Thermoalkalophilic lipase from an extremely halophilic bacterial strain Bacillus atrophaeus FSHM2: Purification, biochemical characterization and application

Article abstract of DOI:10.1080/10242422.2017.1308494

The present study was designed to isolate and identify an extremely halophilic lipase-producing bacterial strain, purify and characterize the related enzyme and evaluate its application for ethyl and methyl valerate synthesis. Among four halophilic isolates, the lipolytic ability of one isolate (identified as Bacillus atrophaeus FSHM2) was confirmed. The enzyme (designated as BaL) was purified using three sequential steps of ethanol precipitation and dialysis, Q-Sepharose XL anion-exchange chromatography and SP Sepharose cation-exchange chromatography with a final yield of 9.9% and a purification factor of 31.8. The purified BaL (Mw～85 kDa) was most active at 70 °C and pH 9 in the presence of 4?M NaCl and retained 58.7% of its initial activity after 150 min of incubation at 80 °C. The enzyme was inhibited by Cd²⁺ (35.6 ± 1.7%) but activated by Ca²⁺ (132.4 ± 2.2%). Evaluation of BaL's stability in the presence of organic solvents showed that xylene (25%) enhanced the relative activity of the enzyme to 334.2 ± 0.6% after 1 h of incubation. The results of esterification studies using the purified BaL revealed that maximum ethyl valerate (88.5%) and methyl valerate (67.5%) synthesis occurred in the organic solvent medium (xylene) after 48 h of incubation at 50 °C.

Full text of DOI:10.1080/10242422.2017.1308494

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Thermoalkalophilic lipase from an extremely
halophilic bacterial strain Bacillus atrophaeus
FSHM2: Purification, biochemical characterization
and application
Atefeh Ameri, Mojtaba Shakibaie, Mohammad Ali Faramarzi, Alieh Ameri,
Sahar Amirpour-Rostami, Hamid Reza Rahimi & Hamid Forootanfar
To cite this article: Atefeh Ameri, Mojtaba Shakibaie, Mohammad Ali Faramarzi, Alieh Ameri,
Sahar Amirpour-Rostami, Hamid Reza Rahimi & Hamid Forootanfar (2017): Thermoalkalophilic
lipase from an extremely halophilic bacterial strain Bacillus atrophaeus FSHM2: Purification,
biochemical characterization and application, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2017.1308494
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1308494
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Date: 18 April 2017, At: 07:38

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
http://dx.doi.org/10.1080/10242422.2017.1308494
RESEARCH ARTICLE
Thermoalkalophilic lipase from an extremely halophilic bacterial strain
Bacillus atrophaeus FSHM2: Purification, biochemical characterization and
application
Atefeh Ameria, Mojtaba Shakibaiea, Mohammad Ali Faramarzib, Alieh Americ, Sahar Amirpour-Rostamia,
Hamid Reza Rahimid and Hamid Forootanfare
b
aPharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran; Department
of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences,
c
Tehran, Iran; Department of Medicinal Chemistry, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran;
e
dDepartment of Toxicology and Pharmacology, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran; Herbal
and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Iran
ABSTRACT
ARTICLE HISTORY
Received 7 May 2015
Revised 1 March 2017
Accepted 6 March 2017
The present study was designed to isolate and identify an extremely halophilic lipase-producing
bacterial strain, purify and characterize the related enzyme and evaluate its application for ethyl
and methyl valerate synthesis. Among four halophilic isolates, the lipolytic ability of one isolate
(identified as Bacillus atrophaeus FSHM2) was confirmed. The enzyme (designated as BaL) was
purified using three sequential steps of ethanol precipitation and dialysis, Q-Sepharose XL anion-
exchange chromatography and SP Sepharose cation-exchange chromatography with a final yield
of 9.9% and a purification factor of 31.8. The purified BaL (Mwꢀ85 kDa) was most active at 70 ꢁC
and pH 9 in the presence of 4 M NaCl and retained 58.7% of its initial activity after 150min of
incubation at 80 ꢁC. The enzyme was inhibited by Cd2þ (35.6 1.7%) but activated by Ca2þ
(132.4 2.2%). Evaluation of BaL's stability in the presence of organic solvents showed that
xylene (25%) enhanced the relative activity of the enzyme to 334.2 0.6% after 1 h of incubation.
The results of esterification studies using the purified BaL revealed that maximum ethyl valerate
(88.5%) and methyl valerate (67.5%) synthesis occurred in the organic solvent medium (xylene)
after 48 h of incubation at 50 ꢁC.
KEYWORDS
Lipase; thermostability;
Bacillus atrophaeus;
purification; ethyl valerate
Introduction
host microorganisms made these enzymes the second
largest group of industrial biocatalysts after bacterial
amylases (Hasan et al. 2006; Bora et al. 2013).
Modifying oils and fats in food industries (to improve
the texture and flavour of cheese and bread), waste-
water treatment in pulp-, paper- and leather-manufac-
turing processes, as well as synthesis of biopolymers,
biodiesel, flavouring compounds and enantio-specific
pharmaceuticals are among the most important poten-
tial industrial and biotechnological applications of
lipases (Hasan et al. 2006; Salihu and Alam 2015). As
each biotechnological process may require specific
properties, there is great interest in finding lipases har-
bouring unique characteristics such as a wide range of
thermal and pH operational activity, as well as resist-
ance to salt, heavy metals and organic solvents (Salihu
and Alam 2015; Saengsanga et al. 2016).
Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are
biotechnologically valuable biocatalysts characterized
by the ability to catalyse the hydrolysis of long-chain
acylglycerols at the lipid–water interface and liberate
diglycerides, monoglycerides, free fatty acids and gly-
cerol (Guncheva and Zhiryakova 2011; Christopher
et al. 2015). Besides their natural substrates, these ser-
ine hydrolases are also capable to catalyse the transes-
terification, esterification, acidolysis, aminolysis and
alcoholysis of a wide range of acyl donors and nucleo-
philes (Khoobi et al. 2016; Dhake et al. 2013). The spe-
cial properties of microbial-derived lipases including (i)
activity toward organic solvents as a low-water-content
environment, (ii) regio- and enantioselectivity, (iii) cost-
effective production, (iv) good feasibility for immobil-
ization and (v) susceptibility to express in different
CONTACT Mojtaba Shakibaie
Sciences, Kerman, Iran; Hamid Forootanfar
Medical Sciences, Kerman, Iran
shakiba@kmu.ac.ir
Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical
h_forootanfar@kmu.ac.ir Herbal and Traditional Medicines Research Center, Kerman University of
Supplemental data for this article can be accessed here.
ß 2017 Kerman University of Medical Sciences, Kerman, Iran. Published by informa UK Limited, trading as Taylor & Francis Group.

2
A. AMERI ET AL.
Due to the presence of the genus Bacillus in
of collected soil sample was re-suspended in sterile
solution of NaCl (15% w/v, 20 mL) supplemented with
Tween-80 (0.05% v/v). Next, the prepared soil suspen-
sion was filtered through Whatman No. 1 filter paper,
and 0.5 mL of the resulting soil sample filtrate were
streaked on a BHI agar dish supplemented with
Rhodamine B (0.001% w/v), sodium chloride (15% w/v),
and original olive oil (1% v/v). The prepared plates
were subsequently incubated at 60 ꢁC until the bacter-
ial colonies with shiny orange fluorescence halos (an
indicator of lipase activity) under ultraviolet light
(350 nm) were developed (Castro-Ochoa et al. 2005;
Hasan-Beikdashti et al. 2012). The most potent lipase-
producing isolates were repeatedly subcultured to
obtain an axenic culture of each isolate, which was
then preserved in 20% (v/v) glycerol at ꢂ80 ꢁC
(Moshfegh et al. 2013).
The morphological and biochemical specifications
of the selected bacterium were assessed based on the
Bergey's Manual of Systematic Bacteriology (Vos et al.
2009). Furthermore, thermal and salt tolerance proper-
ties were demonstrated by culturing the selected iso-
late at various temperatures (5 ꢂ 75 ꢁC) or in the BHI
agar medium supplemented with various concentra-
tions of NaCl (0 ꢂ 20% w/v), respectively. In addition,
analysis of 16S rDNA gene sequencing was conducted
after amplification of the related ribosomal gene seg-
ment with the primer pair of 27F (50-
AGAGTTTGATCCTGGCTCAG-30) as forward primer and
1525R (50-AAGGAGGTGATCCAGCC-30) as reverse primer
followed by comparison of the obtained sequence
using the BLAST software (Hasan-Beikdashti et al.
2012; Forootanfar et al. 2015).
extreme habitats of the Earth-like volcanic water, des-
ert soil and polar ice, there are many reports about
isolating extremophilic lipases which originated from
the genus Bacillus (Guncheva and Zhiryakova 2011;
Samaei-Nouroozi et al. 2015). For example, Emtenani
et al. (2013) obtained a thermo-alkaliphilic lipase (opti-
mally works at pH 8 and 70 ꢁC) from B. subtilis DR8806
that was isolated from a hot mineral spring. The puri-
fied lipolytic enzyme of B. thermoleovorans CCR11 (iso-
lated from the “El Carrizal” hot springs in Veracruz,
Mexico) worked optimally at 60 ꢁC and pH 9–10
(Castro-Ochoa et al. 2005). Saengsanga et al. (2016)
isolated an alkaline lipase (which exhibited maximal
activity at pH 10) from B. amyloliquefaciens E1PA and
successfully expressed it in an E. coli host.
Characterization of the alkaline thermostable extracel-
lular lipase (stable at 60 ꢁC and pH 8 for 1 h) produced
by B. cereus C7 (isolated from spoiled coconut)
revealed its resistance towards a wide range of bile
salts, chemical surfactants and heavy metals (Dutta
and Ray 2009).
The main aim of the present study was the isolation
and identification of a thermoalkalophilic bacterial
strain capable of producing lipase from environmental
samples followed by the evaluation of the factors
affecting lipase production. The extracellularly pro-
duced lipase was then purified and characterized, and
its ability to synthesize ethyl and methyl valerate was
assessed in both solvent-free and organic solvent
media.
