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High Level Expression of Thermostable Lipase fromGeobacillus sp. Strain T1Thean Chor LEOWa, Raja Noor Zaliha Raja Abdul RAHMANa, Mahiran BASRIa & Abu BakarSALLEHa

a Enzyme and Microbial Technology Research Group, Faculty of Science andEnvironmental Studies, Universiti Putra MalaysiaPublished online: 22 May 2014.

To cite this article: Thean Chor LEOW, Raja Noor Zaliha Raja Abdul RAHMAN, Mahiran BASRI & Abu Bakar SALLEH (2004)High Level Expression of Thermostable Lipase from Geobacillus sp. Strain T1, Bioscience, Biotechnology, and Biochemistry,68:1, 96-103, DOI: 10.1271/bbb.68.96

To link to this article: http://dx.doi.org/10.1271/bbb.68.96

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High Level Expression of Thermostable Lipase from Geobacillus sp. Strain T1

Thean Chor LEOW, Raja Noor Zaliha Raja Abdul RAHMAN,y

Mahiran BASRI, and Abu Bakar SALLEH

Enzyme and Microbial Technology Research Group, Faculty of Science and Environmental Studies,

Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia

Received July 4, 2003; Accepted September 2, 2003

A thermostable extracellular lipase of Geobacillus sp.

strain T1 was cloned in a prokaryotic system. Sequence

analysis revealed an open reading frame of 1,251 bp in

length which codes for a polypeptide of 416 amino acid

residues. The polypeptide was composed of a signal

peptide (28 amino acids) and a mature protein of 388

amino acids. Instead of Gly, Ala was substituted as the

first residue of the conserved pentapeptide Gly-X-Ser-

X-Gly. Successful gene expression was obtained with

pBAD, pRSET, pET, and pGEX as under the control of

araBAD, T7, T7 lac, and tac promoters, respectively.

Among them, pGEX had a specific activity of

30.19Umg�1 which corresponds to 2927.15Ug�1 of wet

cells after optimization. The recombinant lipase had an

optimum temperature and pH of 65�C and pH 9,

respectively. It was stable up to 65�C at pH 7 and active

over a wide pH range (pH 6–11). This study presents a

rapid cloning and overexpression, aimed at improving

the enzyme yield for successful industrial application.

Key words: Geobacillus sp.; thermostable lipase; Gluta-

thione S-transferase (GST) fusion protein;

cloning; overexpression

Lipases or acylglycerol hydrolases (E.C.3.1.1.3) areenzymes that catalyze the hydrolysis of long chaintriglycerides with the formation of diacylglycerides,monoglycerides, glycerol, and free fatty acids at theinterface between the insoluble substrate and water.1)

However, lipases are also capable of catalyzing thereverse reaction of hydrolysis in the formation of estersfrom alcohols and fatty acids2) or via transesterifica-tion.3)

Since microbial extracellular lipases are usually morethermostable than animal or plant lipases, they receivedmuch more attention for their potential use in industriesand diagnostics.4) A major requirement for a commercialenzyme is thermal stability, because thermal denatura-tion is a common cause of enzyme inactivation.5) In

addition, increasing enzyme thermostability wouldallow enzymatic reactions to be done at higher temper-atures; this would help to increase conversion rates,substrate solubility, and to reduce the possibility ofmicrobial growth and the viscosity of the reactionmedium.6) Although thermophiles can be good candi-dates in producing thermostable enzymes, it is oftenimpractical because of the low yield. In addition, hightemperature fermentations may need specialized equip-ment.7) As a consequence, a molecular approach throughgenetic engineering becomes a good alternative toachieve high-level expression towards bulk productioneconomically via prokaryotic systems. So far, severalthermostable lipases have been successfully cloned andexpressed in heterologous hosts intracellularly.8–10)

Expression of foreign protein in prokaryotic systemsis the most widely used approach to achieve high-levelexpression; both for fundamental studies and forcommercial purposes.11) In addition, the fast growthrate and ease of cultivation technology for E. coli makeit suitable for industrial application. However, there aretwo goals need to be accomplished during geneexpression, namely high cell density and high-levelgene expression.12) The expression vector and host areimportant issues for achieving maximal expression ofcloned genes. However, molecular cloning of a foreigngene does not ensure that the gene will be expressedsuccessfully.13) The most difficult problems in bacterialexpression are proteolytic degradation and the produc-tion of proteins that accumulate in misfolded forms,most often as inclusion bodies.11)

