No Job Name


Cloning and heterologous expression of two cold-active lipases
from the Antarctic bacterium Psychrobacter sp. Gpor_189 421..429
Lin Xuezheng,1 Cui Shuoshuo,1 Xu Guoying,2 Wang Shuai,1 Du Ning1 & Shen Jihong1

1 Key Laboratory of Marine Bioactive Substances, First Institute of Oceanography, SOA, 6 Xianxialing Road, Qingdao 266061, China

2 Ocean College, Shandong University at Weihai, 180 Wenhuaxi Road, Weihai Shandong 264209, China

Abstract

Antarctic bacteria producing extracellular lipolytic enzymes with activity at low
temperature were isolated, and the most promising strain, named G, was
identified as a Psychrobacter species based on 16S rDNA sequence alignment.
The genomic DNA of this bacterium was used to construct its plasmid genomic
library into pUC118 plasmid vectors, and to screen the cold-active lipolytic
enzyme genes. Two genes encoding for cold-active lipolytic enzymes, Lip-1452
(with an open reading frame of 1452 bp in length) and Lip-948 (with an open
reading frame of 948 bp in length), were screened. The primary structure of the
two lipases deduced from the nucleotide sequence showed a consensus pen-
tapeptide containing the active serine (Lip-1452, GDSAG, and Lip-948, GNSMG)
and a conserved His-Gly dipeptide in the N-terminal part of the enzyme.
Protein sequence alignment and conserved regions analysis indicated that the
two lipases probably belonged to family IV and family V of the bacterial
lipolytic enzymes, respectively. The upstream and downstream sequences of
the two lipolytic lipases were also obtained. The two lipase genes were cloned
into the expression vector pCold III and integrated into Escherichia coli
BL21 (DE3). The functional expression of both lipase genes by E. coli BL21
(DE3) cells was observed as the formation of clear haloes around colonies on
a 1% (vol/vol) tributyrin plate upon induction with isopropyl-b-D-
thiogalactopyranoside at 5°C. A lipase activity assay showed that the specific
activity of the pCold III+Lip-948 expression system was up to 3.7 U ml-1,
whereas that of pCold III+Lip-1452 was very low.

Keywords
Antarctic; cold-active lipase; gene cloning;

gene expression; Psychrobacter.

Correspondence
Lin Xuezheng, Key Laboratory of Marine

Bioactive Substances, First Institute of

Oceanography, SOA, 6 Xianxialing Road,

Qingdao 266061, China. E-mail:

linxz@fio.org.cn

doi:10.1111/j.1751-8369.2010.00189.x

Enzymes from psychrotrophic and psychrophilic micro-
organisms have received increasing attention because of
their relevance for both basic and applied research.
Research efforts have been stimulated by the recognition
that cold-adapted enzymes might offer novel opportuni-
ties for biotechnological exploitation, based on their high
catalytic activity at low temperatures, low thermostability
and unusual specificities. Cold-active enzymes with huge
biotechnological potentials include protease, lipase,
amylase and cellulase in the detergent industry,
b-galactosidase in the dairy industry, dehydrogenase as a
biosensor in environmental protection, oxidase in biore-
mediation, and many enzymes, e.g., methylase and
aminotransferase, in biotransformation (Alquati et al.
2002; Cieśliński et al. 2005; Parra et al. 2008).

Bacteria produce different classes of lipolytic enzymes,
including carboxylesterases (EC 3.1.1.1) that hydrolyse

water-soluble esters and lipases (EC 3.1.1.3) that hydrol-
yse long-chain triacylglycerol substances, catalysing both
the hydrolysis and the synthesis of acylglycerides and
other fatty acid esters (Rosenau et al. 2000; Lee et al.
2004).

