Purification and characterization of a mesophilic organic solvent tolerant lipase produced by Acinetobacter sp. K5b4

Article abstract of DOI:10.1080/10242422.2018.1506445

The extracellular lipase produced by Acinetobacter sp. K5b4 was purified to homogeneity using ultrafiltration (cutoff 30 KDa) followed by gel filtration chromatography on Sephadex G-50. The enzyme was purified to homogeneity with an apparent molecular mas

Full text of DOI:10.1080/10242422.2018.1506445

Biocatalysis and Biotransformation
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Purification and characterization of a mesophilic
organic solvent tolerant lipase produced by
Acinetobacter sp. K5b4
Muhamad O. Al-Limoun, Khaled M. Khleifat, Khalid Y. Alsharafa, Haitham N.
Qaralleh & Suddam A. Alrawashdeh
To cite this article: Muhamad O. Al-Limoun, Khaled M. Khleifat, Khalid Y. Alsharafa,
Haitham N. Qaralleh & Suddam A. Alrawashdeh (2018): Purification and characterization of a
mesophilic organic solvent tolerant lipase produced by Acinetobacter sp. K5b4, Biocatalysis and
Biotransformation, DOI: 10.1080/10242422.2018.1506445
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2018.1506445
RESEARCH ARTICLE
Purification and characterization of a mesophilic organic solvent tolerant
lipase produced by Acinetobacter sp. K5b4
a
a
a
b
Muhamad O. Al-Limoun
, Khaled M. Khleifat
, Khalid Y. Alsharafa
, Haitham N. Qaralleh
and
a
Suddam A. Alrawashdeh
a
b
Biology Department, Mutah University, Mutah, Karak, Jordan; Department of Medical Laboratory Sciences, Mutah University, Mutah,
Karak, Jordan
ABSTRACT
ARTICLE HISTORY
The extracellular lipase produced by Acinetobacter sp. K5b4 was purified to homogeneity using
ultrafiltration (cutoff 30 KDa) followed by gel filtration chromatography on Sephadex G-50. The
enzyme was purified to homogeneity with an apparent molecular mass of 133KDa by SDS-
PAGE. This purification resulted on 10.24 fold with 18.3% recovery. The Km and Vmax of purified
enzyme when using pNPL hydrolysis were 4.0 mM and 73.53 nmol/ml/min, respectively. The
pure enzyme was greatly stimulated in the presence of 20, 40 and 60% (v/v) methanol, DMSO
and acetone whereas, ethanol, acetonitrile and propanol decreased the enzyme activity.
Received 16 February 2018
Accepted 26 July 2018
KEYWORDS
Extracellular lipase;
Acinetobacter sp. K5b4;
purification; characteriza-
tion; pNPL hydrolysis
ꢀ
Maximum enzyme activity was achieved at pH 7.0 and incubation temperature of 27 C. The
ꢀ
enzyme was stable within a pH range of 6.5 to 7 at 27 C for 1 h. The enzyme activity was
2 2 2
enhanced up to 36% by KCl, BaCl , MgCl and CaCl while obviously inhibited (10–20%) by
CoCl , ZnCl , MnCl and CuCl . No inhibitory effects were observed with 1.0 and 5.0 mM of
2
2
2
2
2-mercaptoethanol and EDTA. Similarly, SDS at 1.0 mM does not affect the enzyme activity while
high reduction (80%) was observed at 5.0 mM SDS concentration. The enzyme was active
against p-nitrophenyl esters of C8, C12 and C16 with highest preference to the medium carbon
chain p-nitrophenyl caprylate (C8). The fact that the enzyme displays distinct stability in the
presence of methanol, DMSO and acetone suggests that this lipase is suitable as biocatalyst in
organic synthesis where such hydrophilic organic solvents are used as a reaction media.
1
. Introduction
Previously, the shortage in lipases with specific
characteristics required for manufacturing processes
was one of the limiting factors of their use
Enzymes are biocatalysts playing an important role in
metabolic and biochemical reactions. Microorganisms
in addition to plants and animals produce lipases.
Commonly, microbial lipases confirmed to be more
preferable compared to other higher organisms lipases
due to their mass production and versatility of their
properties (Andualema and Gessesse 2012). Recently,
more and more carefulness is being paid to lipases
produced by bacteria and fungi.
(Yuan et al. 2016). Microbial lipases are to some extent
stable and are able of catalyzing different reactions;
they are of potential concern for various indus-
trial use.
Based on their remarkable characteristics lipases
now categorized in literature as thermostable (Yuan
ꢀ
et al. 2016), cold active (Canak et al. 2015), alkaline
(Wang et al. 2012), acidic (Panizza et al. 2015) and
Lipases are hydrolytic enzymes (triacylglycerol
hydrolases E.C. 3.1.1.3) that catalyze the hydrolysis of
triacylglycerols (TAGs) to glycerol and free fatty acids
organic solvent tolerant lipases (Li et al. 2014). Their
remarkable characteristics were found individually or
as a combination of two or more of them such as
thermostable alkaline lipases (Bhosale et al. 2016) cold
active alkaline lipase (Wang et al. 2011) thermostable
organic solvent tolerant lipases (Mo et al. 2016) and
thermosatble alkaline organic solvent tolerant lipase
(Ahmed et al. 2010). Obviously, biocatalysts with a
(FAs) (Snellman et al. 2002; Ahmed et al. 2010; Kim
et al. 2012). The biotechnological applications using
lipases require suitable enzymes as a biocatalyst with
complementary characteristics. These lipases usually
obtained from the existing commercial lipase prepara-
tions or the biodiversity of their natural sources.
CONTACT Khaled M. Khleifat
alkh_kha@hotmail.com
Biology Department, Mutah University, Mutah, Karak 61710, Jordan
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
M. O. AL-LIMOUN ET AL.
combination of these characteristics were more prefer-
able in industries.
Hanil Science Industrial CO., LTD, South Korea) for
5 min at 5000 rpm.
However, some commercial applications like bio-
diesel production do not require pure lipase prepara-
tions. Suitable lipase purity is mostly required in the
areas such as food processing, detergent, paper and
pulp industry. Therefore, it is necessary to remove
some unwanted impurities like proteins because of
their antagonistic effects on the desired enzyme’s
activity (Moradi et al. 2015). However, purified lipase
preparations are needed for the biocatalytic produc-
tion of fine chemicals, pharmaceuticals and cosmetics
One milliliter of the concentrated enzyme was
applied to a 50 ml Sephadex G-50 column (1.0 ꢁ 65 cm)
equilibrated with 0.05 M sodium phosphate buffer, pH
7.0. The column was eluted with equilibration buffer at
a flow rate of 0.2 ml/min. Fractions of about 2 ml were
collected and assayed for enzyme activity (Winkler and
Stuckmann 1979) and protein content at 280 nm.
Fractions with the highest lipase activity obtained from
gel filtration chromatography were chosen. Protein
samples were analyzed by SDS-PAGE as described by
Laemmli (1970). SDS-PAGE was carried out in 10% (w/v)
homogenous gel consisting of a resolving gel (pH 8.8,
10% w/v, acrylamid) and a stacking gel of 4% (w/v)
acrylamide (pH 6.8). Reagents, buffers, broad range pro-
tein markers in the range of 6.5–200 KDa (Sigma-
Aldrich) and the enzyme sample were prepared accord-
ing to Bollag et al. (1996). Electrophoresis was carried
out at constant voltage of120V for 1 h using Mini-
Protein electrophoresis apparatus (Cleaver Scientific
Ltd, UK). The gel was stained with silver staining
method as described by Bollag et al. (1996).
(Costa-Silva et al. 2014). This study aimed to purify
and characterize the extracellular lipase enzyme pro-
duced by Acinetobacter sp. K5b4 that was isolated
from hydrocarbon contaminated soil samples, Al-karak
province, south of Jordan.
2. Materials and methods
2.1. Bacterial isolate
The bacterial strain used in this study was isolated
from hydrocarbon contaminated soil samples collected
from Al-karak province, south of Jordan. The bacterial
strain was identified based on the biochemical tests
2
.4. Characterization of pure lipase of
TM
(RapID
ONE System, Remel, USA) and 16S rRNA
Acinetobacter sp. K5b4
gene sequencing (GENEWIZ, USA) as Acinetobacter
species (Allimoun et al. 2015).
2
.4.1. Substrate specificity
Lipase specificity toward p-nitrophenyl fatty acid esters
with different carbon chain length was measured
spectrophotometrically (SPUV-19, Sco-TECH, Germany)
using the method of Winkler and Stuckmann (1979).
p-nitrophenylcaprylate (pNPC), p-nitrophenyl laurate
2
.2. Culture medium and growth conditions
For extracellular lipase production from Acinetobacter
sp. K5b4, the bacterial strain was grown in the opti-
mized culture medium and growth conditions
reported earlier (Allimoun et al. 2015). The optimized
culture medium (pH 7.0) composed of (per 100 ml);
(pNPL) and p-nitrophenyl palmitate (pNPP) (Sigma-
Aldrich) as lipase enzyme mono-ester substrates was
alternatively changed in the assay reaction solution.
Hundred microliters of the purified enzyme was added
to 1.90 ml of the assay reaction mixture of each sub-
1.0% (w/v) olive oil, 0.2% (w/v) glycerol, 0.15% (w/v)
yeast extract and 0.05% (w/v) NaCl. The culture
medium was inoculated with 0.4% (v/v) of 0.2 OD6
seed culture and incubated at 30 C and 150 rpm
for 48 h.
00
strate, and the reaction was allowed to proceed for
ꢀ
ꢀ
1
0 min at 30 C. The p-nitrophenol formed from the
hydrolysis action of lipase in each substrate mixture
was measured at 410 nm immediately. Relative activity
was calculated by comparing the enzyme activity in
the three substrates reaction mixtures.
2.3. Purification of extracellular lipase produced
by Acinetobacter sp. K5b4
Before the gel filtration step, the culture broth was
harvested and centrifuged at 5000 rpm for 5 min at
room temperature. Fifteen milliliter of the cell free
supernatant (total activity of 187 mU/ml) were concen-
trated 3X to a final volume of 5 ml using Amicon
Ultra-15 centrifugal filter units (Sigma-Aldrich, USA)
cut off 30 KDa in refrigerated centrifuge (Combi 514 R,
2
.4.2. K and V
determination
max
m
The Michelis–Menten constant (K ) and the maximum
m
velocity (Vmax) were determined by measuring enzyme
activity of the purified lipase at various concentrations
of p-nitrophenyl laurate (pNPL) (300–900 mM) over
40 min. Lipase assay was conducted at various

