Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression and biochemical characterization

Article abstract of DOI:10.1016/j.procbio.2013.08.016

A thermo-alkaliphilic lipase from Bacillus subtilis DR8806 was functionally expressed as an N-terminal 6xHis-tagged recombinant enzyme in Escherichia coli BL21 using pET-28a(+) expression vector. Sequence analysis revealed an open reading frame of 639 bp

Full text of DOI:10.1016/j.procbio.2013.08.016

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Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis
DR8806: Expression and biochemical characterization
Shirin Emtenania, Ahmad Asoodeha,b,∗, Shamsi Emtenania
a Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
b Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 May 2013
Received in revised form 17 August 2013
Accepted 26 August 2013
A thermo-alkaliphilic lipase from Bacillus subtilis DR8806 was functionally expressed as an N-terminal
6xHis-tagged recombinant enzyme in Escherichia coli BL21 using pET-28a(+) expression vector. Sequence
analysis revealed an open reading frame of 639 bp encoding a 212-amino acid protein containing the
well-conserved Ala-His-Ser-Met-Gly motif. One-step purification of the His-tagged recombinant lipase
was achieved using Ni-NTA affinity chromatography with a specific activity of 1364 U/mg. The purified
enzyme with an apparent molecular mass of 26.8 kDa demonstrated the maximum activity at 70 ◦C and
pH 8.0 for hydrolysis of p-nitrophenylbutyrate as substrate. The enzyme activity was strongly inhibited by
divalent ions of heavy metals such as Hg2+ and Cu2+, while retained over 90% of the original activity in the
presence of several reagents including DTNB (5,5ꢀ-dithiobis-(2-nitrobenzoic acid)), SDS (sodium dodecyl
sulfate), urea, DMF (dimethylformamide), DTT (dithiothreitol), glycerol and Triton X-100. While being
considerably stable in organic solvents, imidazolium-based ionic liquids (ILs) had stimulatory effects on
the activity of purified lipase. Remarkable stabilization of enzyme at alkaline pH and in ionic liquids as
well as its thermostability/thermoactivity are among the most fundamental characteristics which offer
great potential for various biotechnological applications including detergent formulation, bioremediation
processes and biotransformation in non-aqueous media.
Keywords:
Cloning
Lipase
Bacillus subtilis DR8806
Biochemical characterization
Ionic liquids
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
appropriate mesophilic hosts [5]. Recently, ionic liquids (ILs) have
gained much attention as an alternative to conventional organic
Lipases (triacylglycerol hydrolases, E.C. 3.1.1.3) are a class of
enzymes capable of hydrolyzing ester bonds in triglycerides at
oil–water interface. Hydrolyzing the triglycerides, lipases also cat-
alyze their synthesis from fatty acids and glycerol. Lipases, which
are among the most widely used hydrolytic enzymes in industry,
have become the object of particular attention for various biotech-
nological applications [1,2]. Thermostable lipases have garnered
wide spread interest for potential application in the detergent,
pharmaceutical, dairy, oil and fat industries due to their extreme
stability at high temperatures and in organic solvents. In addition,
operating bioprocesses at higher temperatures may result in an
increase in diffusion rate and enhanced solubility of hydrophobic
substrates as well as reduction in microbial contamination [3,4].
While harvesting a limited amount of enzymes from thermostable
lipase producing bacteria, high expression level of protein has
been achieved through cloning thermophilic genes into more
solvents particularly in biocatalytic reactions. Owing to their
unique properties including nonvolatility, thermal stability and
ionic conductivity as well as nonflammability, ILs are considered as
environmentally friendly green solvents [6]. The stimulatory effects
of ILs on reactions involving lipases have been reported in previous
studies [7,8]. A thermophilic lipolytic bacterium was previously
isolated from Dig Rostam hot mineral spring in Iran. The strain was
identified as Bacillus subtilis based on the 16S rDNA gene sequence
(GenBank: JF309277) and has been deposited in Iranian Biological
Resource Center under acquisition number of IBRC-M10742 [9].
The aim of the present study was to report the cloning, expres-
sion and purification of recombinant lipase from Bacillus subtilis
DR8806, as well as biochemical properties of the purified enzyme.
2. Materials and methods
2.1. Bacterial strains and plasmids
A lipase producing strain B. subtilis DR8806 isolated from hot mineral spring was
grown in nutrient broth at 37 ◦C. E. coli DH5␣ and BL21 (DE3) were used as cloning
and expression hosts, respectively. The E. coli strains were cultivated in Luria-Bertani
(LB; 1.0% tryptone, 0.5% yeast extract and 1.0% NaCl) medium at 37 ◦C. B. subtilis
DR8806 was served as the source of genomic DNA. The plasmid pTZ57R/T (Fer-
mentas, Maryland, USA) was used for cloning and sequencing of the lipase gene. The
∗
Corresponding author at: Department of Chemistry, Faculty of Sciences, Fer-
dowsi University of Mashhad, Mashhad, Iran. Tel.: +98 511 8795457;
fax: +98 511 8795457.
