International Journal of Biological Macromolecules 123 (2019) 792–800

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Production, purification and biochemical characterization of a

thermoactive, alkaline lipase from a newly isolated Serratia sp. W3
Tunisian strain
Ahlem Eddehech a, Zied Zarai a,⁎, Fatma Aloui a, Nabil Smichi a, Alexandre Noiriel b,
Abdelkarim Abousalham b, Youssef Gargouri a
a
Laboratory of Biochemistry and Enzymatic Engineering of Lipases, National School of Engineers of Sfax, University of Sfax, PB 1173, Km 4 Road Soukra, Sfax, Tunisia
b
Univ Lyon, Université Lyon 1, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires, UMR 5246, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM2), 43, Bd du 11
novembre 1918, F-69622 Villeurbanne cedex, France

a r t i c l e i n f o a b s t r a c t

Article history: A newly isolated Serratia sp. W3 strain was shown to secrete a non-induced lipase in the culture medium. Lipo-
Received 7 September 2018 lytic activity was optimized using the response surface methodology (RSM) and the extracellular lipase from
Received in revised form 9 November 2018 Serratia sp. W3 (SmL) was purified to homogeneity with a total yield of 10% and its molecular mass was estimated
Accepted 9 November 2018
of about 67 kDa by SDS-PAGE. The amino acid sequence of the first 7 N-terminal residues of SmL revealed a high
Available online 12 November 2018
degree of homology with other Serratia lipase sequences. The purified SmL can be considered as thermoactive li-
Keywords:
pase, its maximal specific activity measured at pH 9 and 55 °C was shown to be 625 U/mg and 300 U/mg using
Serratia tributyrin and olive oil emulsion as substrate, respectively. In contrast to other described Serratia lipases, SmL
Lipase was found to be stable at a large scale of pH between pH 5 and pH 12. SmL was also able to hydrolyze its substrate
Optimization, response surface methodology, in presence of various oxidizing agents as well as in presence of surfactants and some commercial detergents.
purification Then, considering the overall biochemical properties of SmL, it can be considered as a potential candidate for in-
Characterization dustrial and biotechnological applications, such as synthesis of biodiesel and in the detergent industry.
Thermo-alkaline © 2018 Elsevier B.V. All rights reserved.

1. Introduction The lipase A from Serratia genus containing = 613–614 amino-acid

Lipases (EC 3.1.1.3) represent an important variety of biotechnolog-
ically valuable enzymes [1,2]. They are widely distributed in nature and
numerous purified varieties are already well-characterized [3–5]. In
fact, the enzymes extracted from microorganisms are the most interest-
ing because of their potential applications in various industries such as
food, dairy, pharmaceuticals, detergents, textile, biodiesel, cosmetics,
synthesis of fine chemicals, agrochemicals, and new polymeric mate-
rials [6,7].
Bacterial lipases are classified into eight different families based on
amino acid sequence and biochemical properties [8]. Family I.3 is repre-
sented by lipases from Serratia marcescens and Pseudomonas fluorescens,
segregated from other lipases not only by their amino acid sequences
but also by their secretion models and biological properties [9]. Serratia
species are gram-negative bacilli belonging to the family of Enterobac-
teriaceae, which are opportunistic to human, plant and insect. These
species are widespread in various biotopes from soil, water, plants and
air.
⁎ Corresponding author.
E-mail address: zaraizied@hotmail.fr (Z. Zarai).
residues and a molecular mass of about 65 kDa was described [10,11].
This enzyme was used to produce a large scale of the racemic 3-(4-
methoxyphenyl) glycidic acid methyl ester, which is a chiral precursor
for diltiazem synthesis, a calcium-channel blocker and coronary vasodi-
lator [12].
Enzyme production by microorganisms is affected by culture me-
dium composition and growth conditions. Response surface methodol-
ogy (RSM) has been strictly adopted to optimize microbial enzyme
production [13,14]. This experimental design method presents a useful
model for studying the effect of several parameters influencing the re-
sponses by varying them simultaneously and at a minimum number
of experimental tests [15].
The lack of industrial scale-up of lipases previously described from
Serratia genus may be due to their relatively low stability and catalytic
activity inadequate to the industrial process conditions (high tempera-
tures, extreme pH values or non-aqueous solvents); hence the impor-
tance of looking for Serratia lipases with properties suitable for
practical applications.
Thermostable lipases are more appropriate to industrial processes
because the reactions might be performed at elevated temperatures
and therefore the structure of enzyme is maintained in extreme

