; Hobson et al. 1993; Ihara et
al. 1995; Yang et al. 2000; Traub et al. 2001). So far, in vivo
expression of functional P. aeruginosa lipase in heterologous
host Escherichia coli has not been achieved, and hence,
engineering of lipases for improved properties has been
limited to homologous host. In homologous host, the
enantioselectivity of P. aeruginosa lipase towards the
598 Appl Microbiol Biotechnol (2010) 85:597–604

hydrolysis of chiral model substrate 2-methyldecanoic acid
p-nitrophenyl ester (Liebeton et al. 2000) and amide hydro-
lysing activity (Fujii et al. 2005) has been improved by
directed evolution. Utilising the knowledge of the crystal
structure of P. aeruginosa lipase (Nardini et al. 2000), the
substrate specificity of this lipase has been improved by
combinatorial active site saturation test, the process known
as CASTing (Reetz et al. 2006).
In the present study, co-expression of both lipA and lipB
from P. aeruginosa B2264 in heterologous host E. coli has
been carried out that resulted in in vivo expression of the
active lipase. The recombinant lipase was purified using
single-step immobilised metal ion affinity chromatography,
and the characteristics of purified enzyme were studied.
Materials and methods
Bacterial strains, plasmid and growth conditions
agar plates supplemented with antibiotics. E. coli BL21
(DE3) cultures were then inoculated in 5 ml of LB medium
supplemented with antibiotics. They were maintained in
15% (v/v) glycerol stocks, frozen in liquid nitrogen and
stored at −80°C. The strains and plasmids used in this study
are listed in Table 1.
DNA manipulations
Chromosomal DNA of P. aeruginosa B2264 was obtained
from cells grown in complex medium at 200 rpm at 30°C
for 24 h as described earlier (Ausubel et al. 1987) with
some modification, as the precipitation of DNA was carried
out using ethanol instead of isopropanol. Plasmid DNA was
isolated by alkaline lysis method (Sambrook et al. 1989).
The oligonucleotide primers used in this study were
obtained from Microsynth, Switzerland. DNA sequencing
was performed at the Department of Biochemistry, Univer-
sity of Delhi South Campus, India.

P. aeruginosa B2264 was a kind gift from National Region
Research Laboratory, Peoria, IL, USA. The E. coli BL21
(DE3) strain (Novagen, USA) was used for expression of
(His)6-tagged lipA. Plasmid pET32Xa/LIC (Novagen) and
pET29a (Novagen) were used for the construction of the
expression system. The antibiotic concentrations used for
the optimum growth of cells were ampicillin (100µg/ml)
and kanamycin (50µg/ml; Hi-Media, India). P. aeruginosa
B2264 strain was activated from cultures on complex
medium plates containing 5 g/l polypeptone, 3 g/l yeast
extract, 5 g/l D-glucose, 0.5 g/l NaCl, 0.5 g/l MgSO4⋅7H2O
and 2% agar. P. aeruginosa B2264 was maintained in 5 ml
of the above complex medium without agar. E. coli BL21
(DE3) was activated from cultures on Luria–Bertani (LB)
Construction of expression plasmids
The lipA (936 bp) and lipB (867 bp) fragments were
amplified by polymerase chain reaction (PCR) separately
using P. aeruginosa B2264 genomic DNA. The primers
designed for amplification of lipA and lipB genes were
based on the reported P. aeruginosa PA01 lipA gene
(NCBI: NC_002516) and lipB gene (NCBI: NP_251553).