Materials and methods
Chemicals
Cultivation of the isolate and evaluation of
factors affecting lipase production
Brain heart infusion (BHI) broth, Rhodamine B and
ethyl valerate were supplied by Merck Chemicals
(Darmstadt, Germany). p-Nitrophenyl palmitate (pNPP),
p-nitrophenol (pNP) and bovine serum albumin (BSA)
were provided by Sigma-Aldrich (St. Louis, MO). SP
Sepharose and Q-Sepharose were purchased from
Pharmacia (Uppsala, Sweden). All other applied sub-
stances and solvents were of the highest purity
available.
To determine the growth curve and time course of lip-
ase production the selected isolate was inserted into
500-mL Erlenmeyer flasks including 150 mL of basal
medium composed of (g/L) mannose, 5; olive oil, 10;
NaCl, 200; (NH4)3PO4, 2; NaH2PO4, 2; NaNO3, 2; and
FeSO4.7H2O, 0.04 and incubated at 60 ꢁC while slowly
rotated for 72 h. Samples (1 mL) were taken at regular
intervals and their OD600 was determined after remov-
ing the remaining olive oil (Gupta et al. 2007).
Thereafter, the bacterial cells were separated by centri-
fugation (10,000 g, 10 min), and consequently, the lipo-
lytic activity was assessed as elucidated in the
following section.
Sample collection, isolation and identification of
halophilic bacterium capable to produce lipase
Fifteen salty and hypersaline soil samples were gath-
ered from different sites of Dasht-e Lut (30ꢁ3601800N,
59ꢁ0400400E) near Kerman, Iran, where the land surface
can reach maximum temperatures of 70.7 ꢁC. Each 1 g
The effects of carbon sources (fructose, galactose,
glucose, glycerol, lactose, maltose, mannitol, starch
and sucrose all at final concentrations of 0.5% w/v),

BIOCATALYSIS AND BIOTRANSFORMATION
3
nitrogen supplements containing yeast extract, tryp-
tone, peptone, urea, ammonium chloride and ammo-
nium sulphate (all at final concentrations of 0.5% w/v),
lipase inducers including hazelnut oil, castor oil, coco-
nut oil, sesame oil, sunflower oil, sweet oil, Tween-80
and Triton X-100 (all at final concentrations of 1% v/v),
and metal cations of MgSO4.7H2O, CaCl2.2H2O, KCl,
FeCl3 and ZnSO4.7H2O (at final concentrations of
1 mM) on lipase production were assessed by individu-
ally adding each factor to the basal media (as men-
tioned earlier) followed by cultivating the isolate for
16 h and determining lipolytic activity.
same buffer at a flow rate of 120 mL/h to remove the
unbound proteins and the bound proteins were eluted
by increasing the NaCl concentration (0–500 mM) step-
wise. Next, the previous step's pooled active fractions
were loaded on the SP Sepharose column (16 mm
ꢃ130 mm) equilibrated using phosphate buffer
(50 mM, pH 6.5). Thereafter, the column was washed
with the same buffer and the bound proteins were
eluted using NaCl gradient (0–500 mM) in the same
buffer. After each purification step, the fractions
obtained were analysed for lipolytic activity and pro-
tein content (A280), and the specific activity, as well as
the purification factor and yield, were calculated.
The subunit molecular mass of the purified lipase
and the homogeneity of each purification step were
determined by sodium dodecyl sulphate polyacryl-
amide gel electrophoresis (SDS–PAGE) using 10%
gel based on the method described by Laemmli
(1970). The N-terminal sequencing of the purified
lipase was assessed by Seqlab Co. (Gottingen,
Germany).
Lipase assay and protein estimation
The previously described method of Hasan-Beikdashti
et al. (2012) was applied to measure lipolytic activity.
Briefly, 0.1 mL of the sample supernatant (as lipase
solution) and 0.8 mL of phosphate buffer (0.1 M, pH
7.8) containing 0.4% of Triton X-100 and NaCl (1 M)
were mixed with the lipase substrate (pNPP solution,
0.01 M) and the prepared mixture was consequently
incubated at 60 ꢁC for 30 min under mild shaking.
Afterward, the absorbance of the reaction mixture was
recorded at 410 nm using a Shimadzu double beam
spectrophotometer (UV-1800, Shimadzu Corporation,
Tokyo, Japan). The amount of enzyme that catalysed
the formation of 1 lmol of pNP per min from pNPP
was defined as one unit (U) of lipolytic activity. In
order to determine the protein concentration,
Bradford's colorimetric method was applied (Bradford
1976) using BSA as the standard.
Lipase characterization
Influence of pH and temperature on activity and
stability
The pH-activity profile of the purified lipase was dem-
onstrated after performing lipase assay in the presence
of a lipase substrate (pNPP) prepared in a 100 mM
solution of the following buffers: citrate buffer (pH
3 ꢂ 5), phosphate buffer (pH 6 ꢂ 8) and carbonate buf-
fer (pH 9 ꢂ 10) all containing NaCl (1 M) under the pre-
viously mentioned assay conditions. The pH stability
profile of the enzyme was evaluated by incubating the
purified lipase at 60 ꢁC for 300 min in the above-men-
tioned buffers (pH ranges of 3 ꢂ 11), and the percent-
age of residual enzyme activities was determined.