E-mail address: asoodeh@um.ac.ir (A. Asoodeh).
1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.08.016
Please cite this article in press as: Emtenani S, et al. Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression
and biochemical characterization. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.08.016

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target gene was cloned into pET-28a(+) expression vector (Novagen, USA) under the
control of strong T7 promoter to allow the insert to be maintained and expressed in
E. coli host. To screen the transformants, medium was supplemented with ampicillin
or kanamycin at a final concentration of 100 ␮g/mL and 50 ␮g/mL, respectively.
glycine–HCl buffer pH 2.0–3.0, sodium acetate buffer pH 3.5–5.5, sodium phosphate
buffer pH 6.0–7.5, Tris-HCl buffer pH 8.0–9.5, Na2HPO4–NaOH buffer pH 10. The
pH stability of the lipase was determined after 60 min of pre-incubation at differ-
ent pH values (2.0–10.0) at 50 ◦C. The residual lipolytic activity was determined
under standard assay conditions. All enzyme assay experiments were conducted in
triplicates.
2.2. Cloning and sequencing of the lipase gene
2.6.2. Effect of temperature on lipase activity and stability
A pair of oligonucleotide primers was synthesized based on coding sequences
available for the B. subtilis lipase gene. The sequence of the forward primer LipF
was 5ꢀ-CGCGGATCCGCGATTATGAAATTTGTAAAAAGAAGG-3ꢀ and that of the reverse
primer LipR was 5ꢀ-CCCAAGCTTGGGTCATTAATTCGTATTCTGGCC-3ꢀ. Genomic DNA
from B. subtilis DR8806 was prepared by the method developed by Sambrook
and Russell [10]. The lipase gene was amplified from genomic DNA using a set
of primers (LipF and LipR) with incorporated restriction enzymes BamHI/HindIII
(Takara, Dalian, China), allowing the directional in-frame ligation of the amplified
fragment into pET-28a(+) vector. The purified amplicon was cloned into the T/A
cloning vector pTZ57R/T, in accordance with the manufacturer’s instructions. Com-
petent cells of E. coli were prepared by using a conventional CaCl2 method [10].
Transforming into the E. coli DH5␣ competent cells using heat shock method [10],
the cells were cultivated on LB-agar medium containing ampicillin (100 ␮g/mL). The
plasmid DNA was isolated with Fermentas plasmid DNA isolation kit (Fermentas,
Maryland, USA) following the manufacturer’s instructions. Colony PCR using specific
primers, restriction enzyme analysis and sequencing after plasmid extraction were
performed to confirm the presence of the target gene. The nucleotide sequence of
B. subtilis DR8806 lipase gene was determined and submitted to GenBank database.
The insert was subcloned in pET-28a(+) vector by digesting the pTZ57R/T cloning
vector containing the lipase gene with BamHI/HindIII followed by ligation to the
previously digested expression vector using T4 DNA ligase (Takara, Dalian, China).
The resulting expression construct was confirmed by lipase gene amplification and
double restriction digestion. The recombinant pET-28a(+) vector was employed for
the expression of the lipase gene in E. coli BL21 (DE3) competent cells. The afore-
mentioned construct placed the insert in frame with N-terminal region coding for
six His residues facilitating the protein purification process.
The effect of temperature on the activity of lipase was determined by monitoring
the enzyme activity at various temperatures in the range of 30–80 ◦C for 30 min in
Tris-HCl buffer, pH 8.0. The influence of temperature on lipase stability was analyzed
by incubating the enzyme solution at above-mentioned temperatures for 60 min
followed by the evaluation of the remaining activity according to standard assay
method. To determine the enzyme half-life, the cloned lipase was kept at different
temperatures ranging from 60 ◦C to 80 ◦C and the residual activity was assessed at
30 min intervals over a total period of 150 min.
2.6.3. Effect of metal ions on the purified lipase activity
The effects of various metal ions (Mg2+, Cu2+, Mn2+, Ca2+, K+, Na+, Fe2+, Zn2+, Co2+
,
Pb2+, Ba2+, Cd2+ and Hg2+) at 1 and 5 mM concentrations on the enzyme activity
was assayed at 70 ◦C, pH 8.0 using p-nitrophenylbutyrate as substrate, upon pre-
incubation of the purified lipase in each compound for 30 min. The enzyme activity
of control sample (recombinant lipase without any metal ion) was taken as 100%.