For amplification of lipA, sense LPlipA and antisense
RPlipA were used, and for lipB amplification, sense LPlipB
and antisense RPlipB were used (Table 1). Each reaction
mixture (50µl) contained 300 ng of P. aeruginosa B2264
genomic DNA, 50 pmol of sense and antisense primers,
10X reaction buffer, 1.5 mM MgCl2, 5% DMSO, 200µM

Table 1 Strains, plasmids and primers used for this study

Strain or plasmid or oligonucleotide Relevant characteristic or sequence (5′-3′) Source

Strains
P. aeruginosa Wild-type strain NRRL B2264 NRRL
E. coli BL21 (DE3) F− ompT hsdSB(rB− mB−) gal dcm (DE3) Novagen
Plasmids
pET 32Xa/LIC T7 promoter-driven high-efficiency protein expression Novagen
and sequencing vector; encodes Apr
pET29a T7 promoter-driven high-efficiency protein expression Novagen
and sequencing vector; encodes Kmr
pET32lipA pET32Xa/LIC derivative carrying lipA gene of P. aeruginosa B2264 This study
pET29alipB pET29a carrying lipB gene of P. aeruginosa B2264 This study
Primers
LPlipA CAT GCC ATG GCC ATG AAG AAG AAG TCT CTG CTC CCC Amplification of lipA, NcoI site
RPlipA CCG CTC GAG CTA CAG GCT GGC GTT CTT CAG Amplification of lipA, XhoI site
LPlipB CAT GCC ATG GCC GTG AAG AAA ATC CTC CTG Amplification of lipB, NcoI site
RPlipB CCG CTC GAG TCA GCG CTG CTC GGC CTG G Amplification of lipB, XhoI site
Appl Microbiol Biotechnol (2010) 85:597–604
of each dNTP (MBI Fermentas) and 3 U of Taq polymerase
(MBI Fermentas). The PCR for lipA consisted of an initial
denaturation for 5 min at 94°C. Thirty cycles were run,
each cycle consisting of 1 min of denaturation at 94°C,
1 min of annealing at 59°C and 1 min of extension at 72°C
followed by a final extension of 10 min at 72°C. For
amplification of lipB, step down PCR programme was run,
which consisted of four steps with annealing temperature of
63°C declining to 57°C at 2°C interval. The resultant PCR
product and expression vectors pET32Xa/LIC and pET29a
were then digested with NcoI and XhoI. The digested PCR
product was purified by Qiagen PCR purification kit using
the manufacturer’s instructions. Digested plasmids were
purified by phenol chloroform extraction followed by
ethanol precipitation. lipA and lipB were then ligated with
pET32Xa/LIC and pET29a, respectively, in the ligation
mixture containing 100 ng of vector and 200 ng of insert
using 1 U of T4 DNA ligase (MBI Fermentas) in a final
volume of 10µl which was incubated overnight at 16°C.
Competent E. coli BL21 (DE3) cells prepared by CaCl2
method were transformed using the heat shock and cold
treatment (Sambrook et al. 1989). The transformed colonies
were screened by colony PCR followed by double digestion
of the constructed plasmid (pETlipA and pETlipB).
Functional expression of recombinant lipase in E. coli
The E. coli BL21 (DE3) strains harbouring pETlipA or
pETlipB or both pETlipA and pETlipB were grown at 37°C
and 220 rpm. When optical density at 610 reached 0.6 to 1.0,
they were induced with 1 mM of isopropyl-β-D-thiogalacto-
pyranoside (IPTG). After 3 h, both uninduced and induced
cells (0.5 ml) were harvested by centrifugation (8,000×g,
5 min at 4°C) and expression was checked by running both
uninduced and induced cells on 12% sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE;
Laemmli 1970). The pETlipA contains ampicillin resistance
marker and pETlipB contains kanamycin resistance marker;
therefore, LB medium containing 100µg/ml ampicillin and
50µg/ml kanamycin was used for further experiments. For
the optimization of inducer concentration, E. coli BL21
(DE3) strain containing both pETlipA and pETlipB was
grown in 50 ml of LB at 30°C and 220 rpm. The inoculum
(1%, v/v) was transferred in 100 ml LB medium. The cells
were induced at an optical density of 0.8 at 610 nm with
different concentrations of IPTG ranging from 100 to 1000
µM and were grown for 10 h after induction. For the
optimization of optical density at the time of induction, cells
were grown at 220 rpm at 30°C. After 14 h, 1% (v/v)
inoculum was transferred to 100 ml LB medium, and cells
were induced with 400µM IPTG at different optical densities
ranging from 0.6 to 1.6 at 610 nm and were grown for 10 h
after induction. For the optimization of temperature, cells
599
were grown at different temperatures, i.e. 25°C, 30°C and
37°C, in 50 ml of LB for 14 h. Inoculum (1%, v/v) was
transferred to 100 ml LB medium. The cells were induced
with 400µM IPTG at an optical density of 1.4 at 610 nm and
were grown for 10 h after induction.