In order to estimate the effect of temperature on
the activity of the purified enzyme, the lipolytic activ-
ity was measured at temperatures of 4 ꢁC, 20 ꢁC, 30 ꢁC,
37 ꢁC, 45 ꢁC, 60 ꢁC, 70 ꢁC, 80 ꢁC, 90 ꢁC and 100 ꢁC. The
temperature stability pattern of the purified lipase was
demonstrated by pre-incubating the enzyme solution
at temperatures of 50 ꢁC, 55 ꢁC, 60 ꢁC, 65 ꢁC, 70 ꢁC,
75 ꢁC and 80 ꢁC for 300 min then measuring the
residual lipase activity each 30 min (Cao et al. 2012;
Daoud et al. 2013).
Purification of the lipase
All purification steps were performed at 4 ꢁC. First, the
bacterial strain was cultivated in the basal medium
(previously defined) for 16 h, and the lipase-containing
culture broth was then prepared by two steps of
removing the produced biomass using centrifugation
(6000 g, for 10 min) followed by precipitation of the
remaining fatty acids assisted by addition of a CaCl2
solution (0.4 M) and subsequent centrifugation at 4 ꢁC
and 12,000 g for 20 min. The prepared cell-free culture
broth was consequently brought to 80% saturation
with pre-chilled ethanol, and the precipitated proteins
were collected by centrifugation at 12,000 g for
25 min. The resulting precipitate was dissolved in Tris-
HCl buffer (100 mM, pH 8) and dialysed against the
same buffer. The concentrated sample was applied
onto a Q-Sepharose XL column (16 mm ꢃ 130 mm)
equilibrated with Tris-HCl buffer (50 mM, pH 8). The
column was washed with a five-column volume of the
Effect of metal ions, enzyme inhibitors and surfac-
tants on lipase activity
The effects of different metal ions including Mg2þ, Kþ,
Ca2þ, Cu2þ, Agþ, Co2þ, Cd2þ, Mn2þ, Zn2þ and Fe3þ (all

4
A. AMERI ET AL.
at final concentrations of 10 mM), enzyme inhibitors
dithiothreitol (DTT, 0.5 mM) and EDTA (2.5 mM), as well
as surfactants containing SDS (1 mM), Triton X-100
(1%), Tween-40 (1%), and Tween-80 (1%) on the activ-
ity of the purified lipase was determined by separately
adding each factor into the assay mixture and measur-
ing the residual lipase activity under standard assay
conditions.
Lipase-mediated synthesis of methyl and ethyl
valerate
Ester synthesis was performed in 50-mL stoppered con-
ical flasks in aqueous and organo-aqueous medium
based on the procedure reported by Khoobi et al.
(2015). For the aqueous medium, the reaction mixture
(final volume of 10 mL) containing valeric acid solution
in methanol or ethanol (acid/alcohol molar ratios of 2:1,
1:1, 1:2, 1:3, 1:4 and 1:5) was prepared and the purified
lipase of B. atrophaeus FSHM2 (10 U/mL, in Tris buffer
100 mM pH 9) was added. The reaction was subse-
quently incubated at 50 ꢁC and 100 rpm for 1, 2, 4, 6, 12,
24, 36 and 48 h. In the solvent-containing medium, a
solution including 4 mmol of methanol or ethanol and
1 mmol of valeric acid in anhydrous organic solvents of
n-hexane or xylene (final volume of 10 mL) was pre-
pared. Then, the purified lipase (10 U/mL, in Tris buffer
100 mM pH 9) was inserted into the prepared reaction
mixture and allowed to stand with agitation at 50 ꢁC on
a rotary shaker (100 rpm) for 1, 2, 4, 6, 12, 24, 36 and
48 h. Afterwards, interval samples of 500 lL were period-
ically withdrawn, extracted using n-hexane and subjected
to ester formation analyses using a gas chromatography
apparatus (Agilent, 7890A) equipped with a flame ioniza-
tion detector (FID, Agilent) and a capillary column (HP
Innowax 30 m length ꢃ 0.32 lm i.d. ꢃ 0.5 lm thickness).
The temperature range of 60 ꢂ 210 ꢁC (with temperature
rate of 10 ꢁC/min) was used while the carrier gas nitro-
gen (flow rate of 30 mL/min), and the temperature of
the injector and detector were set to 250 ꢁC. Control
experiments were carried out by inserting the inactivated
lipase (using trichloro acetic acid 10%) into the reaction
mixture. The ester yield was then calculated based on
comparing the retention time and peak sample area
with the standard (Kraai et al. 2008).
Effect of NaCl concentration on lipase activity and
stability
Different concentrations of NaCl (1 ꢂ 5 M) were added
to the standard reaction mixture (as described earlier)
containing lipase, and the residual activity was meas-
ured to examine the influence of NaCl on the lipase
activity. In addition, the salt stability of the purified lip-
ase was assessed by pre-incubating the enzyme in the
presence of different NaCl concentrations (1 ꢂ 5 M) for
300 min at 60 ꢁC followed by determining the remain-
ing lipolytic activity compared to the control (the puri-
fied lipase in the absence of NaCl).
