Biosci. Biotechnol. Biochem., 68 (1), 96–103, 2004
High Level Expression of Thermostable Lipase from Geobacillus sp. Strain T1 Thean Chor L EOW, Raja Noor Zaliha Raja Abdul R AHMAN,y Mahiran B ASRI, 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-SerX-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 30.19 Umg 1 which corresponds to 2927.15 Ug 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; Glutathione S-transferase (GST) fusion protein; cloning; overexpression
Lipases or acylglycerol hydrolases (E.C.3.1.1.3) are enzymes that catalyze the hydrolysis of long chain triglycerides with the formation of diacylglycerides, monoglycerides, glycerol, and free fatty acids at the interface between the insoluble substrate and water.1) However, lipases are also capable of catalyzing the reverse reaction of hydrolysis in the formation of esters from alcohols and fatty acids2) or via transesterification.3) Since microbial extracellular lipases are usually more thermostable than animal or plant lipases, they received much more attention for their potential use in industries and diagnostics.4) A major requirement for a commercial enzyme is thermal stability, because thermal denaturation is a common cause of enzyme inactivation.5) In y
addition, increasing enzyme thermostability would allow enzymatic reactions to be done at higher temperatures; this would help to increase conversion rates, substrate solubility, and to reduce the possibility of microbial growth and the viscosity of the reaction medium.6) Although thermophiles can be good candidates in producing thermostable enzymes, it is often impractical because of the low yield. In addition, high temperature fermentations may need specialized equipment.7) As a consequence, a molecular approach through genetic engineering becomes a good alternative to achieve high-level expression towards bulk production economically via prokaryotic systems. So far, several thermostable lipases have been successfully cloned and expressed in heterologous hosts intracellularly.8–10) Expression of foreign protein in prokaryotic systems is the most widely used approach to achieve high-level expression; both for fundamental studies and for commercial purposes.11) In addition, the fast growth rate and ease of cultivation technology for E. coli make it suitable for industrial application. However, there are two goals need to be accomplished during gene expression, namely high cell density and high-level gene expression.12) The expression vector and host are important issues for achieving maximal expression of cloned genes. However, molecular cloning of a foreign gene does not ensure that the gene will be expressed successfully.13) The most difficult problems in bacterial expression are proteolytic degradation and the production of proteins that accumulate in misfolded forms, most often as inclusion bodies.11) Previously, 29 putative lipase producers were isolated from Palm Oil Mill Effluent in Malaysia. Among them, the isolate T1, with the highest lipase production of 0.15 Uml1 , was selected for further study. It was identified as Geobacillus sp. strain T1. In this paper, we report the rapid cloning of thermostable lipase through the PCR technique. Manipulation of thermostable T1 lipase gene expression was done through prokaryotic
To whom correspondence should be addressed. Fax: +603-89430913; E-mail:
[email protected] 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
Heat-stable Lipase from Geobacillus sp. Strain T1
systems under the regulation of various kinds of promoters.
Materials and Methods Bacterial strains and plasmids. Geobacillus sp. strain T1 was grown in a nutrient broth at 60 C and E. coli Top 10 [F0 mcrA, (mrr-hsdRMS-mcrBC), 80lacZM15, 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 at 37 C. pBAD (Invitrogen; Groningen, Netherlands), pRSET C (Invitrogen; Groningen, Netherlands), pET22b(+) (Novagen; Darmstadt, Germany) and pGEX-4T1 (Amersham Bioscience; United Kingdom, England) were used for cloning, sequencing, and expression. DNA manipulation. Genomic DNA from Geobacillus sp. Strain T1 was prepared by the method of Sambrook et al.14) with some modification. Plasmid DNA was isolated with a QIAGEN miniprep spin kit (QIAGEN; Hilden, Germany) according to the manufacturer’s instructions. The PCR product was purified with a GeneClean Kit (Qbiogene; Carlsbad, USA) as described by the supplier. Competent cells of E. coli Top 10 and BL21strains were prepared by using a conventional CaCl2 method.14) Cloning of the thermostable lipase gene. A pair of degenerate primers was designed on the basis of the conserved region among lipases from B. stearothermophilus, B. thermocatenulatus, and B. thermoleovorans available in databases. The primers used were CJH-F1: 50 -AGS RTG ATG AAA KGC TGY GGG CTK ATG K-30 and CJH-R1: 50 -KYW TTA AGG CYG CAA RCT CGC CA-30 . The open reading frame (ORF) of the thermostable lipase gene was amplified from the genomic DNA of Geobacillus sp. strain T1 with Taq DNA polymerase (MBI Fermentas; St. Leon-Rot, Germany) with the following PCR conditions: an initial denaturation step at 94 C for 4 min, 35 cycles at 94 C for 1 min, annealing at 60 C for 2 min, and extension at 72 C for 1 min, except for the final extension of 7 min, and preservation at 4 C. The PCR product was electrophoresed on a 1% (w/v) agarose gel and purified with a GeneClean Kit (Qbiogene; Carlsbad, USA). The purified DNA was cloned with the TOPO TA pBAD vector (Invitrogen; Groningen, Netherlands) according to the manufacturer’s instructions, used to transform E. coli Top 10 competent cells, and plated on LB agar plate (100 g/ml ampicillin). Positive clones were first screened with a tributyrin-LB agar plate9) (100 g/ml ampicillin) further streaked onto Triolein15) and Rhodamine-LB agar16) (100 g/ml ampicillin) and analyzed by PCR.