Previously, 29 putative lipase producers were isolatedfrom Palm Oil Mill Effluent in Malaysia. Among them,the isolate T1, with the highest lipase production of0.15Uml�1, was selected for further study. It wasidentified as Geobacillus sp. strain T1. In this paper, wereport the rapid cloning of thermostable lipase throughthe PCR technique. Manipulation of thermostable T1lipase gene expression was done through prokaryotic

y To whom correspondence should be addressed. Fax: +603-89430913; E-mail: rnzaliha@fsas.upm.edu.my

Abbreviations: sp., spesies; GST, Glutathione S-transferase; B. stearothermophilus, Bacillus stearothermophilus; B. thermoleovorans, Bacillus

thermoleovorans; B. thermocatenulatus, Bacillus thermocatenulatus; E. coli, Escherichia coli; w/o, without; ORF, open reading frame; IPTG,

isopropyl �-D-thiogalactoside; mM, millimolar; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel

electrophoresis

Biosci. Biotechnol. Biochem., 68 (1), 96–103, 2004

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systems under the regulation of various kinds ofpromoters.

Materials and Methods

Bacterial strains and plasmids. Geobacillus sp. strainT1 was grown in a nutrient broth at 60�C and E. coli Top10 [F0 mcrA, �(mrr-hsdRMS-mcrBC), �80lacZ�M15,�lacX74, deoR, recA1, araD139, �(ara-leu)7697,galU, galK, rpsL(StrR), endA1, nupG], BL21(De3)[F� ompT hsdSB(rB

�mB�) gal dcm (DE3)] and

BL21(De3)plysS [F� ompT hsdSB(rB�mB

�) gal dcm(DE3) pLysS(CmR)] were grown in LB medium at37�C. pBAD (Invitrogen; Groningen, Netherlands),pRSET C (Invitrogen; Groningen, Netherlands),pET22b(+) (Novagen; Darmstadt, Germany) andpGEX-4T1 (Amersham Bioscience; United Kingdom,England) were used for cloning, sequencing, andexpression.

DNA manipulation. Genomic DNA from Geobacillussp. Strain T1 was prepared by the method of Sambrooket al.14) with some modification. Plasmid DNA wasisolated with a QIAGEN miniprep spin kit (QIAGEN;Hilden, Germany) according to the manufacturer’sinstructions. The PCR product was purified with aGeneClean Kit (Qbiogene; Carlsbad, USA) as describedby the supplier. Competent cells of E. coli Top 10 andBL21strains were prepared by using a conventionalCaCl2 method.14)

Cloning of the thermostable lipase gene. A pair ofdegenerate primers was designed on the basis of theconserved region among lipases from B. stearothermo-philus, B. thermocatenulatus, and B. thermoleovoransavailable in databases. The primers used were CJH-F1:50-AGS RTG ATG AAA KGC TGY GGG CTK ATGK-30 and CJH-R1: 50-KYW TTA AGG CYG CAA RCTCGC CA-30. The open reading frame (ORF) of thethermostable lipase gene was amplified from thegenomic DNA of Geobacillus sp. strain T1 with TaqDNA polymerase (MBI Fermentas; St. Leon-Rot,Germany) with the following PCR conditions: an initialdenaturation step at 94�C for 4min, 35 cycles at 94�Cfor 1min, annealing at 60�C for 2min, and extension at72�C for 1min, except for the final extension of 7min,and preservation at 4�C.

The PCR product was electrophoresed on a 1% (w/v)agarose gel and purified with a GeneClean Kit (Qbio-gene; Carlsbad, USA). The purified DNA was clonedwith the TOPO TA pBAD vector (Invitrogen; Gronin-gen, Netherlands) according to the manufacturer’sinstructions, used to transform E. coli Top 10 competentcells, and plated on LB agar plate (100�g/ml ampi-cillin). Positive clones were first screened with atributyrin-LB agar plate9) (100�g/ml ampicillin) furtherstreaked onto Triolein15) and Rhodamine-LB agar16)

(100�g/ml ampicillin) and analyzed by PCR.