Organic solvents stability of the purified lipase
The effect of organic solvents with different log p
(Table 1) on the lipase stability was investigated by
inserting each applied organic solvent into the enzyme
solution to attain a final concentration of 25% (v/v)
and incubating the prepared mixture for 1 h at 60 ꢁC.
The remaining organic solvents were then removed
using centrifugation at 12,000 g for 5 min (4 ꢁC), and
the residual activity was subsequently measured as
previously described.
Table 1. Stability of the purified lipase of B. atrophaeus
FSHM2 towards organic solvents (25% v/v).
Statistical analyses
Organic solvent
Log p
Relative activity (%)
Three replicates of each above-mentioned experiment
were conducted, and the obtained results were
reported as mean SD. In order to calculate the statis-
tical significance (probability values less than 0.05)
between mean values, an independent sample t-test
and one-way analysis of variance (ANOVA) with
Dunnett's T3 post hoc test were applied using SPSS
software (version 15.0, SPSS Inc., Chicago, IL).
Control
Methanol
Ethanol
ꢂ
100.0 0.0
42.6 1.2a
83.7 0.8a
69.6 0.5a
117.8 0.9a
97.6 1.6
ꢂ0.76
ꢂ0.24
2.9
n-Octanol
n-Butanol
n-Heptane
Xylene
n-Hexane
Isoamylalcohol
Isopropyl alcohol
Chloroform
1,4-Dioxane
DMF
0.89
4.0
3.1
3.5
1.3
334.2 0.6a
153.2 1.4a
121.4 0.9a
113.1 1.3
7.7 0.2a
0.28
2.0
ꢂ0.27
53.3 0.5a
53.4 0.5a
23.4 1.1a
100 0.6
ꢂ1
Acetone
Dichloromethane
DMSO
ꢂ0.23
1.25
ꢂ1.35
Results and discussion
56.4 0.7a
Isolation and identification of the selected
lipase-producing bacterial strain
The reaction mixture was prepared by insertion of the purified lipase into
each organic solvent solution (ratio of 3:1) followed by incubation at
60 ꢁC for 1 h and subsequent measuring remained lipolytic activity.
Experiments were performed in triplicates and significance (a) was
attained after ANOVA with Dunnett’s T3 post hoc test.
Screening of the thermo-halophilic bacterial strains (4
isolates acquired from 15 gathered samples) for lipase

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 1. Time course of lipase production and growth curve of B. atrophaeus FSHM2 cultivated in a basal medium containing
NaCl (20% w/v) and olive oil (1% v/v).
activity revealed that only one isolate (designated as
M2) produced the related shiny fluorescence halos
(under UV light, 350 nm) around their colonies when
grown at 60 ꢁC on the Rhodamine B containing BHI
agar medium (supplemented with olive oil, 1% w/v
and NaCl, 15% w/v) that indicated lipase production.
The biochemical characterization results demonstrated
that the selected microorganism was a Gram-positive,
non-motile and rod-shaped bacterium that formed a
white to pale yellow colony on the BHI agar medium
and was capable to grow at a temperature range of
40–75 ꢁC and in the presence of a high level of sodium
chloride (2.5–20%). Isolate M2 represented positive
reactions for oxidase and catalase but was not able to
hydrolyse gelatin and starch. It was able to produce
acid from maltose, sucrose, lactose, mannitol, glycerol,
galactose, fructose and glucose. Comparison of the
obtained sequence of the amplified 16S rDNA gene
using the BLAST software revealed 99% similarity with
Bacillus atrophaeus. The 1420 bp gene sequence was
then submitted to GenBank (accession number,
KF682367).
The time course of lipase production, alteration of
OD600, and pH changes in the culture broth of B. atro-
phaeus FSHM2 during a 72-h incubation period at
60 ꢁC (Figure 1) indicated that maximal lipolytic activity
(4995 U/L) occurred after 16 h of incubation where
OD600 of culture broth reached 1.43.
The ability of Bacillus strains to secrete extremo-
philic lipases has been previously reported (Guncheva
and Zhiryakova 2011). For example, Saengsanga et al.
(2016) launched an alkaline lipase (optimal pH of 10)
produced by B. amyloliquefaciens E1PA that was iso-
lated from lipid-rich food waste. Dutta and Ray (2009)
reported that the alkaline thermostable extracellular
lipase of B. cereus C7 (isolated from spoiled coconut)
was maximally produced after 24 h of cultivation.
Evaluation of factors affecting lipase production
Due to the critical role of the culture medium's chem-
ical components on lipase productivity and the growth
of lipase-producing microbial strains (Hasan-Beikdashti
et al. 2012; Bora et al. 2013), the effects of carbon and
nitrogen supplementation, metal ions and lipase
inducers on lipase activity of B. atrophaeus FSHM2
were investigated in the present study. Glucose, hazel-
nut oil, and calcium ions positively affected lipase pro-
duction by increasing production to 13,850 U/L,
6279 U/L and 5513 U/L in comparison with the basal
medium (4987 U/L, p ꢄ .05). However, urea exhibited
negative effect by decreasing lipase production to
4446 U/L compared to that of the basal medium
(4987 U/L, p ꢄ .05).