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DNA sequencing. The recombinant plasmid was sequenced with an ABI PRISM 377 DNA automated sequencer (Applied Biosystems, USA). The nucleotide sequence of the T1 lipase gene was identified and deposited into Genbank under the accession number AY260764. Expression of the thermostable T1 lipase gene. Subcloning of the T1 lipase gene was done by designing various sets of primers which incorporated suitable restriction enzyme sites. The restriction enzyme sites used for subcloning are as follow: restriction enzyme sites BamH1/EcoR1 at pGEX-4T1, Pst1/HindIII at pRSET C, and EcoR1/HindIII at pET22b(+) which involved primers pGEX-For: 50 -GAA GGG ATC CGT GAT GAA ATG CTG TCG GAT TAT G-30 and pGEXRev: 50 -AAT AGA ATT CTT AAG GCT GCA AGC TCG CCA A-30 ; PH-For: 50 -GAA GCT GCA GGT GAT GAA ATG C-30 and PH-Rev: 50 -AAT AAA GCT TTT AAG GCT GCA A-30 ; and EH1-For: 50 -AGA AGA ATT CCG TGA TGA AAT GCT GTC GGA-30 and EH1-Rev: 50 -AAT AAA GCT TTT AAG GCC GCA AAC TCG CCA-30 , respectively. The ligated plasmid was used to transform E. coli strains and screened with tributyrin LB agar plates containing appropriate antibiotics. E. coli strains harboring recombinant plasmids were grown in 1 L blue cap bottles containing 200 ml of LB medium supplemented with 100 g/ml ampicillin on a rotary shaker (200 rpm) at 37 C. E. coli BL21(De3)plysS was cultured in the presence of 35 g/ml chloramphenicol and 100 g/ml ampicillin. The cultures harboring recombinant plasmids pET22b(+), pRSET C, and pGEX-4T1 were induced with 1 mM of isopropyl--D-thiogalactopyranoside (IPTG) at OD600 nm 0.5 for 8 h. The culture harboring the recombinant plasmid pBAD was induced with 0.02% of L-arabinose under the same conditions. Cultures (10 ml) were harvested by centrifugation and resuspended with 2 ml of 50 mM of potassium phosphate buffer (pH 7.0) before sonication (Branson 250 sonifier: output 2, duty cycle 30 and min 2) and cleared by centrifugation (12,000 rpm, 20 min). The clear crude lysate was put through a lipase assay.17) Assay of lipase activity. The lipase activity was assayed colorimetrically.17) Culture filtrate (1 ml) was shaken with 2.5 ml of olive oil (70% oleate residues) emulsion (1:1, v/v) and 20 l of 0.02 M CaCl2 in a water bath shaker at an agitation rate of 200 rpm. The emulsion was prepared by mixing together an equal volume of olive oil (Bertoli, Italy) and 50 mM phosphate buffer with a magnetic stirrer for 10 min. The reaction mixture was shaken for 30 min at 50 C. The enzyme reaction in the emulsion system was stopped by adding 6 N HCl (1 ml) and isooctane (5 ml), followed by mixing using a vortex mixer for 30 s. The upper isooctane layer (4 ml) containing the fatty acid was transferred to a test tube for analysis. Copper reagent (1 ml) was added and
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BTL BST BTC CJH-F1
: : : :
-6 -3 -3
BTL BST BTC CJH-R1
:1227 :1230 :1230 :
5'-AGCGTGATGAAATGCTGTCGGGTTATGTTTGT-3' 5'-AGGATGATGAAAGGCTGCCGGGTGATGGTTGT-3' 5'-AGGATGATGAAAGGCTGCCGGGTGATGGTTGT-3' ****************************
26 29 29
5'-CCGAGCAGTTGGCGAGCTTGCGGCCTTAAAAC-3' 1254 5'-CCGAGCAACTGGCGAGTTTGCGGCCTTAAAAC-3' 1257 5'-CCGAGCAGTTGGCGAGTTTGCGGCCTTAATGA-3' 1257 ***********************
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.