Lipolytic enzymes are important biocatalysts for
various industrial applications (e.g., ester synthesis,
optical resolution, transesterification and washing)
because, among other characteristics, they lack require-
ments for cofactors, they are remarkably stable in organic
solvents, they have broad substrate specificity, and they
have region- and stereo-selectivity. In the past decade,
increasing attention has been drawn to potential
applications of cold-adapted lipolytic enzymes from
microorganisms populating permanently cold environ-
ments (Pfeffer et al. 2007; Długołecka et al. 2008). These
enzymes are characterized by higher kcat and physiological

Polar Research 29 2010 421–429 © 2010 the authors, journal compilation © 2010 Blackwell Publishing Ltd 421

mailto:linxz@fio.org.cn


efficiency (kcat/km), and by a lower and rather constant km
at temperatures close to 0°C (Yang et al. 2008). Several
recent papers reviewed cold-active microbial lipases
(Joseph et al. 2007; Joseph et al. 2008). A dozen genes
encoding cold-active lipolytic enzymes have been cloned
from different species and environmental metagenomes
(Choo et al. 1998; Quyen et al. 1999; Alquati et al. 2002;
Ryu et al. 2006; Zhang et al. 2007; Długołecka et al. 2008;
Parra et al. 2008; Yang et al. 2008; Jeon et al. 2009).

From water samples collected off King George Island,
Antarctica, we isolated dozens of cold-adapted microor-
ganisms that can degrade lipids at low temperature (5°C).
One of the promising lipolytic strains, named “G”, was
identified as a Psychrobacter species by molecular identifi-
cation based on 16S rDNA. In this study, we report the
cloning and sequencing of two lipase genes from the
strain Psychrobacter sp. G., and their heterologous expres-
sion in Escherichia coli BL21 (DE3).

Material and methods

Samples and isolation of lipolytic
enzyme-producing strains

Surface (0–20 cm) water samples were collected at
62°12′39″S, 58°54′41″W, on 9 January 2008 in the waters
off south-western King George Island, one of the South
Shetland Islands, using a water sampler (model WB-PM,
Beijing Purity Instrument Co., Beijing, China). The tem-
perature and salinity of the samples were 1.9–2.5°C and
33.1–33.8, respectively. The screening medium comprised
5 g of peptone, 1 g of yeast extract, 10 ml of tributyrin
and 1000 ml of seawater. The strains producing lipolytic
enzymes were detected by the formation of clear haloes
around the colony at 5°C. The lipolytic enzyme activity
assay was performed with a substrate of olive oil at 35°C,
as described by Yang et al. (2004).

Strains and vectors

Of the lypolytic strains detetcted, one (G) was selected for
further analysis on account of its higher extracellular
lipase activity at low temperature, and its low enzymatic
optimum temperature. The strain was identified as Psy-
chrobacter sp. by molecular identification based on 16S
rDNA alignment, as described by Yang et al. (2004).

Escherichia coli DH5a and BL21 (DE3) were used as host
strains for DNA manipulation and gene expression,
respectively. Plasmid vectors pUC118 HincII/BAP (code
D3322; Takara Biotechnology Co., Otsu, Japan) and
pCold III (code 3363; Takara Biotechnology) were used as
vectors for gene cloning and heterologous expression,
respectively.

Cloning of the lipolytic enzyme genes

The chromosomal DNA from Psychrobacter sp. G was iso-
lated using a Genomic DNA Prep Kit (model number
DP302-02; Tiangen Biotech, Beijing, China), following
the instructions in the manufacturer’s manual. The chro-
mosomal DNA was partially digested with Sau3AI and
2–6-kb fragments were purified from a 0.8% agarose gel
using an Agarose Gel DNA Purification Kit v2.0 (model
number DP209-02; Tiangen Biotech, Beijing, China).
These DNA fragments were ligated into pUC118 HincII/
BAP with a DNA Ligation Kit v2.0 (model number
D6022; Takara Biotechnology Co.).