Lipase activity was measured as previously described.
2.6.4. Effect of organic solvents and ionic liquids on lipase performance
The effect of organic solvents on lipase activity was determined following pre-
incubation of enzyme for 30 min at 25 ◦C under 150 rpm shaking in the presence
of methanol, acetone, hexane, heptane, toluene, ethanol, chloroform, isopropanol,
diethyl alcohol, butanol and isoamyl alcohol at concentrations of 10% v/v and 20%
v/v. The incubation was conducted in closed screw cap tubes with silicone rubber
gasket in order to prevent evaporation of the enzyme reaction.
The influence of imidazolium-based ionic liquids (ILs) on lipase activity was
investigated after a 30 min-preincubation of the purified enzyme with different con-
centrations of ILs (ranging from 2 to 10% v/v) at 50 ◦C and pH 8.0. The remaining
activity was analyzed by spectrophotometric method under the assay condition.
The lipolytic activity was determined by triplicate experiments.
2.3. Expression of the lipase gene
A transformant of E. coli BL21 harboring the recombinant plasmid (pET-28a(+)-
lip) was cultured in LB medium containing 50 ␮g/mL kanamycin and incubated
overnight at 37 ◦C with 150 rpm shaking. The pre-culture was inoculated (1% v/v)
into 200 mL fresh LB medium supplemented with kanamycin at 37 ◦C until the
OD600 reached 0.5. To induce the expression of recombinant lipase, isopropyl-␤-
d-thiogalactopyranoside (IPTG) was added to the medium at a final concentration
of 1 mM and the growing culture was incubated at 25 ◦C for a further 8 h-period. Fol-
lowing induction, the aliquots were harvested by centrifugation (12,000 g, 20 min,
2.6.5. Influence of various effectors on enzyme activity
The effect of a variety of chemical reagents (1 and 5 mM) on the enzyme activ-
ity was investigated by pre-incubating the lipase for 30 min at 70 ◦C in 50 mM
Tris-HCl buffer (pH 8.0) containing following chemical agents; oxidizing agents:
ammonium persulfate, potassium iodide and H2O2, reducing agents: ascorbic acid
and ␤-mercaptoethanol, chelating agents: sodium citrate and EDTA (ethylenedi-
aminetetraacetic acid), detergents: SDS, CTAB (cetyltrimethylammonium bromide)
and Triton X-100, additives: PEG 4000 (polyethylene glycol) and glycerol, inhibitors:
PMSF (phenyl methyl sulfonyl fluoride), DTT, DMF, urea, DTNB, sodium fluoride,
mercuric chloride and phenanthroline. The activity of the enzyme without additives
was assumed as 100%.
4
◦C) and the pellets were resuspended in 10 mL lysis buffer (NaH2PO4 50 mM NaCl,
0.3 M imidazole 10 mM and 1 mM PMSF, pH 8.0). Induced cells were disrupted by
sonication for six 30 s burst with a 30 s cooling period between each burst, the
cell-free extracts were obtained by centrifugation.
2.4. Purification of the recombinant lipase enzyme
2.6.6. Effect of commercial detergents on enzyme stability
The stability of the recombinant lipase in commercial enzyme-containing pow-
der/liquid detergents was investigated. The solid detergents utilized in this study
were as follows: Barf (Paxan, Iran), Vash (Henkel, Germany), Softlan (Pakshoo, Iran),
Persil (Henkel, Germany) washing powder and handwash powder, Pril (Henkel,
Germany), Shoma (TolyPers, Iran), Finish (Reckitt Benckiser, Canada) and Darya
(TolyPers, Iran). The liquid detergents tested were Goli (Paxan, Iran), Persil (Henkel,
Germany), Ave (Pakshoo, Iran) and Ganj (RaminGostar, Iran). Dissolving in tap water,
the solid detergents were prepared at a final concentration of 5 mg/L, while a 100-
fold dilution of liquid detergents was performed to simulate washing conditions. To
inactivate the endogenous enzymes in aforementioned detergents, diluted deter-
gent preparations were pre-incubated for 30 min at 80 ◦C before the addition of
recombinant lipase. The enzyme was added to powder/liquid detergent solution fol-
lowing incubation at 70 ◦C for 60 min, followed by activity measurement. To allow
further comparison, the effect of commercial detergents on the stability of a com-
mercial lipase (porcine pancreatic lipase, PPL) was also studied under the same
experimental conditions.