https://doi.org/10.1016/j.ijbiomac.2018.11.050
0141-8130/© 2018 Elsevier B.V. All rights reserved.
 A. Eddehech et al. / International Journal of Biological Macromolecules 123 (2019) 792–800
conditions. Further advantages include increased solubility of lipid sub-
strates in water, faster reaction and reduced possible risk of contamina-
tion [16,17].Therefore, the overall purpose of this work is to identify a
new strain Serratia sp. W3 isolated from palm leaf and optimize the cul-
ture conditions for a maximum production of a thermo-active, alkaline
and detergent-stable lipase. Further, the obtained lipase purification
and biochemical characterization was sought to respond to biotechno-
logical application requirements.
2. Materials and methods
2.1. Bacterial strains, plasmids, and media
Tributyrin (99%, puriss.) and benzamidine were obtained from Fluka
(Buchs, Switzerland). Sodium deoxycholic acid (NaDC), sodium
taurodeoxycholic acid (NaTDC), casein peptone, yeast extract and
ethylene diamine tetraacetic acid (EDTA) were purchased from Sigma
Chemical (St. Louis, USA); Arabic gum was from Mayaud Baker LTD
(Dagenham, United Kingdom); protein molecular mass marker and
supports of chromatography used for lipase purification: Sephacryl
S-100, monoQ-Sepharose and DEAE-cellulose gels were from Amersham;
acrylamide and electrophoresis grade were from BDH (Poole, United
Kingdom); pH-stat was from Metrohm (Switzerland).
2.2. Source of strains
In order to study some of their enzymatic potentials, a set of bacterial
strains, isolated from several different Tunisian biotopes of which we
quote date palm leaf, located in the Nefta oasis north of “Chott Djerid”,
and industrial effluents from the Sfax region were the subject of a qual-
itative test followed by a quantitative one in order to screen their lipo-
lytic activities for potentiality to degrade olive oil.
2.3. Isolation and screening of lipolytic bacterium
Initial screening of lipolytic microorganisms from various Tunisian
biotopes was carried out using a plate assay in a medium containing tri-
acylglycerol and the fluorescent dye Rhodamine B [18]. The solid me-
dium contains 1‰ olive oil, 1% nutrient broth, 1% NaCl, 1.5 g agar and
1‰ Rhodamine B. The culture plates were incubated at 37 °C, and colo-
nies giving orange fluorescence halos around them, upon UV irradiation,
were regarded as putative lipase producers [18]. Among the variable
isolates, W3 showed the maximum zone formation of 10 mm on
tributyrin medium at 37 °C of incubation after 24 h. Qualitatively
screened lipolytic bacteria W3 were subjected to quantitative screening
by assaying their enzyme activity under submerged fermentation with
medium A containing: 17 g/L peptone, 5 g/L yeast extract, 2.5 g/L
K2HPO4, 5 g/L NaCl, pH 7, and finally selected for further studies.
2.4. Identification of the isolated strain
The bacteria were identified by morphological and physiological
characterizations, including Gram reaction, motility, cell morphology
growth under anaerobic conditions, catalase and oxidase production,
as well as other tests included in the species description. The biochem-
ical tests were conducted using the API 20NE system. The identification
was further improved via the 16S rRNA gene sequencing method.
Briefly, genomic DNA of W3 was extracted from bacterial colonies by
a set buffer method and amplified using (5′ CCGAATTCGTCGACAACAG
AGTTTGATCCTGGCTCAG 3′) and reverse (5′ CCCGGGATCCAAGCTTAAG
GAGGTGATCCAGCC 3′) universal primers. PCR amplification was pro-
grammed to carry out an initial denaturation step at 94 °C for 3 min,
30 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for
1 min and elongation at 72 °C for 2 min, followed by a final amplification
step at 72 °C for 3 min. The 16S rRNA gene sequence was compared with
793
sequences available in the nucleotide database using the BLAST nucleo-
tide algorithm at the NCBI.
2.5. Lipase activity measurement
Lipase activity was measured titrimetrically at pH 9 and 55 °C with a
pH-stat, under standard conditions described previously [19], using
tributyrin or olive oil emulsions as substrates in the presence of 3 mM
CaCl2 and 1 mM NaDC. One unit of lipase activity corresponds to 1
μmol of fatty acid liberated per minute under standard conditions. Pro-
tein concentration was determined as described by Bradford [20] using
bovine serum albumin (BSA) as the reference protein.
2.6. Optimization of enzyme production
2.6.1. Preliminary studies
Preliminary studies have been carried out in order to select the best
nitrogen and carbon sources based on the classical method ‘one variable
at a time’. Different conditions were tested with several sources of car-
bon (casein, glucose, maltose, esters (Tween 80 and Tween 20), triglyc-
erides (olive oil and soya oil)) and different organic and inorganic
nitrogen sources (urea, yeast extract, soy flour, NH4Cl).
One factor approach experiment was adopted to study the effect of
incubation temperature, incubation time and agitation speed on en-
zyme production (Fig. S1). Obtained results revealed that the lipolytic
activity reached its maximum at 30 °C and 200 rpm. The follow-up of li-
polytic activity's kinetics showed that the beginning of the growth
phase was at 32 h. The production of the lipase was triggered from the
beginning of the growth phase and reached its maximum activity at
the end of the exponential phase corresponding to 32 h of culture.
The experiments were made in 250 mL Erlenmeyer flasks with a
useful volume of 50 mL on a nutrient broth (medium A) from a 16-h
old pre-culture (Optical Density (OD) at 600 nm = 0.08), the pH was
set at 7. The culture was incubated aerobically during 32 h on a rotary
shaker set at 200 rpm and at a temperature of 30 °C.
We adopted a planning experimental methodology to enhance the
production of SmL by Serratia sp.W3. These include a first screening
by Plackett-Burman design and an optimization by a Box Benkhen
Design.
2.6.2. Plackett - Burman designs
According to the preliminary study, it was assumed that the extra-
cellular lipase production depended on seven parameters (yeast extract,
peptone, tryptone, NaCl, K2HPO4, glucose and pH). In order to deter-
mine which of these potential parameters had a statistically significant
effect on the enzyme synthesis, a Plackett-Burman (PB) design with
15 experiments was carried out. The variables were analyzed at three
levels of their values: high (+1), baseline (0) and low (−1). The base-
line level (0) corresponded to central values of the screening design.
The levels attributed to each variable were determined based on results
of preliminary study (data not shown).
2.6.3. Box Benkhen
The factors selected based on the screening design as having a signif-
icant effect on lipolytic activity were subjected to further optimization
with RSM using a Box–Benkhen experimental design. The variables
were prescribed into three levels, coded −1, 0 and 1. Consequently, all
the involved factors' level combinations were constructed.
2.7. Production and purification of Serratia sp. W3 lipase
The culture medium from Serratia sp. W3 was prepared under opti-
mal conditions for 32 h of incubation at 30 °C and 200 rpm. Cells were
discarded by centrifugation (25 min, 8470 g) and the resulting crude en-
zyme solution (500 mL) was precipitated with solid ammonium sulfate
(65% saturation) at 4 °C.
794 A. Eddehech et al. / International Journal of Biological Macromolecules 123 (2019) 792–800

