Download Chaperone-dependent gene expression of organic

Process Biochemistry 45 (2010) 346–354
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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Chaperone-dependent gene expression of organic solvent-tolerant lipase from
Pseudomonas aeruginosa strain S5
Syarul Nataqain Baharum a,1, Raja Noor Zaliha Raja Abdul Rahman a,*, Mahiran Basri b, Abu Bakar Salleh a
a
b
Faculty Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 11 August 2009
Received in revised form 7 October 2009
Accepted 8 October 2009
The gene coding for the intracellular organic solvent-tolerant lipase of Pseudomonas aeruginosa strain S5
was isolated from a genomic DNA library and cloned into pRSET. The cloned sequence included two open
reading frames (ORF) of 1575 bp for the first ORF (ORF1), and 582 bp for the second ORF (ORF2). The
ORF2, known as chaperone, plays an important role in the expression of the S5 gene. The ORF2 is located
downstream of lipase gene, and functions as the act gene for ORF1. The conserved pentapeptide, Gly-XSer-X-Gly, is located in the ORF1. A sequence coding for a catalytic triad that resembles that of a serine
protease, consisting of serine, histidine, and aspartic acid or glutamic acid residues, was present in the
lipase gene. Expression of the S5 lipase gene in E. coli resulted in a 100-fold increase in enzyme activity
9 h after induction with 0.75 mM IPTG. The recombinant protein revealed a size of 60 kDa on SDS-PAGE.
The Lip S5 gene was stable in the presence of 25% (v/v) n-dodecane and n-tetradecane after 2 h
incubation at 37 8C.
ß 2009 Elsevier Ltd. All rights reserved.
Keywords:
Organic solvent-tolerant lipase
Chaperone
Pseudomonas aeruginosa
Shot-gun cloning
Expression
Phylogenetic tree analysis
1. Introduction
Many researchers have found that among lipases of various
origins (animal, plant and microorganism), those from bacteria,
especially from Pseudomonas species, exhibit the highest versatility, reactivity and stability in catalyzing reactions in the organic
phase. For example, Ogino et al. [1] and Isken et al. [2], reported
that they were able to isolate lipolytic enzymes, from the organic
solvent-tolerant Pseudomonas strains LST-03 and S12.
According to Quyen et al. [3], the lipase genes can be divided
into three groups, designated classes I to III and based on
homology. Class III is only distantly related to the other classes.
Pseudomonas lipases of classes I and II, including the broadly used
lipases of P. cepacia, and P. glumae (class II), as well as those of P.
aeruginosa (class I), need a chaperone; a gene that is located
downstream of the lipase gene, for efficient secretion and folding of
active lipase. The deduced amino acid sequences of the chaperones
for these lipase genes belong to two homology groups.
A variety of lipase-encoding genes from different Pseudomonas
sp. have been cloned and sequenced. However, the expression of
* Corresponding author. Tel.: +603 8946 7592; fax: +603 8946 7593.
E-mail address: [email protected] (Raja Noor Zaliha Raja Abdul
Rahman).
1
Present address: Institute of Systems Biology, Universiti Kebangsaan Malaysia,
43600 UKM Bangi, Selangor, Malaysia.
1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2009.10.008
subfamily I.1 and I.2 Pseudomonas lipases is hampered by the fact
that a lipase chaperone is necessary for the correct folding of the
proteins to their enzymatically active forms [4].
Previously, we reported on an organic solvent-tolerant lipase
secreted by S5, a benzene, toluene, ethyl-benzene and p-xylene
(BTEX) degrader strain identified as Pseudomonas aeruginosa. Studies
of the effects of nutritional and physical factors on enzyme activity,
as well as purification of the enzyme, were also reported [5–7]. To our
knowledge, however, limited studies have reported on the expression of enzymatically active Pseudomonas lipases in E. coli systems. In
those reports that are published, expression of the lipases levels are
very low without the helper gene or chaperone protein [8].
The cloning and expression of an organic solvent-tolerant
lipase, in the presence of the activation gene, was also reported by
Ogino et al. [9,10]; however, no stability test of the lipase in organic
solvents was conducted.
In this paper, we report on the cloning of an organic solventtolerant lipase from a genomic library, as well as the role of ORF2 as
a chaperone in the activation of the S5 lipase gene in Escherichia
coli. Furthermore, a test of the organic solvent tolerance of the
lipase in the present of lipase-specific foldase was performed.
2. Materials and methods
2.1. Bacterial strains and plasmids
Pseudomonas aeruginosa strain S5 was used in this study and grown under the
conditions reported by Baharum et al. [5]. The bacterial pure culture was submitted
S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354
to the German collection of microorganisms and cell culture (DSMZ), and was
assigned the accession number DSM 17160, as reported by Rahman et al. [7]. In
order to prepare competent cells, a single colony of Escherichia coli Top 10 cells from
fresh LB agar was inoculated in LB (5 ml), and incubated at 37 8C with shaking at
200 rpm until an A600 of 0.4–0.5 was obtained. Competent cells were prepared, and
transformation of E. coli was performed according to the methods described by
Sambrook et al. [11]. Genomic DNA was extracted using DNeasy tissue kits by
Qiagen, USA, following the directions of the supplier. Meanwhile, plasmid DNA was
extracted using QIAprep Spin Miniprep Kits, according to the manufacturer’s
instructions.