The cells were harvested by centrifuging at 8,000×g for
5 min at 4°C after 10 h of growth after induction. The pellet
was washed with 50 mM Tris–HCl buffer (pH 8.0)
containing 5 mM CaCl2 and was resuspended in 5 ml of
the same buffer. The cells were then disrupted by sonication
using 15 bursts of 18 kHz each for 30 s alternated by 1 min
of intermittent cooling on ice. Cell debris was removed by
centrifugation at 13,520×g for 30 min at 4°C.
Sequence analysis
The amino acid sequences deduced from the lipA and lipB
gene of P. aeruginosa was analysed by BLAST using the
NCBI web site (http://www.ncbi.nlm.nih.gov/BLAST/),
and CLUSTAL W analysis was performed using the DNA
Star programme.
Purification of soluble recombinant protein
The cell hydrolysate obtained from 1 l culture after
sonication was subjected to protamine sulphate treatment
for the removal of nucleic acids and was then passed
through a 0.45-µm syringe-end filtre prior to loading on
His-Tag column. The lysate was loaded onto HIS-SelectTM
HF nickel affinity gel column (Sigma Chemicals Co., St.
Louis, MO, USA) equilibrated with 50 mM Tris–HCl
buffer (pH8.0) containing 5 mM CaCl2, 500 mM NaCl and
10 mM imidazole. The column was then washed exten-
sively with 50 mM Tris–HCl buffer (pH8.0) containing
5 mM CaCl2, 500 mM NaCl and 30 mM imidazole to
remove the unspecific and unbound proteins. The lipase
was eluted using a linear gradient of 10–400 mM imidazole
in Tris–HCl buffer (pH8.0) containing 5% (v/v) glycerol at
a flow rate of 0.2 ml/min, and fractions of 1 ml were
collected. The protein concentrations were determined by
Bradford assay (Bradford 1976). The active fractions were
pooled and separated on denaturing polyacrylamide gel
electrophoresis (SDS-PAGE; Laemmli 1970).
Lipase assay
The lipase activity was determined using para-nitrophenyl
palmitate (pNPP) as substrate (Winkler and Stuckmann
1979) with slight modifications. Solution A was prepared
by dissolving pNPP (0.015 g) in 5 ml of 2-propanol at
37°C, and solution B was prepared by dissolving 0.045 g of
gum acacia and 0.9 ml of triton X-100 in 0.01 M potassium
phosphate buffer, pH8.0. Solution A and solution B were
600
then mixed to form substrate solution. Substrate and
enzyme were pre-incubated at 37°C for 5 min before the
reaction. Appropriate dilution of enzyme (0.1 ml) was
added to substrate (2.4 ml) and incubated at 37°C for
10 min. The reaction was quenched by adding 2 ml of
acetone. Reaction mixture was centrifuged at 3,400×g for
5 min at 4°C. Absorbance of p-nitrophenol released was
recorded at 410 nm. One unit of enzyme activity corre-
sponds to 1µmol of p-nitrophenol released per minute. The
molar extinction coefficient of pNP at neutral pH was
11,023 M−1 cm−1. Appropriate controls were taken for
correcting the effect of pH, imidazole and temperature on
pNP extinction and pNPP autohydrolysis at extreme pH
and temperatures. Protein concentrations were determined
by Bradford assay (Bradford 1976).
Characterization of lipase
The molecular weight of LipA was determined by SDS-
PAGE (Laemmli 1970) using protein molecular weight
marker (Bangalore Genei, India). To determine the opti-
mum pH of recombinant lipase, the activity of recombinant
lipase was estimated between pH3.6 and 11.0. The pH
stability of the enzyme was studied by assaying residual
activity of the enzyme after incubation at 37°C for 1 h in
the following buffers: 100 mM sodium acetate (pH3.6, 4.6
and 5.6); 100 mM potassium phosphate (pH6.0 and 7.0);
100 mM Tris–HCl (pH8.0 and 9.0); 100 mM sodium
carbonate buffer (pH10.0 and 11.0).