DNA sequencing. The recombinant plasmid wassequenced with an ABI PRISM 377 DNA automatedsequencer (Applied Biosystems, USA). The nucleotidesequence of the T1 lipase gene was identified anddeposited into Genbank under the accession numberAY260764.

Expression of the thermostable T1 lipase gene.Subcloning of the T1 lipase gene was done by designingvarious sets of primers which incorporated suitablerestriction enzyme sites. The restriction enzyme sitesused for subcloning are as follow: restriction enzymesites BamH1/EcoR1 at pGEX-4T1, Pst1/HindIII atpRSET C, and EcoR1/HindIII at pET22b(+) whichinvolved primers pGEX-For: 50-GAA GGG ATC CGTGAT GAA ATG CTG TCG GAT TAT G-30 and pGEX-Rev: 50-AAT AGA ATT CTT AAG GCT GCA AGCTCG CCA A-30; PH-For: 50-GAA GCT GCA GGT GATGAA ATG C-30 and PH-Rev: 50-AAT AAA GCT TTTAAG GCT GCA A-30; and EH1-For: 50-AGA AGAATT CCG TGA TGA AAT GCT GTC GGA-30 andEH1-Rev: 50-AAT AAA GCT TTT AAG GCC GCAAAC TCG CCA-30, respectively. The ligated plasmidwas used to transform E. coli strains and screened withtributyrin LB agar plates containing appropriate anti-biotics. E. coli strains harboring recombinant plasmidswere grown in 1 L blue cap bottles containing 200ml ofLB medium supplemented with 100�g/ml ampicillinon a rotary shaker (200 rpm) at 37�C. E. coliBL21(De3)plysS was cultured in the presence of35�g/ml chloramphenicol and 100�g/ml ampicillin.The cultures harboring recombinant plasmidspET22b(+), pRSET C, and pGEX-4T1 were inducedwith 1mM of isopropyl-�-D-thiogalactopyranoside(IPTG) at OD600 nm � 0.5 for 8 h. The culture harboringthe recombinant plasmid pBAD was induced with 0.02%of L-arabinose under the same conditions. Cultures(10ml) were harvested by centrifugation and resuspend-ed with 2ml of 50mM of potassium phosphate buffer(pH 7.0) before sonication (Branson 250 sonifier: output2, duty cycle 30 and min 2) and cleared by centrifuga-tion (12,000 rpm, 20min). The clear crude lysate wasput through a lipase assay.17)

Assay of lipase activity. The lipase activity wasassayed colorimetrically.17) Culture filtrate (1ml) wasshaken with 2.5ml of olive oil (70% oleate residues)emulsion (1:1, v/v) and 20�l of 0.02M CaCl2 in awater bath shaker at an agitation rate of 200 rpm. Theemulsion was prepared by mixing together an equalvolume of olive oil (Bertoli, Italy) and 50mM phosphatebuffer with a magnetic stirrer for 10min. The reactionmixture was shaken for 30min at 50�C. The enzymereaction in the emulsion system was stopped by adding6N HCl (1ml) and isooctane (5ml), followed by mixingusing a vortex mixer for 30 s. The upper isooctane layer(4ml) containing the fatty acid was transferred to a testtube for analysis. Copper reagent (1ml) was added and

Heat-stable Lipase from Geobacillus sp. Strain T1 97

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again mixed with a vortex mixer for 30 s. The reagentwas prepared by adjusting the solution of 5% (w/v)copper (II) acetate-1-hydrate to pH 6.1 with pyridine.The absorbance of the upper layer was read at 715 nm.Lipase activity was measured by measuring the amountof free fatty acid released from the standard curves offree fatty acids. One unit of lipase activity was definedas the amount of enzyme releasing 1�mole of fatty acidper minute.

Electrophoresis. SDS-PAGE was done on 12% run-ning gels by using the method of Laemmli.18) A broadrange of protein standard (MBI Fermentas; St. Leon-Rot, Germany) was used as a molecular mass marker.