2.2. Lipase gene isolation by PCR
In order to clone the DNA encoding a partial sequence of the S5 lipase gene, two
oligonucleotide primers: forward F1, 50 -ATG CTG CCT ATA GAC CTG GGT C-30 ;
reverse R1, 30 -ACC CGC ACG CGC TAT CCC AT-50 ; were designed and synthesized on
the basis of the conserved nucleotide sequence from the P. aeruginosa PAO1 LipC.
2.3. Genomic DNA library construction (MboI partial digestion)
In order to prepare a clonable size of genomic DNA, small scale reactions were
carried out to digest the DNA into 2–10 kb fragments [12]. The plasmid was
digested with BamH1 in order to generate compatible ends for ligation with
genomic DNA partially digested with MboI, as described by Wu et al. [12], with
slight modification. The cleaved and desphosphorylated products were then
purified as described by Sambrook et al. [11].
Ligation of genomic DNA partially digested with MboI with pRSET digested with
BamHI, was performed as described by Sambrook et al. [11].
2.4. Effect of different concentration of IPTG on lipase secretion
E. coli containing the recombinant plasmid was induced with different
concentrations of IPTG ranging from 0 mM to 1 mM at OD600 of 0.5 h for 6 h. Ten
ml of culture was pelleted by centrifugation (12,000 rpm, 10 min) and subjected to
intracellular lipase assay. SDS-PAGE was used to analyze the expressed protein bands.
2.5. Effect of induction times on lipase secretion
Induction times ranging from 0 to 40 h at 3 h time intervals were tested for lipase
secretion. Ten ml of culture was harvested every 3 h and subjected to the
intracellular lipase assay. SDS-PAGE was used to analyze the expressed protein
bands.
2.6. Organic solvent-tolerance test
A culture containing the recombinant plasmid and induced with 0.75 mM IPTG,
was harvested after 8 h of induction and tested for organic solvent tolerance as
previously described [5–7].
2.7. Nucleotide sequence
The nucleotide sequence of the S5 lipase has been assigned GenBank accession
number AY738723.
347
3.2. Cloning of organic solvent-tolerant lipase gene with the pDrive
cloning vector.
After PCR amplification of the DNA fragment, the purified PCR
product was ligated into the pDrive cloning vector and the
recombinant plasmid was transformed into E. coli TOP 10 cells. The
plasmids revealed sizes of 4 kb to 5 kb after gel electrophoresis.
The recombinant plasmids were amplified with the primers For
PGL and Rev PGL to amplify the insert. All recombinant clones were
found to contain a 1.7 kb DNA fragment after gel electrophoresis.
The recombinant clone was sequenced, and was found to
contain an open reading frame (ORF) comprising 1575 nucloetides
corresponding to 448 amino acids (Fig. 1). The sequence for a
conserved pentapeptide, Gly-X-Ser-X-Gly, was located at position
602–616. The conserved pentapeptide of this enzyme consisted of
amino acids glycine-histidine-serine-glutamine-glycine. This pentapeptide is similar to the one common to serine protease family
members [13]. The G-X-S-X-G conserved pentapeptide was similar
to that found in most Pseudomonas lipases, including Pseudomonas
KWI-56 [14], Pseudomonas B11-1 [15] and P. aeruginosa [16].
3.3. Cloning and expression of the S5 lipase gene with different
expression vectors
In order to express the S5 lipase gene in an Escherichia coli
expression system, the purified PCR fragment (1575 bp) was
cloned into pTrcHis, an expression vector, and recombinant
plasmids were transformed into E. coli Top 10. The recombinant
plasmids were extracted and were 4–5 kb on 1% (w/v) agarose gels.
Plasmids were digested with EcoR1 to release the insert, and found
to contain a 1.7 kb DNA fragment after gel electrophoresis.
E. coli containing a recombinant clone was grown in LB–
ampicillin broth. The culture was harvested after 6 h of induction
with IPTG, and was assayed for lipase activity. A sample was
analyzed by SDS-PAGE. However, the lipase activity was low and
no expression band was detected on the SDS-PAGE.
Due to the low activity, and the absence of an expression band
on the SDS-PAGE, the DNA fragment was cloned into the pQE-30AU
expression vector. The positive recombinant clones were grown in
LB–ampicillin broth, and assayed for lipase activity. The result was
similar to that obtained with the S5/pTrcHis/recombinant plasmids. The lipase activity produced by the recombinant plasmids
was low, and no expression band was detected by SDS-PAGE.