The optimum temperature of lipase was determined at
various temperatures (0°C to 80°C) using pNPP as substrate
at pH9.0. The heat stability of the enzyme was studied by
assaying the residual activities after 1-h incubation at various
temperatures (0°C to 80°C) at pH9.0.
The Michaelis–Menten constant (Km) and maximum
velocity for the reaction (Vmax) with pNPP (range from
0.07 to 20 mM) were calculated by the Lineweaver–Burk
plot.
Nucleotide sequence accession number
The lipA and lipB DNA sequences of P. aeruginosa B2264
have been deposited in the GenBank database under
accession numbers EU376533 and EU376534, respectively.
Results
Cloning and overexpression of lipA and lipB
The co-expression of pETlipA and pETlipB was performed
in E. coli to obtain functional lipase. The lipA gene
Appl Microbiol Biotechnol (2010) 85:597–604
(936 bp) was amplified by PCR using P. aeruginosa
B2264 chromosomal DNA as template. pETlipA was
constructed by inserting the lipA gene under the control of
strong T7 promoter in pET32Xa/LIC in frame and in
translational fusion with S-Tag, His-Tag and Trx-Tag. The
pETlipA construct was confirmed by digestion of pETlipA
with NcoI and XhoI, which showed the release of a 1-kb
product. E. coli BL21 (DE3) containing pETlipA after
induction with IPTG produced a polypeptide of 48 kDa
(Fig. 1a). Similarly, lipB gene (867 bp) was amplified using
P. aeruginosa B2264 chromosomal DNA as template. The
lipB gene was inserted in pET29a under the control of T7
promoter and in translational fusion with S-tag and His-tag,
and the plasmid was designated pETlipB. E. coli BL21
(DE3) containing pETlipB upon induction with IPTG
produced a polypeptide of 35 kDa (Fig. 1a).
Sequence analysis
The amino acid sequences deduced from the lipA gene of
P. aeruginosa B2264 showed 99.7% identity with P.
aeruginosa LST-03 lipase, 99.4% identity with P. aeruginosa
PA01 and P. aeruginosa IGB83 lipase, 98.4% identity
with lactonising lipase of Pseudomonas sp. 109, 76.2%
identity with Pseudomonas mendocina lipase, 77.2% identity
with Pseudomonas stutzeri A1501 lipase, 57.6% identity
with Vibrio cholerae AM 19226 lipase, 59.5% identity with
Oceanobacter sp. RED65 lipase and 37.6% identity with
Burkholderia cepacia lipase. Although recombinant lipase
from P. aeruginosa B2264 was similar to P. aeruginosa
PA01, it differed in V156I, I204V.
The amino acid sequences deduced from the lipB gene of
P. aeruginosa showed 100% identity with P. aeruginosa
LST-03 LipB, 99.3% identity with P. aeruginosa PA01
LipB, 66.1% identity with P. mendocina LipB, 54.2%
identity with P. stutzeri A1501 LipB, 33.3% identity with
Oceanobacter sp. RED65 LipB, 30.2% identity with
Acinetobacter sp. ADP1 LipB and 23.3% identity with B.
cepacia G63 LipB.
Co-expression of lipA and lipB and optimization
of expression of functional lipase
Both the lipA (936 bp) and lipB (867 bp) were inserted
under the influence of strong T7 promoter in pET32Xa/
LIC and pET29a vectors, respectively. Upon induction
with IPTG, E. coli BL21 (DE3) containing pETlipA and
pETlipB expressed both polypeptides LipA (48 kDa) and
LipB (35 kDa) as shown in SDS-PAGE gel (Fig. 1b). Co-
expression of lip A and lipB resulted in the expression of
functional lipase which was confirmed by the activity
test.