Characterization of crude T1 lipase. The effects oftemperature on enzyme activity and stability were testedat temperatures ranging from 40 to 80�C at 5�C intervalsfor 30min. The crude recombinant lipase was also keptat a wide range of pH values ranging from pH 4–12 thentested for pH activity and stability determination. Thebuffer systems used were 50mM acetate buffer (pH 4–6),potassium phosphate buffer (pH 6–8), Tris-HCl buffer(pH 8–9), glycine-NaOH buffer (pH 9–11), andNa2HPO3/NaOH buffer (pH 11–12). The temperatureand pH stability were assayed by incubating the crudefusion lipase at various temperatures and pH for 30minbefore testing.

Results and Discussion

PCR cloning of thermostable lipase gene from Geo-bacillus sp. strain T1The characterization of sequence similarity in dis-

tantly related proteins has proved useful for under-standing the evolution of gene families. Nowadays,knowledge of bacterial lipolytic enzymes is increasing ata rapid and exciting rate. Bacterial lipolytic enzymes areclassified based on a comparison of the amino acidsequences and biological properties. Thermostable li-pases from thermophilic bacteria are grouped togetherinto subfamily 5 of family I, which consisted of true

lipases.19) Thermostable lipases are highly conservedwithin the sequences of ORF and more variable up anddownstream from the ORF. With the limited informationon other thermostable lipases, it is possible to clonelipases derived from the same family.

The thermostable T1 lipase gene was amplified fromgenomic DNA of Geobacillus sp. strain T1 by designinga pair of degenerate primers through alignment ofreported sequences of thermostable lipase gene from adatabase (Fig. 1). Cloning of the amplified gene(�1; 260 bp) to TOPO TA pBAD vector (Invitrogen;Groningen, Netherlands) was facilitated by single 30-Toverhang of the vector and a single 30-A overhang of thePCR product. The Taq DNA polymerase conferred 30-Aoverhang to the PCR product, while the TOPO TAcloning vector was supplied linearized with a single 30-Toverhang to enable direct cloning and expression of thePCR product. Primary screening was done with tribu-tyrin-LB agar (100�g/ml ampicillin) due to its sensi-tivity conferred for detection. However, tributyrin is nota good substrate for the lipases because the hydrolysis oftributyrin can be catalyzed by esterases which do notshow true lipase activity.2) Some more specific triolein-LB and Rhodamine B-LB agar plates were used toconfirm the true lipase activity. The positive clone notonly formed a clearing zone on tributyrin-LB agar platesbut also conferred intense blue color and orangefluorescence on triolein-LB agar plates and RhodamineB-LB agar plates (data not shown).

Nucleotide sequence and deduced amino acid se-quence of T1 lipase

Sequencing of the T1 lipase gene showed that it was1,251 bp in length, which codes for 416 amino acids(Fig. 2). The deduced molecular mass and pI werecalculated to be 46.309 kDa and 6.36, respectively. TheShine-Dalgarno sequence, �35 and �10 promoter re-gions upstream from initiation codon ATG at position1 were predicted online (http.www.fruitfly.orgseqtoolspromoter.html). The putative signal peptide cleav-age site was located at between Ala-28 and Ala-29 whenpredicted by using the SignalP V2.0 world wide web

BTL : -6 5'-AGCGTGATGAAATGCTGTCGGGTTATGTTTGT-3' 26BST : -3 5'-AGGATGATGAAAGGCTGCCGGGTGATGGTTGT-3' 29BTC : -3 5'-AGGATGATGAAAGGCTGCCGGGTGATGGTTGT-3' 29CJH-F1 : ****************************

BTL :1227 5'-CCGAGCAGTTGGCGAGCTTGCGGCCTTAAAAC-3' 1254BST :1230 5'-CCGAGCAACTGGCGAGTTTGCGGCCTTAAAAC-3' 1257BTC :1230 5'-CCGAGCAGTTGGCGAGTTTGCGGCCTTAATGA-3' 1257CJH-R1 : ***********************

Fig. 1. Sequences Alignment of Reported Thermostable Lipases from Database through CLUSTALW Multiple Sequence Alignment from Biology

Workbench.

Symbols used are: BTL, B. thermoleovorans (AF134840); BST, B. stearothermophilus (U78785) and BTC, B. thermocatenulatus (X95309).

The asterisks showed the sequences of degenerated primers.

