Chaperone-dependent gene expression of organic solvent-tolerant lipase fromPseudomonas aeruginosa strain S5

Syarul Nataqain Baharum a,1, Raja Noor Zaliha Raja Abdul Rahman a,*, Mahiran Basri b, Abu Bakar Salleh a

a Faculty Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysiab Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Process Biochemistry 45 (2010) 346–354

A R T I C L E I N F O

Article history:

Received 11 August 2009

Received in revised form 7 October 2009

Accepted 8 October 2009

Keywords:

Organic solvent-tolerant lipase

Chaperone

Pseudomonas aeruginosa

Shot-gun cloning

Expression

Phylogenetic tree analysis

A B S T R A C T

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-X-

Ser-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.

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1. Introduction

Many researchers have found that among lipases of variousorigins (animal, plant and microorganism), those from bacteria,especially from Pseudomonas species, exhibit the highest versati-lity, reactivity and stability in catalyzing reactions in the organicphase. For example, Ogino et al. [1] and Isken et al. [2], reportedthat they were able to isolate lipolytic enzymes, from the organicsolvent-tolerant Pseudomonas strains LST-03 and S12.

According to Quyen et al. [3], the lipase genes can be dividedinto three groups, designated classes I to III and based onhomology. Class III is only distantly related to the other classes.Pseudomonas lipases of classes I and II, including the broadly usedlipases of P. cepacia, and P. glumae (class II), as well as those of P.

aeruginosa (class I), need a chaperone; a gene that is locateddownstream of the lipase gene, for efficient secretion and folding ofactive lipase. The deduced amino acid sequences of the chaperonesfor 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: rnzaliha@biotech.upm.edu.my (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 � 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 factthat a lipase chaperone is necessary for the correct folding of theproteins to their enzymatically active forms [4].

Previously, we reported on an organic solvent-tolerant lipasesecreted by S5, a benzene, toluene, ethyl-benzene and p-xylene(BTEX) degrader strain identified as Pseudomonas aeruginosa. Studiesof the effects of nutritional and physical factors on enzyme activity,as well as purification of the enzyme, were also reported [5–7]. To ourknowledge, however, limited studies have reported on the expres-sion of enzymatically active Pseudomonas lipases in E. coli systems. Inthose reports that are published, expression of the lipases levels arevery low without the helper gene or chaperone protein [8].

The cloning and expression of an organic solvent-tolerantlipase, in the presence of the activation gene, was also reported byOgino et al. [9,10]; however, no stability test of the lipase in organicsolvents was conducted.

In this paper, we report on the cloning of an organic solvent-tolerant lipase from a genomic library, as well as the role of ORF2 asa chaperone in the activation of the S5 lipase gene in Escherichia

coli. Furthermore, a test of the organic solvent tolerance of thelipase 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) 346–354 347

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.

3. Results

3.1. Lipase gene isolation by PCR

The genomic DNA from strain S5 was amplified by PCR, usingthe primers described, with 60 8C as the annealing temperature in30 cycles. A 900 bp PCR product was detected. The purified PCRproduct was sent for sequencing. Based on the sequencing analysis,the PCR successfully amplified the lipase gene (964 bp), whichshowed high homology (91%) with PAO1 Lip C. The size of the PCRproduct, however, was too small to code for the lipase, with amolecular weight of 60 kDa as reported by Rahman et al. [6].

To amplify a longer PCR fragment of the lipase gene, a secondbatch of oligonucleotide primers were designed and synthesizedon the basis of the conserved nucleotide sequence from the otherPseudomonas sp. lipase gene. These primers were: For PGL: 50-ATGGTC AGA TCG ATG CGT TCC A-30; and Rev PGL: 50-TCA CAG GCC CTGCAG CTT GA-30. The sequences of the primers were chosen basedon a BLAST result available database that showed high similaritywith the PCR product of 900 bp; the primers successfully amplifieda PCR product of 1700 bp.

3.2. Cloning of organic solvent-tolerant lipase gene with the pDrive

cloning vector.

After PCR amplification of the DNA fragment, the purified PCRproduct was ligated into the pDrive cloning vector and therecombinant plasmid was transformed into E. coli TOP 10 cells. Theplasmids revealed sizes of 4 kb to 5 kb after gel electrophoresis.The recombinant plasmids were amplified with the primers ForPGL and Rev PGL to amplify the insert. All recombinant clones werefound to contain a�1.7 kb DNA fragment after gel electrophoresis.