3.4. Lipase gene isolation from a genomic DNA library
3. Results
3.1. Lipase gene isolation by PCR
The genomic DNA from strain S5 was amplified by PCR, using
the primers described, with 60 8C as the annealing temperature in
30 cycles. A 900 bp PCR product was detected. The purified PCR
product was sent for sequencing. Based on the sequencing analysis,
the PCR successfully amplified the lipase gene (964 bp), which
showed high homology (91%) with PAO1 Lip C. The size of the PCR
product, however, was too small to code for the lipase, with a
molecular weight of 60 kDa as reported by Rahman et al. [6].
To amplify a longer PCR fragment of the lipase gene, a second
batch of oligonucleotide primers were designed and synthesized
on the basis of the conserved nucleotide sequence from the other
Pseudomonas sp. lipase gene. These primers were: For PGL: 50 -ATG
GTC AGA TCG ATG CGT TCC A-30 ; and Rev PGL: 50 -TCA CAG GCC CTG
CAG CTT GA-30 . The sequences of the primers were chosen based
on a BLAST result available database that showed high similarity
with the PCR product of 900 bp; the primers successfully amplified
a PCR product of 1700 bp.
The S5 lipase gene was isolated using a genomic DNA library
strategy. From a genomic DNA library, about 8000 recombinant
clones were produced. Out of these, five positive clones, which
produced halo zones on the tributyrin–ampicillin agar plates due
to the hydrolysis of tributyrin in the medium, were detected.
The recombinant plasmids were tested for the production of
intracellular lipase activity in E. coli. The clones were screened on
tributyrin/Amp agar plates, triolein/Amp agar plates and rhodamine B/Amp agar plates. All the plates showed positive results. The
plasmids were digested with BamHI to release the inserts, and a
clone was identified that contained a 10 kb DNA fragment after gel
electrophoresis. To further confirm the presence of the lipase gene,
the recombinant plasmid was amplified with PCR and a PCR
product of 1700 bp was obtained.
3.5. Restriction mapping of the putative lipase gene
The recombinant plasmid was digested with several restriction
enzymes (RE) to determine the restriction sites in the insert.
Digestion with BamHI and PstI produced a linear band on agarose
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S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354
Fig. 1. The nucleotide sequence of the lipase gene from P. aeruginosa S5 in the absence of chaperone. Note: start and stop codon was underlined. The conserved pentapeptide
was bolded.
gels (1% w/v). Therefore, these RE sites were not present in the
insert. Digestion with EcoRI produced two fragments; one of
5500 bp and one of 1800 bp. Meanwhile, double digestion with a
combination of EcoRI and PstI produced three bands of 5000 bp,
3000 bp and 1500 bp. Digestion with a combination of BamHI and
PstI produced three fragments (4000 bp, 1300 bp and 700 bp).
Double digestion with EcoRI and BamHI produced three bands of
4000 bp, 1700 bp and 1000 bp. Based on this information, a
restriction map was constructed (Fig. 2) to assist with further
subcloning experiments.
Most researchers have reported that the size of Pseudomonas
lipase genes is between 1000 bp and 3000 bp [13,14,17]. Based on
these findings, fragments of 1000–3000 bp were isolated and
cloned into pRSET. Recombinant clones that carried the 1000 bp,
1300 bp, 1500 bp, 1700 bp or 1800 bp fragments did not produce
halos on tributyrin–amp agar plates, and thus did not contain the
entire lipase gene. However, recombinant clones that carried the
3000 bp fragment produced positive results on tributyrin–amp,
triolein and rhodamine agar plates. The recombinant plasmid was
isolated and sequenced.
Fig. 2. Restriction Mapping scheme of the DNA coding P. aeruginosa S5 lipase from
lipS5. Note: the lipolytic phenotype was determined by halo formation on
tributyrin–amp agar.
S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354
3.6. Nucleotide sequence analysis
The recombinant clone containing the putative lipase gene was
sequenced, and was found to contain two open reading frames
(ORF); the first comprised 1575 bp (ORF1), and the second was
582 bp (ORF2, located downstream from ORF1) (Fig. 3). The
nucleotide sequence of ORF1 agreed with the sequence of the S5
lipase gene previously amplified with PCR. The ORF1 has three
possible methionines as transcription starting points, at positions
241, 288 and 254. The initial ATG codon at position 241 is the most
likely to be the start codon, since the presence of a typical Shine–
Dalgarno (SD) sequence in the gene is 9 bases upstream of the ATG
codon.
The second ORF (ORF2) was detected 132 nucleotides downstream from the stop codon of ORF1. The second ORF (ORF2),
detected after shot-gun cloning, functions as the act gene for ORF1
to express the lipase activity.
The sequence for the conserved pentapeptide, Gly-X-Ser-X-Gly,
is located in ORF1. The S5 lipase conserved pentapeptide is similar
to other Pseudomonas lipases, as shown in Fig. 4. The sequence
alignment of four lipases showed that all of these lipases have
conserved pentapeptides like G-X-S-X-G except lip3 (LST03) lipase.