98 T. C. LEOW et al.

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server (http.www.cbs.dtu.dkservicesSignalP-2.0.html).20) Therefore, the deduced mature lipase wascomposed of 388 amino acids, which correspond to amolecular mass of 43.195 kDa. Like others Bacilluslipases, an Ala replaces the first Gly residue in theconserved Gly-Xaa-Ser-Xaa-Gly, which is conservedamong microbial and mammalian lipases. Figure 3shows an Ala residue replacing Gly in all thermostablelipases from Bacillus spp. as compared to lipases fromother genus such as Psychrobacter immobilis B10,21)

Pseudomonas fluorescens B52,22) and Staphylococcushaemolyticus.23) PSI-BLAST of an amino acids se-quence showed high homology of T1 lipase with otherthermostable lipases from B. thermoleovorans,8) B.thermocatenulatus,10) and B. stearothermophilus.9)

Expression of T1 lipase geneA good combination of expression system and host is

necessary to obtain high-level expression. Nowadays, E.coli remains a valuable organism for overproduction of

GCGGTGATGGAACGCTGCCATGACATAGTGCATCACCCTCTCGCTGTCGGCGAGAAACGA*********************** AATATGACGGCACAAAAGTGCATTTTTCTCTTTCCCTCATCAGAAAACCCGACAATTGCC GGGACTGAATAGCCTGATTATTTAGTATAGAATATTCAGGTAATTCGGAATAAAGGGGTT