The recombinant clone was sequenced, and was found tocontain an open reading frame (ORF) comprising 1575 nucloetidescorresponding to 448 amino acids (Fig. 1). The sequence for aconserved pentapeptide, Gly-X-Ser-X-Gly, was located at position602–616. The conserved pentapeptide of this enzyme consisted ofamino acids glycine-histidine-serine-glutamine-glycine. This pen-tapeptide is similar to the one common to serine protease familymembers [13]. The G-X-S-X-G conserved pentapeptide was similarto 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) wascloned into pTrcHis, an expression vector, and recombinantplasmids were transformed into E. coli Top 10. The recombinantplasmids were extracted and were 4–5 kb on 1% (w/v) agarose gels.Plasmids were digested with EcoR1 to release the insert, and foundto 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 inductionwith IPTG, and was assayed for lipase activity. A sample wasanalyzed by SDS-PAGE. However, the lipase activity was low andno expression band was detected on the SDS-PAGE.

Due to the low activity, and the absence of an expression bandon the SDS-PAGE, the DNA fragment was cloned into the pQE-30AUexpression vector. The positive recombinant clones were grown inLB–ampicillin broth, and assayed for lipase activity. The result wassimilar to that obtained with the S5/pTrcHis/recombinant plas-mids. The lipase activity produced by the recombinant plasmidswas low, and no expression band was detected by SDS-PAGE.

3.4. Lipase gene isolation from a genomic DNA library

The S5 lipase gene was isolated using a genomic DNA librarystrategy. From a genomic DNA library, about 8000 recombinantclones were produced. Out of these, five positive clones, whichproduced halo zones on the tributyrin–ampicillin agar plates dueto the hydrolysis of tributyrin in the medium, were detected.

The recombinant plasmids were tested for the production ofintracellular lipase activity in E. coli. The clones were screened ontributyrin/Amp agar plates, triolein/Amp agar plates and rhoda-mine B/Amp agar plates. All the plates showed positive results. Theplasmids were digested with BamHI to release the inserts, and aclone was identified that contained a 10 kb DNA fragment after gelelectrophoresis. To further confirm the presence of the lipase gene,the recombinant plasmid was amplified with PCR and a PCRproduct of 1700 bp was obtained.

3.5. Restriction mapping of the putative lipase gene

The recombinant plasmid was digested with several restrictionenzymes (RE) to determine the restriction sites in the insert.Digestion with BamHI and PstI produced a linear band on agarose

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.

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.

S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354348

gels (1% w/v). Therefore, these RE sites were not present in theinsert. Digestion with EcoRI produced two fragments; one of5500 bp and one of 1800 bp. Meanwhile, double digestion with acombination of EcoRI and PstI produced three bands of 5000 bp,3000 bp and 1500 bp. Digestion with a combination of BamHI andPstI produced three fragments (4000 bp, 1300 bp and 700 bp).Double digestion with EcoRI and BamHI produced three bands of4000 bp, 1700 bp and 1000 bp. Based on this information, arestriction map was constructed (Fig. 2) to assist with furthersubcloning experiments.

Most researchers have reported that the size of Pseudomonas

lipase genes is between 1000 bp and 3000 bp [13,14,17]. Based onthese findings, fragments of 1000–3000 bp were isolated andcloned into pRSET. Recombinant clones that carried the 1000 bp,1300 bp, 1500 bp, 1700 bp or 1800 bp fragments did not producehalos on tributyrin–amp agar plates, and thus did not contain theentire 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 wasisolated and sequenced.

S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354 349

3.6. Nucleotide sequence analysis

The recombinant clone containing the putative lipase gene wassequenced, and was found to contain two open reading frames(ORF); the first comprised 1575 bp (ORF1), and the second was582 bp (ORF2, located downstream from ORF1) (Fig. 3). Thenucleotide sequence of ORF1 agreed with the sequence of the S5lipase gene previously amplified with PCR. The ORF1 has threepossible methionines as transcription starting points, at positions241, 288 and 254. The initial ATG codon at position 241 is the mostlikely to be the start codon, since the presence of a typical Shine–Dalgarno (SD) sequence in the gene is 9 bases upstream of the ATGcodon.

The second ORF (ORF2) was detected 132 nucleotides down-stream from the stop codon of ORF1. The second ORF (ORF2),detected after shot-gun cloning, functions as the act gene for ORF1to 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 similarto other Pseudomonas lipases, as shown in Fig. 4. The sequencealignment of four lipases showed that all of these lipases haveconserved 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 anyamino acid) that is similar to the one common to serine proteasefamilies. A catalytic triad resembling that of serine protease andconsisting of serine (Ser 130), histidine (His 289), aspartic acid (Asp226) or glutamic acid residues, is present in this lipase gene. Siomiet al. [13] suggested that the serine residue in this triad isimportant for catalysis.