The resultant plasmids were introduced into E. coli
DH5a, providing a genomic library containing
2.1 ¥ 105 cfu/ml recombinant E. coli clones. The colonies
producing cold-active lipolytic enzymes were detected by
the formation of clear haloes around the colonies on
lysogeny broth (LB) agar plates supplemented with
ampicillin (50 mg ml-1), 0.1 mM isopropyl b-D-
thiogalactopyranoside (IPTG) and 1% (vol/vol) tributyrin
at low temperature (5°C) (Yang et al. 2008). The recom-
binant plasmids from the lipolytic enzyme positive clone
were extracted and sequenced.

Construction of the expression plasmid of Lip-1452
and Lip-948

An E. coli expression vector, pCold III, was used to express
the lipolytic enzymes in E. coli. Based on the upstream
and downstream sequences of the known lipolytic
enzyme gene sequences, the specific primers for poly-
merase chain reaction (PCR) amplification were designed
and synthesized as follows: primer F1 5′-CTGTAGGA
GCTCATGTCTAACTCAACAGTACTAT-3′, primer R1
5′-CATAAATCTAGAATCAATC TTACAGGTACCAA-3′
for Lip-1452, and primer F2 5′-CTGTAGGAGCTCATGC
TATTAAAACGCC T-3′ and primer R2 5′-CATAAA
TCTAGATTACGCCTTAAAACATCA-3′ for Lip-948 were
used to generate a PCR product carrying a SacI restriction
site at its 5′ end and an XbaI restriction site at its 3′ end.

Lipase gene expression in E. coli

Escherichia coli BL21 (DE3) transformed with the expres-
sion plasmid pCold III+Lip-1452 or pCold III+Lip-948 was
grown overnight with shaking (200 revolutions per min)
in 10 ml LB. Expression was performed in LB medium
(supplemented with 0.2% glucose) with the cold-shock
vectors, in accordance with the manufacturer’s instruc-
tions. When OD600 was 0.5, the culture solution was
refrigerated at 15°C and left to stand at the same tem-
perature for 30 min. After adding IPTG to a final

Cloning and expression of cold-active lipases L. Xuezheng et al.

Polar Research 29 2010 421–429 © 2010 the authors, journal compilation © 2010 Blackwell Publishing Ltd422



concentration of 0.5 mmol L-1, cells were cultured at
15°C for 24 h. Cells were harvested and ruptured by
ultrasonic lysis in 25 mmol L-1 sodium phosphate buffer.
The cell debris (insoluble fraction) was removed by cen-
trifugation at 12 000 ¥ g for 20 min, and the supernatant
was used for lipolytic enzyme assay and sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
analysis.

Lipolytic enzyme assay and SDS-PAGE analysis

The lipolytic enzyme assay was performed with tributyrin
as substrate at 35°C, as described by Pfeffer et al. (2007).
One unit (U) of enzymatic activity was defined as the
quantity of enzyme that liberates 1 mmol of fatty acids per
minute.

SDS-PAGE was run essentially as described by
Sambrook & Russel (2001). The samples were dissolved
in Laemmli’s sample buffer and were heated at 95°C for
5 min and cooled before being applied to a gel. The SDS
gel was composed of a 5% (weight/vol) stacking gel and
a 15% (weight/vol) resolving gel. The samples were sub-
jected to electrophoresis first at 80 V on the stacking gel
and then 120 V on the resolving gel. After electrophore-
sis, the gel was fixed and stained with 0.1% (weight/vol)
Coomassie brilliant blue R-250.