BIOCATALYSIS AND BIOTRANSFORMATION
3
substrate concentrations at pH 7.0 and temperature of
on the maximum enzyme activity at the optimum pH
being 100%.
ꢀ
2
5 ± 1 C. The initial linear rate of product formation,
initial velocity (v ), of the purified lipase calculated by
The pH stability was determined by incubating the
enzyme solution separately at different pH values cov-
ering the range of pH 6.5–8.5. The pH of the pure
enzyme preparation was adjusted to pH 6.5, 7.0, 7.5,
o
adding 100 ll of the purified lipase into 1.9 ml of the
assay solutions. The absorbance at 410 nm was meas-
ured every 2 min for a 40 min in a spectrophotometer
8
.0 and 8.5 using 1 N HCl or NaOH. The pure enzyme
(
SPUV-19, Sco-TECH, Germany) to determine the
ꢀ
preparations was then incubated at 30 C for 1 h and
sample of 100 ml was withdrew every 20 min to deter-
mine the residual lipase enzyme activity.
amount of p-nitrophenol formed. The actual amount
of p-nitrophenol formed per unit time, given as nano-
moles per minute (nmol/min), was calculated by con-
verting the absorbance value at 410 nm to an actual
concentration (nmol) by comparing the absorbance at
2
.4.5. Effect of temperature on the activity and sta-
bility of the purified lipase
4
10 nm wave length with the p-nitrophenol standard
curve. The concentration of p-nitrophenol formed dur-
ing the course of the reaction was then graphed as a
In order to determine the optimum temperature of
Acinetobacter sp. K5b4 pure lipase activity, the assay
reaction mixture was incubated for 10 min at (24, 27,
function of time. Initial velocity (v ) was then obtained
o
ꢀ
from the slope on p-nitrophenol concentration versus
time graph. The kinetic parameters (Km and Vmax) were
30, 33 and 35 C). Hundred microliter of the purified
enzyme was added to the assay reaction mixture, and
reaction was allowed to proceed for 10 min at the
respective temperatures. The thermostability of the
purified enzyme was investigated at different tempera-
calculated by plotting 1/V against 1/[S] in Lineweaver
o
Burk plot.
ꢀ
tures covering the range from 30 to 60 C. 100 ll of
2.4.3. Effect of hydrophilic organic solvents on
the purified lipase was incubated separately at the
lipase activity
ꢀ
temperature of 30, 35, 40, 45, 50, 55 and 60 C for 1 h.
The effects of different concentrations of different
hydrophilic organic solvents on the activity of the
pure lipase preparation of Acinetobacter sp. K5b4 were
studied. Different concentrations (20, 40 and 60% (v/v)
of dimethyl sulfooxide (DMSO), methanol, ethanol,
acetone, acetonitrile and propanol were used in the
assay reaction mixture. Hundred microliter of the pure
enzyme was added to the assay reaction mixture and
Hundred microliters of the samples were withdrawn
every 20 min to determine the residual activity of the
purified enzyme.
2
.4.6. Effect of metal salts on the purified
enzyme activity
One mM of CoCl , KCl, BaCl, CaCl , ZnCl , MnCl ,
2
2
2
2
MgCl and CuCl metal salts were added to the assay
2
2
the reaction was allowed to proceed for 10 min at
reaction mixture in order to determine the effect of
metals on the purified lipase enzyme activity. Hundred
microliter of the purified enzyme was added to
ꢀ
3
0 C. The p-nitrophenol formed from the hydrolysis
action of lipase was measured at 410 nm immediately.
Relative activity was calculated by comparing the
enzyme activity of the control experiment with the
enzyme activity in the presence of the tested
organic solvents.
1
.90 ml of the assay reaction mixture, and the reaction
ꢀ
was allowed to proceed for 10 min at 27 C. Relative
activity was calculated by comparing the activity from
the control reaction with the activity from the reaction
with the presence of metal salts.
2.4.4. Effect of pH on the activity and stability of
the purified lipase
2
.4.7. Effect of various detergents and chemical
agents on the purified lipase activity
The effect of pH on the purified lipase activity was
investigated at various pH values; 6, 6.5, 7, 7.5 and 8.
The pH of the assay reaction mixtures were varied
using different buffers, 100 mM citrate buffer for pH
Three chemical agents with different concentrations
were investigated for their effect on lipase activity.
EDTA, SDS and 2-Mercaptoethanol were added to the
reaction mixture to the final concentration of 1 and
5 mM. Hundred microliter of the purified enzyme was
added to 1.90 ml of the assay reaction mixture, and
the reaction was allowed to proceed for 10 min at
6
–6.5, phosphate buffer for pH 7–7.5 and Tris-HCl buf-
fer for 7.5–8. Hundred microliter of the purified
enzyme was added to the assay reaction mixture, and
reaction was allowed to proceed for 10 min at the
respective pH. Relative activity was calculated based
ꢀ
27 C. Relative enzyme activity was determined by