The N-terminally-attached His-tag lipase was purified under native condi-
tions using the immobilized metal ion affinity chromatography (IMAC) column
(Qiagen, CA, USA). The crude enzyme preparation was loaded onto the Ni-
nitrilotriacetate (Ni-NTA) column previously equilibrated with native binding buffer
(50 mM NaH2PO4, 300 mM NaCl and 10 mM imidazole, pH 8.0). The column was
washed sequentially with native wash buffer containing 20 mM imidazole. Finally,
the bound target proteins were eluted with elution buffer (50 mM NaH2PO4, 300 mM
NaCl and 250 mM imidazole, pH 8.0) at a flow rate of 1 ml min−1. The purification
procedure was carried out at 4 ◦C.
2.5. Lipase assay and protein determination
Lipase activity was measured spectrophotometrically at 405 nm using 0.01 M
p-nitrophenylbutyrate (pNPB) (Sigma–Aldrich, USA) as a substrate following incu-
bation at 50 ◦C for 30 min and pH 8.0 (Tris-HCl buffer). One unit of lipase activity was
defined as the amount of enzyme needed to liberate 1 ␮mol of p-nitrophenol per
minute under standard assay conditions [2]. To determine the protein concentra-
tion, Bradford method was conducted using bovine serum albumin (Sigma–Aldrich,
USA) as the standard [11]. The homogeneity of the purified enzyme and also the
performance of affinity purification were analyzed on a 12% (w/v) SDS-PAGE poly-
acrylamide gel [12]. Protein bands were visualized by Coomassie brilliant blue R-250
staining. A protein standard (Vivantis, CA, USA) in the range of 10.5–175 kDa was
used as molecular mass marker.
2.6.7. Determination of substrate specificity
Lipase substrate specificity was analyzed using the spectrophotometric assay,
using 0.01 M p-nitrophenyl acetate (C2), butyrate (C4) and palmitate (C16) dissolved
in ethanol as substrates.
3. Results and discussion
2.6. Enzyme characterization
3.1. Cloning and sequence analysis of the lipase gene
2.6.1. Effect of pH on lipase activity and stability
To evaluate optimum pH of the recombinant enzyme, the lipolytic activity was
assayed over a pH range from 2.0 to 10.0 using the following buffers (50 mM):
The sequence of the lipase gene expressed in-frame with an N-
terminal region coding for six His residues has been deposited in
Please cite this article in press as: Emtenani S, et al. Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression
and biochemical characterization. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.08.016

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Fig. 1. Comparison of the amino acid sequences of various lipases originated from genus Bacillus. The amino acids of catalytic triad (His, Ser and Glu) are marked with
asterisks. Oxyanion hole (box I) and conserved pentapeptide (box II) are illustrated.
the GenBank under the accession number KC262177. The sequence
of the putative lipase gene contains a complete ORF of length 639 bp
which encodes a 212 amino acid precursor with a molecular mass
of 22.8 kDa and a theoretical pI of 9.72. The putative signal pep-
tide cleavage site was predicted to be located between Ala-31 and
Ala-32 using SignalP 4.0 software [13]. Lipases typically have a Ser-
Asp-His catalytic triad in which the active site serine is located
within the middle of the conserved pentapeptide Gly-X-Ser-X-Gly
[14]. The catalytic site of recombinant lipase comprises the triad
of Ser110, Glu166 and His189. According to multiple alignment of
Bacillus lipase sequences obtained from GenBank, B. subtilis DR8806
lipase sequence exhibited the conserved consensus motif initiated
with Ala (Ala-His-Ser-Met-Gly) instead of Gly. The alignment of
various sequences of thermostable Bacillus lipases revealed a high
degree of sequence similarity at both DNA and amino acid levels
(Fig. 1).
homogenous protein as represented by SDS-PAGE (Fig. 2). With the
insertion of a 3.8 kDa extension region belonging to expression vec-
tor, the predicted molecular mass for deduced protein sequence of
recombinant lipase was estimated to be approximately 26.8 kDa.
Although thermophilic bacteria could be ideal candidates as ther-
mostable lipase producers, only a minute amount of enzyme can
be obtained which may not be suitable for industrial application
[3]. As shown in Table 1, the fusion enzyme was ultimately puri-
fied three-fold with a yield of 57% resulting in an increased lipase
specific activity from 456.3 (crude enzyme) to 1364 U/mg (purified
recombinant lipase).
3.3. pH profile and stability
The effect of pH on enzyme activity and stability was examined
in the pH range of 2.0–10.0. Illustrated in Fig. 3, the enzyme was
active in a broad range of pH (pH 5.0–10.0) with an optimum pH of
8.0 in 50 mM Tris-HCl buffer. In spite of being highly active within a
broad pH range, a sharp decline in lipase activity was monitored at
pH below 5.0, at which almost none or low activity was observed.