Results

Isolation and identification of psychrotrophic
strains with cold-active lipolytic activity

From nearly 100 strains, 26 strains producing lipolytic
enzymes were detected by the formation of clear haloes
around the colonies. Strain G was selected as the most
promising producer of lipolytic enzymes because it had
the highest lipolytic activity (12.8 U ml-1 culture) among
the 26 strains above when they were cultured at 5°C for
72 h in Zobell 2216E medium (YSI China Ltd, Beijing,
China), supplemented with 1% Tween 80 (ICI America,
Bridgewater, NJ, USA). The optimal temperature of the
lipolytic enzyme was about 35°C. The strain was identi-
fied as a Psychrobacter species based on 16S rDNA
alignment. The almost-complete 16S rDNA sequence of
strain G (FJ386502), which consisted of 1406 bp, was
analysed using the nucleotide–nucleotide basic algorithm
alignment search tool (BLASTN) program for comparing
DNA sequences. It was found to be more than 99% iden-
tical to hundreds of different species Psychrobacter, such as
Psychrobacter cryohalolentis K5 (AY660685), Psychrobacter
sp. DVS12b (AY864464) and Psychrobacter sp. ice-oil-471
(DQ521392).

Cloning of the lipolytic enzyme genes

Among approximately 20 000 recombinant colonies from
the genomic library of Psychrobacter sp. G, 10 lipolytic
enzyme positive transformants were identified on the
basis of the formation of clear haloes around the colony
on a tributyrin plate at 5°C, which indicated that they
might encode cold-active lipolytic enzymes. From these
10 colonies, two different lipolytic genes that contained
an open reading frame of 1452 bp (Lip-1452, GU247898;
Fig. 1) and 948 bp (Lip-948, GU247897; Fig. 2) were
obtained. They encoded 315 and 483 amino acids, giving
calculated molecular weights of about 53 and 34 kDa,
respectively.

Sequence analysis

The protein–protein basic algorithm alignment search
tool (BLASTP) was used to search for homologies to other
amino acid sequences deposited in the National Center
for Biotechnology Information database (accessible at
http://www.ncbi.nlm.nih.gov). The highest homology
(98.9%) of Lip-1452 was achieved with the a/b-hydrolase
fold-3 from P. cryohalolentis K5 (YP_581719). It was 65.3%
identical to the cold-active esterase from Pseudomomas sp.
St1 (AF260707), and 54.5% identical to the lipase from
Psychrobacter sp. 2–17 (ABR12515). The two conserved
regions, HGGGF and GDSAG, were identified in Lip-1452
(Figs. 1, 3).

Based on the conserved catalytic triad and overall iden-
tity of the amino acid sequences, Lip-1452 was identified
as a member of the hormone-sensitive lipase (HSL)
group, namely family IV of the bacterial lipases (Fig. 3).
Lip-1452 probably had a catalytic triad (Ser-His-Asp), and
contained an oxyanion hole to stabilize the tetrahedral
intermediate (Parra et al. 2008). Based on alignment
using MEGALIGN 5.0 (DNAStar, Madison, WI, USA), the
two conserved regions, HGGGF and GDSAG, could be
identified in the amino acid sequences of Lip-1452. The
primary structure of the pentapeptide GDSAG started at
Gly297, and Ser299 was probably also a member of the
triad. Perhaps Asp414 and His444 were the second and
third members of the triad, respectively. The conserved
Asp was in a consensus sequence LDXL, and the His
constituting the triad was preceded by a Pro and followed
by a Gly. All these characters indicated that Lip-1452 was
a member of the HSL group of bacterial lipases (Arpigny
et al. 1999; Parra et al. 2008).

Dozens of homologues of Lip-948 were obtained
through BLASTP analysis. The highest homology
(97.8%) was the a/b hydrolase fold from P. cryohalolentis
K5 (YP_579291). It was 82.5% identical to the triacylg-
lycerol lipase from Moraxella sp. (AF260707). It was also

Cloning and expression of cold-active lipasesL. Xuezheng et al.

Polar Research 29 2010 421–429 © 2010 the authors, journal compilation © 2010 Blackwell Publishing Ltd 423

http://www.ncbi.nlm.nih.gov


very similar to the triacylglycerol lipase from Psychrobacter
sp. 7195 (80.6%, CAJ76164) and Psychrobacter immobilis
(79.6%, Q92104). The two conserved regions, HGFGG
and GNSMG, were identified in Lip-948 and are shown in
Figs. 2 and 4.