again mixed with a vortex mixer for 30 s. The reagent was 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 amount of free fatty acid released from the standard curves of free fatty acids. One unit of lipase activity was defined as the amount of enzyme releasing 1 mole of fatty acid per minute. Electrophoresis. SDS-PAGE was done on 12% running gels by using the method of Laemmli.18) A broad range of protein standard (MBI Fermentas; St. LeonRot, Germany) was used as a molecular mass marker. Characterization of crude T1 lipase. The effects of temperature on enzyme activity and stability were tested at temperatures ranging from 40 to 80 C at 5 C intervals for 30 min. The crude recombinant lipase was also kept at a wide range of pH values ranging from pH 4–12 then tested for pH activity and stability determination. The buffer systems used were 50 mM acetate buffer (pH 4–6), potassium phosphate buffer (pH 6–8), Tris-HCl buffer (pH 8–9), glycine-NaOH buffer (pH 9–11), and Na2 HPO3 /NaOH buffer (pH 11–12). The temperature and pH stability were assayed by incubating the crude fusion lipase at various temperatures and pH for 30 min before testing.
lipases.19) Thermostable lipases are highly conserved within the sequences of ORF and more variable up and downstream from the ORF. With the limited information on other thermostable lipases, it is possible to clone lipases derived from the same family. The thermostable T1 lipase gene was amplified from genomic DNA of Geobacillus sp. strain T1 by designing a pair of degenerate primers through alignment of reported sequences of thermostable lipase gene from a database (Fig. 1). Cloning of the amplified gene (1; 260 bp) to TOPO TA pBAD vector (Invitrogen; Groningen, Netherlands) was facilitated by single 30 -T overhang of the vector and a single 30 -A overhang of the PCR product. The Taq DNA polymerase conferred 30 -A overhang to the PCR product, while the TOPO TA cloning vector was supplied linearized with a single 30 -T overhang to enable direct cloning and expression of the PCR product. Primary screening was done with tributyrin-LB agar (100 g/ml ampicillin) due to its sensitivity conferred for detection. However, tributyrin is not a good substrate for the lipases because the hydrolysis of tributyrin can be catalyzed by esterases which do not show true lipase activity.2) Some more specific trioleinLB and Rhodamine B-LB agar plates were used to confirm the true lipase activity. The positive clone not only formed a clearing zone on tributyrin-LB agar plates but also conferred intense blue color and orange fluorescence on triolein-LB agar plates and Rhodamine B-LB agar plates (data not shown).