The high enzyme activity at alkaline pH was probably due to
Ala-Gly (first Gly-residue) substitution in the consensus sequence
Gly-X-Ser-X-Gly [15]. The optimum pH of B. subtilis DR8806 lipase
was identical to that of other thermophilic lipases from B. subtilis
EH 37 [16], Bacillus sphaericus 205y [17], Bacillus sp. L2 [18] and
a lipolytic enzyme from B. subtilis DR8806 [19]. The pH stability
of enzyme showed that it was stable (>90% of lipase activity) at
pH values ranging from 8.0 to 10.0 upon a 60-min treatment at
50 ◦C in Tris-HCl buffer. The enzyme was fairly stable at pH 5 with
57% residual activity. Nevertheless, the lipase stability decreased
sharply when the pH was below 5.0. Lipases which are stable
3.2. Overexpression and purification of the recombinant lipase
Competent E. coli BL21 (DE3) cells were transformed with appro-
priate recombinant pET-28a(+) expression system encoding the
lipase gene with the His-tag sequence in an N-terminal config-
uration. The insertion of target fragment into expression vector
in the correct direction and reading frame was firstly confirmed
by lipase amplification using specific linker primers. Cloning of
lipase gene into pET-28a(+) vector was also confirmed by dou-
ble digestion. The expression of lipase was carried out by the
induction of IPTG-inducible bacteriophage T7 promoter using 1 mM
IPTG. The enzyme was expressed as an amino-terminal 6x-His
tag to assist the protein purification using Ni-NTA column. One-
step IMAC-purification of the recombinant lipase resulted in a
Please cite this article in press as: Emtenani S, et al. Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression
and biochemical characterization. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.08.016

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Fig. 2. SDS-PAGE analysis of recombinant ␣-lipase. (a) M marker, lanes 1–3: cell fractions at 2 h (lane 1), 4 h (lane 2) and 8 h (lane 3) post induction with 1 mM IPTG, (b) M
marker, lane 1 purified recombinant lipase using Ni-NTA affinity chromatography.
Table 1
Purification steps of His-tagged recombinant lipase using Ni-NTA column.
Purification step
Specific activity (U/mg)
Total protein (mg)
Total activity (U)
Purification (fold)
Yield (%)
Crude
Ni-NTA column
456.3
1364
100
19
45,557
26,000
1
3
100
57
under alkaline conditions are considered as promising candidates
for removable of fat stains in detergent formulation [20].
as a mesophilic enzyme [19]. The recombinant enzyme retained
90% and 87.5% of its initial activity after incubation at 75 ◦C and
80 ◦C for 1 h, respectively. A recombinant lipase from Bacillus ther-
moleovorans ID-1 with an optimum temperature of 75 ◦C retained
50% and 30% of its initial activity after 1-h incubation at 60 ◦C and
70 ◦C, respectively. The results indicated that the enzyme might be
valuable in lipid processing industry, which requires the enzyme
to be stable above 50 ◦C [2]. The half-life of the purified lipase was
calculated to be 145 min at its optimum temperature. The enzyme
retained 50% of its original activity after 92 min incubation at 80 ◦C,
pH 8.0. An extra-cellular lipase produced by Bacillus licheniformis
MTCC 6824 showed a half-life (t1/2) of 48 min at 60 ◦C [21]. The
half-life of Bacillus stearothermophilus MC 7 lipase was found to
be 30 min at 70 ◦C [22]. Considering its appropriate properties, the
recombinant alkalothermophilic enzyme will play a dominant role
3.4. Effect of temperature on enzyme activity and stability
The effect of temperature on lipase activity was measured at dif-
ferent temperatures ranging from 30 ◦C to 80 ◦C at 5 ◦C intervals for
30 min (Fig. 4). The temperature profile of lipase is of great impor-
tance for high temperature operational bioprocesses, since the
enzyme was active over a wide range of temperatures (30–80 ◦C),
with an optimum activity at 70 ◦C. The optimum temperature of
enzyme was in agreement with other thermostable lipases from
Bacillus sp. L2 [18] and Geobacillus sp. T1 [3]. An esterase from
Bacillus subtilis DR8806 showed an optimal temperature at 50 ◦C
120
100
80
120
100
80
60
60
40
Temperature activity
pH profile
40
Temperature stability
20
20
0
pH stabilty
0
4
5
6
7
8
9
10
11
25 30 35 40 45 50 55 60 65 70 75 80 85
pH
Temperature ( º C)
Fig. 3. Effect of pH on the activity and stability of B. subtilis DR8806 recombinant
lipase. Effect of pH on lipase activity was investigated by assaying its activity at
various pH values. The enzyme stability was determined by incubating the enzyme
at 50 ◦C for 60 min at different pH values. Each data point depicts the mean of three
independent assays.