- 35 - 10

CAGTCGATTCGAGGGGAGGAGAAGGAGAGCGTGATGAAATGCTGTCGGATTATGTTTGTG

RBS M K C C R I M F V

TTGCTCGGATTATGGTTTGTGTTCGGCCTATCGGTCCCGGGAGGGCGGACGGAAGCGGCA

L L G L W F V F G L S V P G G R T E A ↑A

TCCCTACGCGCCAATGATGCACCGATTGTGCTTCTCCATGGGTTTACCGGATGGGGACGA

S L R A N D A P I V L L H G F T G W G R

GAGGAAATGTTTGGATTCAAGTATTGGGGCGGCGTGCGCGGCGATATCGAACAATGGCTG

E E M F G F K Y W G G V R G D I E Q W L

AACGACAACGGTTATCGAACGTTTACGCTGGCGGTCGGACCGCTCTCGAGCAACTGGGAC

N D N G Y R T F T L A V G P L S S N W D

CGGGCGTGTGAAGCGTATGCTCAGCTTGTCGGCGGGACGGTCGATTATGGGGCAGCCCAT

R A C E A Y A Q L V G G T V D Y G A A H

GCGGCAAAGCACGGCCATGCGCGGTTTGGCCGCACTTATCCCGGCCTGTTGCCGGAATTG

A A K H G H A R F G R T Y P G L L P E L

AAAAGGGGTGGCCGCATCCATATCATCGCCCACAGCCAAGGGGGGCAGACGGCCCGCATG

K R G G R I H I I A H S Q G G Q T A R M

CTTGTCTCGCTCCTAGAGAACGGAAGCCAAGAAGAGCGGGAGTACGCCAAGGCGCATAAC

L V S L L E N G S Q E E R E Y A K A H N

GTGTCGTTGTCACCGTTGTTTGAAGGTGGACATCATTTTGTGTTGAGTGTGACGACCATC

V S L S P L F E G G H H F V L S V T T I

GCCACTCCTCATGACGGGACGACGCTTGTCAACATGGTTGATTTCACCGATCGCTTTTTT

A T P H D G T T L V N M V D F T D R F F

GACTTGCAAAAAGCGGTGTTGGAAGCGGCGGCTGTCGCCAGCAACGTGCCGTACACGAGT

D L Q K A V L E A A A V A S N V P Y T S

CAAGTATACGATTTTAAGCTCGACCAATGGGGACTGCGCCGCCAGCCGGGTGAATCGTTC

Q V Y D F K L D Q W G L R R Q P G E S F

GACCATTATTTTGAACGGCTCAAGCGCTCCCCTGTTTGGACGTCCACAGATACCGCCCGC

D H Y F E R L K R S P V W T S T D T A R

TACGATTTATCCGTTTCCGGAGCTGAGAAGTTGAATCAATGGGTGCAAGCAAGCCCGAAT

Y D L S V S G A E K L N Q W V Q A S P N

ACGTATTATTTGAGTTTCTCTACAGAACGGACGTATCGCGGAGCGCTCACAGGCAACCAT

T Y Y L S F S T E R T Y R G A L T G N H

TATCCCGAACTCGGAATGAATGCATTCAGCGCGGTCGTATGCGCTCCGTTTCTCGGTTCG

Y P E L G M N A F S A V V C A P F L G S

TACCGCAATCCGACGCTCGGCATTGACGACCGATGGTTGGAGAACGATGGCATTGTCAAT

Y R N P T L G I D D R W L E N D G I V N

ACGGTTTCCATGAACGGTCCAAAGCGTGGATCAAGCGATCGGATCGTGCCGTATGACGGG

T V S M N G P K R G S S D R I V P Y D G

ACGTTGAAAAAAGGGGTTTGGAATGATATGGGAACGTACAACGTCGACCATTTGGAAATC

T L K K G V W N D M G T Y N V D H L E I

ATCGGCGTTGACCCGAATCCGTCATTTGATATTCGCGCCTTTTATTTGCGGCTTGCCGAG

I G V D P N P S F D I R A F Y L R L A E

CAGTTGGCGAGCTTGCAGCCTTAAAACGAGTATTTTGCGAAAAAGCCATCTCGATCGGAT

Q L A S L Q P -

GGCTCTTTTTATGGAAAAGTTCCCATGGGCAGCGCGCTTTGCCCTCCACCCGGGGATGAA

ATTCACGCGTCAGTCCTGCATGTTCACGGAAAAGTCTCATGGGCGACTCGCTCCCCTAAA

CCCGGGGATGAAAATGACTATCAAGAAGTATGCATGTTCACGGAAAAGGTCCATGGGCAA

CGGTTTGCCCTCTACCCAAGGATAAAAATGACCCATCCCAGCACGTGCATTTTCACAGAA

GCGCATTATGTTGTATGACGGCCGCGGATGTCCGATGGTATAATAAGGGTAAAGCAAGCG

GATGGGGGAATGGACGTGAAGCCGCCCGAGCGGCAAAAGGAACGGTATACGTATCAAGAT

TATGTCAAGTGGGACGGACGGTGGGAGCTGATCAACGGCGTGCCCTACAACATGGCACCG

ACCCCTTCATTTGTCCGCCAGTCTATCGTCGG *************************

Fig. 2. The Nucleotide and Amino Acid Sequences of the T1 Lipase Gene from Geobacillus sp. Strain T1.

The predicted promoter region (�10 and �35 promoter) and ribosome binding site (RBS) are underlined. The head arrow indicates the signal

peptide cleavage site of the T1 lipase gene. The inverted repeat sequence downstream is indicated by horizontal arrows. A conserved

pentapeptide among thermostable lipases is indicated by the box. The asterisk indicated the primers used for full length T1 lipase gene cloning.

The T1 lipase sequence has been submitted to the GenBank database under the accession number AY260764.

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recombinant proteins intracellularly.11) However, inspite of the extensive knowledge of the genetics andmolecular biology of E. coli, not every gene can beexpressed effectively in this organism. Basically, geneexpression could be achieved through high-level geneexpression by increasing the amount of expressedprotein up to 50% of the total cell protein through genemanipulation and proper choosing of the vector and hostcombination. Alternatively, fed-batch fermentation canfulfill the requirement for high cell density to improvethe level of expression from a certain volume ofcultures. In this study, a thermostable T1 lipase genefrom Geobacillus sp. strain T1 was expressed withpBAD, pRSET, pET, and pGEX under the regulation ofpromoters araBAD, T7, T7 lac, and tac, respectively(Table 1). These systems allow regulation of the gene of

interest under the control of different promoters, whichallows chemically inducible, high level expression.