Lip-948 probably belonged to family V, based on
sequence alignment, especially for the two conserved
regions HGFGG and GNSMG. Perhaps Ser142, Asp264
and His291 composed the catalytic triad (Figs. 2, 4). It
probably contained a signal peptide (Met1-Ala27), which
indicated that Lip-948 might be a secreted protein.

Lipase expression in E. coli

The Lip-1452 and Lip-948 sequences were separately
inserted into the expression vector pCold III, and recom-
binant plasmid was constructed. E. coli BL21 (DE3) cells
were then transformed with the recombinant plasmid
and induced with 0.1 mM IPTG at 15°C. The formation of
clear haloes around both Lip-1452 and Lip-948 colonies on
an LB tributyrin plate indicated that there were func-
tional expression of lipolytic enzymes in the expression
system above.

The total proteins from the fermentation culture
medium were analysed using SDS-PAGE (Figs. 5, 6). New
protein bands with molecular masses of approximately 53
and 34 kDa, which were consistent with the molecular
mass deduced from the nucleotide sequence, were pro-
duced by recombinant E. coli harbouring pCold III+Lip-
1452 and pCold III+Lip-948, respectively. Although lipase
activity of the two expression systems was detected on a
tributyrin plate, when the specific activity was measured
with the method used by Pfeffer et al. (2007), the enzyme
activity of recombinant E. coli harbouring pCold III+Lip-
1452 was relatively low, whereas the specific activity of
the expression system of pCold III+Lip-948 was 3.7 U ml-1

culture (Table 1).

Fig. 1 Nucleotide sequence of Lip-1452 from
Psychrobacter sp. G, and its deduced amino

acid sequence below.

Table 1 Summary of enzyme activity of lipase gene expressed in different
systems.

Vector

Halo formation on LB

tributyrin plate

Specific activity

(U ml-1)

pCold III No None detected

pCold III+Lip-1452 Yes None detected
pCold III+Lip-948 Yes 3.7

Cloning and expression of cold-active lipases L. Xuezheng et al.

Polar Research 29 2010 421–429 © 2010 the authors, journal compilation © 2010 Blackwell Publishing Ltd424



Discussion

Our work describes two lipolytic enzyme genes from the
plasmid genomic library of Psychrobacter sp. G, collected
from seawater in Antarctica. Simple overexpression in E.
coli was used to determine the enzymatic activity of sub-
clones generated from an original active pUC118 clone.
The strain was identified as a Psychrobacter species for its
close similarity at the 16S rDNA level to hundreds of
species of the Psychrobacter genus. The specific lipolytic
activity was 12.8 U ml-1, with a substrate of olive oil
emulsion, when it was cultured in Zobell 2216E medium
supplemented with 1% Tween 80.

The genomic library of Psychrobacter sp. G was con-
structed in pUC118 HincII/BAP, and was introduced into
E. coli DH5a. Among 10 colonies forming clear haloes on
a tributyrin agar plate, two lipase genes with different
opening reading frames were identified. One open
reading frame consisted of 1452 (Lip-1452) nucleotides
that encoded 483 amino acids with a molecular mass of
approximately 53 kDa and a pI of 5.4. The other open
reading frame consisted of 948 (Lip-948) nucleotides that
encoded 315 amino acids with molecular weights of
34 kDa and a pI of 8.0.

Arpigny et al. (1999) were the first to classify bacterial
lipolytic enzymes into eight families based on differences
in amino acid sequences and biological properties.

Family I, the largest group, was further divided into
seven subfamilies (I.1–I.7; Angkawidjaja & Kanaya
2006). The primary structure of Lip-1452 and Lip-948
indicated that they were probably members of families
IV and V, respectively, of the bacterial lipolytic enzymes.
The amino acid sequence alignment of Lip-1452 indi-
cated the two conserved regions, HGGGF and GDSAG,
were identified in this sequence. Perhaps its catalytic
triad consisted of Ser238, Asp414 and His444 (Fig. 3).
The primary structure of this protein showed that it was
a member of family IV, namely the HSL group (Arpigny
et al. 1999).