The precipitate obtained after centrifugation was then resuspended Table 1

in 25 mM sodium acetate, pH 5.4 containing 25 mM NaCl and 2 mM The various media components included in PB experiments and their corresponding
higher (+1), medium (0), and lower (−1) concentration levels.
benzamidine (buffer A) and insoluble material was removed by
centrifugation at 8470 g during 5 min. The obtained sample (10 mL) Variables Level
was loaded on a Sephacryl S-100 column (2.5 cm × 150 cm) pre- −1 0 +1
equilibrated with buffer A. It was then eluted with the same buffer
Yeast extract 0 0.25 0.5
at a flow rate of 30 mL/min. The lipase activity was checked as Peptone 0 1 2
previously described and the elution profile of proteins was monitored Tryptone 0 0.25 0.5
at 280 nm. NaCl 0 0.25 0.5
The active fractions were applied to the second step of purification K2HPO4 0 0.25 0.5
Glucose 0 0.5 1
on Mono Q Sepharose anion exchanger (2 cm × 20 cm) column pre- pH 7 7.5 8
equilibrated with buffer A. Adsorbed material was then eluted with a
linear NaCl gradient (200 mL of 150–500 mM in buffer A) at a flow
rate of 60 mL/h. The active fractions were eluted between 250 and
400 mM NaCl, collected and concentrated with ultrafiltration disk The effect of surfactant agents (10%, v/v for 1 h at 40 °C) on Sml sta-
membranes with a cut-off of 50 KDa. The samples were washed three bility was also checked. Residual SmL activity was measured at pH 9 and
times with 3 × 50 mL of buffer B (25 mM Tris–HCl, pH 8) to remove 55 °C.
NaCl. The enzyme solution was finally applied to a DEAE-Cellulose
anion exchanger pre-equilibrated in buffer B using Amersham Biosci- 2.9.4. Stability of SmL in organic solvents
ences AKTA FPLC System. The column (1.5 × 5 cm) was washed with The SmL stability in organic solvents was determined by mixing
50 mL of the same buffer. No lipase activity was detected in the washing purified lipase with increasing concentrations (10%, 50%) of different
flow. Adsorbed material was eluted with a linear NaCl gradient (100 mL solvents (dimethylsulfoxide, methanol, ethanol, isopropanol and
of 20–500 mM in buffer B) at a rate of 60 mL/h. The lipase activity was acetone) for 24 h of incubation. The mixture was incubated after vortex
eluted between 200 and 300 mM NaCl. Active and pure fractions were at room temperature with shaking. Samples were withdrawn periodi-
stored at 20 °C until used for biochemical characterization. cally to determine the residual activity under standard conditions
using tributyrin as substrate.

2.8. Gel electrophoresis and N-terminal sequence analysis

3. Results and discussion
The purified lipase was analyzed electrophoretically by sodium do-
3.1. Lipase production
decyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) on 15%
gel according to the Laemmli method [21]. The N-terminal amino-acid
According to the screening of isolates on Rhodamine B agar plate, two
sequence was determined by automated Edman's degradation using
lipase-producing bacteria were obtained. Only one, namely W3, showed a
an Applied Biosystems protein sequencer [22].
lipolytic activity of 6 U/mL on tributyrin and 4 U/mL on an olive oil emul-
sion via a quantitative enzymatic test using the pH stat technique.
2.9. Biochemical characterization Various preliminary tests were carried out with a view to choosing
the best components of the medium (carbon source, phosphorus ni-
2.9.1. Effect of pH and temperature on SmL activity and stability trates, etc.). In light of results, peptone and yeast extracts were found
Investigation for the optimum pH of purified lipase activity was car- the most suitable for a better lipase production by W3. In fact, they
ried out in various buffers at different pH (5–10.5) at 50 °C. The pH sta- are sources of amino acids and bacteria growth factors; and also contain
bility of the SmL was determined by incubating the lipase in buffer cofactors and vitamins essential for the development of bacteria and the
solutions with different pH values [3–12] at 100 mM for 2 h at 4 °C. production of metabolites.
The residual lipase activity was determined, after centrifugation,
under standard assay conditions. 3.2. Identification of the strain
The optimal temperature for the purified lipase was determined by
performing the enzymatic assay on pH stat at a heat range from 30 to The 1000 nucleotides amplified by PCR were sequenced and the re-
65°C) at pH 9. Thermal lipase stability was determined by incubating sults exhibited a 99% identity with different species belonging to Serra-
the enzyme solution at different temperatures (30–60 °C) and pH 5.4 tia genus. Eventually, the sequence was submitted in the genomic
for 60 min. The residual lipase activity was determined, after centrifuga-
tion, under standard assay method, the non-heated lipase, which was
Table 2
left at room temperature, was considered as the control (100% of enzy-
ANOVA analysis for Sml from Serratia sp. W3 activity in Box Benkhen design experiments.
matic activity).
Source Sum of df Mean F value Prob N F
squares square
2.9.2. Effect of metal ions on the SmL activity
Model 262.30 9 29.14 10.46 0.0094 Significant
Divalent metal cations play an important role in the structure and A-Peptone 55.13 1 55.13 19.78 0.0067
function of proteins. Various metal ion (Mg2+, Fe2+, Cu2+, Ca2+) re- B-Yeast extract 1.53 1 1.53 0.55 0.4919
quirement in SmL activity was tested at pH 9 and 55 °C with pH-stat. C-Tryptone 34.03 1 34.03 12.21 0.0174
AB 1.00 1 1.00 0.36 0.5753
Experiments were performed as described above using tributyrin as
AC 12.25 1 12.25 4.39 0.0902
the substrate in the presence of increasing metal ion concentrations. BC 10.56 1 10.56 3.79 0.1091
A^2 24.25 1 24.25 8.70 0.0319
B^2 43.63 1 43.63 15.65 0.0108
2.9.3. Effect of detergent on SmL activity and stability C^2 99.36 1 99.36 35.65 0.0019
In order to investigate the effect of bile salts on SmL activity, the Residual 13.94 5 2.79
hydrolysis rate of the tributyrin by the SmL was measured at 55 °C Lack of fit 13.94 3 4.65
and pH 9 in the presence of 3 mM CaCl2 and with increasing NaDC Pure error 0.00 2 0.00
Total 276.23 14
concentration.
 A. Eddehech et al. / International Journal of Biological Macromolecules 123 (2019) 792–800 795

database as a new Serratia sp. W3 Tunisian cultivar under the following
accession number MH762127.
3.3. Optimization of enzyme production
Bacterial ability to produce lipases is highly dependent on the strain
and bacterial medium composition influencing the enzyme synthesis
and its activity. In this study, we aimed to select the most influential fac-
tors using the PB approach among a large number of variables.
Reducing the number of parameters to be taken into account in
modeling makes it possible to minimize the coefficients to be identified
in the models and consequently the experiments to be carried out for
this operation. According to this assumption, six medium components
(yeast extract, peptone, tryptone, NaCl, K2HPO4, glucose) and one oper-
ational parameter (pH) were tested using a 15-run matrix. These