Results and Discussion PCR cloning of thermostable lipase gene from Geobacillus sp. strain T1 The characterization of sequence similarity in distantly related proteins has proved useful for understanding the evolution of gene families. Nowadays, knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. Bacterial lipolytic enzymes are classified based on a comparison of the amino acid sequences and biological properties. Thermostable lipases from thermophilic bacteria are grouped together into subfamily 5 of family I, which consisted of true
Nucleotide sequence and deduced amino acid sequence of T1 lipase Sequencing of the T1 lipase gene showed that it was 1,251 bp in length, which codes for 416 amino acids (Fig. 2). The deduced molecular mass and pI were calculated to be 46.309 kDa and 6.36, respectively. The Shine-Dalgarno sequence, 35 and 10 promoter regions upstream from initiation codon ATG at position 1 were predicted online (http.www.fruitfly.orgseq toolspromoter.html). The putative signal peptide cleavage site was located at between Ala-28 and Ala-29 when predicted by using the SignalP V2.0 world wide web
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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
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G
G
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T
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A↑A
TATCCCGAACTCGGAATGAATGCATTCAGCGCGGTCGTATGCGCTCCGTTTCTCGGTTCG Y
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A
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A
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TCCCTACGCGCCAATGATGCACCGATTGTGCTTCTCCATGGGTTTACCGGATGGGGACGA S
L
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A
N
D
A
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I
V
L
L
H
G
F
T
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G
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GAGGAAATGTTTGGATTCAAGTATTGGGGCGGCGTGCGCGGCGATATCGAACAATGGCTG E
E
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G
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AACGACAACGGTTATCGAACGTTTACGCTGGCGGTCGGACCGCTCTCGAGCAACTGGGAC N
D
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T
F
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A
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CGGGCGTGTGAAGCGTATGCTCAGCTTGTCGGCGGGACGGTCGATTATGGGGCAGCCCAT R
A
C
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A
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GCGGCAAAGCACGGCCATGCGCGGTTTGGCCGCACTTATCCCGGCCTGTTGCCGGAATTG A
A
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AAAAGGGGTGGCCGCATCCATATCATCGCCCACAGCCAAGGGGGGCAGACGGCCCGCATG K
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G
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I
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TACCGCAATCCGACGCTCGGCATTGACGACCGATGGTTGGAGAACGATGGCATTGTCAAT Y
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ACGGTTTCCATGAACGGTCCAAAGCGTGGATCAAGCGATCGGATCGTGCCGTATGACGGG T
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ACGTTGAAAAAAGGGGTTTGGAATGATATGGGAACGTACAACGTCGACCATTTGGAAATC T
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ATCGGCGTTGACCCGAATCCGTCATTTGATATTCGCGCCTTTTATTTGCGGCTTGCCGAG I
G
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CAGTTGGCGAGCTTGCAGCCTTAAAACGAGTATTTTGCGAAAAAGCCATCTCGATCGGAT Q
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GGCTCTTTTTATGGAAAAGTTCCCATGGGCAGCGCGCTTTGCCCTCCACCCGGGGATGAA
CTTGTCTCGCTCCTAGAGAACGGAAGCCAAGAAGAGCGGGAGTACGCCAAGGCGCATAAC L
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GTGTCGTTGTCACCGTTGTTTGAAGGTGGACATCATTTTGTGTTGAGTGTGACGACCATC V
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GCCACTCCTCATGACGGGACGACGCTTGTCAACATGGTTGATTTCACCGATCGCTTTTTT A
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GACTTGCAAAAAGCGGTGTTGGAAGCGGCGGCTGTCGCCAGCAACGTGCCGTACACGAGT D
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CAAGTATACGATTTTAAGCTCGACCAATGGGGACTGCGCCGCCAGCCGGGTGAATCGTTC Q
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ATTCACGCGTCAGTCCTGCATGTTCACGGAAAAGTCTCATGGGCGACTCGCTCCCCTAAA CCCGGGGATGAAAATGACTATCAAGAAGTATGCATGTTCACGGAAAAGGTCCATGGGCAA CGGTTTGCCCTCTACCCAAGGATAAAAATGACCCATCCCAGCACGTGCATTTTCACAGAA GCGCATTATGTTGTATGACGGCCGCGGATGTCCGATGGTATAATAAGGGTAAAGCAAGCG GATGGGGGAATGGACGTGAAGCCGCCCGAGCGGCAAAAGGAACGGTATACGTATCAAGAT TATGTCAAGTGGGACGGACGGTGGGAGCTGATCAACGGCGTGCCCTACAACATGGCACCG ACCCCTTCATTTGTCCGCCAGTCTATCGTCGG *************************
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GACCATTATTTTGAACGGCTCAAGCGCTCCCCTGTTTGGACGTCCACAGATACCGCCCGC D
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TACGATTTATCCGTTTCCGGAGCTGAGAAGTTGAATCAATGGGTGCAAGCAAGCCCGAAT Y
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ACGTATTATTTGAGTTTCTCTACAGAACGGACGTATCGCGGAGCGCTCACAGGCAACCAT T
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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.