4
M. O. AL-LIMOUN ET AL.
comparing the enzyme activity in the presence and
absence of various agents.
K5b4 was carried out using Amicon Ultra-15 centrifu-
gal filter unit cutoff 30 KDa (Sigma-Aldrich). The results
of this step showed that the crude enzyme prepar-
ation was approximately concentrated 3X as
2658.5 mU total activity of the crude lipase was
detected in 5 ml concentrate as compared to the total
activity of 2806.5 mU in 15 ml crude filtrate. In add-
ition, the specific activity of the crude enzyme prepar-
ation was intensified from 256.3 to 1296.8 mU/mg
protein with recovery percentage of 94.7 and 5.05
purification folds (Table 1). One milliliter of the con-
centrated partially pure enzyme was loaded into
Sephadex G-50 (1 cm ꢁ65 cm). The flow rate of the
eluted enzyme solution was 0.2 ml per min and con-
2
.4.8. Enzyme assay and protein content
Lipase activity was measured spectrophotometrically
SPUV-19, Sco-TECH, Germany) as described by Winkler
(
and Stuckmann (1979). Five hundred micromolar
of p-nitrophenyl laurate were dissolved in 10 ml of
dimethyl sulfooxide (DMSO) and emulsified in 90 ml of
1
00 mM phosphate buffer (pH 7.0), 0.1% (w/v) polyvinyl
alcohol (PVA) and 0.4% (w/v) Triton X-100. Hundred
microliter of the enzyme was added to 1.9 ml of the
ꢀ
reaction mixture. After 10 min of incubation at 27 C,
the color change was measured at 410 nm and com-
pared with the p-nitrophenol standard curve. One unit
ꢀ
ducted at room temperature (25 ± 1 C). Enzyme activ-
(
U) of lipase activity was defined as the amount of the
ities were detected in coming early fractions 8, 9, 10
and 11 (Figure 1). The specific activity was 2626.2
enzyme that released 1 mmol of p-nitrophenol per min.
Protein content in the enzyme preparation (crude and
concentrated) was determined based on Lowry method
(
mU/mg). The enzyme solutions from pooled fractions
which gave the highest purification fold (F8, 9, 10 and
1) were analyzed for protein homogeneity using SDS-
(Lowry et al. 1951). While protein content in fractions
1
obtained from Sephadex G-50 column was determined
at 280 nm (SPUV-19, Sco-TECH, Germany) by comparing
the readings with the BSA standard curve at 280 nm.
PAGE electrophoresis. The purified enzyme resolved
into a single band on an SDS-PAGE gel (Figure 2) had
an estimated molecular weight of 113 KDa. Table 1
also shows that, the specific activity of the filtrate of
the enzyme solution in fractions 8–11 increased from
3. Results and discussion
2
56.3mU/mg protein (crude enzyme) to 2626.2mU/mg
3.1. Extracellular lipase purification
protein. The results also showed that the gel filtration
step was resulted in 10.2 purification fold with 18.3%
recovery (Fraction 8–11).
A principal step for concentrating and partial purifica-
tion of the extracellular lipase from Acinetobacter sp.
Table 1. Purification table of the Acinetobacter sp. K5b4 lipase enzyme.
Volume (ml) Total activity (mU) Total protein (mg) Specific activity (mU/mg) Yield (%) Purification (fold)
Crude enzyme
Amicon tube (3X)
G-50 Sephadex gel filtration (F8-11)
15
5
8
2806.5
2658.5
513.6
10.95
2.05
0.44
256.3
1296.8
2626.2
100
94.7
18.3
1
5.05
10.24
Figure 1. Chromatography of lipase from Acinetobacter sp. K5b4 on Sephadex G-50.