Fig. 1. Response surface plot of SmL production showing the mutual interaction between (A) yeast extract (X1) and peptone (X2) concentrations at constant value of tryptone (0.25%),
(B) tryptone (X3) and yeast extract (X1) concentrations with peptone fixed at 1%, (C) tryptone (X3) and peptone (X2) concentrations at constant yeast extract value (0.25%).
796 A. Eddehech et al. / International Journal of Biological Macromolecules 123 (2019) 792–800

parameters were studied at three levels of value (Table 1). The PB re-
sults indicated a variation of lipase activity in the range from 0 to
6 U/mL in fifteen trials. This wide range proved the importance of this
step in selecting the most involved factors and fixing the level of less in-
fluential ones.
The lipolytic activity data were statistically analyzed within a PB de-
sign using the Design Expert software to estimate t-value represented
by the Pareto-chart diagram, the sum of squares and percentage of con-
tribution of each factor in the response (data not shown). The factor
contribution rates and the Pareto chart data which present, in rod
form and in ascending order, the t-value of the effects of different pa-
rameters studied showed that the most influential factors are peptone,
yeast extract, tryptone and pH.
In fact, wheat peptone, yeast extract and tryptone concentration
had a positive effect on lipase production, while the pH exhibited a
negative one. When the sign of the effect of the tested variable is
positive, the response is greater at a high level of the parameter,
and vice versa.
Thus, on a set of 7 parameters estimated as potentially influential, 4
factors had a significant involvement in the observed responses and the
statistically insignificant variables, i.e. glucose, K2HPO4 and NaCl, were
discarded in the successive optimization stage.
3.3.2. Graphical interpretation of the mathematic model
The fitted model was used to draw curves that reflect the indepen-
dent effect of peptone, yeast extract and tryptone concentration in cul-
ture medium on lipolytic activity. The surface curves (Fig. 1) show that a
high lipolytic activity can be reached when using high concentrations of
both peptone and tryptone.
3.3.3. Optimum point
The response optimizer in Design Expert software was used to find
the optimum value of the variables for maximum lipolytic activity by
Serratia sp. W3. The optimum value of the variables in actual unit was
predicted as 14.9 g/L casein peptone, 3.7 g/L tryptone, 2.2 g/L yeast ex-
tract, 3 g/L NaCl, 5 g/L glucose, the pH being set at 7 and the temperature
at 30 °C with the predicted maximum lipolytic activity of 12.93 U/mL.
The organism produced 13 U/mL, thus confirming the validity. The li-
pase production yield (13 U/mL) was absolutely more important than
the one obtained during the preliminary study (5 U/mL). Thus, lipase
A 1.2 35
1.0 30

Absorbance (280 nm)

Activity (U.mL-1)
25
0.8
20
3.3.1. Lipolytic activity optimization using response surface methodology 0.6
(RSM) 15
Peptone, yeast extract, tryptone and pH, the most influential factors 0.4
10
in lipolytic activity, were optimized using a Box Behnken design 0.2 5
(Table S1). For a plan with 4 factors, 27 experiments were necessary.
In order to reduce this number, the pH was discarded and the three 0.0 0
other factors were chosen as critical variables affecting lipase produc- 10 20 30 40 50 60 70 80
tion. For each experiment, the remaining factors were maintained at Fraction number
KH2PO4 0% (negative effect −0.5 and low contribution percentage
1.11), glucose 0.5%, NaCl 0.5% and pH 7. 0.25 25
The mathematical model in terms of coded factors is expressed as
B [NaCl] (M)
follows: 0.5
0.20 20
Absorbance (280 nm)

Activity (U.mL-1)
0.15 15
R1 ¼ þ12:00 þ 2:63  A þ 0:44  B þ 2:06  C−0:50  A  B þ 1:75  A
 C−1:63  B  C−2:56  A2 –3:44  B2 –5:19  C2 0.10 10

0.05 5
where R1 is the estimated lipolytic activity and A, B and C the coded
values for peptone, yeast extract and tryptone, respectively. 0.15
0.00 0
Statistical significance of model equations was evaluated by ANOVA 0 10 20 30 40
based on Fisher's test. The analysis of the variance shows that the sum of Fraction number
the squared deviations (SS) evaluated with 14 degrees of freedom (dof)
is divided into two sums of squares. The first, estimated at 9 dof, is due 0.14 14
C [NaCl] (M)
to regression (to factors) which encompasses the effects of factors and 0.12 0.50 12
Absorbance (280 nm)

interactions between them. The second, estimated with 5 ddl, is attrib-

0.10 10
Activity (U.mL-1)

uted to the residual variation.

The ANOVA of the regression model (Table 2) demonstrates that the 0.08 8
model is highly significant. This is evident from the calculated F-value 0.06 0.25 6
(Fmodel = 10.46) and probability value (p = 0.0094). 0.04 4
The individual effect of peptone (p = 0.0067), and tryptone (p =
0.02 2
0.0174) and the interaction effect of peptone versus peptone (0.0319),
0.00
tryptone versus tryptone (p = 0.0019), yeast extract versus yeast ex- 0.00 0
tract (0.0108) and peptone versus tryptone (0.0902) were found to be 0 5 10 15 20 25 30
Fraction number
the most significant factors influencing lipolytic activity. These results
were confirmed by the Student's t-test (α = 0.05).
Fig. 2. Purification of SmL. (A) Chromatography profile of SmL from on Sephacryl S-100 gel
The fit of the model was also expressed by the correlation coefficient filtration column. Protein elution was performed with buffer A at a flow rate of 30 mL/min
R2 found to be 0.95, indicating that 95% of the variability in the response as described in Material and Methods. Fractions containing SmL activity were pooled and
(lipolytic activity) could be explained by the model which proves a high loaded on Mono-Q Sepharose column (B). The peak of lipase activity emerged at 300 mM
significance of the adjustment. The closer the values of R2 to 1, the better NaCl. The pooled active and dialyzed SmL fractions from the Mono-Q Sepharose column
were applied to a DEAE cellulose column (5 × 1.5 cm) (C). Non-fixed proteins were
the model would explain the variability between the experimental and washed out with the same buffer in the absence of NaCl. The elution of the adsorbed
the model predicted values. Adjusted R2 (0.86) confirms the good proteins was performed with a linear gradient of NaCl (0–500 mM). The flow rate was
agreement between the experimental and the predicted results. 60 mL/h and the fraction size was 1 mL.
 A. Eddehech et al. / International Journal of Biological Macromolecules 123 (2019) 792–800 797

kDa 1 2 3 Table 4
N-terminal sequence comparison of SmL (present study) with SM6, ES-2 and ECU 1010
lipases.a,b

SmL --MGIFSYKDLDEKASKALFSDALAI Present study

SM6 SHMGIFSYKDLDENASKALFSDALAI [26]

ECU 1010 --MGIFSYKDLDENASKALFSDALAI [24]

97 ES-2
a
--MGIFSYKDLDENASKALFSDALAI [24]

Amino acid sequences for comparison were obtained using the program BLAST-P

67 (NCBI, NIH, USA) database.

b
SmL residue not identical with the sequences of other Serratia lipases is indicated in
red letter.

43 CaCl2, 1 mM NaDC at pH 9 and 55 °C. These data indicate that the en-
zyme has a preference for short-chain triacylglycerol substrates. No
phospholipase activity was detected when using egg PC as the substrate
in the same experimental conditions.