Lipases have a sequence motif of Gly-X-Ser-X-Gly (where X is any
amino acid) that is similar to the one common to serine protease
families. A catalytic triad resembling that of serine protease and
consisting of serine (Ser 130), histidine (His 289), aspartic acid (Asp
226) or glutamic acid residues, is present in this lipase gene. Siomi
et al. [13] suggested that the serine residue in this triad is
important for catalysis.
3.7. Phylogenetic analysis of S5 lipase
The G + C content of a gene encoding a protein may reveal the
origin of the protein. The G + C content was calculated using
FramePlot 2.3.2 software available at http://www.watson.nih.go.jp. The G + C content of this organic solvent-tolerant lipase gene
was 65.6%, which is very close to that reported for P. aeruginosa,
66.0% [17] to 66.6% [18]. According to Ogino et al. [17], the genes of
Pseudomonas sp. have a high G + C content in the wobble base
position, which serves as a check on the location and accuracy of
the protein-coding sequence. Most work has reported high G + C
contents of Pseudomonas sp. lipase genes [13,19,20]. According to
West and Iglewski [21], a high G + C content is typical for DNA of P.
aeruginosa. The high G + C content lipase gene may code for a more
stable enzyme than low G + C genes do.
A phylogenetic tree was constructed based on the sequence
similarity among the group of species in order to gain information
regarding their evolutionary relationship. The phylogenetic tree
for S5 lipase was constructed through multiple sequence alignments with other microbial lipases. The phylogenetic tree for S5
lipase is presented in Fig. 5. The S5 lipase gene was closely related
with the P. aeruginosa KWI-56 lipase (lip act) and slightly distant
from the P. aeruginosa PK-12CS lipase gene. The S5 lipase gene was
distantly related to genes for other reported organic solventtolerant lipases, such as LST-03 [17], and B. sphaericus 205y [22].
This lipase was also distant from the Lip C lipases (PAO1 and
U75975), neither of which have the chaperone or act gene.
3.8. Signal peptide prediction and hydrophobicity profile of S5 lipase
The signal peptide of S5 lipase was predicted using the SignalP
2.0 World Wide Web server at http.www.cbs.dtu.dkservicesSignalP-2.0.html. These data presented the values of the C-, S- and
Y-scores for the signal peptide and cleavage site for S5 lipase. The
sharp peak indicated the C-score at position 36 between amino
acids VHA-AT. This peak corresponded to the change in the value of
349
the S-score from a high to a low value. The cleavage site is located
at position 35–37, indicated by the maximal Y-score.
The hydrophobicity profile of the S5 lipase gene was analyzed
using ProtScale tools on the Expasy Molecular Biology Server by
the method of Kyte and Doolittle [23]. The minimum and
maximum hydrophobicity of S5 lipase were 3.322 and 2.444,
respectively. The results revealed five major hydrophobic grooves,
which were residues TAALVALAAS LPVHA (21–35), KGLI (59–63),
QVEEVLALTG (111–120), WRRCSRWS (234–241), and KYPVVLVIC
(370–378).
3.9. Amino acid composition
The amino acid composition of S5 lipase was analyzed with the
ProtParam Tool from the Expasy Molecular Biology. Out of 679
amino acids in the deduced sequence of the S5 lipase from P.
aeruginosa S5, the total number of negatively charged residues
(Asp + Glu) was 37, while the total number of positively charged
residues (Arg + Lys) was 154. There were a total of 207 charged
residues (Arg, Asp, Glu, His and Lys), representing 30.5% of the total
amino acid composition. The number of hydrophobic residues (Ala,
Ile, Phe, Leu, Met, Pro, Val and Trp) is 223 amino acids, or 32.9% of
the total. The number of uncharged residues (Asn, Cys, Gln, Gly, Ser,
Thr and Tyr) is 249 amino acids, or 36.6% of the total composition.
The total atomic composition of this lipase is 10592 atoms,
including carbon (3173), hydrogen (5260), nitrogen (1200), oxygen
(921) and sulfur (38), and represented by a formula of C3173 H5260
N1200 O921 S38. The aliphatic index is defined as the relative volume
of a protein occupied by aliphatic side chains (alanine, valine,
isoleucine and leucine). It is an index to measure the stability of a
globular protein. The aliphatic index for the S5 lipase is 49.28,
based on the deduced sequence of the polypeptide chain. The lipS5
has an instability index of 87.32. As an enzyme is considered
unstable when the instability index is more than 40 [24], the lipase
could be classified as unstable.