BIOCATALYSIS AND BIOTRANSFORMATION
5
Several lipases from different Acinetobacter strains
have been previously purified to homogeneity. In their
purification process, lipases from Acinetobacter strains
usually treated with different chromatography resins in
single, two or multistep procedure. Their molecular
mass were reported and generally estimated to be
between 20 and 65 KDa which is much less than the
molecular mass of Acinetobacter sp. K5b4 lipase that
reported in this work. An exception is the earliest study
made by Breuil and Kushner (1975) who estimated the
molecular mass of the lipase enzyme of psychrophilic
Acinetobacter sp. O16 to be more or equal to 200 KDa.
A summary of the molecular weights reported in the lit-
eratures of the purified lipases from different
Acinetobacter species is shown in Table 2.
nitrophenyl mono-acyle ester were investigated in this
experiment. Substrate specificity results of the investi-
gated lipase with various p-nitrophenyl esters showed
that, medium carbon chain p-nitrophenyl fatty acid
esters served as good substrates for the enzyme, and
p-nitrophenylcaprylate (C8) was the best among all the
substrates tested (Figure 3). On the other hand, the
enzyme showed similar preference to the p-nitrophenyl
mono-acyle ester p-nitrophenyl laurate (C12) and p-
nitrophenyl palmitate (C16). In parallel to our results
the lipase enzyme produced by Acinetobacter sp. XMZ-
2
6 and Acinetobacter sp. RAG-1 were found to preferen-
tially attacks medium length acyl chains C6 and C8
Snellman et al. 2002; Zheng et al. 2011). However, due
(
to the instability of p-nitrophenylcaprylate (C8) sub-
strate, p-nitrophenyl laurate (C12) was used in further
experiment as enzyme substrate in the assay solution.
3.2. Characterization of pure lipase of
Acinetobacter sp. K5b4
3
.2.2. K and V
m max
3
.2.1. Substrate specificity
Michelis–Menten constant (K ) and the maximum vel-
m
Substrate preferences of Acinetobacter sp. K5b4 pure
lipase for carbon chain length of acyl group in the p-
ocity (Vmax) were determined by measuring enzyme
activity of the purified lipase at various concentrations
Figure 2. SDS-PAGE of the collected fractions with lipase activity from Acinetobacter sp. (F8) Fraction 8, (F9) Fraction 9, (F10)
Fraction 10, (F11) Fraction 11, (C) crude enzyme solution and (M) Broad range protein marker (6.5–200 KDa).
Table 2. Molecular weights of the purified lipases from various Acinetobacter species.
Microorganism
(KDa)
References
Acinetobacter radioresistens CMC-1
Acinetobacter sp. KM109
Acinetobacter calcoacetius LP009
Acinetobacter sp. RAG-1
Acinetobacter lwoffii 16C-1
Acinetobacter sp. SY-01
Acinetobacter sp. ES-1
Acinetobacter sp.
Acinetobacter baumannii BD5
Acinetobacter baylyi
45
62
23
33
45
37.2
32
32
35
30
53
37.186
60
46
Hong and Chang (1998)
Mitsuhashi et al. (1999)
Pratuangdejkul and Dharmsthiti (2000)
Snellman et al. (2002)
Kim and Park (2002)
Han et al. (2003)
Lee et al. (2006)
Saisubramanian et al. (2008)
Park et al. (2009)
Uttatree et al. (2010)
Wang et al. (2011)
Kim et al. (2012)
Jagtap and Chopade (2015)
Moradi et al. (2015)
Acinetobacter johnsonii LP28
Acinetobacter venetians V28
Acinetobacter sp.
Acinetobacter sp.
Acinetobacter sp. O16
Acinetobacter sp. K5b4
200
113
Breuil and Kushner (1975)
In this study