3.7. Phylogenetic analysis of S5 lipase

The G + C content of a gene encoding a protein may reveal theorigin of the protein. The G + C content was calculated usingFramePlot 2.3.2 software available at http://www.watson.nih.-go.jp. The G + C content of this organic solvent-tolerant lipase genewas 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 ofPseudomonas sp. have a high G + C content in the wobble baseposition, which serves as a check on the location and accuracy ofthe protein-coding sequence. Most work has reported high G + Ccontents of Pseudomonas sp. lipase genes [13,19,20]. According toWest 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 morestable enzyme than low G + C genes do.

A phylogenetic tree was constructed based on the sequencesimilarity among the group of species in order to gain informationregarding their evolutionary relationship. The phylogenetic treefor S5 lipase was constructed through multiple sequence align-ments with other microbial lipases. The phylogenetic tree for S5lipase is presented in Fig. 5. The S5 lipase gene was closely relatedwith the P. aeruginosa KWI-56 lipase (lip act) and slightly distantfrom the P. aeruginosa PK-12CS lipase gene. The S5 lipase gene wasdistantly related to genes for other reported organic solvent-tolerant lipases, such as LST-03 [17], and B. sphaericus 205y [22].This lipase was also distant from the Lip C lipases (PAO1 andU75975), 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 SignalP2.0 World Wide Web server at http.www.cbs.dtu.dkservices-SignalP-2.0.html. These data presented the values of the C-, S- andY-scores for the signal peptide and cleavage site for S5 lipase. Thesharp peak indicated the C-score at position 36 between aminoacids VHA-AT. This peak corresponded to the change in the value of

the S-score from a high to a low value. The cleavage site is locatedat position 35–37, indicated by the maximal Y-score.

The hydrophobicity profile of the S5 lipase gene was analyzedusing ProtScale tools on the Expasy Molecular Biology Server bythe method of Kyte and Doolittle [23]. The minimum andmaximum 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 theProtParam Tool from the Expasy Molecular Biology. Out of 679amino 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 chargedresidues (Arg + Lys) was 154. There were a total of 207 chargedresidues (Arg, Asp, Glu, His and Lys), representing 30.5% of the totalamino acid composition. The number of hydrophobic residues (Ala,Ile, Phe, Leu, Met, Pro, Val and Trp) is 223 amino acids, or 32.9% ofthe 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 volumeof a protein occupied by aliphatic side chains (alanine, valine,isoleucine and leucine). It is an index to measure the stability of aglobular protein. The aliphatic index for the S5 lipase is 49.28,based on the deduced sequence of the polypeptide chain. The lipS5has an instability index of 87.32. As an enzyme is consideredunstable when the instability index is more than 40 [24], the lipasecould be classified as unstable.

The amino acid comparison with other lipases was studied bycomparing the composition (percentages) of amino acids of S5with other lipases from various microorganisms. Amino acidcomposition among lipases are presented in Table 1. In the S5lipase, Ala, Arg, Gly and Ser are most abundant, while, Met, Phe andTyr are less abundant. Meanwhile, Arg is more abundant in the S5and KWI-56 lipases. On the other hand, Ala, Gly, Leu and Val areabundant in ten lipases reported earlier. Most of the lipases have alow percentage of Cys. In 1992, Dartois et al. [25], suggested thatproteins lacking cysteine, or with a low content of cysteine, aregenerally more flexible molecules, whose tertiary structure relieson weaker bonds. Furthermore, in the case of lipase, the lack ofcysteine may allow conformational changes necessary for enzy-matic 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 studiedby inducing recombinant bacterial cultures with increasing IPTGconcentrations. The highest lipase activity (15.1 U/ml) wasdetected 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 lipaseactivity was enhanced by 70% compared to no induction with0.5 mM of IPTG. The lipase activity was slightly decreased to13.9 U/ml at 1.00 mM of IPTG. The optimization of the expressionof recombinant lipase was carried out by varying the inductiontimes with 0.75 mM IPTG. Using this IPTG concentration, at 1 hafter induction, the lipase activity was increased to 6.39 U/ml from

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) 346–354350

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) 346–354 351

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).

S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354352

0.04 U/ml at 0 time induction (data not shown). The lipase activitywas increased drastically after 3 h to about 16 U/ml and 27.7 U/mlafter 6 h time induction.