Appl Microbiol Biotechnol (2010) 85:597–604
Fig. 1 SDS-PAGE gel stained with Coomassie brilliant blue R-250
showing expression of recombinant proteins. a Lane 1 Protein
molecular weight marker. Lane 2 Whole cell protein from E. coli
BL21 (DE3) harbouring pETlipA uninduced. Lane 3 Whole cell
protein from E. coli BL21 (DE3) harbouring pETlipA 3 h after
induction. Lane 4 Whole cell protein from E. coli BL21 (DE3)
harbouring pETlipB uninduced. Lane 5 Whole cell protein from E.
coli BL21 (DE3) harbouring pETlipB 3 h after induction. b Lane 1
Protein molecular weight marker. Lanes 2, 4 Whole cell protein from
E. coli BL21 (DE3) harbouring pETlipA and pETlipB uninduced.
Lanes 3, 5 Whole cell protein from E. coli BL21 (DE3) harbouring
The conditions were optimised to improve the expression
of functional lipase. The parameters chosen were addition of
5 mM CaCl2, IPTG concentration ranging from 50 to 1,000
µM, temperatures (25°C, 30°C and 37°C) and optical density
at 610 nm at the time of induction ranging from 0.6 to 1.6.
Optimised conditions were 400µM IPTG, optical density of
1.4 at 610 nm and 25°C. Under optimised conditions, the
lipase activity was improved by approximately sevenfold, i.e.
from 1.8±0.3 to 13±1.3 U/ml. The expression of lipase was
also quantified in soup and pellet based on the band intensity
(total pixel) of selected bands and analysing in MATLAB
(using image processing toolbox). The intensity of Lip A in
601
pETlipA and pETlipB 3 h after induction. c 12% SDS-PAGE showing
the level of recombinant P. aeruginosa lipase in the supernatant and
pellet fraction. The induced cells were pelleted and broken down by
sonication in 50 mM Tris–HCl buffer, pH8.0. Lane 1 Marker. Lane 2
Soup obtained from 0.75 ml of culture. Lane 3 Pellet obtained from
0.75 ml of culture. d SDS-PAGE gel showing purified recombinant P.
aeruginosa B2264 lipase. The recombinant His-Tag lipase was
purified by immobilised metal ion chromatography. Lane 1 A medium
molecular weight marker kit (Bangalore Genei, India). Lane 2 Purified
recombinant P. aeruginosa lipase. The position of recombinant P.
aeruginosa lipase is indicated by an arrow
soup (962 pixels) and pellet (777 pixels) was in a ratio 1.24:1
(Fig. 1c).
Purification of active recombinant lipase
The purification of the recombinant lipase was carried out by a
single-step purification method using immobilised metal ion
affinity chromatography (His-tag chromatography). The re-
combinant lipase was purified from cell hydrolysate (specific
activity 14 U/mg protein) to 16-fold with specific activity of
225 U/mg protein with yield of 35%. The purified lipase was
separated as a single band in SDS-PAGE (Fig. 1d).
602
Characterization of recombinant lipase
The relative molecular weight of recombinant lipase was
found to be 48 kDa using SDS-PAGE (Fig. 1d). The
purified recombinant lipase was active over a pH range of
7.0 to 9.0, and optimum activity was obtained at pH9.0
(Fig. 2a). The stability of the recombinant lipase was
checked by using pNPP as substrate between pH3.0 and
11.0. The recombinant lipase was stable between pH5.0
and 9.0 for 1 h at 37°C (Fig. 2a). The effect of temperature
on purified recombinant lipase was studied by activity
assay at various temperatures ranging from 0°C to 80°C,
and the recombinant lipase was found to be active between
30°C and 60°C and has maximum activity at 37°C
(Fig. 2b). The recombinant lipase was found to be stable
up to 45°C at pH9.0 for 1 h (Fig. 2b).
Km and Vmax values for recombinant P. aeruginosa lipase
using pNPP as substrate were found to be 151.5±29µM
and 217±22.5µmol min−1 mg−1 protein. The kinetic data
on P. aeruginosa differed due to the different substrates
employed by different researchers. Km and Vmax for P.
aeruginosa Pse A lipase were 70.4 mM and 2.24 mmol
min−1 mg−1 towards pNPP as substrate (Gaur et al. 2008).