6
M. O. AL-LIMOUN ET AL.
Figure 3. p-nitrophenyl Mono-acyl ester substrate specificity of Acinetobacter sp. K5b4 pure lipase enzyme.
of p-nitrophenyl laurate (pNPL) (300–900 mM) over
0 min (Figure 4). The kinetic parameters (Km and
respectively, as compared with the control. Similar to
4
methanol, the addition of DMSO to the enzyme assay
in the concentrations of 20, 40 and 60% (v/v) caused a
rise in the hydrolysis activity by 46.4, 175.6 and
267.6%, respectively relative to control. The addition
of acetone to the assay solution was also showed an
enhancement in the enzyme activity over the studied
concentrations. At 20, 40 and 60% (v/v) concentra-
tions, the enzyme exhibited stimulation in the enzyme
activity of 223, 203 and 24% (v/v) in comparing with
the control. Alternatively, the addition of ethanol,
acetonitrile and propanol to the assay solution inhibit
the enzyme activities (except at the 20% (v/v) of
acetonitrile that cause 143% boosting over the con-
trol) in the range between 20 and 95% as compared
with the control.
Vmax) were calculated from a Lineweaver-Burk plot fit
by linear regression. Our results showed that the
Km and Vmax values for the pure lipase enzyme
produced by Acinetobacter sp. K5b4, using pNPL as
substrate, were 4.0 mM and 73.53 nmol/ml/min,
respectively.
Practically, straightforward comparison of the kin-
etic parameters of other lipases from different species
of the same genus reported in the literature is trivial.
This is partially due to the differences in the assay
conditions, the substrate used, concentration range
and the nature of the enzyme. However, few reports
were found in the literature reporting some of the kin-
etic parameters of certain pure enzymes produced by
Acinetobacter strains. Kim and Park (2002) detected
the Km and Vmax of the purified 45 KDa esterase
enzyme produced by Acinetobacter lwoffii 16C-1, using
p-nitrophenyl butyrate as substrate, as 11 lM and
The extracellular lipases extracted from various
Acinetobacter strains retain their initial activity
between 70 and 75% after exposure to different
organic solvents. For example, Acinetobacter sp. RAG-1
-1
1
31.6 lM min mg of protein, respectively. In another
(Snellman et al. 2002), the thermostable alkaline lipase
study, the Km value of the cold-adapted lipase enzyme
from Acinetobacter sp. XMZ-26, using p-nitrophenyloc-
tanoate as substrate, found to be 0.075 ± 0.008 mM
of Acinetobacter sp. EH28 (Ahmed et al. 2010), the
cold adapted lipase of Acinetobacter sp. XMZ-26
(
Zheng et al. 2011), Acinetobacter sp. XMZ-26 (Zheng
et al. 2012) and Acinetobacter venetians V28 (Kim et al.
012). Besides, several lipases produced by different
(Zheng et al. 2011, 2012).
2
3.2.3. Effect of hydrophilic organic solvents on
Acinetobacter strains were reported as organic solvent
tolerant enzymes. For instance, the alkaline lipase of
Acinetobacter radioresistens (CMC-1) exhibited activa-
tion in presence of 40% (v/v) dimethylsulfoxide and
20% (v/v) dimethylformamide (Hong and Chang 1998).
The lipase of the benzene tolerant Acinetobacterbaylyi
found to be highly resistance to many organic
solvents especially benzene and isoamyl alcohol
lipase activity
Relative activities of Acinetobacter sp. K5b4 pure lipase
was studied in the presence of different concentra-
tions of various water miscible organic solvents
(Figure 5). In the presence of 20, 40 and 60% (v/v) of
water miscible organic solvent methanol, the enzyme
activity was enhanced by a 79, 169 and 49%,

BIOCATALYSIS AND BIOTRANSFORMATION
7
Figure 4. p-nitrophenol concentration (nmol) versus time graph obtained by measuring enzyme activity of the purified lipase at
various concentrations of p-nitrophenyl laurate (pNPL) (300–900 mM) over 40 min.
(
Uttatree et al. 2010). The lipase enzyme of
solvents and do not require special stabilization; they
will be very useful industrially. The solvent-stable
enzyme produced by Acinetobacter sp. K5b4 in this
study will be very useful as catalysts for reactions in
the presence of organic solvents.
Acinetobacter sp. reported by Khoramnia et al. (2011)
shows increase in the activity in presence of methanol
(
114.7%), acetonitrile (105.9%), ethanol (153.4%), acet-
one (133.9%), 2-propanol (135.9%), ethyl acetate
(111.4%) and hexane (114.0%).
Hydrophilic solvents, such as acetone are usually
3
.2.4. The effect of pH on pure lipase activity
unsuited with enzyme activity because they strip off
water from the enzymes, leading to the unfolding of
the molecule with the exposure of the inner hydro-
phobic residues (Tsuzuki et al. 2003). Conversely,
enzymes naturally stable in the presence of organic
and stability
The effects of the pH, on the activity of pure lipase pro-
duced by Acinetobacter sp. K5b4 were studied. The
results (Figure 6(a)) showed that the pure lipase was

8
M. O. AL-LIMOUN ET AL.
Figure 5. Relative activities was calculated based on the enzyme activity in the control experiment (in absence of organic solvent)
being 100%.
Figure 6. Effect of pH on Acinetobacter sp. K5b4 pure lipase enzyme (a) activity and (b) stability. Relative enzyme activity was cal-
culated based on the maximum enzyme activity at the optimum pH being 100%. Residual activity was calculated based on the 0
time activity being 100%.
active at different pH environments ranging from pH
.0 to pH 8.0. Maximal activity of the enzyme was
obtained at pH 7.0. However, a few reports were exists
for lipases from Acinetobacter strains that showed max-
imum activity at acidic pH such as the lipase produced
by Acinetobacter sp. which shows maximum activity at
6