30 The lipase activity of this protein was further confirmed by zymo-

gram assay under native conditions (data not shown). Only one band
of the enzyme showing triacylglycerol hydrolysis was revealed and
that fits well with the molecular mass of purified lipase.

20 3.5. N-terminal sequence analysis of SmL

The SmL NH2-terminal sequencing allowed the identification of 7

Fig. 3. SDS-PAGE analysis of purified SmL in a 12% polyacrylamide gel. Lane 1, molecular
mass markers, lanes 2 and 3: DEAE-Cellulose purified lipase fractions.
activity was multiplied by a factor of 2.6-fold. The expected result
(13 U/mL) was very close to the experimental result (12.93 U/mL). By
optimizing the medium composition and the culture conditions, not
only the production of lipase was enhanced but also the cost of enzyme
production was reduced.
3.4. Purification of SmL
The SmL was purified as described in the “Materials and methods”
section. The extracellular lipase was purified to homogeneity from the
culture medium of Serratia sp. W3. The enzyme was precipitated with
65% saturation of ammonium sulfate used as a starting material for fur-
ther purification. The precipitate was solubilized in minimum buffer A
and loaded to Sephacryl S-100 gel filtration column. Protein elution
was performed with the same buffer. Fractions containing SmL activity
(Fig. 2A) were pooled and loaded on Mono-Q Sepharose column. The
peak of lipase activity emerged at 300 mM NaCl (Fig. 2B). Fractions
containing lipase activity were finally gathered and applied to a DEAE
cellulose column equilibrated with buffer B. The elution profile of the
SmL obtained after this step is shown in Fig. 2C. Pure SmL was eluted
between 250 and 350 mM NaCl.
SDS-PAGE analysis showed that the pure enzyme exhibited one
band corresponding to a molecular mass of about 67 kDa (Fig. 3).
Many lipases from Serratia had a molecular mass of 62–67 kDa with pI
4.5–5.8 [23,24]. The purification flow sheet is shown in Table 3. The spe-
cific activity of the pure enzyme reached 625 U/mg and 300 U/mg using
TC4 and olive oil as substrate, respectively, in the presence of 3 mM
residues, I-F-S-Y-K-D-L. This N-terminal sequence exhibited a high de-
gree of homology with lipases of the same previously characterized
genus (Table 4) [23,24] and with that of a thermostable lipase from P.
fluorescens SIK W1 [25].
3.6. Biochemical characterization
3.6.1. Effects of pH on SmL activity and stability
The pH stability of the SmL was determined by pre-incubating the
enzyme over a wide pH range of 3–12 for 1 h at room temperature
and as shown in Fig. 4A, the SmL was highly stable over a broad pH
range and maintained 100% of its maximal activity between pH 5.0
and 7.0 and 60% at pH 12. Our results differ from those reported in the
literature for related lipases. For instance, Zaki and Saeed (2012) [26]
showed that maximum stability of lipase from one strain of Serratia
marcescens was at pH 8; but under pH 5, the lipase lost about 50% of
its activity. Abdon (2003) [27] reported lipase stability of psychrophilic
Serratia marcescens between 8 and 9 while maximum stability of lipase
from S. grimisii was reported to be between of 7–9 [28].
Moreover, the pH activity profile of the purified lipase is shown in
Fig. 4B. The results showed that the purified enzyme displayed activity
over a broad range of pH [7–11], with an optimum at pH 9 and at
pH 10.5 the enzyme keeps more than 60% of its maximum activity.
Under the same experimental conditions, Matsumae and Shibatani
(1994) [23] reported that the lipase activity of Serratia marcescens
Sr41 8000 is maximal at pH 8; while at pH 9, the enzyme retains less
than 40% of its activity is totally cancelled at pH 10, while Gao et al.
(2004) [29] and Bachkatova and Severina (1980) [30] showed that op-
timum pH for lipase activity was 6.5 and 6.3, respectively from two
other Serratia genus strains.

Table 3
Flowsheet of the purification procedure of SmL. See Materials and methods for details. One unit (U) corresponds to micromole of fatty acid released per minute using tributyrin as substrate
under the experimental conditions used. Activity measurements are described in Materials and methods.

Purification step Total protein (mg) Total activity (U) Specific activity (U/mg) Yield (%) Purification (fold)

Culture supernatant 1280 5100 3.98 100 1

(NH4)2SO4 (70%) 273 1700 6.2 33 1.55
Sephacryl S-100 column 6 1200 198 23.52 50
Mono-Q Sepharose column 1.75 720 410 13.72 103
DEAE cellulose column 0.72 450 625 10 157
798 A. Eddehech et al. / International Journal of Biological Macromolecules 123 (2019) 792–800

3.6.2. Effect of temperature on SmL activity and stability showed that the lipase was active at temperatures ranging from 30 °C to
The effect of temperature on SmL stability was determined by mea- 60 °C and activity increased significantly with temperature to reach its
suring the residual SmL activity after incubation of the pure enzyme at maximum value at 55 °C. This high activity at 55 °C may be explained
various temperatures (Fig. 4 C). The thermal stability profile of the puri- by the presence of calcium ions in the assay medium which could stabi-
fied enzyme showed that the lipase was inactivated at high tempera- lize the conformation of the enzyme.
tures (above 55 °C). These results show that SmL is a thermo-active enzyme, unlike all
The enzyme retained more than 50% or 30% of its initial activity after the Serratia lipases previously described by Bachkatova and Severina
30 min of incubation at 50 °C and 60 °C, respectively. These results are in (1980) [30], which showed that the highest lipase activity was from S.
accordance with earlier works [27] that found that lipase activity from marcescens strain 345 at 45 °C. In addition, Abdon (2003) [27] reported
S. marcescens is not as thermostable as other bacterial lipases. However, an optimal temperature of lipase activity of one S. marcescens strain at
according to previous studies, calcium ions could enhance the thermo- 37 °C, and Immanuel et al. (2008) [31] revealed an optimal temperature
stability of the family I.3 lipase [12]. This was confirmed with our extra- of lipase activity of S. rubidaea between 25 and 35 °C. In fact,
cellular lipase (this study). In fact, the effect of Ca2+ supplementation at extremozymes can be used in industrial reactions that are not feasible
a final concentration of 3 mM was clearly noticed almost in the whole at ambient temperature. High temperature is preferable in many chem-
range of temperatures compared to that without the addition of the ical reactions to guarantee higher solubility of substrates, better mixing,
ions. The SmL is able to retain more than 70% of its initial activity in faster reaction rate, lower viscosity, and decreased risk of microbial con-
the presence of calcium ions even at 60 °C. Hence, calcium ions appar- tamination [32].
ently play a key role in maintaining the three-dimensional conforma-
tion of SmL at extreme temperatures. 3.6.3. Effect of metal ions on SmL activity
The effect of temperatures in SmL activity was also studied by Different ions (Ca2+, Cu2+, Mg2+ and Fe2+) were studied for their
assaying the enzyme activity at different temperatures (Fig. 4D). Results influence on the purified Serratia sp. W3 lipase activity. As shown in