Discussion
In general, the lipase from Pseudomonas sp. when overex-
pressed in E. coli get accumulated in the form of inclusion
bodies, as they require lipase-specific chaperone for folding
A120 120
100 100
% Remaining Activity
% Relative Activity
% Relative Activity
80 80
60 60
40 40
20 20
0 0
3 5 7 9 11
pH
Fig. 2 a Effect of pH on the activity and stability of purified
recombinant P. aeruginosa B2264 lipase: The activity of purified P.
aeruginosa B2264 lipase was measured at 37°C using pNPP as the
substrate in the buffers ranging from pH3.6 to 11. The relative activity
(triangle) was calculated taking lipase activity at pH9.0 as 100%
activity under similar conditions. The pH stability of the enzyme was
determined after incubating recombinant P. aeruginosa lipase at 37°C
for 1 h in the buffers ranging from pH3.6 to 11. The remaining
activities (square) of recombinant P. aeruginosa B2264 lipase are
shown taking lipase activity at pH9.0 as 100% activity under similar
Appl Microbiol Biotechnol (2010) 85:597–604
into active conformation. This problem has been addressed
earlier by in vitro folding of lipases (Oshima-Hirayama et
al. 1993; Ahn et al. 1997; Amada et al. 2000; Kojima et al.
2003) and expression of lipase in homologous hosts
(Liebeton et al. 2000; Omori et al. 2005). In vitro folding
has limitations of engineering protein by directed evolu-
tion and the expression in homologous host has problems
in the purification of enzyme as Pseudomonas is a path-
ogenic organism. In addition, for large scale production E.
coli is known to be better host (Quyen et al. 1999). Thus,
to address these issues, in the present study, lipase was
functionally expressed in heterologous host E. coli. E. coli
BL21 (DE3) containing pETlipA after induction with
IPTG produced a polypeptide of 48 kDa (Fig. 1a), which
is in agreement with the calculated molecular mass of
predicted amino acid sequence (47.59 kDa). The resulting
recombinant fusion protein LipA is likely to consist of the
481 amino acids with an N-terminal fusion of 169 amino
acids (~19 kDa) corresponding to the S-tag epitope, His-
tag epitope and Trx-tag epitope and a unique Factor Xa
cleavage site, and its total calculated molecular mass is
expected to be 47.59 kDa. Thus, the molecular weight of
Lip A is 29 kDa and the fusion partner is ~19 kDa, which
is in agreement with the previous reports. E. coli BL21
(DE3) containing pETlipB upon induction with IPTG
produced a polypeptide of 35 kDa (Fig. 1a), which is in
agreement with the calculated molecular mass of predicted
amino acid sequence (34.98 kDa). The resulting recombi-
nant fusion protein LipB is likely to consist of the 318
amino acids with an N-terminal fusion of 29 amino acids
B 120 120
100 100
% Remaining Activity
80 80
60 60
40 40
20 20
0 0
0 20 40 60 80
Temperature (oC)
conditions. b The optimum temperature and heat stability of purified
recombinant P. aeruginosa B2264 lipase: The lipase activity was
measured at various temperatures using pNPP as substrate at pH9.0.
The relative activities (triangle) are shown taking 100% activity at pH
9.0 and 37°C. The heat stability of the enzyme was determined by
incubating recombinant P. aeruginosa B2264 lipase for 1 h at various
temperatures at pH9.0. The remaining activities (square) were
measured by using pNPP as substrate taking 100% activity at pH9.0
and 37°C
Appl Microbiol Biotechnol (2010) 85:597–604
corresponding to the S-tag epitope and a unique thrombin
cleavage site.
E. coli BL21 (DE3) cells containing pETlipA have not
shown any lipase activity. This may be attributed to the
formation of inclusion bodies via non-specific association
of hydrophobic patches on the surface of folding inter-
mediates (Ventura 2005). Therefore, various conditions
were optimised for the expression of lipase. E. coli BL21
(DE3) containing pETlipA was induced with IPTG concen-
trations ranging from 50 to 1,000μM at an O.D. of 0.8 and
37°C to get controlled expression. But the protein remained
as aggregates and no lipase activity was obtained. The use
of low growth temperature generally results in higher yields
and improved biological activity of the soluble protein even
for folding-reluctant species (Strandberg and Enfors 1991;
Ferrer et al. 2004; Vera et al. 2007). Therefore, the growth
temperature of lipase production in E. coli BL21 (DE3)
cells harbouring pETlipA was reduced from 37°C to 25°C,
but no lipase activity was obtained. These results are
consistent with the earlier reports of Pseudomonas lipases
which were overexpressed in E. coli but accumulated in
insoluble form (Oshima-Hirayama et al. 1993; Ahn et al.