Evidence points to Lip-1452 and Lip-948 belonging
to a large superfamily called the “a/b hydrolase” super-
family. The members of this superfamily share the
“nucleophilic elbow”, which is a characteristic sequence
motif, Gly-Xaa-Ser-Xaa-Gly, for most esterases and
lipases. The Ser residue in this motif constitutes a “cata-
lytic triad” with Asp and His residues that are placed in
the specific order serine-aspartic acid-histidine in the
polypeptide (Suzuki et al. 2003). Lip-1452 was character-
ized by the motif GDSAG and Lip-948 was characterized
by GNSMG. Lip-1452 had the catalytic triad in the specific
order Ser299-Asp414-His444, and Lip-948 had the cata-
lytic triad Ser142-Asp264-His291.

The activity of the recombinant lipase produced by the
expression system was relatively low, consistent with

Fig. 2 Nucleotide sequence of Lip-948 from
Psychrobacter sp. G, and its deduced amino

acid sequence below.

Cloning and expression of cold-active lipasesL. Xuezheng et al.

Polar Research 29 2010 421–429 © 2010 the authors, journal compilation © 2010 Blackwell Publishing Ltd 425



most previous findings (Suzuki et al. 2003; Pfeffer et al.
2007; Długołecka et al. 2008; Parra et al. 2008). Most of
the lipase expressed in E. coli seemed to exist as an
inclusion body. There are reports that lipases are not
fully processed by some species of E. coli, and lipases
aggregate as a result of their own hydrophobicity (Yang
et al. 2008). Both Lip-1452 and Lip-948 were expressed

in E. coli; however, functional heterologous expression
was low, especially for Lip-1452. Because the heterolo-
gous expression of lipase in E. coli cells yielded inclusion
bodies with no catalytic activity, renaturation studies
had to be executed to obtain a catalytically active form
of the enzyme (Suzuki et al. 2003). However, the
expression in an active form of lipase belonging to sub-

Fig. 3 Alignment of amino acid sequences of Lip-1452 with homologous lipases. YP_581719, a/b-hydrolase fold-3 from Psychrobacter cryohalolentis K5;
AF260707, cold-active esterase from Pseudomonas sp. St1; ABR12515, lipase from Pseudomonas sp. 2–17; P24484, lipase from Moraxella sp.

Cloning and expression of cold-active lipases L. Xuezheng et al.

Polar Research 29 2010 421–429 © 2010 the authors, journal compilation © 2010 Blackwell Publishing Ltd426



families I.1 and I.2 depends on a chaperone protein
named lipase-specific foldase, the gene of which is
located downstream of the lipase gene for the efficient
secretion and folding of active lipase (Arpigny et al.
1999; Quyen et al. 1999), whereas the folding of sub-
family I.3 lipases does not require the assistance of any
molecular chaperone (Angkawidjaja & Kanaya 2006).
For example, a lipase belonging to subfamily I.1 or I.2
was heterologously expressed in E. coli, and the chemi-
cal refolding of inactive lipase in the absence of its
chaperone yielded only 25 U mg-1, whereas in a simple
and rapid in vitro refolding procedure with its modified
and truncated chaperone, functionally active lipase was
obtained with a specific activity of up to 4850 U mg-1

and a yield of 31 400 U g-1 of E. coli wet cells (Quyen

et al. 1999). Iizumi et al. (1991) reported that the acti-
vator gene (act), existing downstream of the lipase (lip)
of Pseudomonas fragi, enhanced the heterologous expres-
sion of lip in E. coli. When the lip was expressed in E. coli
using lac promoter on the pUC plasmid vector, the lipase
activity of E. coli carrying both the lip and act was 200
times greater than that carrying only lip.