BIOCATALYSIS AND BIOTRANSFORMATION
9
ꢀ
(50 C) (Han et al. 2003), Acinetobacter sp. ES-1,
pH 6.0 (Khoramnia et al. 2011). Enzymes that showed
their maximal activity at neutral pH (7.0) include lipase
from Acinetobacter calcoacetius LP009 and Acinetobacter
sp. ES-1 (Pratuangdejkul and Dharmsthiti 2000; Lee
et al. 2006). However, several Acinetobacter lipases were
reported with maximal activity at alkaline pH such as
the lipase produced by Acinetobacter radioresistens
CMC-1, Acinetobacter sp. KM109, Acinetobacter sp.
RAG-1, Acinetobacter lwoffii 16C-1, Acinetobacter species
ꢀ
ꢀ
(40 C) (Lee et al. 2006), Acinetobacter sp. (37 C)
ꢀ
(Saisubramanian et al. 2008), Acinetobacter sp. (40 C)
(Kasana et al. 2008), Acinetobacter baumannii
ꢀ
BD5 (35 C) (Park et al. 2009), Acinetobacter sp. EH28
ꢀ
ꢀ
(50 C) (Ahmed et al. 2010), Acinetobacter baylyi (60 C)
(Uttatree et al. 2010), Acinetobacter johnsonii LP28
ꢀ
ꢀ
(30 C) (Wang et al. 2011), Acinetobacter sp. XMZ-26
ꢀ
ꢀ
(15 C) (Zheng et al. 2011), Acinetobacter sp. (45 C)
SY-01,
Acinetobacter
sp.,
Acinetobacter
lwoffii
(Khoramnia et al. 2011), Acinetobacter sp. XMZ-26 (55 C)
CR9, Acinetobacter baumannii BD5, Acinetobacter sp.
EH28, Acinetobacter baylyi, Acinetobacter johnsonii LP28,
Acinetobacter sp. XMZ-26 and Acinetobacter venetians
V2 (Hong and Chang 1998; Mitsuhashi et al. 1999; Kim
and Park 2002; Snellman et al. 2002; Han et al. 2003;
Kasana et al. 2008; Saisubramanian et al. 2008;
Park et al. 2009; Ahmed et al. 2010; Uttatree et al.
(Zheng et al. 2012) and Acinetobacter venetians V28
(40 C) (Kim et al. 2012).
ꢀ
Thermostability test showed that the pure lipase
enzyme of Acinetobacter sp. K5b4 was unstable over
all the studied temperatures. The residual activity of
the pure enzyme after 1 h incubation at 30, 35, 40, 45,
ꢀ
50 and 55 C calculated to be 80.5, 71.6, 65.6, 52.1,
2
010; Wang et al. 2011; Zheng et al. 2011; Kim
33.3 and 15.6%, respectively (Figure 7(b)). Regarding
thermostability, most of the published reports
showed that the most lipase enzymes produced by
Acinetobacter strains either to be thermostable
enzymes or cold active lipases. Thermostable enzymes
includes the lipases produced by Acinetobacter sp.
et al. 2012).
Purified Acinetobacter sp. K5b4 lipase was able to
retain 100% of the original activity after 1-h incubation
at pHs 6.5 and 7.0. On the other hand, the enzyme
activity was drastically minimized at pHs 7.5, 8.0 and
ꢀ
8
.5 by 64.3, 48.5 and 36%, respectively after 1 h incu-
RAG-1 (70 C) (Snellman et al. 2002), Acinetobacter cal-
ꢀ
bation (Figure 6(b)). Generally speaking Acinetobacter
lipases showed stability at extensive range of pH val-
ues such as the lipases produced by Acinetobacter sp.
KM109 (pH range of 6–8) (Mitsuhashi et al. 1999),
Acinetobacter calcoacetius LP009 (pH range of 4.0–8.0)
coacetius
LP009
(45 C)
(Pratuangdejkul
and
ꢀ
Dharmsthiti 2000), Acinetobacter sp. EH28 (50 C)
(Ahmed et al. 2010), Acinetobacter baylyi (60–80 C)
ꢀ
(Uttatree et al. 2010), Acinetobacter johnsonii LP28
ꢀ
ꢀ
(20–50 C) (Wang et al. 2011) and Acinetobacter
(Pratuangdejkul and Dharmsthiti 2000), Acinetobacter
sp. (70 C) (Khoramnia et al. 2011). Lipases produced
sp. RAG-1 (pH range of 5.8–9.0) (Snellman et al. 2002),
Acinetobacter species SY-01 (pH range of 9–11)
by Acinetobacter strains with medium to low
temperature stability were includes Acinetobacter sp.
KM109 lipase that was stable at or below 35 C
ꢀ
(Han et al. 2003), Acinetobacter baylyi (pH range
of 6.0–9.0) (Uttatree et al. 2010), Acinetobacter johnso-
nii LP28 (pH range of 8.0–11.0) (Wang et al. 2011),
Acinetobacter sp. (pH range of 6–11) (Khoramnia
et al. 2011).
(Mitsuhashi et al. 1999), Acinetobacter baumannii BD5
cold adapted lipase that maintained 28% of the ori-
ꢀ
ꢀ
ginal activity at 35 C and full activity at 0 C
(Park et al. 2009) and the cold adapted lipase
of Acinetobacter sp. XMZ-26 lipase that remained
ꢀ
ꢀ
3.2.5. The effect of temperature on the pure enzyme
most of its activity between 5 C and 35 C
(Zheng et al. 2011).
activity and stability
The effect of incubation temperature on Acinetobacter sp.
K5b4 pure lipase activity showed that the enzyme was
active at cramped area of temperature values ranging
3
.2.6. Effect of metals on the purified
enzyme activity
ꢀ
from 24 to 35 C (Figure 7(a)). Maximum hydrolysis activity
The enzyme activity was enhanced (0–36%) by KCl,
BaCl , MgCl2 and CaCl2 while obviously inhibited
ꢀ
of the pure enzyme detected at 27 C. However, various
2
Acinetobacter strains lipases were stated to have optimal
(10–20%) byCoCl , ZnCl , MnCl and CuCl (Figure 8).
2
2
2
2
ꢀ
temperatures ranged from 15 to 60 C. These includes
The effect of metal ions on lipase activity of
Acinetobacter was studied by several researchers.
The previous results showed neither perfect
agreement nor disagreement with our results. They
showed that the effects of these metals on the differ-
ent Acinetobacter lipases are either dependent or
ꢀ
Acinetobacter sp. KM109 (45 C) (Mitsuhashi et al. 1999),
ꢀ
Acinetobacter calcoacetius LP009 (50 C) (Pratuangdejkul
and Dharmsthiti 2000), Acinetobacter sp. RAG-1
ꢀ
ꢀ
(
(
55 C) (Snellman et al. 2002), Acinetobacter lwoffii 16C-1
37 C) (Kim and Park 2002), Acinetobacter species SY-01