The amino acid comparison with other lipases was studied by
comparing the composition (percentages) of amino acids of S5
with other lipases from various microorganisms. Amino acid
composition among lipases are presented in Table 1. In the S5
lipase, Ala, Arg, Gly and Ser are most abundant, while, Met, Phe and
Tyr are less abundant. Meanwhile, Arg is more abundant in the S5
and KWI-56 lipases. On the other hand, Ala, Gly, Leu and Val are
abundant in ten lipases reported earlier. Most of the lipases have a
low percentage of Cys. In 1992, Dartois et al. [25], suggested that
proteins lacking cysteine, or with a low content of cysteine, are
generally more flexible molecules, whose tertiary structure relies
on weaker bonds. Furthermore, in the case of lipase, the lack of
cysteine may allow conformational changes necessary for enzymatic activity.
4. Optimization and expression of P. aeruginosa lipase in E. coli
4.1. Effect of IPTG concentration and induction time
The effect of the IPTG concentration in media has been studied
by inducing recombinant bacterial cultures with increasing IPTG
concentrations. The highest lipase activity (15.1 U/ml) was
detected after 6 h induction with 0.75 mM IPTG (data not shown).
The lipase activity without any induction by IPTG was 6.5 U/ml,
and at 0.25 mM of IPTG the activity was 8.9 U/ml. The lipase
activity was enhanced by 70% compared to no induction with
0.5 mM of IPTG. The lipase activity was slightly decreased to
13.9 U/ml at 1.00 mM of IPTG. The optimization of the expression
of recombinant lipase was carried out by varying the induction
times with 0.75 mM IPTG. Using this IPTG concentration, at 1 h
after induction, the lipase activity was increased to 6.39 U/ml from
350
S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354
Fig. 3. Nucleotide sequence of the lipase gene from P. aeruginosa S5 in the presence of chaperone. The Shine–Dalgarno ribosome binding site, start and stop codon were
underlined. The conserved pentapeptide was bolded. The residues that thought to comprise the active center of the S5 lipase were highlighted in the box.
S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354
351
Fig. 4. Comparison of the amino acid sequence of active site among lipases. The amino acids in the box indicate the active site G-X-S-X-G. The residues that thought to
comprise the active center of various lipases were highlighted in the box. The sequences were obtained from GenBank. The abbreviations used were lips5/act: S5 lipase, lipact:
P. aeruginosa KWI-56 (S77842), lipC_PAO1: P. aeruginosa lip C PAO1 (AE004894) and lip3_OST: P. aeruginosa lip3 LST 03 (AB125368).
S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354
352
gradually at 27 h, with a 20% loss in lipase activity between 18 h
and 27 h. A gradual drop of expression level in a later phase could
be explained by the outgrowth of non-induced cells containing the
plasmid [26].
Expression of the protein was confirmed on SDS-PAGE stained
with Coomassie Blue. The molecular mass of the target protein was
determined by comparing it with a protein marker. A protein band
was observed at 60 kDa (data not shown) after 8 and 9 h of
induction. Clear correlation was observed between the intensity of
the expressed protein and the different induction times. The size of
the protein band was in agreement with the size of the S5 lipase
(60 kDa), as determined in earlier studies [6]. These results suggest
that the S5 lipase gene was successfully cloned and expressed in an
E. coli system, with a high level of protein expression.
4.2. Stability of recombinant lipase in various organic solvents
Fig. 5. The phylogenetic tree analysis of S5 lipase gene. Dendogram shows sequence
relationship between P. aeruginosa S5 lipase with other microbial lipases. The
abbreviations used were lipS5/act: P. aeruginosa S5 (AY787823), lip act:
Pseudomonas sp. KWI-56 (S77842), lip3_OST: P. aeruginosa Lip3 (AB125368),
LipC_PAO1: P. aeruginosa PAO1 LipC (AE004894), U75975LipC: P. aeruginosa LipC
(U75975), PK-12CS: P. mendocina PK-12CS (AY091666), Bsp_42: Bacillus sp. 42
(AY787835), B.s205y: B. sphaericus 205y (AF453713), C.v_lipA: Chromobacterium
viscosum LipA (AJ295616), and A.c_lipA: Acinetobacter calcoaceticus RAG-1
(AF047691).
0.04 U/ml at 0 time induction (data not shown). The lipase activity
was increased drastically after 3 h to about 16 U/ml and 27.7 U/ml
after 6 h time induction.
At 7 h after induction, the lipase activity was as approximately
equal to that observed at 6 h after induction. The recombinant
protein was optimally expressed at 8 h after induction with IPTG.
The lipase activity at this induction time was 34.4 U/ml, and
slightly decreased to 31 U/ml and 30.57 U/ml after 9 and 18 h after
induction, respectively. The protein expression level decreased
Based on previous studies [5–7], S5 lipase was identified as an
organic solvent-tolerant enzyme. In order to confirm that the
organic solvent-tolerant lipase gene was successfully cloned, the
stability of the Top 10/S5/pRSET lipase in the presence and absence
of organic solvents was determined.