Fig. 4. Effect of temperature on the enzyme activity and stability. The purified lipase
was assayed at different temperatures (30–80 ◦C). For the stability test, the enzyme
was incubated for 60 min at various temperatures, pH 8.0. The lipolytic activity was
determined by triplicate experiments.
Please cite this article in press as: Emtenani S, et al. Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression
and biochemical characterization. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.08.016

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Table 2
Table 3
Effect of metal ions on enzyme activitya.
Effect of organic solvents on the purified recombinant lipasea.
Metal salts
Relative activity (%) 1 mM
5 mM
Organic solvent
Relative activity (%) 10% v/v
20% v/v
Control
MgCl2
CuCl2
MnCl2
CaCl2
KCl
100.0
54
36
53
52
45
39
33
87
100
54
78
45
12
100.0
51
20
69
44
50
57
46
47
43
42
54
64
0
Control
Methanol
Acetone
Hexane
Heptane
Toluene
Ethanol
Chloroform
Isopropanol
Diethyl alcohol
Butanol
100
96
90
83
85
94
75
102
88
96
93
97
100
92
92
98
108
85
92
101
90
91
96
NaCl
FeCl2
ZnCl2
CoCl2
PbCl2
BaCl2
CdCl2
HgCl2
Isoamyl alcohol
92
a
The enzyme was pre-incubated at 25 ◦C for 30 min with different organic sol-
vents at 10% and 20% v/v prior to lipase assay. A sample reaction without organic
solvent was taken as control.
a
The lipase activity was monitored under the standard assay conditions following
incubation with various metal ions at 1 and 5 mM concentrations. The percent lipase
activity for control was taken as 100%.
140
120
100
in industrial applications including biopolymer/biodiesel synthesis
as well as the production of pharmaceuticals, cosmetic, flavor and
commercial laundry detergents [20].
80
E(MM)Br
3.5. Effect of metal ions on lipase activity
60
B(MM)Br
40
The effect of metal ions on the activity of the cloned lipase is rep-
resented in Table 2. The influence of metal ions could be attributed
to the change in the solubility and behavior of ionized fatty acids
upon complex formation with metal ions and the direct inhibition
of enzyme catalytic function [22]. The results demonstrated that
the enzyme was dramatically inactivated by Hg2+ and Cu2+, sug-
gesting the ability of these ions to alter enzyme conformation and
direct inhibition of the catalytic site. Pb2+, Zn2+ and Ba2+ ions led to a
moderate reduction in enzyme activity at 5 mM concentration. The
lipase enzyme was also inactivated by 50% in the presence of KCl,
while retained almost 100% activity after being exposed to CoCl2
at a concentration of 1 mM. A lipase from B. stearothermophilus MC
7 was inhibited by divalent ions of heavy metals, completely by
Cu2+ and strongly by Fe2+ and Zn2+ [22]. The lipolytic activity of
OST-lipase from B. sphaericus 205y was stimulated in the presence
of Ca2+ and Mg2+ and inhibited to the extent of 90% and 94% when
ZnSO4 and FeCl3 were used, respectively [17]. Partial inhibition of
H(MM)Br
20
0
B(MM)Cl
0%
2%
4%
6%
8%
10%
Ionic liquids (%)
Fig. 5. Influence of various imidazolium-based ionic liquids with 2–10% (v/v) con-
centrations on the activity of recombinant lipase from B. subtilis DR8806. Values
presented are the means of triplicate analyses.
acetate [21]. Organic solvent-tolerant lipases have gained focus as
potentially valuable enzymes in non-aqueous biocatalysis. Thus,
the stability of recombinant enzyme toward organic solvents is of
great significance particularly for synthesis of esters using lipolytic
enzymes.
Exhibiting the features of both organic and ionic com-
pounds, ILs emerged as remarkably interesting non-aqueous
media for numerous enzymatic reactions such as alcoholysis,
ammoniolysis and perhydrolysis [6]. As displayed in Fig. 5,
the recombinant enzyme was also investigated for its lipolytic
activity against different 1-alkyl-3-methylimidazolium-based ILs
including 1-ethyl-3-methylimidazolium bromide ([EMIM][Br]),
BP-6 LipA activity was caused by 10 mM concentrations of Fe3+
,
Cu2+, Pb2+ and Zn2+, whereas Hg2+ caused a significant inhibition
of the recombinant enzyme [23].