Enzyme purification, molecular weight estimation
and N-terminal sequencing
In the present study, the produced lipase of B. atro-
phaeus FSHM2 (designated as BaL) was purified in
three sequential steps. First, the cell-free culture broth
was concentrated using ethanol precipitation (80%)
and dialysis (39.8% recovery and 8.2 purification factor)
and then two chromatographic steps employing anion
(19.4% yield and purification factor of 20.7) and cation
(9.9% recovery and 31.8 purification factor)-exchange
media were successfully applied for lipase purification
(Table 2). The homogeneity of each purification step is

6
A. AMERI ET AL.
Table 2. Purification steps for extracellular lipase produced by B. atrophaeus FSHM2.
Purification step
Total activity (U) Total protein (mg) Specific activity (U/mg) Recovery (%) Purification (factor)
Centrifuged culture broth (crude)
173.0
68.9
33.6
17.2
94.3
4.7
0.9
1.8
14.7
37.3
57.3
100.0
39.8
19.4
9.9
1
8.2
20.7
31.8
0–80% ethanol precipitation and dialysis
Q-Sepharose XL anion-exchange chromatography
SP Sepharose cation-exchange chromatography
0.3
Table 3. Comparison of the N-terminal amino acid sequences of different Bacillus derived lipases.
Microorganism
N-terminal amino acid sequencea
Accession number
B. atrophaeus FSHM2
Bacillus atrophaeus
Bacillus atrophaeus
Bacillus atrophaeus UCMB-5137
Bacillus cereus
M
M
M
M
M
M
K
K
K
K
K
K
Q
N
N
N
N
N
R
R
R
R
R
R
V
V
V
V
V
V
N
N
N
N
N
N
P
P
P
P
P
P
D
E
E
E
E
E
L
L
L
L
L
L
L
L
L
L
L
L
Q
Q
Q
Q
Q
Q
G
G
G
G
G
G
L
L
L
L
L
L
E
M
M
M
M
M
M
Present study
GI:498487057
GI:751416253
GI:830324800
GI:872530815
GI:983300457
E
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Bacillus cereus
aidentical amino acids are shown as bold letter.
illustrated in Figure S1, and the molecular weight of
the purified lipase was estimated to be approximately
85 kDa.
Olusesan et al. (2011) purified a thermostable lipase
(molecular weight, 45 kDa) of the extremophilic B. sub-
tilis NS8 using three sequential steps of ultrafiltration,
anion-exchange chromatography (DEAE-Toyopearl),
and size exclusion chromatography (Sephadex G-75)
with the final purification factor of 500 and yield of
16%. The solvent-tolerant lipase (molecular weight,
69 kDa) of B. sphaericus MTCC 7542 was purified after
two consecutive steps of ammonium sulphate precipi-
tation (purification factor of 1.35 and yield of 74%)
and DEAE–Sepharose anion-exchange chromatography
(purification factor of 17.33 and yield of 5.7%)
(Tamilarasan and Kumar 2012). A literature review
revealed that the molecular weight of lipases origi-
nated from Bacillus strains usually vary from 19 kDa (B.
subtilis 168) (Lesuisse et al. 1993) to 108 kDa (Bacillus
sp. GK 8) (Dosanjh and Kaur 2002; Guncheva and
Zhiryakova 2011).
Figure 2. Influences of pH on the activity (j) and stability (ꢁ)
of the purified lipase of B. atrophaeus FSHM2.
lost 7.5% and 24.4% of its maximal activity after 5 h of
incubation at pH 7.8 and pH 10, respectively. Similar
results were reported by Chen et al. (2007) who
observed that more than 70% of the original activity
of the purified lipase of B. cereus C71 (optimum pH of
9) was retained after 3 h of incubation over a pH range
of 8.5–10. In general, the optimum pH of most
Bacillus-originated lipases falls on the alkaline side
(Guncheva and Zhiryakova 2011).
As presented in Table 3, the N-terminal amino acid
sequence of BaL was MKQRVNPDLLQGLEM, which
showed 86.7% similarity to B. atrophaeus and B. cereus
lipases.
After demonstrating lipase activity at various tem-
peratures, it was determined that the BaL showed
maximal activity at 70 ꢁC (Figure 3(a)) and altering the
temperature to 80 ꢁC, 90 ꢁC and 100 ꢁC decreased the
lipolytic activity to 88.2%, 71.5% and 43.2% of the
maximum, respectively. At 37 ꢁC and 45 ꢁC, the enzyme
showed approximately half of its maximal activity
(Figure 3(a)). The stability curve of the BaL (Figure
3(b)) revealed that the enzyme lost 51.3% and 58.3%
of its maximum activity after 150 min of incubation at
80 ꢁC and 75 ꢁC, respectively. However, the BaL
retained 92.5% of its original activity after 300 min of
incubation at 55 ꢁC (Figure 3(b)). Lee et al. (1999)
BaL characterization
Influence of pH and temperature on BaL activity
and stability
According to the relative activity profile of the BaL
(Figure 2) at different pH levels, the optimum pH of
the enzyme was 9 where the maximum stability of the
enzyme was also observed at this pH. The lipolytic
activity was gradually decreased by decreasing the pH
from 9 to 3 (15.8%) (Figure 2). Determining the pH sta-
bility of the BaL (Figure 2) revealed that the enzyme

BIOCATALYSIS AND BIOTRANSFORMATION
7
Table 4. Obtained results of relative activity of the purified
lipase from B. atrophaeus FSHM2 in the presence of metal
ions, enzyme inhibitors and detergents.