The enzyme was treated for 2 h in 25% (v/v) organic solvents,
and assayed for lipase activity. log P values of organic solvents
were adapted from Laane et al. [27]. As shown in Table 2, the
enzyme was active in the presence of the organic solvents, with
log P values of 2.0–7.6; however, the enzyme activity decreased
after prolonged incubation with all of the organic solvents, except
n-decane, n-dodecane and n-tetradecane, which resulted in log P
values higher than 5.0. The stability of the S5 lipase in n-decane, ndodecane and n-tetradecane was 1.4, 1.8 and 1.2 times higher,
respectively, than in the absence of the solvents.
S5 lipase was unstable in polar organic solvents, such as 1decanol, 1-octanol, ethyl acetate and benzene, with log P values
below 4.0. This enzyme lost 94% and 89% its activity in ethyl
acetate and 1-octanol, respectively, compared to the activity
observed in the absence of organic solvents. Surprisingly, this lipS5
was also unstable in n-hexane and cyclohexane. However, the
enzyme activity was activated in benzene, chloroform and toluene
with log P between 2.0 and 2.5.
Table 1
Amino acid composition of S5 lipase and lipases from other organisms.
Percentage of amino acids (%)
Residue
S5
KWI-56
Lip3
LipC
PK-12CS
42
205y
LipA
RAG-1
017
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
Total
10.6
20.3
3.7
2.4
3.1
2.7
3.1
8.5
2.4
2.7
4.4
2.4
1.5
0.6
7.1
11.1
5.7
2.4
1.5
3.8
100
16.5
25.9
0.4
7.3
2.9
2.9
4.3
11.4
3.8
1.1
3.5
0.1
0.2
0.8
5.2
3.5
2.1
0.4
0.5
6.6
100
11.1
7.6
3.2
4.5
0.3
4.8
7.3
7.0
2.5
3.2
14.0
2.9
1.5
4.1
6.7
4.5
2.2
0.6
2.5
9.2
100
12
3.6
4.5
4.3
0.6
3.9
4.5
10.4
2.6
2.3
10.0
2.9
1.6
3.9
4.5
7.4
6.5
0.6
2.6
11.3
100
9.6
4.1
5.1
4.1
0.9
2.6
4.1
11.9
2.3
4.8
10.2
3.2
2.6
2.9
4.5
10.3
5.1
0.6
3.5
7.0
100
7.9
6.7
4.6
5.5
0.9
2.9
4.8
10.0
3.1
3.1
9.3
2.9
1.9
4.8
4.6
6.5
6.0
2.6
4.6
7.0
100
5.8
1.8
4.3
6.3
0.2
4.3
2.3
6.3
1.8
8.0
9.1
7.1
3.3
4.3
6.3
7.0
5.5
2.5
4.5
9.3
100
14.2
4.2
4.2
4.7
0.5
4.7
1.4
10.6
2.5
2.8
9.5
1.7
1.3
2.2
3.4
8.1
10.0
1.1
2.8
10.3
100
9.8
2.4
6.2
3.8
1.1
4.4
1.7
11
2.1
3.0
9.7
3.3
2.1
3.3
4.1
9.8
6.2
0.8
4.1
10.9
100
9.3
3.8
3.5
4.5
0.6
4.8
3.2
10.9
3.5
5.4
11.5
2.6
1.3
2.2
3.8
8.3
5.6
0.6
3.8
9.3
100
Notes: The sequences for calculation of amino acid composition were obtained from GenBank. The abbreviations used were S5: P. aeruginosa S5 (AY787823), KWI-56:
Pseudomonas sp. KWI-56 (S77842), Lip3: P. aeruginosa Lip3 (AB125368), Lip C: P. aeruginosa PAO1 LipC (AE004894), PK-12CS: P. mendocina PK-12CS (AY091666), 42: Bacillus
sp. 42 (AY787835), 205y: B. sphaericus 205y (AF453713), Lip A: C. viscosum LipA (AJ295616), RAG-1: A. calcoaceticus RAG-1 (AF047691) and 017: Vibrio cholerae 017 (Y00557).
S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354
Table 2
LipS5 lipase activity in various organic solvents.
Organic solvents
log Pa
None
Ethyl acetate
Benzene
Chloroform
Toulene
1-Octanol
Cyclohexane
n-Hexane
1-Decanol
n-Decane
n-Dodecane
n-Tetradecane
n-Hexadecane
–
0.68
2.0
2.0
2.5
2.9
3.2
3.6
4.0
5.6
6.6
7.6
8.8
Specific activity (U/mg)b
30 min
2h
1095.74 0.013
596.27 0.018
1421.28 0.020
1375.00 0.013
1388.30 0.023
1430.32 0.010
1466.00 0.015
1552.13 0.012
1393.62 0.016
1336.17 0.019
1324.47 0.023
1158.00 0.018
947.34 0.019
569.14 0.017
38.83 0.018
607.00 0.018
658.00 0.019
667.02 0.015
76.60 0.019
291.00 0.018
225.00 0.013
157.45 0.016
947.87 0.018
1171.28 0.019
769.68 0.020
381.38 0.018
Note: The enzyme and organic solvent was mixed in a 3:1 ratio and the mixture was
incubated at 37 8C with shaking at 150 rpm for 2 h and assayed for the remaining
lipase activity.
a
Adapted from Laane et al. [27].
b
Data are means standard deviation of three determinations. All the data shows
significantly different (P < 0.05; by Turkey test).