Initially, a thermostable T1 lipase gene was clonedand expressed in the pBAD expression system down-stream of the araBAD promoter for tightly-regulatedexpression in E. coli Top 10 with 0.02% of L-arabinoseas inducer. The active recombinant lipase obtained wasaround 8.76Umg�1 after 8 h of induction time. The T1lipase gene was further expressed with pRSET C (T7promoter) and the specific activity of around6.41Umg�1 and 6.35Umg�1 of protein was achievedin E. coli BL21(De3) and E. coli BL21(De3)plysS,respectively when induced with 1mM IPTG under thesame conditions. However, growth was severely im-paired by protein expression with this system because ofthe leaky system conferred by the T7 promoter. There-fore, a more tightly regulated expression systempET22b(+) under the control of the T7 lac promoterwas further tested for T1 lipase gene expression. Itcarries an N-terminal pelB signal sequence for potentialperiplasmic localization. Unfortunately, this particularexpression system had only a low level of expressionwith the host E. coli BL21(De3) (0.79Umg�1) and E.coli BL21(De3) plysS (2.54Umg�1) upon inductionwith 1mM IPTG. Next, expression of the T1 lipase genewas done under the regulation of the tac promoter as afusion protein (Fig. 4). This expression system greatlyincreased the level of expression in E. coli BL21(De3)(15.49Umg�1) and E. coli BL21(De3)plysS (20.02Umg�1) when induced at OD600 nm � 0.5 with 1mMof IPTG for 8 h. The expression level obtained by thepGEX system in E. coli BL21(De3) plysS was around 3-and 7-fold higher than pRSET and pET systems underthe same conditions. Based on this, the pGEX systemwas chosen for further study. Enzymatic assay showedthat 1.5-fold increases in lipase expression were encoun-

Geobacillus sp. strain T1 135 I H I I A H S Q G G Q 145

B. thermoleovorans ID-1 135 I H I I A H S Q G G Q 145

B. stearothermophilus L1 136 V H I I A H S Q G G Q 146

B. thermocatenulatus 136 V H I I A H S Q G G Q 146

Psychrobacter immobilis B10 136 I H V G G N S M G G A 146

Pseudomonas fluorescens B52 201 V V V S G H S L G G L 211

Staphylococcus haemolyticus 432 I H L I G H S M G G Q 442

Fig. 3. Conserved Region of Several Bacterial Lipases.

Open box indicates conserved pentapeptide of bacterial lipases. Amino acid sequences were obtained from the following sources. B.

thermoleovorans ID-1 (AF134840), B. stearothermophilus L1 (U78785), B. thermocatenulatus (X95309), Psychrobacter immobilis B10

(X67712), Pseudomonas fluorescens B52 (M863560) and Staphylococcus haemolyticus (AF096928).

Table 1. Expression of T1 Lipase Gene with Various Expression

Systems

ExpressionPromoters Hosts

Expression level

systems (Umg�1)

pBAD araBAD E. coli Top 10 8.76

pRSET C T7 E. coli BL21(De3) 6.41

E. coli BL21(De3)plysS 6.35

pET22b(+) T7 lac E. coli BL21(De3) 0.79

E. coli BL21(De3)plysS 2.54

pGEX-4T1 tac E. coli BL21(De3) 15.48

E. coli BL21(De3)plysS 20.02

Note: Crude cell lysate were prepared with a sonicator. The soluble fractions

were assayed colorimetrically with olive oil as substrate. One unit of lipase

activity was defined as 1�mole of liberated fatty acid per minute the under

the assay conditions. The cultures were induced at an OD600 nm � 0.5 with

0.02% of L-arabinose (pBAD) and 1mM of IPTG (pRSET C, pET22b(+),

pGEX-4T1) at 37�C for 8 h.

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tered with 0.05mM of IPTG as inducer at 8 h ofinduction time with specific activity around30.19Umg�1, which corresponds to 2927.15Ug�1 ofwet cells (Fig. 5A).

A comparison study of expressed protein amongdifferent lipases was difficult due to the variation inassay methods and units of expression. T1 lipase geneexpression in the pGEX system was higher than theexpression level obtained by a thermostable lipase fromBacillus thermoleovorans ID-1 in the two-plasmidssystem of pET22b(+) and pGP1-2.8) The expressionlevel of lipase from Bacillus thermoleovorans ID-1 wasaround 320U/g cells, which is 9 times lower than theexpression of T1 lipase in the pGEX system. In anotherstudy, intracellular expression of Bacillus licheniformisin pET20b(+) upon induction with IPTG at 30�C wasaround 300 units from 50ml culture,24) which wasslightly lower than the T1 lipase gene expression ofaround 585.43 units from the same volume of culture.