Acknowledgements

This work was financially supported by China’s National
High-Tech R & D Program (2007AA091905) and the Basic
Scientific Research Operation Fund of China (GY02-
2007-T11).

Fig. 4 Alignment of amino acid sequences of Lip-948 with homologous lipases. YP_579291, a/b-hydrolase fold from Psychrobacter cryohalolentis K5;
P24640, triacylglycerol lipase from Moraxella sp.; CAJ76164, triacylglycerol lipase from Psychrobacter sp. 7195; Q02104, triacylglycerol lipase from

Psychrobacter immobilis.

Cloning and expression of cold-active lipasesL. Xuezheng et al.

Polar Research 29 2010 421–429 © 2010 the authors, journal compilation © 2010 Blackwell Publishing Ltd 427



References

Alquati C., Gioia L.D., Santarossa G., Alberghina L., Fantucci
P. & Lotti M. 2002. The cold-active lipase of Pseudomonas
fragi: heterologous expression, biochemical characterization
and molecular modeling. European Journal of Biochemistry
269, 3321–3328.

Angkawidjaja C. & Kanaya S. 2006. Family I.3 lipase:
bacterial lipase secreted by the type I secretion system.
Cellular and Molecular Life Sciences 63, 2806–2817.

Arpigny J.L. & Jaeger K.E. 1999. Bacterial lipolytic enzymes:
classification and properties. Biochemistry Journal 343,
177–183.

Choo D.W., Kurihara T., Suzuki T., Soda K. & Esaki N. 1998.
A cold-adapted lipase on an aladkan psychrotroph,
Pseudomonas sp. strain B11-1: gene cloning and enzyme
purification and characterization. Applied Environmental
Microbiology 64, 484–491.

Cieślinśki H., Kur J., Białkowska A., Baran I., Makovski K. &
Turkiewicz M. 2005. Cloning, expression, and purification
of a recombinant cold-adapted b-galactosidase from
Antarctic bacterium Pseudoalteromonas sp. 22b. Protein
Expression and Purification 59, 27–34.

Długołecka A., Cieśliński H., Turkieewicz M., Białkowska
A.M. & Kur J. 2008. Extracellular secretion of
Pseudoalteromonas sp. cold-adapted esterase in Escherichia
coli in the presence of Pseudoalteromonas sp. components of
ABC transport system. Protein Expression and Purification 62,
179–184.

Iizumi T., Nakamura K., Shimada Y., Sugihara A., Tominaga
Y. & Fukase T. 1991. Cloning, nucleotide sequencing, and
expression in Escherichia coli of a lipase and its activator
genes from Pseudomonas sp. KWI-56. Agricultural Biology
and Chemistry 55, 2349–2357.

Jeon J.H., Kim J.T., Kim YJ., Kim H.Y., Lee H.S., Kang S.G.,
Kim S.J. & Lee J.H. 2009. Cloning and characterization
of a new cold-active lipase from a deep-sea sediment
metagenome. Applied Microbiology and Biotechnology 81,
865–874.

Joseph B., Ramteke P.W., Thomas G. & Shrivastava N. 2007.
Standard review cold-active microbial lipases: a versatile
tool for industrial applications. Biotechnology and Molecular
Biology Review 2, 39–48.

Joseph B., Ramteke P.W., Thomas G. 2008. Cold-active
microbial lipases: some hot issues and recent
developments. Biotechnology Advances 26, 457–
470.

Lee S.W., Won K., Lim H.K., Kim J.C., Choi G.A. & Cho K.Y.
2004. Screening for novel lipolytic enzymes from
uncultured soil microorganisms. Applied Microbiology and
Biotechnology 65, 720–726.

Parra L.P., Reyes F., Acevedo J.P., Salazar O., Andrews B.A.
& Asenjo J.A. 2008. Cloning and fusion expression of a
cold-active lipase from marine Antarctic origin. Enzyme and
Microbial Technology 42, 371–377.