3.6. Effect of organic solvents and ionic liquids on lipase
The influence of various organic solvents at concentrations of
10% and 20% v/v on lipase activity was assessed under standard
conditions (Table 3). The recombinant enzyme was remarkably sta-
ble toward organic solvents, retaining more than 95% of its activity
in the presence of isoamyl alcohol, methanol, hexane, butanol and
diethyl alcohol. A relative activity of 102% was apparent in the pres-
ence of chloroform. The lipolytic activity was enhanced at higher
concentrations of heptane showing stimulatory effect of the sol-
vent on the enzyme. Other solvents did not promote the lipolytic
activity, however, high lipolytic activity was observed at lower con-
centrations of the organic solvents. The exposure of BF-3 lipase
to organic solvents elucidated that the enzyme was activated by
methanol, ethanol and toluene (10% and 20% v/v) whereas the
stability of enzyme was drastically reduced with increasing the
concentration of organic solvents [24]. Moreover, the activity of an
alkaline metallolipase from B. licheniformis MTCC 6824 was found
to be considerably decreased in chloroform, acetonitrile and ethyl
1-n-butyl-3-methylimidazolium
bromide
([BMIM][Br]),
1-hexyl-3-methylimidazoliumbromide ([HMIM][Br]) and 1-
butyl-3-methylimidazolium chloride ([BMIM][Cl]). In agreement
with previously reported studies, ILs had promoting influence on
lipase activity [7,8]. Besides, the enzyme activity was improved
with the increase in the concentration of the ionic liquid. 1-Butyl-
3-methylimidazolium chloride ([BMIM][Cl]) exhibited the highest
promoting effect among all investigated ILs in the present study.
The stimulatory impact of Br-containing ILs on enzyme activity
was as follows [HMIM][Br] > [BMIM][Br] > [EMIM][Br]. Similar
to previous reports [7,8], the lipase activity was enhanced with
the increase in alkyl chain length of the imidazolium ring of ILs.
The effect of the elongation of alkyl side chain on the hydrolytic
activity seems to be in correlation with the hydrophobic nature of
the alkyl group, improving the ability of IL to obstruct the lipase
non-polar active site [7]. Lipase catalyzed-reactions have benefited
most from the exploitation of ILs [6]. The results indicated that the
Please cite this article in press as: Emtenani S, et al. Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression
and biochemical characterization. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.08.016

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S. Emtenani et al. / Process Biochemistry xxx (2013) xxx–xxx
Table 4
Effect of various chemical reagents on recombinant lipase activitya.
Reagent molecule
Relative activity (%)
Oxidizing agents
Ammonium persulfate
Potassium iodide
Reducing agents
Ascorbic acid
␤-Mercaptoethanol
Chelating agents
Sodium citrate
EDTA
1 mM
86
81
5 mM
80
99
70
77
55
106
39
88
46
82
Detergents
SDS
10% (w/v)
79
20% (w/v)
93
CTAB
Triton X-100
Additives
100
100
1% (w/v)
94
100
98
2% (w/v)
87
PEG 4000
Glycerol
Inhibitors
DMF
Urea
106
1 mM
87
88
87
95
5 mM
95
99
79
Fig. 6. Effect of different commercial solid/liquid detergents on lipase activity. The
enzyme was first treated with detergent and the residual activity was measured
under assay conditions. The activity of control samples in the absence of detergents
was taken as 100%. Values presented are the means of triplicate analyses.
PMSF
DTT
90
94
DTNB
102
63
79
90
0
61
72
to withstand relatively harsh washing conditions (alkaline pH, ele-
vated temperatures as well as surfactant/additive resistance) is a
prerequisite of industrial enzymes used in detergent preparation.
Sodium fluoride
Mercuric chloride
Phenanthroline
82
a
The recombinant lipase was incubated with different effectors for 30 min under
optimal conditions. The residual activity was measured under the standard enzyme
assay condition.
3.8. Stability of enzyme toward commercial solid/liquid
detergents
The compatibility of recombinant lipase with solid/liquid
household laundry and automatic dishwashing detergents was
investigated following pre-incubation of the purified enzyme with
a broad range of commercial detergents. As represented in Fig. 6,
the enzyme was extremely stable in the presence of powder and
liquid detergents, remaining 100% of its activity toward Shoma, Barf
and Finish and more than 95% in the presence of Pril, Persil, Softlan
and Ganj after 1 h incubation at 70 ◦C. Interestingly, the recom-
binant lipase was more stable than the porcine pancreatic lipase
(PPL), which was completely inhibited by commercial detergents
used including Pril, Sotlan, Barf, Ave and Sotlan (data not shown).