In fact, expression of T1 lipase as a fusion protein isfeasible for simple detection and purification. Protease-

deficient host E. coli BL21 host strains, which are lonand ompT proteases deficient, were used for expressionto reduce degradation of the fusion protein. As shown inFig. 5B, the molecular mass of fusion protein and freeGST tag was around 63 kDa and 26 kDa, respectivelywhen analyzed with SDS-PAGE.

Characterization of crude recombinant lipaseThe crude fusion lipase had an optimum temperature

of 65�C when olive oil was used as substrate and wasstable up to 65�C for 30min (Fig. 6) when assayed at pH7.0 for 30min, which is somewhat higher than lipasesfrom Bacillus stearothermophilus L1 and Bacillusthermocatenulatus.9,10) As shown in Fig. 7A, an opti-mum pH of the recombinant lipase was around pH 9. It

Fig. 4. Constructions of Recombinant Plasmid pGEX/T1.

Thermostable T1 lipase gene was subcloned into pGEX-4T1

vector for fusion protein expression.

0

10

20

30

40

w/o 0.025 0.05 0.1 0.5 1 1.5 2

IPTG (mM)

Spec

ific

act

ivit

y (U

/mg)

A

GST

Fusionprotein

M 1 2 3 4 5 6 7 8 9

116

66.2

45

35

25

18.4

kDa

B

Fig. 5. Expressed Fusion Protein at Various Inducer (IPTG) Con-

centrations.

A) Enzymatic assay of recombinant T1 lipase. (open column), E.

coli BL21(De3)plysS harboring recombinant plasmid pGEX/T1.

The culture was grown in 1L blue cap bottle containing 200ml of

LB medium containing 100�g/ml ampicillin and 35�g/ml chlor-

amphenicol at 37�C under a shaking rate of 200 rpm. It was induced

with different concentrations of IPTG (w/o, 0.025, 0.05, 0.1, 1, 1.5

and 2mM) at OD600 nm � 0.5. B) SDS-PAGE (12%) of expressed T1

lipase. M: standard protein markers were �-galactosidase (116 kDa),bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate

dehydrogenase (35 kDa), restriction endonuclease Bsp 98l (25 kDa),

and �-lactoglobulin (18.4 kDa), without IPTG (lane 1), 0.025mM

(lane 2), 0.050mM (lane 3), 0.100mM (lane 4), 0.500mM (lane 5),

1mM (lane 6), 1.5mM (lane 7), 2mM (lane 8) and GST (lane 9).

Arrows indicate GST fusion protein and GST.

Heat-stable Lipase from Geobacillus sp. Strain T1 101

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was stable over wide ranges of pH ranging from pH 6–11. The activity was, however, drastically decreased atpH above 11 (Fig. 7B). The optimum pH of T1 lipase(pH 9) was nearly identical to lipases from otherthermophilic bacteria such as Bacillus thermoleovoransID-1, Bacillus stearothermophilus L1, and Bacillusthermocatenulatus8–10) (pH 8–9) but lower than lipasesfrom mesophilic counterpart such as Bacillus lichen-iformis21) (pH 10.5).T1 thermostable lipase was highly active at alkaline

pH (up to pH 11) and stable and widely active attemperatures up to 65�C. These properties indicate itspotential when used in detergent formulation. Furthergenetic manipulation by site-directed mutagenesis of thecloned gene opens new possibilities for the introductionof pre-designed changes, resulting in the production oftailor-made lipases with novel and desirable properties.Furthermore, response surface methodology25) in de-scribing the relationship between tested variables andexpression level, could be used to optimize thermostableT1 lipase gene expression during scale-up fermentationto develop a more accurate bioprocess for industrialapplication in a shorter time.PCR cloning is a feasible way to clone genes of

interest from the same family of lipases. High-levelexpression could be achieved through molecular ap-proaches with various expression systems. Fusionprotein allows the rapid purification and detection ofexpressed protein. This study showed that the pGEXexpression system conferred a higher level of expressionover the expression vectors pBAD, pRSET C, andpET22b(+).

Acknowledgments

This research was supported financially by theMinistry of Science, Technology, and Environment,Malaysia (09-02-04-0336-EA001).

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0

50

100

4 5 6 8 10 11 12

pH

Rel

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e ac

tivi

ty (

%)

7 9

A

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50

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