server (http.www.cbs.dtu.dkservicesSignalP-2.0. html).20) Therefore, the deduced mature lipase was composed of 388 amino acids, which correspond to a molecular mass of 43.195 kDa. Like others Bacillus lipases, an Ala replaces the first Gly residue in the conserved Gly-Xaa-Ser-Xaa-Gly, which is conserved among microbial and mammalian lipases. Figure 3 shows an Ala residue replacing Gly in all thermostable lipases from Bacillus spp. as compared to lipases from other genus such as Psychrobacter immobilis B10,21)
Pseudomonas fluorescens B52,22) and Staphylococcus haemolyticus.23) PSI-BLAST of an amino acids sequence showed high homology of T1 lipase with other thermostable lipases from B. thermoleovorans,8) B. thermocatenulatus,10) and B. stearothermophilus.9) Expression of T1 lipase gene A good combination of expression system and host is necessary to obtain high-level expression. Nowadays, E. coli remains a valuable organism for overproduction of
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Geobacillus sp. strain T1
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I H I I A H S Q G G Q
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B. thermoleovorans ID-1
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B. stearothermophilus L1
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V H I I A H S Q G G Q
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B. thermocatenulatus
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V H I I A H S Q G G Q
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Psychrobacter immobilis B10
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I H V G G N S M G G A
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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).
recombinant proteins intracellularly.11) However, in spite of the extensive knowledge of the genetics and molecular biology of E. coli, not every gene can be expressed effectively in this organism. Basically, gene expression could be achieved through high-level gene expression by increasing the amount of expressed protein up to 50% of the total cell protein through gene manipulation and proper choosing of the vector and host combination. Alternatively, fed-batch fermentation can fulfill the requirement for high cell density to improve the level of expression from a certain volume of cultures. In this study, a thermostable T1 lipase gene from Geobacillus sp. strain T1 was expressed with pBAD, pRSET, pET, and pGEX under the regulation of promoters araBAD, T7, T7 lac, and tac, respectively (Table 1). These systems allow regulation of the gene of
Table 1. Expression of T1 Lipase Gene with Various Expression Systems Expression Promoters systems
Hosts
Expression level (Umg1 )
pBAD
araBAD
E. coli Top 10
8.76
pRSET C
T7
E. coli BL21(De3) E. coli BL21(De3)plysS
6.41 6.35
pET22b(+) T7 lac
E. coli BL21(De3) E. coli BL21(De3)plysS
0.79 2.54
pGEX-4T1 tac
E. coli BL21(De3) E. coli BL21(De3)plysS
15.48 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 1 mM of IPTG (pRSET C, pET22b(+), pGEX-4T1) at 37 C for 8 h.
interest under the control of different promoters, which allows chemically inducible, high level expression. Initially, a thermostable T1 lipase gene was cloned and expressed in the pBAD expression system downstream of the araBAD promoter for tightly-regulated expression in E. coli Top 10 with 0.02% of L-arabinose as inducer. The active recombinant lipase obtained was around 8.76 Umg1 after 8 h of induction time. The T1 lipase gene was further expressed with pRSET C (T7 promoter) and the specific activity of around 6.41 Umg1 and 6.35 Umg1 of protein was achieved in E. coli BL21(De3) and E. coli BL21(De3)plysS, respectively when induced with 1 mM IPTG under the same conditions. However, growth was severely impaired by protein expression with this system because of the leaky system conferred by the T7 promoter. Therefore, a more tightly regulated expression system pET22b(+) under the control of the T7 lac promoter was further tested for T1 lipase gene expression. It carries an N-terminal pelB signal sequence for potential periplasmic localization. Unfortunately, this particular expression system had only a low level of expression with the host E. coli BL21(De3) (0.79 Umg1 ) and E. coli BL21(De3) plysS (2.54 Umg1 ) upon induction with 1 mM IPTG. Next, expression of the T1 lipase gene was done under the regulation of the tac promoter as a fusion protein (Fig. 4). This expression system greatly increased the level of expression in E. coli BL21(De3) (15.49 Umg1 ) and E. coli BL21(De3)plysS (20.02 Umg1 ) when induced at OD600 nm 0.5 with 1 mM of IPTG for 8 h. The expression level obtained by the pGEX system in E. coli BL21(De3) plysS was around 3and 7-fold higher than pRSET and pET systems under the same conditions. Based on this, the pGEX system was chosen for further study. Enzymatic assay showed that 1.5-fold increases in lipase expression were encoun-
Heat-stable Lipase from Geobacillus sp. Strain T1
101
A Specific activity (U/mg)
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0.025
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B 116 Fusion protein
66.2 45 35
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Fig. 4. Constructions of Recombinant Plasmid pGEX/T1. Thermostable T1 lipase gene was subcloned into pGEX-4T1 vector for fusion protein expression.