At 7 h after induction, the lipase activity was as approximatelyequal to that observed at 6 h after induction. The recombinantprotein was optimally expressed at 8 h after induction with IPTG.The lipase activity at this induction time was 34.4 U/ml, andslightly decreased to 31 U/ml and 30.57 U/ml after 9 and 18 h afterinduction, respectively. The protein expression level decreased

Table 1Amino acid composition of S5 lipase and lipases from other organisms.

Percentage of amino acids (%)

Residue S5 KWI-56 Lip3 LipC PK-1

Ala 10.6 16.5 11.1 12 9.6

Arg 20.3 25.9 7.6 3.6 4.1

Asn 3.7 0.4 3.2 4.5 5.1

Asp 2.4 7.3 4.5 4.3 4.1

Cys 3.1 2.9 0.3 0.6 0.9

Gln 2.7 2.9 4.8 3.9 2.6

Glu 3.1 4.3 7.3 4.5 4.1

Gly 8.5 11.4 7.0 10.4 11.9

His 2.4 3.8 2.5 2.6 2.3

Ile 2.7 1.1 3.2 2.3 4.8

Leu 4.4 3.5 14.0 10.0 10.2

Lys 2.4 0.1 2.9 2.9 3.2

Met 1.5 0.2 1.5 1.6 2.6

Phe 0.6 0.8 4.1 3.9 2.9

Pro 7.1 5.2 6.7 4.5 4.5

Ser 11.1 3.5 4.5 7.4 10.3

Thr 5.7 2.1 2.2 6.5 5.1

Trp 2.4 0.4 0.6 0.6 0.6

Tyr 1.5 0.5 2.5 2.6 3.5

Val 3.8 6.6 9.2 11.3 7.0

Total 100 100 100 100 100

Notes: The sequences for calculation of amino acid composition were obtained from G

Pseudomonas sp. KWI-56 (S77842), Lip3: P. aeruginosa Lip3 (AB125368), Lip C: P. aerugino

sp. 42 (AY787835), 205y: B. sphaericus 205y (AF453713), Lip A: C. viscosum LipA (AJ29561

gradually at 27 h, with a 20% loss in lipase activity between 18 hand 27 h. A gradual drop of expression level in a later phase couldbe explained by the outgrowth of non-induced cells containing theplasmid [26].

Expression of the protein was confirmed on SDS-PAGE stainedwith Coomassie Blue. The molecular mass of the target protein wasdetermined by comparing it with a protein marker. A protein bandwas observed at 60 kDa (data not shown) after 8 and 9 h ofinduction. Clear correlation was observed between the intensity ofthe expressed protein and the different induction times. The size ofthe protein band was in agreement with the size of the S5 lipase(60 kDa), as determined in earlier studies [6]. These results suggestthat the S5 lipase gene was successfully cloned and expressed in anE. coli system, with a high level of protein expression.

4.2. Stability of recombinant lipase in various organic solvents

Based on previous studies [5–7], S5 lipase was identified as anorganic solvent-tolerant enzyme. In order to confirm that theorganic solvent-tolerant lipase gene was successfully cloned, thestability of the Top 10/S5/pRSET lipase in the presence and absenceof 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 solventswere adapted from Laane et al. [27]. As shown in Table 2, theenzyme was active in the presence of the organic solvents, withlog P values of 2.0–7.6; however, the enzyme activity decreasedafter prolonged incubation with all of the organic solvents, exceptn-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, n-dodecane 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 1-decanol, 1-octanol, ethyl acetate and benzene, with log P valuesbelow 4.0. This enzyme lost 94% and 89% its activity in ethylacetate and 1-octanol, respectively, compared to the activityobserved in the absence of organic solvents. Surprisingly, this lipS5was also unstable in n-hexane and cyclohexane. However, theenzyme activity was activated in benzene, chloroform and toluenewith log P between 2.0 and 2.5.

2CS 42 205y LipA RAG-1 017

7.9 5.8 14.2 9.8 9.3

6.7 1.8 4.2 2.4 3.8

4.6 4.3 4.2 6.2 3.5

5.5 6.3 4.7 3.8 4.5

0.9 0.2 0.5 1.1 0.6

2.9 4.3 4.7 4.4 4.8

4.8 2.3 1.4 1.7 3.2

10.0 6.3 10.6 11 10.9

3.1 1.8 2.5 2.1 3.5

3.1 8.0 2.8 3.0 5.4

9.3 9.1 9.5 9.7 11.5

2.9 7.1 1.7 3.3 2.6

1.9 3.3 1.3 2.1 1.3

4.8 4.3 2.2 3.3 2.2

4.6 6.3 3.4 4.1 3.8

6.5 7.0 8.1 9.8 8.3

6.0 5.5 10.0 6.2 5.6

2.6 2.5 1.1 0.8 0.6

4.6 4.5 2.8 4.1 3.8

7.0 9.3 10.3 10.9 9.3

100 100 100 100 100

enBank. The abbreviations used were S5: P. aeruginosa S5 (AY787823), KWI-56:

sa PAO1 LipC (AE004894), PK-12CS: P. mendocina PK-12CS (AY091666), 42: Bacillus

6), RAG-1: A. calcoaceticus RAG-1 (AF047691) and 017: Vibrio cholerae 017 (Y00557).

Table 2LipS5 lipase activity in various organic solvents.

Organic solvents log Pa Specific activity (U/mg)b

30 min 2 h

None – 1095.74� 0.013 569.14� 0.017

Ethyl acetate 0.68 596.27� 0.018 38.83� 0.018

Benzene 2.0 1421.28� 0.020 607.00� 0.018

Chloroform 2.0 1375.00� 0.013 658.00� 0.019

Toulene 2.5 1388.30� 0.023 667.02� 0.015

1-Octanol 2.9 1430.32� 0.010 76.60� 0.019

Cyclohexane 3.2 1466.00� 0.015 291.00� 0.018

n-Hexane 3.6 1552.13� 0.012 225.00� 0.013

1-Decanol 4.0 1393.62� 0.016 157.45� 0.016

n-Decane 5.6 1336.17� 0.019 947.87� 0.018

n-Dodecane 6.6 1324.47� 0.023 1171.28� 0.019

n-Tetradecane 7.6 1158.00� 0.018 769.68� 0.020

n-Hexadecane 8.8 947.34� 0.019 381.38� 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).

S.N. Baharum et al. / Process Biochemistry 45 (2010) 346–354 353

5. Discussion

In this study, we cloned and expressed a new organic solvent-tolerant lipase from P. aeruginosa strain S5 using a shot-gun cloningapproach, after several attempts with PCR cloning failed. Previousfailures to express the S5 lipase might have been due to the factthat the Pseudomonas lipase gene needs a helper or activator genein the E. coli systems. According to Iizumi et al. [14], thePseudomonas sp. KWI-56 lipase activity was enhanced in E. coli

by the function of the activator gene that exists downstream of thelipase gene. This finding was similar to that of Iizumi and Fukase[28]. These researchers proposed that the act gene, which isdownstream of the lip gene, is required for the expression of lipaseactivity as a trans-acting factor. In addition, Ogino et al. [9], alsoreported the presence of a lipase-specific foldase involved in theoverexpression of LST-03 lipase in E. coli. However, the details ofthe activation mechanism, and the function of the act gene wereunknown.

Recently, the crystal structure of the lipase and lipase-specificfoldase complex from Burkholderia glumae was determined;however, the mechanism was still not fully clarified [29]. Thelipase activator protein may be a kind of chaperone that mediatesthe folding of the lipase protein. Pseudomonas lipases that areclassified as class I or II need a helper protein, named chaperone,modulator or activator by different authors, that helps in thecorrect folding and thus allows them to be active [30].

Some Gram-negative bacteria have special mechanisms toavoid the degradation of cell components, such as intracellularproteins, periplasmic proteins and membranes, by the endogenousattack of extracellular hydrolyzing enzymes and toxins. Thesynthesis 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 intermolecularchaperone. The Pseudomonas lipase is first produced as a zymogen,and becomes active as a result of the function of an additional geneproduct. The activation of Pseudomonas lipase involves conforma-tional changes in the protein molecule [28].

We believe that this study is the only attempt that has beenmade to investigate the enzyme activity of a solvent-stable lipaseand its chaperone in the presence of organic solvents. Furthermore,Ogino et al. [9,10] reported solely on the activation of the lipasewith a specific foldase, without investigating the stability of thislipase and foldase complex in organic solvents.

In conclusion, we have identified a new organic solvent-tolerant lipase that is activated in E. coli by the function of achaperone, or helper gene. This gene is present in ORF2, whichexists downstream from the lipase gene. We also verified that theS5 lipase was active in the presence of organic solvents.

Acknowledgements

This work was financially supported by Grant No. 09-02-04-0002 from the Malaysian Ministry of Science, Technology andInnovation (MOSTI). The authors acknowledge the contribution ofRobert Coe from the University of Sheffield, United Kingdom, forhis help in correcting the English language of the manuscript.

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