120.0 700

100.0
A 600 B
Specific activity (U/mg)
Residual activity (%)

500
80.0
400
60.0
300
40.0
200
20.0 100
0.0 0
2 4 6 8 10 12 2 4 6 8 10 12
pH
pH
700 120
600 C 100
D
Specific activity (U/mg)

Residual activity (%)

500 80
400
60
300
40
200
Sml (0 mM Ca2+)
20
100 Sml (3 mM Ca2+)

0 0
30 40 50 60 70 30 40 50 60 70
Temperature (°C) Temperature (°C)

700 700
Ca2+
600 Mg2+ E F
Specific activity (U/mg)

600
Specific activity (U/mg)

Cu2+
500 Fe2+ 500
400 400
300 300
200 200
100 100

0 0
0 2 4 6 8 10 12 0 2 4 6 8 10
[NaDC] mM
Ions concentrations (mM)

Fig. 4. pH effect on the SmL activity (A) and stability (B): The enzyme activity was determined by measuring the activity at various pH and after incubation the pure SmL for 1 h in different
buffer solutions at various pH ranging from 3 to 12. The activity of the enzyme at pH 5.4 was taken as 100%. (C) Temperature effect on enzyme activity and stability (D): SmL activity was
tested at various temperatures and after 30 min of incubation in the presence and absence of CaCl2 at different temperatures. The activity of the non-heated enzyme was taken as 100%. In
all experiments, lipase activity was measured under standard conditions using TC4 as substrate. (E) Effect of the concentration of metal ions on SmL activity. Lipase activity was measured
at increasing concentrations of metal ions using tributyrin as substrate in the presence of 1 mM NaDC. The star indicates the enzymatic activity measured in the absence of any metal ions
traces and in the presence of 10 mM EDTA. (F) Effect of increasing concentration of bile salts (NaDC) on lipase activity in presence of 3 mM CaCl2 using the tributyrin as substrate as
substrate. Each point represents the mean of three independent experiments.
 A. Eddehech et al. / International Journal of Biological Macromolecules 123 (2019) 792–800
Fig. 4E, calcium ions play the role of a cofactor for the SmL. The presence
of this ion in the assay medium was shown to be the most effective in
increasing lipase activity to reach a maximum of 625 U/mg using
tributyrin as substrate at 55 °C and at pH 9. Indeed, previous studies
have reported that the lipases from Serratia genus are defined as
calcium-stimulated and are already known to contain a Ca2+ binding
site near the active site that affects the stability and activity of the li-
pases. Furthermore, the addition of Ca2+ drastically enhances the lipase
activities. [33,34].
Magnesium ion is also demonstrated to be a for SmL catalysis
which reaches a maximum level of hydrolysis (480 U/mg) in the
presence of 3 mM of Mg2+ in the assay medium. However, Cu2+
and Fe2+ seem to be SmL activity inhibitors. In fact, in absence of
these metal ions and in presence of 10 mM EDTA, our enzyme
shows its maximum specific activity of the order of 300 U/mg. This ac-
tivity decreases by increasing the concentration of Cu2+ and Fe2+. Our
data are in accordance with the results published by Zaki and Saeed
(2012) [26] which exhibited that Zn2+ and Cu2+ ions depressed the
Serratia marcescens N3 lipase activity while Mg2+ and Ca2+ were
found to stimulate it. The same results were found by Pogori et al.
(2008) [35] with an extracellular lipase from Rhizopus chinensis while
Yu et al. (2007) [36] proved that Ba2+ and Mn2+ are the two main stim-
ulators of Yarrowia lipolytica lipase.
3.6.4. Effect of detergents on SmL activity and stability
Aware of the importance of surfactants to the preparation of emul-
sions for lipase assays and their characterization, we studied the effect
of a commercial detergent (NaDC) on the SmL activity. As shown in
Fig. 4F, the presence of bile salts seems to enhance the SmL activity up
to a limit concentration of 1 mM, which reaches an optimum of
625 U/mg. Comparable results were obtained with some bacterial li-
pases as Staphylococcus simulans [37], Staphylocococcus aureus [38]
and fungal lipases as Fusarium solani [39]. This detergent has no inhibi-
tory effect on the SmL activity even at a large concentration of bile salts
(7 mM) unlike what has been described for the lipase of R. oryzae lipase
activity [40].
Besides, the stability of the enzyme in the presence of some
surfactants was studied. As shown in Fig. 5, SmL was highly stable
towards some known surfactants after 1 h incubation at 40 °C and
retained its full activity in the presence of 20 mM Triton TX-100,
Tween 80, NaDC and NaTDC; while in the presence of SDS, the
purified enzyme was noted to be less stable and lost about 75% of
its initial activity. This is in contrast with bibliographical results
where Serratia marcescens ECU1010 lipase was stable in the presence
of different surfactant agents such as Pg400de, Pg250de and Tween-80,
and Triton X series, such as Triton X-45 and Triton X-100, inhibited the
activity seriously [41].
120
100
Relative activity (%)
80
60
40
20
0
Triton X100 SDS NaDC NaTDC
Fig. 5. Lpase stability of SmL in presence of surfactants. SmL preparation was incubated in
presence of surfactants (20 mM), commercial detergents (1%, w/v) and oxidizing agents
(10%, v/v) for 1 h at 40 °C. Residual SmL activity was determined at pH 9 and 55 °C. All
experiments were done in triplicate.
799
Table 5
Stability of Serratia Sp. W3 lipase in the presence of various organic solvents. The enzyme
was incubated 24 h under shaking condition at 25 °C in the presence of one solvent (10%,
v/v) and (10%, v/v), and then the residual activity was determined at pH 9 and 55 °C using
tributyrin as substrate. The control was measured after incubation of the esterase in ab-
sence of organic solvent.
Organic solvents Residual activity (%) Residual activity (%)
(10%, v/v) (50%, v/v)
Methanol 90 17
DMSO 97 23
Ethanol 87 18
Acetone 77 13
Isopropanol 83 26
3.6.5. Effect of organic solvents on SmL stability
The organic solvent tolerance of any of the lipases from Serratia spe-
cies has not been widely reported so far. In this report, the stability of
the purified SmL with respect to certain solvents generally miscible
with water was tested and results were expressed in terms of residual
activity compared to an untreated control. It is well known that micro-
bial lipases are rarely stable in hydrophobic organic solvents. However,
Serratia Sp. W3 lipase has shown extremely high stability (Table 5), at a
concentration of 10% of water-miscible organic solvents. The lipase did
not show a drastic decrease in residual activity after 24 h incubation,
with better stability in the presence of DMSO, an organic solvent widely
used to dissolve proteins to some extent. These data were in agreement
with Zhao et al. (2008) [41] who proved in a previous study that the
DMSO at 10% concentration activated a lipase from Serratia marcescens
ECU1010. Besides, SmL was shown quite stable, remaining active after
24 h of preincubation in the presence of neat hydrophilic solvents
such as acetone and isopropanol, respectively with 83 and 77.0% of ini-
tial activity.
Interestingly, the purified lipase retained activity even after
preincubation at a concentration of 50% of water-miscible organic sol-
vents although the activity level did not exceed 30% of the initial activity
for all the solvents tested. This stability of Serratia sp. W3 lipase against
organic solvents elects it as a potential candidate for application in the
synthesis of esters.
4. Conclusions
In this study, Plackett–Burman and Box–Behnken designs were
employed to optimize the culture conditions for the production of a
novel lipase by a newly isolated Serratia sp. W3 Tunisian cultivar from
palm leaves. Lipase was purified to homogeneity at 157-fold of purity
and showed a specific activity of 625 U/mg and 300 U/mg on tributyrin
and olive oil emulsion, respectively. When fully characterized, the SmL
displayed high stability in a variety of industrially relevant organic
solvents and in the presence of surfactants. Furthermore, SmL displayed
interesting biochemical criteria such as the high stability in a wide pH
range and activity at high temperature and at alkaline conditions,
suggesting that this enzyme may be a suitable candidate for bio-
transformations in the food and pharmaceutical industries.
Additionally, the novelty of Serratia sp. W3 strain, and the lipase ex-
plored here with its unique stability characteristics, makes this enzyme
a potential catalyst for other biotechnological applications such as
synthesis of biodiesel, biodegradable biopolymers for application in
the detergent industry.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2018.11.050.
Tween 80
Acknowledgments
This study was supported by “Ministry of Higher Education Scientific
research Tunisia” through a grant to “Laboratory of Biochemistry and
Enzymatic engineering of Lipases - ENIS”.
800 A. Eddehech et al. / International Journal of Biological Macromolecules 123 (2019) 792–800