tered with 0.05 mM of IPTG as inducer at 8 h of induction time with specific activity around 30.19 Umg1 , which corresponds to 2927.15 Ug1 of wet cells (Fig. 5A). A comparison study of expressed protein among different lipases was difficult due to the variation in assay methods and units of expression. T1 lipase gene expression in the pGEX system was higher than the expression level obtained by a thermostable lipase from Bacillus thermoleovorans ID-1 in the two-plasmids system of pET22b(+) and pGP1-2.8) The expression level of lipase from Bacillus thermoleovorans ID-1 was around 320 U/g cells, which is 9 times lower than the expression of T1 lipase in the pGEX system. In another study, intracellular expression of Bacillus licheniformis in pET20b(+) upon induction with IPTG at 30 C was around 300 units from 50 ml culture,24) which was slightly lower than the T1 lipase gene expression of around 585.43 units from the same volume of culture. In fact, expression of T1 lipase as a fusion protein is feasible for simple detection and purification. Protease-
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Fig. 5. Expressed Fusion Protein at Various Inducer (IPTG) Concentrations. A) Enzymatic assay of recombinant T1 lipase. (open column), E. coli BL21(De3)plysS harboring recombinant plasmid pGEX/T1. The culture was grown in 1 L blue cap bottle containing 200 ml of LB medium containing 100 g/ml ampicillin and 35 g/ml chloramphenicol 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 2 mM) 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), 0.025 mM (lane 2), 0.050 mM (lane 3), 0.100 mM (lane 4), 0.500 mM (lane 5), 1 mM (lane 6), 1.5 mM (lane 7), 2 mM (lane 8) and GST (lane 9). Arrows indicate GST fusion protein and GST.
deficient host E. coli BL21 host strains, which are lon and ompT proteases deficient, were used for expression to reduce degradation of the fusion protein. As shown in Fig. 5B, the molecular mass of fusion protein and free GST tag was around 63 kDa and 26 kDa, respectively when analyzed with SDS-PAGE. Characterization of crude recombinant lipase The crude fusion lipase had an optimum temperature of 65 C when olive oil was used as substrate and was stable up to 65 C for 30 min (Fig. 6) when assayed at pH 7.0 for 30 min, which is somewhat higher than lipases from Bacillus stearothermophilus L1 and Bacillus thermocatenulatus.9,10) As shown in Fig. 7A, an optimum pH of the recombinant lipase was around pH 9. It
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Fig. 6. Effects of Temperature on Enzyme Activity (A) and Stability (B). T1 lipase was assayed at various temperatures ranging ( ) from 40–80 C. For the stability test, T1 lipase was assayed after heat treatment at various temperatures ( ) for 30 min.
was stable over wide ranges of pH ranging from pH 6– 11. The activity was, however, drastically decreased at pH above 11 (Fig. 7B). The optimum pH of T1 lipase (pH 9) was nearly identical to lipases from other thermophilic bacteria such as Bacillus thermoleovorans ID-1, Bacillus stearothermophilus L1, and Bacillus thermocatenulatus8–10) (pH 8–9) but lower than lipases from mesophilic counterpart such as Bacillus licheniformis21) (pH 10.5). T1 thermostable lipase was highly active at alkaline pH (up to pH 11) and stable and widely active at temperatures up to 65 C. These properties indicate its potential when used in detergent formulation. Further genetic manipulation by site-directed mutagenesis of the cloned gene opens new possibilities for the introduction of pre-designed changes, resulting in the production of tailor-made lipases with novel and desirable properties. Furthermore, response surface methodology25) in describing the relationship between tested variables and expression level, could be used to optimize thermostable T1 lipase gene expression during scale-up fermentation to develop a more accurate bioprocess for industrial application in a shorter time. PCR cloning is a feasible way to clone genes of
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Fig. 7. Effect of pH on Enzyme Activity (A) and Stability (B). T1 lipase was assayed at various pHs from pH 4 to pH 12 by a colorimetric method. Symbols used are: ( ), Acetate buffer; ( ), Potassium phosphate buffer; ( ), Tris-HCl buffer; ( ), Glycine buffer; ( ), Na2 HPO4 /NaOH buffer. For the stability test, the T1 lipase was assayed after incubation at various pHs (1:1, v/v) at 65 C for 30 min.
interest from the same family of lipases. High-level expression could be achieved through molecular approaches with various expression systems. Fusion protein allows the rapid purification and detection of expressed protein. This study showed that the pGEX expression system conferred a higher level of expression over the expression vectors pBAD, pRSET C, and pET22b(+).
Acknowledgments This research was supported financially by the Ministry of Science, Technology, and Environment, Malaysia (09-02-04-0336-EA001).
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