Metal ions
Final concentration
Relative activity (%)
Control
Mg2þ
–
100
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
74.4 1.4a
86.7 0.8a
50.3 1.1a
132.4 2.2a
59.6 1.4a
52.1 0.9a
35.6 1.7a
77.3 0.8a
93.8 2.3
53.5 1.5a
Kþ
Cu2þ
Ca2þ
Agþ
Co2þ
Cd2þ
Mn2þ
Zn2þ
Fe3þ
Enzyme inhibitors
EDTA
DTT
2.5 mM
0.5 mM
71.4 1.1a
73.4 2.2a
Detergents
Triton X-100
Tween-80
Tween-40
SDS
1%
1%
1%
47.3 1.6a
42.1 1.3a
8.6 0.9a
2.3 1.5a
1 mM
aANOVA analysis with Dunnett’s T3 post hoc test was applied to deter-
mine the significant values (p value <.05).
mentioned fatty acids (Guncheva and Zhiryakova
2011).
EDTA (as chelating agent) was able to inhibit the
BaL activity (Table 4) which was in accordance with
the results obtained by Kanwar et al. (2006) who
described the negative effect of EDTA on the activity
of the lipase originating by B. coagulans MTCC-6375
(78%). In contrast, the activity of B. pumilus B26 lipase
was not affected in the presence of EDTA (Kim et al.
2002). The negative effect of DTT as a reducing agent
on the activity of the BaL (Table 4) was similar to the
previously reported results of Sabri et al. (2009) who
determined 22.5% of decreased activity of purified lip-
ase originating from Bacillus sp. L2.
The non-ionic surfactants containing Triton X-100,
Tween-80 and Tween-40, as well as the anionic surfac-
tant of SDS significantly decreased the BaL activity to
47.3 1.6%, 42.1 1.3%, 8.6 0.9% and 2.3 1.5,
respectively (Table 4). In contrast, Chen et al. (2007)
achieved 160%, 141% and 128% of relative activity of
B. cereus C71 lipase in the presence of Triton X-100
(0.5%), Tween-80 (0.5%) and Tween-20 (0.5%),
respectively.
As shown in the salt tolerance profile of the BaL
(Figure 4), the relative activity and stability of the
enzyme were gradually increased by elevating the salt
concentration and maximum relative lipolytic activity
was observed with a 4 M concentration of NaCl
(Figure 4). However, the purified lipase was less active
(31.4%) and less stable (16.7%) in the absence of NaCl
compared with those of 4 M NaCl (p value <.05,
Figure 4). At a higher salt concentration (5 M), both
the relative activity (79.8%) and stability (63.2%) of the
Figure 3. (a) Temperature-activity profile and (b) temperature
stability curve of the purified lipase of B. atrophaeus FSHM2.
reported that the purified lipase of B. thermoleovorans
ID-1 showed maximal activity at 70–75 ꢁC and retained
50% of its maximal activity after 30 min of incubation
at 70 ꢁC. Bradoo et al. (1999) observed that 50% of the
initial activity of the thermotolerant lipase from B.
stearothermophilus SB-1 remained after 15 min of incu-
bation at 100 ꢁC.
Effect of metal ions, enzyme inhibitors, detergents
and salt concentration on BaL activity
As presented in Table 4, except for calcium, which
enhanced the relative activity of the BaL to
132.4 2.2% (p value <.05), all of the other heavy
metal ions tested significantly declined the BaL activity
(Table 4). Chen et al. (2007) reported that the purified
lipase of B. cereus C71 was inhibited by all the heavy
metal ions tested. In general, the positive effect of
Ca2þ on the hydrolysis process of the lipases, espe-
cially lipases originating from the Bacillus genus, are
ascribed to (i) the structural role of this divalent cation
and (ii) suppression of the inhibitory activity of the
formed fatty acids (released during enzymatic hydroly-
sis) due to the formation of insoluble Ca-salts of the

8
A. AMERI ET AL.
Figure 4. Influence of NaCl concentrations on the activity and
stability of the purified lipase. Lipase activity was measured
using pNPP as a lipase substrate containing different NaCl con-
centrations, while enzyme stability was demonstrated after 5 h
of incubation of the enzyme in the presence of NaCl and
ꢅ
measuring of residual activity. Significance (p value <.05) was
determined after the ANOVA analysis of the attained results
(n ¼ 3) compared with those of control (enzyme reaction mix-
ture in the presence of 4 M NaCl).
BaL significantly dropped compared to the 4 M NaCl
concentration (p value <.05) (Figure 4). A similar pat-
tern occurred when the reaction mixture of lipase
assay was prepared in the absence of NaCl (p value
<.05) (Figure 4). It seems that the presence of salt up
to the critical concentration of 4 M is beneficial for the
hydrolytic activity of the BaL. Similar results were
achieved by Balaji and Jayaraman (2014) where a halo-
philic lipase producer (Bacillus sp. VITL8) was isolated
from oil-contaminated areas. They found that more
than 55% of the original activity of the produced lip-
ase remained at 60 ꢁC and pH 10 in the presence of
3 M NaCl (Balaji and Jayaraman 2014).
Figure 5. Lipase-catalysed synthesis of (a) ethyl valerate and
(b) methyl valerate in solvent-free and organic solvent-contain-
ing media. Data are shown as mean SD (n ¼3).
presence of all studied organic solvents except for
diethyl ether that increased the lipase activity to
120%. Benzene (30%) and hexane (30%) increased the
residual activity of the purified lipase of Bacillus sp.