5. Discussion
In this study, we cloned and expressed a new organic solventtolerant lipase from P. aeruginosa strain S5 using a shot-gun cloning
approach, after several attempts with PCR cloning failed. Previous
failures to express the S5 lipase might have been due to the fact
that the Pseudomonas lipase gene needs a helper or activator gene
in the E. coli systems. According to Iizumi et al. [14], the
Pseudomonas sp. KWI-56 lipase activity was enhanced in E. coli
by the function of the activator gene that exists downstream of the
lipase gene. This finding was similar to that of Iizumi and Fukase
[28]. These researchers proposed that the act gene, which is
downstream of the lip gene, is required for the expression of lipase
activity as a trans-acting factor. In addition, Ogino et al. [9], also
reported the presence of a lipase-specific foldase involved in the
overexpression of LST-03 lipase in E. coli. However, the details of
the activation mechanism, and the function of the act gene were
unknown.
Recently, the crystal structure of the lipase and lipase-specific
foldase complex from Burkholderia glumae was determined;
however, the mechanism was still not fully clarified [29]. The
lipase activator protein may be a kind of chaperone that mediates
the folding of the lipase protein. Pseudomonas lipases that are
classified as class I or II need a helper protein, named chaperone,
modulator or activator by different authors, that helps in the
correct folding and thus allows them to be active [30].
Some Gram-negative bacteria have special mechanisms to
avoid the degradation of cell components, such as intracellular
proteins, periplasmic proteins and membranes, by the endogenous
attack of extracellular hydrolyzing enzymes and toxins. The
synthesis of proteins as inactive precursors, or so-called zymogens,
is one mechanism against such attacks. Zymogens are, as a rule,
activated by modifications, or by the action of an intermolecular
chaperone. The Pseudomonas lipase is first produced as a zymogen,
and becomes active as a result of the function of an additional gene
product. The activation of Pseudomonas lipase involves conformational changes in the protein molecule [28].
We believe that this study is the only attempt that has been
made to investigate the enzyme activity of a solvent-stable lipase
and its chaperone in the presence of organic solvents. Furthermore,
Ogino et al. [9,10] reported solely on the activation of the lipase
with a specific foldase, without investigating the stability of this
lipase and foldase complex in organic solvents.
353
In conclusion, we have identified a new organic solventtolerant lipase that is activated in E. coli by the function of a
chaperone, or helper gene. This gene is present in ORF2, which
exists downstream from the lipase gene. We also verified that the
S5 lipase was active in the presence of organic solvents.
Acknowledgements
This work was financially supported by Grant No. 09-02-040002 from the Malaysian Ministry of Science, Technology and
Innovation (MOSTI). The authors acknowledge the contribution of
Robert Coe from the University of Sheffield, United Kingdom, for
his help in correcting the English language of the manuscript.
References
[1] Ogino H, Miyamoto K, Ishikawa H. Organic solvent-tolerant bacterium which
secretes organic solvent-stable lipolytic enzyme. Appl Environ Microbiol
1994;60(10):3884–6.
[2] Isken S, Derks A, Wolffs PFG, de Bont JAM. Effect of organic solvents on the yield of
solvent-tolerant Pseudomonas putida S12. Appl Environ Microbiol 1999;65
(6):2631–5.
[3] Quyen DT, Dannert CS, Schimd RD. 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. Appl
Environ Microbiol 1999;65(2):787–94.
[4] Rosenau F, Jaeger K. Bacterila lipases from Pseudomonas: regulation of gene
expression and mechanisms of secretion. Biochimie 2000;82:1023–32.
[5] Baharum SN, Salleh AB, Razak CNA, Rahman RNZA, Basri M, Rahman MBA.
Organic solvent tolerant lipase by Pseudomonas sp. strain S5: stability of
enzyme in organic solvent and physical factors affecting its production.
Ann Microbiol 2003;53:75–83.
[6] Rahman RNZRA, Baharum SN, Salleh AB, Basri M. High yield purification of an
organic solvent tolerant lipase from Pseudomonas sp. strain S5. Anal Biochem
2005;341:267–74.
[7] Rahman RNZA, Baharum SN, Basri M, Salleh AB. S5 Lipase: an organic solvent
tolerant enzyme. J Microbiol 2006;44(6):583–90.
[8] Zhang A, Gao R, Dao N, Xie G, Gao G, Cao S. Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens
JCM5963. J Mol Catal B Enzym 2009;56:78–84.