Conflict of interest [20] M.M. Bradford, Rapid and sensitive method for the quantitation of microgram quan-
tities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72
(1976) 248–254.
The authors declare no conflict of interest. [21] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of
bacteriophage T4, Nature 227 (1970) 680–685.
[22] R.M. Hewick, M.W. Hunkapiller, L.E. Hood, W.J. Dreyer, J. Biol. Chem. 256 (1981)
References 7990–7997.
[23] H. Matsumae, T. Shibatani, Purification and characterization of the lipase from Serra-
[1] R. Verger, “Interfacial activation” of lipases: facts and artifacts, Trends Biotechnol. 15
tia marcescens Sr41 8000 responsible for asymmetric hydrolysis of 3-phenylglycidic
(1997) 32–38.
acid esters, J. Ferment. Bioeng. 77 (2) (1994) 152–158.
[2] R.D. Schmid, R. Verger, Lipases: interfacial enzymes with attractive applications,
[24] R. Meier, T. Dripper, V. Svensson, Karl-Erich Jaeger, Ulrich Baumann, A calcium-
Angew. Chem. Int. Ed. 37 (1998) 1609–1633.
gated lid and a large _-roll sandwich are revealed by the crystal structure of extra-
[3] H. Horchani, N. Ben Salem, Z. Zarai, A. Sayari, Y. Gargouri, M. Chaabouni, Enzymatic
cellular lipase from Serratia marcescens, J. Biol. Chem. 282 (2007) 31477–31483.
synthesis of eugenol benzoate by immobilized Staphylococcus aureus lipase: optimi-
[25] G.H. Chung, Y.P. Lee, G.H. Jeohn, O.J. Yoo, J.S. Rhee, Cloning and nucleotide sequence
zation using response surface methodology and determination of antioxidant activ-
of thermostable lipase gene from Pseudomonas fluorescens SIK W1, Agric. Biol. Chem.
ity, Bioresour. Technol. 101 (2010) 2809–2817.
55 (1991) 2359–2365.
[4] H. Horchani, I. Aissa, S. Ouertani, Z. Zarai, A. Sayari, Staphylococcal lipases: biotech-
[26] N.H. Zaki, S.E. Saeed, Production, purification and characterization of extra cellular
nological applications, J. Mol. Catal. B Enzym. 76 (2012) 125–132.
lipase from Serratia marcescens and its potential activity for hydrolysis of edible
[5] Z. Zarai, M. BouAli, A. Fendri, H. Louati, H. Mejdoub, Y. Gargouri, Purification and bio-
oils, J. Al Nahrain Univ. 15 (2012) 94–102.
chemical properties of Hexaplex trunculus digestive lipase, Process Biochem. 47 (12)
[27] A.M. Abdon, Purification and partial characterization of Psychrotrophic Serratia
(2012) 2434–2439.
marcescens lipase, J. Dairy Sci. 86 (2003) 127–132.
[6] R.K. Saxena, P.K. Ghosh, R. Gupta, W.S. Davidson, S. Bradoo, R. Gulati, Microbial li-
[28] A.M. Abdou, Studies on some Gram-negative proteolytic and lipolytic microorgan-
pases: potential biocatalyst for the future industry, Curr. Sci. 77 (1999) 101–115.
isms in milk and milk products, Ph.D Thesis Zagazig Univ. (Benha branch), Kaliobyia,
[7] K.E. Jaeger, T. Eggert, Lipases for biotechnology, Curr. Opin. Biotechnol. 13 (2002)
Egypt1997.
390–397.
[29] L. Gao, J. Xu, Z.Z. Lin, Optimization of Serratia marcescens lipase production for
[8] J. Arpigny, K. Jaeger, Bacterial lipolytic enzymes: classification and properties,
enantioseletive hydrolysis of 3-Phenylglycidic acid ester, J. Ind. Microbiol.
Biochem. J. 343 (1999) 177–183.
Biotechnol. 31 (11) (2004) 525–530.
[9] K. Amada, M. Haruki, T. Imanaka, M. Morikawa, S. Kanaya, Overproduction in
[30] N.A. Bachkatova, L.O. Severina, Isolation and charaterization of intracellular lipase
Escherichia coli, purification and characterization of a family I.3 lipase from Pseudo-
from Serratia marcescens. 345, Prik. Biokhim Mikobiol. 16 (3) (1980) 315–326.
monas sp. MIS38, Biochim. Biophys. Acta 1478 (2000) 201–210.
[31] G. Immanuel, A. Jwbadhas, A. Palaresam, Investigation of lipase production by milk
[10] Z.D. Long, J.H. Xua, L.L. Zhao, J. Pan, S. Yang, L. Hua, Overexpression of Serratia
isolate, Serratia rubidaea, Food Technol. Biotechnol. 46 (1) (2008) 60–65.
marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen,
[32] S. Mechri, M. Kriaa, M. Ben Elhoul Berrouina, M. Omrane Benmrad, N. Zaraî Jaouadi, H.
J. Mol. Catal. B Enzym. 47 (2007) 105–110.
Rekik, K. Bouacem, A. Bouanane-Darenfed, A. Chebbi, S. Sayadi, M. Chamkha, S. Bejar, B.