1
0
M. O. AL-LIMOUN ET AL.
Figure 7. Effect of incubation temperature on Acinetobacter sp. K5b4 pure lipaseenzyme (a) activity and (b) stability.Relative
enzyme activity was calculated based on the maximum enzyme activity at the optimum temperature being 100%.Residual activity
was calculated based on the 0 time activity being 100%.
independent on the enzyme nature and the
Acinetobacter species studied. For example,
Acinetobacter lwoffii CR9 lipase was enhanced in the
et al. 2011), Acinetobacter sp. XMZ-26 lipase activity
þ2
þ2
was enhanced by the presence of Ca , Mn
Ba (Zheng et al. 2012).
and
þ2
þ2
þ2
þ2
þ2
þ2
presence of Cu , Mo , Mg , Zn , whereas Ca
had inhibitory effect (Kasana et al. 2008), Acinetobacter
3
.2.7. Effect of various detergents and chemical
þ2
þ2
baumannii BD5 lipase was activated by Ca , Mg
agents on the purified lipase activity
þ2
þ2
þ2
and Mn , whereas Zn and Cu inhibited it (Park
The effect of the 1.0 and 5 mM of 2-Mercaptoethanol,
EDTA and SDS on the lipolytic activity was negligible
et al. 2009), Acinetobacter baylyi lipase activity was
2
þ
2þ
completely inhibited in the presence of Fe , Mn
,
2
þ
(Figure 9). At 5.0 mM concentration, the enzyme still
whereas the activity was enhanced by Mg and no
2
þ
resistance to the presence of 2-Mercaptoethanol and
EDTA maintained 100% of the activity compared to
the control. The resistance of the lipase enzyme to the
action of the reducing agents 2-Mercaptoethanol
which favor an S–S to –SH interchange suggests that
the enzyme is not containing disulfide linkages
important for their catalytic activity. In the presence of
significant effect was found in the presence of Ca
and Li (Uttatree et al. 2010), Acinetobacter johnsonii
þ
Lp28 enzyme activity was promoted in the presence
þ
2þ
þ
þ2
þ2
of Na , Ca , K , Mg . On the other hand, Ba
,
þ2
þ3
þ2
Mn , Cr and Co did not affect the enzyme activ-
ity. Whereas, the presence of Al , Cu , Fe , Fe
and Zn
þ3
þ2
þ2
þ3
þ2
were reduced the enzyme activity (Wang

BIOCATALYSIS AND BIOTRANSFORMATION
11
Figure 8. Effects of different metal ions on the hydrolysis activity of Acinetobacter sp. K5b4 pure lipase enzyme.
Figure 9. Pure lipase enzyme relative activities in presence of 1 and 5 mM of 2-Mercaptoethanol, EDTA and SDS.
1
.0 and 5.0 mM of the metal chelating agent EDTA the
of methanol, DMSO and acetone. Further application
of this enzyme in organic synthesis of fine chemicals
and its enantioselectivity is highly recommended in
future research.
enzyme showed high stability which indicating that
the enzyme is not a metalloenzyme (Ping et al. 2018).
On the other hand, high reduction in the enzyme
activity was observed in the presence of 5.0 mM of the
anionic surfactant SDS that reached to 80% decrease
in the enzyme hydrolysis activity.
Disclosure statement
The authors declare no commercial or financial conflict
of interest.
4. Conclusion
Biotechnological applications based on enzymatic bio-
transformation were endlessly growing sector in bio-
technology. Various applications of lipases demand a
compatible biocatalyst such as organic solvent tolerant
enzymes. Water miscible organic solvents are highly
denaturing and at the same time are advantageous
reaction media. Hydrophilic organic solvent tolerant
lipases are an interesting biocatalyst. Acinetobacter sp.
K5b4 lipase enzyme being remarkably hydrophilic
organic solvent tolerant enzyme can be potentially an
excellent biocatalyst in organic synthesis in water mis-
cible organic solvents as reaction media. The enzyme
was not only able to maintain its activity but also
exhibited enhancement in presence of up to 60% (v/v)
ORCID
Muhamad O. Al-Limoun
015-7562
http://orcid.org/0000-0003-
3
Khaled M. Khleifat
Khalid Y. Alsharafa
Haitham N. Qaralleh
http://orcid.org/0000-0002-0216-3175
http://orcid.org/0000-0002-1533-5001
http://orcid.org/0000-0002-
9
658-0040
References
Ahmed EH, Raghavendra T, Madamwar D. 2010. An alkaline
lipase from organic solvent tolerant Acinetobacter sp.
EH28: application for ethyl caprylate synthesis. Bioresour
Technol. 101:3628–3634.