1997; Amada et al. 2000; Kojima et al. 2003). Further-
more, it has been shown that Pseudomonas lipases require
lipase specific chaperone to fold into active conformation
(Jorgensen et al. 1991; Oshima-Hirayama et al. 1993;
Hobson et al. 1993; Ihara et al. 1995; Yang et al. 2000;
Traub et al. 2001). In view of this lipB, which encodes for
lipase-specific chaperone in P. aeruginosa was coex-
pressed along with lipA. E. coli BL21 (DE3) containing
both pETlipA and pETlipB showed lipase activity, though
the activity was low. Next, conditions for co-expression
were optimised for controlled expression by varying IPTG
concentrations, induction at lower temperatures, induction
at different O.D. and addition of Ca2+ (5 mM). Ca2+ was
added, as in an earlier report, calcium-dependent reacti-
vation of P. aeruginosa TE3285 lipase has been shown by
its specific modulator protein LipB (Shibata et al. 1998).
P. aeruginosa B2264 lipase was active over a pH and
temperature range.
Other lipases of P. aeruginosa that have been actively
overexpressed in E. coli include lip3 (34.8 kDa) and lip8
(42 kDa; Ogino et al. 2004a, b). These lipases do not
require any chaperone for folding (Ogino et al. 2004a, b).
Ogino et al. (2007) have earlier demonstrated cloning and
expression of lipase from P. aeruginosa LST-03. They have
mainly focused on in vitro activation of overexpressed and
solubilised lipase (of which signal peptide was deleted) by
lipase-specific foldase. In their co-expression experiment,
lip3 was expressed under the control of T7 promoter,
whereas lif9 was expressed under the control of a weak
promoter. Therefore, the amount of expression of P.
aeruginosa LST-03 Lip3 was higher than Lif9 expression.
603
Thus, the expressed amount of Lip9 in insoluble fraction
was more than in the soluble fraction.
For the optimum folding, both LipA and LipB are
required to be expressed in equal amounts, as lipase and
lipase-specific foldase form 1:1 complex (Hobson et al.
1993; Rosenau et al. 2004). Therefore, in our study, LipA
and LipB were co-expressed in equal amounts under the
control of strong T7 promoter, resulting in the active
expression of P. aeruginosa LipA more in the soluble
fraction than in the insoluble fraction. Thus, in our work,
the focus was in vivo folding of lipase during co-
expression. P. aeruginosa B2264 LipA showed 99.6 %
identity with P. aeruginosa LST-03 Lip3 with one amino
acid variation, i.e. I204V. P. aeruginosa B2264 LipB also
showed 99.6% identity with P. aeruginosa LST-03 d52Lif9
with one amino acid variation, i.e. T249A. Interestingly, the
thermostability of P. aeruginosa B2264 lipase was found to
be higher than Lip3 and Lip8, as both the lipases were
inactivated when incubated at 45°C for 10 min as compared
to P. aeruginosa B2264 lipase, which was completely
stable up to 40°C and retained its 25% stability up to 50°C.
In our study, under optimised condition, the productivity
of native enzyme in P. aeruginosa was 323 U L−1 h−1. The
productivity of recombinant P. aeruginosa in E. coli
carrying both lipA and lipB genes was 812 U L−1 h−1.
The lipase obtained from P. aeruginosa B2264 was more
thermostable and thus may be employed for their applica-
tions in detergents. In addition, this strategy of co-
expression of both lipA and lipB in E. coli may be
employed for the directed evolution of lipases from
Pseudomonas sp. requiring foldases for its activity.
Acknowledgements Bhawna Madan gratefully acknowledges senior
research fellowship award from the Council of Scientific and
Industrial Research, New Delhi, India. This work was partially
supported by a grant from Ministry of Human Resource Development,
Government of India to one of the authors (PM).
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