Pfeffer J., Rusnak M., Hansen C., Rhlid R.B., Schmid R.D. &
Maurer S.C. 2007. Functional expression of lipase A from
Candida antarctica in Escherichia coli—a prerequisite for

Fig. 5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) analysis of pCold III+Lip-1452 in Escherichia coli BL21 (DE3); M,
marker; lane 1, uninduced cell of pCold III+Lip-948; lane 2, total protein of
pCold III+Lip-1452 after 24-h induction; lane 3, supernatant after super-
sonic lysis; lane 4, pellet; lane 5, total protein of pCold III in BL21 after

induction; lane 6, uninduced cell of pCold III. The arrow points to the

predicted protein.

Fig. 6 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) analysis of pCold III+Lip-948 in Escherichia coli BL21 (DE3); M,
marker; lane 1, pellet; lane 2, supernatant after supersonic lysis; lane 3,

total protein of pCold III+Lip-1452 after 24-h induction; lane 4, uninduced
cell of pCold III+Lip-948; lane 5, total protein of pCold III in BL21 after
induction; lane 6, uninduced cell of pCold III. The arrow points to the

predicted protein.

Cloning and expression of cold-active lipases L. Xuezheng et al.

Polar Research 29 2010 421–429 © 2010 the authors, journal compilation © 2010 Blackwell Publishing Ltd428



high-throughput screening and directed evolution. Journal
of Molecular Catalysis B: Enzymatic 45, 62–67.

Quyen D., Schmidt-Dannert C., Schmid R.D. 1999.
High-level formation of active Pseudomonas cepacia lipase
after heterologous expression of the encoding gene and its
modified chaperone in Escherichia coli and rapid in vitro
refolding. Applied and Environmental Microbiology 65,
787–794.

Rosenau F. & Jaeger K.-E. 2000. Bacterial lipases form
Pseudomonas: regulation of gene expression and
mechanisms of secretion. Biochimie 82, 1023–1032.

Ryu H.S., Kim H.K., Choi W.C., Kim M.H., Park S.Y., Han
N.S., Oh T.K. & Lee J.K. 2006. New cold-adapted lipase
from Photobacterium lipolyticum sp. nov. that is closely
related to filamentous fungal lipases. Applied Microbiology
and Biotechnology 70, 321–326.

Sambrook J. & Russel D.W. 2001. Molecular cloning: a
laboratory manual. Woodbury, NY: Cold Spring Harbor
Laboratory Press.

Suzuki T., Nakayama T., Choo D.W., Hirano Y., Kurihara T.,
Nishino T. & Esaki N. 2003. Cloning, heterologous
expression, renaturation, and characterization of a
cold-adapted esterase with unique primary structure from
a psychrotroph Pseudomonas sp. strain B11-1. Protein
Expression and Purification 30, 171–178.

Yang X.X., Lin X.Z., Bian J., Sun X.Q. & Huang X.H. 2004.
Screening and phylogenetic analysis of Antarctic bacteria
producing low-temperature lipase. Acta Oceanologica Sinica
23, 717–723.

Yang X.X., Lin X.Z., Fan T.J., Bian J. & Huang X.H. 2008.
Cloning and expression of lipP, a gene encoding a
cold-adapted lipase from Moritella sp. 2-5-10-1. Current
Microbiology 56, 194–198.

Zhang P. & Zeng R. 2007. Cloning, expression, and
characterization of a cold-adapted lipase gene from an
Antarctic deep-sea psychrotrophic bacterium,
Psychrobacter sp. 7195. Journal of Microbial Biotechnology 17,
604–610.

Cloning and expression of cold-active lipasesL. Xuezheng et al.

Polar Research 29 2010 421–429 © 2010 the authors, journal compilation © 2010 Blackwell Publishing Ltd 429