1
2
M. O. AL-LIMOUN ET AL.
Allimoun MO, Ananzeh MR, Khleifat KM. 2015. Screening
selection and optimization of extracellular methanol and
ethanol tolerant lipase from Acinetobacter sp. K5b4. Int J
Biosci. 6:44–56.
Andualema B, Gessesse A. 2012. Microbial lipases and their
industrial applications: review. Biotechnology 11:100–118.
Bhosale H, Shaheen U, Kadam T. 2016. Characterization of a
hyperthermostable alkaline lipase from Bacillus sonorensis
and its application for biodiesel production using
Jatropha oil. Extremophiles. 18:171–178.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein
measurement with pholin phenol reagent. J Biol Chem.
193:265–275.
Mitsuhashi K, Yamashita M, Hwan Y, Ihara F, Nihira T,
Yamada Y. 1999. Purification and characterization of a
novel extracellular lipase catalyzing hydrolysis of oleyl
benzoate from Acinetobacternov sp. strain KM109. Biosci.
Biotechnol. 63:1959–1964.
Mo Q, Liu A, Guo H, Zhang Y, Li M. 2016. A novel
thermostable and organic solvent-tolerant lipase
from Xanthomonasoryzaepv oryzae YB103, screening,
purification and characterization. Extremophiles. 20:
4R. Enzyme Res. 2016:4170684.
Bollag DM, Rozycki MD, Edelstein SJ. 1996. Protein methods.
New York, NY: Wiley; p. 57–82.
Breuil C, Kushner DJ. 1975. Partial purification and
characterization of the lipase of a facultatively psychro-
philic bacterium (Acinetobacter O16). Can J Microbiol. 21:
4
34–441.
1
57–165.
ꢀ
Canak I, Berkics A, Bajcsi N, Kovacs M, Belak A, Tepari ꢁc R,
Maraz A, Mr ꢀs a V. 2015. Purification and characterization of
Moradi S, Razavi SH, Mousavi SM, Gharibzahedi SMT. 2015.
Optimization and partial purification of a high-activity lip-
ase synthesized by a newly isolated Acinetobacter from
offshore waters of the Caspian Sea under solid-state
fermentation. RSC Adv. 5:12052–12061.
Panizza P, Cesarini S, Diaz P, Giordano SR. 2015. Saturation
mutagenesis in selected amino acids to shift Pseudomonas
sp. acidic lipase Lip I. 3 substrate specificity and activity.
Chem Commun. 51:1330–1333.
a
novel cold-active lipase from the yeast Candida
zeylanoides. J Mol Microbiol Biotechnol. 25:403–411.
Costa-Silva TA, Souza CRF, Oliveira WP, Said S. 2014.
Characterization and spray drying of lipase produced by
the endophytic fungus Cercospora kikuchii. Braz J Chem
Eng. 31:849–858.
Han SJ, Back JH, Yoon MY, Shin PK, Cheong CS, Sung MH,
Hong SP, Chung IY, Han YS. 2003. Expression and charac-
Park IH, Kim SH, Lee YS, Lee SC, Zhou Y, Kim CM, Ahn SC,
Choi YL. 2009. Gene cloning, purification, and character-
terization of
a
novel enantioselective lipase from
Acinetobacter species SY-01. Biochimie 85:501–510.
ization of
Acinetobacter baumannii BD5. J Microbiol Biotechnol.
9:128–135.
a
cold-adapted lipase produced by
Hong MC, Chang MC. 1998. Purification and characteriza-
tion of an alkaline lipase from
Acinetobacter radioresistens CMC-1. Biotechnol Lett. 20:
027–1029.
a newly isolated
1
Ping L, Yuan X, Zhang M, Chai Y, Shan S. 2018. Improvement
of extracellular lipase production by a newly isolated
Yarrowia lipolytica mutant and its application in the bio-
synthesis of L-ascorbyl palmitate. Int J Biol Macromol.
1
Jagtap SC, Chopade BA. 2015. Purification and characteriza-
tion of lipase from Acinetobacter haemolyticus TA 106 iso-
lated from human skin. Songklanakarin J Sci Technol.
1
06:302–311.
37:7–13.
Pratuangdejkul J, Dharmsthiti S. 2000. Purification and
characterization of lipase from psychrophilic Acinetobacter
calcoaceticus LP009. Microbiol Res. 155:95–100.
Saisubramanian N, Sivasubramanian S, Nandakumar N,
Indirakumar B, Chaudhary NA, Puvanakrishnan R. 2008.
Two step purification of Acinetobacter sp. lipase and its
evaluation as a detergent additive at low temperatures.
Appl Biochem Biotechnol. 150:139–156.
Snellman EA, Sullivan ER, Colwell RR. 2002. Purification
and properties of the extracellular lipase, LipA, of
Acinetobacter sp. RAG-1. Eur J Biochem. 269:5771–5779.
Tsuzuki W, Ue A, Nagao A. 2003. Polar organic solvent added
to an aqueous solution changes hydrolytic property of lip-
ase. Biosci Biotechnol. 67:1660–1666.
Kasana RC, Kaur B, Yadav SK. 2008. Isolation and identifica-
tion of a psychrotrophic Acinetobacter sp. CR9 and charac-
terization of its alkaline lipase.
8:207–212.
J Basic Microbiol.
4
Khoramnia A, Ebrahimpour A, Beh BK, Lai OM. 2011.
Production of a solvent, detergent, and thermotolerant
lipase by a newly isolated Acinetobacter sp. in submerged
and solid-state fermentations.
011:702179.
J Biomed Biotechnol.
2
Kim HE, Park KR. 2002. Purification and characterization of
an esterase from Acinetobacter lwoffii I6C-1. Curr
Microbiol. 44:401–405.
Kim Y-O, Heo YL, Kim H-K, Nam B-H, Kong HJ, Kim D-G, Kim
W-J, Kim B-S, Jee Y-J, Lee S-J. 2012. Gene cloning and
Uttatree S, Winayanuwattikun P, Charoenpanich J. 2010.
Isolation and characterization of a novel thermophilic-
organic solvent stable lipase from Acinetobacter baylyi.
Appl Biochem Biotechnol. 162:1362–1376.
Wang HK, Shao J, Wei YJ, Zhang J, Qi W. 2011. A novel low-
temperature alkaline lipase from Acinetobacter johnsonii
LP28 suitable for detergent formulation. Food Technol
Biotechnol. 49:96–102.
characterization of
Acinetobacter venetianus V28.
2:1245–1252.
a
cold-adapted esterase from
J
Microbiol Biotechnol.
2
Laemmli UK. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature.
227:680–685.
Lee KW, Bae HA, Shin GS, Lee YH. 2006. Purification
and catalytic properties of novel enantioselective
lipase from Acinetobacter sp. ES-1 for hydrolysis of
Wang HK, Zhong S, Ma H, Zhang J, Qi W. 2012. Screening
and characterization of a novel alkaline lipase from
Acinetobacter calcoaceticus 1-7 1solated from bohai bay in
(S)-ketoprofen ethyl ester. Enzyme Microb Technol.
38:443–448.
Li X, Qian P, Wu SG, Yu HY. 2014. Characterization of an
china for detergent formulation. Braz
J
Microbiol.
organic solvent-tolerant lipase from Idiomarina sp. W33
43:148–156.

BIOCATALYSIS AND BIOTRANSFORMATION
13
Winkler UK, Stuckmann M. 1979. Glycogen, hyaluronate, and
some other polysaccharides greatly enhance the forma-
tion of exolipase by Serratia marcescens. J Bacteriol.
Zheng X, Chu X, Zhang W, Wu N, Fan Y. 2011. A novel cold-
adapted lipase from Acinetobacter sp. XMZ-26: gene clon-
ing and characterisation. Appl Microbiol Biotechnol.
90:971–980.
138:663–670.
Yuan D, Lan D, Xin R, Yang B, Wang Y. 2016. Screening and
characterization of a thermostable lipase from marine
Streptomyces sp. strain W007. Biotechnol Appl Biochem.
Zheng X, Wu N, Fan Y. 2012. Characterization of a novel lip-
ase and its specific foldase from Acinetobacter sp. XMZ-26.
Process Biochem. 47:643–650.
63:41–50.