Lip2 by 207% and 201%, respectively (Nawani and
Kaur 2007). Guncheva and Zhiryakova (2011), who
reviewed the catalytic properties of the lipases origi-
nating from the genus Bacillus, described that these
biocatalysts are very stable in the presence of organic
solvents. This stability was especially notable with non-
polar solvents due to their ability to shift the equilib-
rium of lipase conformation from closed to open form
and modify the substrates and products solubility in
the reaction medium (Guncheva and Zhiryakova,
2011).
Stability of the BaL in the presence of organic
solvents
Due to the importance of organic solvent stability of
enzymes in various industrial processes the stability of
the BaL in the presence of fourteen common organic
solvents harbouring different log p values (ꢂ1.35 to
4.0) was also evaluated in the present study. As shown
in Table 1, the relative activity of the BaL was signifi-
cantly enhanced in the presence of n-butanol
Application of BaL for esterification
(117.8 0.9%),
xylene
(334.2 0.6%),
n-hexane
(153.2 1.4%), isoamyl alcohol (121.4 0.9%) and iso-
propyl alcohol (113.1 1.3%), while other applied sol-
vents declined the residual BaL activity after 1 h of
incubation at 60 ꢁC (Table 1). Bradoo et al. (1999)
reported that the relative activity of the produced lip-
ase B. atrophaeus was negatively affected by the
The results of the BaL application for synthesis of
ethyl valerate (Figure 5(a)) and methyl valerate
(Figure 5(b)) clearly indicated that the organo-
aqueous solvent medium is more appropriate for lip-
ase-assisted esterification reaction compared to the
aqueous medium. Among the four applied organic

BIOCATALYSIS AND BIOTRANSFORMATION
9
Conclusions
BaL, a thermoalkalophilic and extracellular lipase (opti-
mum temperature of 70 ꢁC and pH 9), was successfully
purified from a cell-free culture broth of an extremely
halophilic bacterial strain (B. atrophaeus FSHM2) using
three consecutive purification steps. Approximately
92.5% of the BaL initial activity was retained after incu-
bating the purified lipase at 55 ꢁC for 5 h. The relative
activity of the BaL (approximate Mw of 85 kDa) was
considerably enhanced in the presence of NaCl (4 M),
Ca2þ (10 mM) and the organic solvent xylene (25%).
Ethyl valerate (conversion, 88.5%) and methyl valerate
(conversion, 67.5%) were synthesized after BaL-cata-
lysed esterification in the xylene containing medium
for 48 h.
Figure 6. Influence of molar ratio of valeric acid/ethyl or
methyl alcohol on esterification percentage assisted by the
purified lipase of B. atrophaeus FSHM2 in a xylene containing
medium at 50 ꢁC.
Disclosure statement
solvents, xylene was the most efficient one which
increased the esterification percentages of ethyl val-
erate and methyl valerate to 88.5% and 67.5%,
respectively, after 48 h of incubation in the presence
of BaL (Figure 5(a,b)). In contrast, the solvent-free
system yielded esterification percentages of 19.3%
and 22% for methyl valerate and ethyl valerate,
respectively (Figure 5(a,b)) under the same condi-
tions. Analysis of the reaction mixture containing
inactivated lipase (as a negative abiotic control)
revealed an esterification percentage of 4%. In the
study of Kumar et al. (2006), who applied the puri-
fied lipase B. coagulans BTS-3 for esterification of
ethanol and propionic acid, it was observed that the
esterification reaction was optimal in the presence of
n-hexane at 55 ꢁC. In general, the presence of an
optimum amount of water is essential for lipase-cata-
lysed esterification reactions due to water's critical
role as a lubricant for mobility of the enzyme in
catalytic action (Sivaramakrishnan and Muthukumar
2014). On the other hand, excessive water can shift
the esterification equilibrium towards hydrolysis of
the produced ester (Dhake et al. 2013). As the molar
ratio of acid/alcohol is another parameter affecting
esterification reaction yield, (Khoobi et al. 2015), the
present study also evaluated this factor. The results
(Figure 6) revealed that the highest esterification per-
centages of both ethyl valerate (85.6%) and methyl
valerate (66.8%) occurred at the molar ratio of 1:4
(v/v) acid to alcohol. Kumar et al. (2013), who
applied the immobilized lipase Bacillus sp. DVL2 for
the production of ethyl oleate, determined that the
molar ratio of 1:1 (v/v) oleic acid and ethanol was
the most appropriate ratio to achieve the highest
ester formation (63%).
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
Funding
This work was financially supported by the grant 94/1691
from Herbal and Traditional Medicines Research Center,
Kerman University of Medical Sciences, Kerman, Iran.
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