[9] Ogino H, Katou Y, Akagi R, Mimitsuka T, Hiroshima S, Gemba Y, et al. Cloning
and expression of gene, and activation of an organic solvent-stable lipase from
Pseudomonas aeruginosa LST-03. Extremophiles 2007;11:809–17.
[10] Ogino H, Inoue S, Akagi R, Yasuda M, Doukyu N, Ishimi K. Refolding of a
recombinant organic solvent-stable lipase, which is overexpressed and forms
and inclusion body, and activation with lipase-specific foldase. Biochem Eng J
2008;40:507–11.
[11] Sambrook J, Frisch ER, Maniatis T. Molecular cloning: a laboratory manual.
New York: Cold Spring Harbor Laboratory, Cold Spring Harbour; 1989.
[12] Wu W, Welsh MJ, Kaufman PB, Zhang HH. Method in gene biotechnology.
Florida: CRC Press LLC; 1997.
[13] Siomi MC, Yoshikawa K, Hirayama NO, Yamamoto K, Sogabe Y, Nakatani T,
et al. Purification, molecular cloning and expression of lipase from Pseudomonas aeruginosa. Arch Biochem Biophys 1992;296(2):505–13.
[14] Iizumi T, Nakamura K, Shimada Y, Sugihara A, Tominaga Y, Fukase T. Cloning,
nucleotide sequencing and expression in Escherichia coli of a lipase and its
activator genes from Pseudomonas sp. KWI-56. Agric Biol Chem 1991;55(9):
2349–57.
[15] Choo D, Kurihara WT, Suzuki T, Soda K, Esaki N. A cold adapted lipase of an
Alaskan psychrotroph, Pseudomonas sp. strain B11-1: gene cloning and
enzyme purification and characterization. Appl Environ Microbiol 1998;64
(2):486–91.
[16] Wilhelm S, Tommassen J, Jaeger KE. A novel lipolytic enzyme located in the outer
membrane of Pseudomonas aeruginosa. J Bacteriol 1999;181(22):6977–86.
[17] Ogino H, Hiroshima S, Hirose S, Yasuda M, Ishimi K, Ishikawa H. Cloning,
expression and characterization of a lipase gene (lip3) from Pseudomonas
aeruginosa LST-03. Mol Gen Genom 2004;271:189–96.
[18] Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warener P, Hickey MJ, et al.
Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000;406:959–64.
[19] Duchene MA, Schweizer F, Lottspeich G, Krauss M, Marget K, Vogel BU, et al.
Sequence and transcriptional strata site of the Pseudomonas aeruginosa outer
membrane porin protein F gene. J Bacteriol 1988;170:155–62.
[20] Zylstra GJ, Olsen RH, Ballou DP. Cloning, expression and regulation of the
Pseudomonas cepacia protocatechuate 3,4-di-oxygenase genes. J Bacteriol
1989;171:5907–14.
[21] West SHE, Iglewski BH. Codon usage in Pseudomonas aeruginosa. Nucleic Acids
Res 1988;16:9323–35.
[22] Hun CJ, Rahman RNZ, Salleh AB, Basri M. A newly isolated organic solvent
tolerant Bacillus sphaericus 205y producing organic solvent-stable lipase. J
Biochem Eng 2003;15:147–51.
354
S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354
[23] Kyte J, Doolittle RF. A simple method for displaying the hydrophatic character
of a protein. J Mol Biol 1982;157:105–32.
[24] Guruprasad K, Reddy BV, Pandit MW. Correlation between stability of a protein
and its dipeptide composition: A novel approach for predicting in vivo stability of
a protein from its primary sequence. Protein Eng 1990;4(2): 155–61.
[25] Dartois V, Baulard A, Schanck K, Colson C. Cloning, nucleotide sequence and
expression in Escherichia coli of a lipase gene from Bacillus subtilis 168. Biochim
Biophys Acta 1992;1131:253–60.
[26] Hahm DH, Kim SH, Pan J, Rhee JS. Maximum yield of foreign lipase in
Escherichia coli HB101 limited by duration of protein expression. J Ferment
Bioeng 1995;79(3):236–41.
[27] Laane C, Bocren S, Vos K, Veeger C. Rules of optimization of biocatalysis in
organic solvent. J Biotech Bioeng 1987;30:81–7.
[28] Iizumi T, Fukase T. Role of the gene encoding lipase activator from Pseudomonas sp. strain KW1-56 in vitro activation of lipase. Biosci Biotechnol
Biochem 1994;58(6):1023–7.
[29] Pauwels K, Lustig A, Wyns L, Tommassen J, Savvides SN, Van Gelder P.
Structure of a membrane-based steric chaperone in complex with its lipase
substrate. Nat Struct Mol Biol 2006;13:374–5.
[30] Yang J, Kobayashi K, Iwasaki Y, Nakano H, Yamane T. In vitro analysis of roles of
a disulfide bridge and a calcium binding site in activation of Pseudomonas sp.
strain KWI-56 lipase. J Bacteriol 2000;182(2):295–302.