[11] M. Mohammadi, Z. Sepehrizadeh, A. Ebrahim-Habibi, A.R. Shahverdi, M.A. Faramarzi,
Jaouadi, Optimized production and characterization of a detergent-stable protease from
N. Setayesh, Bacterial expression and characterization of an activerecombinant li-
Lysinibacillus fusiformis C250R, Int. J. Biol. Macromol. 101 (2017) 383–397.
pase A from Serratia marcescens with truncated C-terminalregion, J. Mol. Catal. B
[33] H. Akatsuka, E. Kawai, K. Omori, S. Komatsubara, T. Shibatani, T. Tosa, The lipA gene
Enzym. 120 (2015) 84–92.
of Serratia marcescens which encodes an extracellular lipase having no N-terminal
[12] R. Meier, T. Drepper, V. Svensson, K.-E. Jaeger, U. Baumann, A calcium-gatedlid and a
signal peptide, J. Bacteriol. 176 (1994) 1949–1956.
large _-roll sandwich are revealed by the crystal structure of extracellular lipase
[34] K. Amada, M. Haruki, T. Imanaka, M. Morikawa, S. Kanaya, Overproduction in
from Serratia marcescens, J. Biol. Chem. 282 (2007) 31477–31483.
Escherichia coli, purification and characterization of a family I: 3 lipase from Pseudo-
[13] A. Shyamala Devi, K. Chitra Devi, R. Rajendiran, Optimization of lipase production
monas sp. MIS38, Biochim. Biophys. Acta Biomembr. 1478 (2000) 201–210.
using Bacillus subtilis by response surface methodology, Int. J. Biol. Biomol. Agric.
[35] M. Yu, Q. Shaowei, T. Tan, Purification and characterization of the extracellular lipase
Food Biotechnol. Eng. 6 (N:9) (2012).
Lip 2 from Yarrowia lipolytica, J. Bacteriol. 138 (2007) 663–670.
[14] C. Luciane Maria, P. Andreiza Lazzarotto, B. Silvia, L. Raquel Aparecida, D.M. Marieli,
[36] N. Pogori, A. Cheikyoussef, D. Wang, Production and biochemical characterization of
R. Christian Oliveira, B. Telma, V.C. Elita, Surface response methodology for the opti-
an extra cellular lipase from Rhizopus chinensis CCTCC M201021, Biotechnology 7
mization of lipase production under submerged fermentation by filamentous fungi,
(2008) 710–717.
Braz. J. Microbiol. 47 (2016) 461–467.
[37] A. Sayari, N. Agrebi, S. Jaoua, Y. Gargouri, Biochemical and molecular characteriza-
[15] R. Kaushik, R.G. Marwah, P. Gupta, S. Saran, L. Saso, V.S. Parmar, R.K. Saxena, Optimi-
tion of Staphylococcus simulans lipase, Biochimie 83 (2001) 863–871.
zation of lipase production from Aspergillus terreus by response surface methodol-
[38] H. Horchani, H. Mosbah, N. Ben Salem, Y. Gargouri, A. Sayari, Biochemical and mo-
ogy and its potential for synthesis of partial glycerides under solvent free
lecular characterization of a thermoactive, alkaline and detergent-stable lipase
conditions, Indian J. Microbiol. 50 (4) (2010) 456–462.
from a newly isolated Staphylococcus aureus strain, J. Mol. Catal. B Enzym. 56
[16] Y.R. Abdel-Fattah, Optimization of thermostable lipase production from
(2009) 237–245.
athermophilic Geobacillus sp: using Box-Behnken experimental design, Biotechnol.
[39] R. Jallouli, F. Khrouf, A. Fendri, T. Mechichi, Y. Gargouri, S. Bezzine, Purification and
Lett. 24 (2002) 1217–1222.
biochemical characterization of a novel alkaline (Phospho)lipase from a newly iso-
[17] P. Rathi, V. Goswami, V. Sahai, R. Gupta, Statistical medium optimization
lated Fusarium solani strain, Appl. Biochem. Biotechnol. 168 (2012) 2330–2343.
andproduction of a hyperthermostable lipase from Burkholderia cepacia in
[40] A. Ben Salah, A. Sayari, R. Verger, Y. Gargouri, Kinetic studies of Rhizopus oryzae li-
abioreactor, J. Appl. Microbiol. 93 (2002) 930–936.
pase using monomolecular film technique, Biochimie 83 (6) (2001) 463–469.
[18] G. Kouker, K.E. Jaeger, Specific and sensitive plate assay for bacterial lipases, Appl.
[41] Z. Li-Li, X. Jian-He, Z. Jian, P. Jiang, W. Zhi-Long, Biochemical properties and potential
Environ. Microbiol. 53 (1) (1987) 211–213.
applications of an organic solvent tolerant lipase isolated from Serratia marcescens
[19] D.A. Six, E.A. Dennis, The expanding superfamily of phospholipase A2 enzymes: clas-
ECU1010, Process Biochem. 43 (2008) 626–633.
sification and characterization, Biochim. Biophys. Acta 1488 (5) (2000) 1–19.