Additionally, similar to the recombinant lipase, PPL was totally sta-
ble toward Finish and Persil solid detergents. The stability of the
recombinant lipase against commercial laundry detergents makes
it a potential candidate in household detergent formulation.
cloned lipase had greater activity and stability in ionic liquids than
in traditional organic solvents.
3.7. Effect of additives, inhibitors, oxidizing and reducing agents
on lipolytic activity
As illustrated in Table 4, the purified lipase exhibited high sta-
bility toward a variety of known reagents, retaining more than 90%
of its activity after pre-incubation with DTNB, SDS, urea, DMF, DTT,
glycerol and Triton X-100. The presence of oxidizing agents such as
ammonium persulfate and potassium iodide in the reaction mix-
ture resulted in a moderate decrease in lipase hydrolytic activity.
The decreased enzyme activity might be due to partial oxidation
of His residue in catalytic triad. The enzyme was slightly affected
when treated with reducing agents; 30% and 23% decrease in the
activity of recombinant lipase were observed in the presence of
ascorbic acid and ␤-mercaptoethanol, respectively. Partial inacti-
vation of the lipase in the presence of PMSF probably was observed
due to modification of an essential serine residue in the enzyme cat-
alytic site. CTAB and Triton X-100 detergents had no effect on the
purified lipase activity. Addition of 1% v/v glycerol and 1 mM DTNB
to the enzyme mixture gradually enhanced the lipolytic activity
in comparison to the control. Sodium fluoride showed moderate
inhibitory effect (37%) on lipase activity at 1 mM concentration,
but caused a complete block of activity at 5 mM concentration.
The lipase was moderately stable toward EDTA, exhibiting only a
13% decrease in its initial activity after 30 min pre-incubation. Con-
tradictory results have been published in literatures regarding the
effect of different additives on lipase activity. The enzyme activity
of Bacillus cereus BF-3lipase was drastically diminished in the pres-
ence of SDS and PMSF [24]. Additionally, the aforementioned agents
strongly inhibited a recombinant lipase from thermophilic Bacillus
sp. L2 at 1 and 10 mM concentrations. Decreasing the lipase activity
in the presence of a chelating agent could be as a result of its influ-
ence on the interfacial area between the substrate and lipase and
also the necessity of metal ions for preserving the enzyme stability
and activity [18]. Accordingly, the capability of B. subtilis DR8806
3.9. Substrate specificity
The enzyme specificity was studied toward p-nitrophenylesters
of different alkyl chain lengths (C2, C4 and C16). The highest
hydrolytic activity was obtained for p-nitrophenylbutyrate, indi-
cating a clear preference of the enzyme for short acyl chain
lengths. Moreover, p-nitrophenylacetate was hydrolyzed quite
well, whereas p-nitrophenoylpalmitate with longer chain length
(C16) was converted slightly. Enzymes belonging to subfamily I.4
of lipases are the small alkaline proteins with preference toward
short-chain substrates [25]. Similar results have been reported
for Bacillus sp. BP-6 LipA and a thermophilic lipase from B. ther-
moleovorans ID-1 [2,15], although other reported lipases have
shown preference for esters with longer fatty acids when assayed
against p-nitrophenyl derivatives [22,24].
4. Conclusion
In retrospect, a thermo-alkaliphilic lipase gene from B. Subtilis
DR8806 was successfully cloned into pET-28a(+) vector. The
success of extracellular expression of an alkalophilic organic lipase
and the simple purification method developed promises many
Please cite this article in press as: Emtenani S, et al. Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression
and biochemical characterization. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.08.016

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S. Emtenani et al. / Process Biochemistry xxx (2013) xxx–xxx
7
biotechnological advantages. The pH profile of recombinant lipase
of B. subtilis DR8806 is similar to other reported extracellular
esterase previously purified from this isolate, but it shows some
differences in properties with respect to temperature activity
and stability, metal ions, stability toward surfactants and organic
solvents. The results clearly demonstrated that the enzyme was
not only stable but also moderately activated in aqueous solutions
of imidazolium-based ionic liquids. Therefore, the replacement of
organic solvents with ILs, which are considered as recyclable clean
solvents or catalysts of green chemistry, may lead to significant
development of lipase hydrolytic activity. Owing to high stability
at elevated temperatures, a broad range of pH and resistant
against organic solvents/ionic liquids, the recombinant lipase can
potentially be applied in the fields of detergents, oil and fat, dairy,
and pharmaceutical industries.
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Acknowledgments
The support for this investigation was provided by Iran National
Science Foundation (INSF) (Grant number: 91058286) and is grate-
fully acknowledged by the authors.
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Please cite this article in press as: Emtenani S, et al. Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression
and biochemical characterization. Process Biochem (2013), http://dx.doi.org/10.1016/j.procbio.2013.08.016