Purification and biochemical characterization of an extracellular lipase from Pseudomonas fluorescens MTCC 2421

Article abstract of DOI:10.1021/jf803797m

An extracellular lipase produced by Pseudomonas fluorescens MTCC 2421 was purified 184.37-fold with a specific activity of 424.04 LU/mg after anion exchange and gel exclusion chromatography. The enzyme is a homomeric protein with an apparent molecular mass of 65.3 kDa. The lipase exhibited hydrolytic resistance toward triglycerides with longer fatty acyl chain length containing unsaturation as evident from the lower V_max (0.23 mM/mg/min) of the lipase toward glycerol trioleate (C_18:1n9) compared with the fatty acid triglycerides having short to medium carbon chain lengths (C _18:0-12:0, V_max 0.32-0.51 mM/mg/min). This indicates a preferential specificity of the lipase toward cleaving shorter carbon chain length fatty acid triglycerides. The lipase exhibited optimum activity at 40 °C and pH 8.0, respectively. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the thermostability of lipase with a significantly high residual activity (100%) after 30 min at 40 °C, as compared to 90.6% after incubation with Ca²⁺ alone. The lipase activity was inhibited by Cu²⁺ and Fe²⁺ (42 and 48%,respectively) at 10 mM. The enzyme lost 31% of its initial activity by 0.001 mM EDTA and 42% by 0.1 mM EDTA. Significant reduction in lipase activity was apparent by 2-mercaptoethanol and phenylmethanesulfonyl fluoride at diluted concentration (0.001 mM), thereby indicating an important role of sulfhydryl groups in the catalytic mechanism.

Full text of DOI:10.1021/jf803797m

J. Agric. Food Chem. 2009, 57,
DOI:10.1021/jf803797m
3859
Purification and Biochemical Characterization
of an Extracellular Lipase from Pseudomonas
fluorescens MTCC 2421
KAJAL CHAKRABORTY* AND R. PAULRAJ
Marine Biotechnology Division, Central Marine Fisheries Research Institute, Ernakulam North P.O., P.B. No.
1603, Cochin 682018, Kerala, India
An extracellular lipase produced by Pseudomonas fluorescens MTCC 2421 was purified 184.37-fold
with a specific activity of 424.04 LU/mg after anion exchange and gel exclusion chromatography. The
enzyme is a homomeric protein with an apparent molecular mass of 65.3 kDa. The lipase exhibited
hydrolytic resistance toward triglycerides with longer fatty acyl chain length containing unsaturation as
evident from the lower Vmax (0.23 mM/mg/min) of the lipase toward glycerol trioleate (C18:1n9) compared
with the fatty acid triglycerides having short to medium carbon chain lengths (C18:0-12:0, Vmax 0.32-
0.51 mM/mg/min). This indicates a preferential specificity of the lipase toward cleaving shorter carbon
chain length fatty acid triglycerides. The lipase exhibited optimum activity at 40 °C and pH 8.0,
respectively. A combination of Ca2+ and sorbitol induced a synergistic effect on the thermostability of
lipase with a significantly high residual activity (100%) after 30 min at 40 °C, as compared to 90.6% after
incubation with Ca2+ alone. The lipase activity was inhibited by Cu2+ and Fe2+ (42 and 48%,
respectively) at 10 mM. The enzyme lost 31% of its initial activity by 0.001 mM EDTA and 42% by
0.1 mM EDTA. Significant reduction in lipase activity was apparent by 2-mercaptoethanol and
phenylmethanesulfonyl fluoride at diluted concentration (0.001 mM), thereby indicating an important
role of sulfhydryl groups in the catalytic mechanism.
KEYWORDS: Lipase; P. fluorescens MTCC 2421; sorbitol; Ca2+; fatty acids; glycerol trioleate
INTRODUCTION
reported to secrete a number of extracellular enzymes, lipases
being the major class. There are reports of Pseudomonas
fluorescens producing an alkaline lipase (9, 10). P. fluorescens
SIK W1 was found to produce a thermostable lipase, which
has high lipolytic activity for short- to medium-chain triacyl-
glycerols (11 ). Lipases were found to discriminate between
different fatty acids and exhibit less hydrolytic activity toward
ester bonds of polyunsaturated fatty acids (PUFAs) (3, 12).
The unique substrate and/or stereospecificity of bacterial
lipase was used for selective enhancement of targeted fatty
acids in triglycerides from fish oils (13 ).
Biochemical characterization of lipases is a preliminary
requirement to identify their unique position and/or stereo-
specificity, which will guide us to use them as candidate
biocatalysts for concentrating a specific class of fatty
acids. In the present study, P. fluorescens MTCC 2421 was
chosen for purification and characterization of an extracellu-
lar lipase. We report methods for the purification of an
extracellular metallolipase from P. fluorescens MTCC 2421
culture broth by physical precipitation followed by chroma-
tography on anion exchanger and molecular sieve and its
biochemical characterization. The said lipase has been char-
acterized with respect to its substrate specificity intended
for use to fractionate specific fatty acids with respect to
different acyl lengths (C12-18) and unsaturation in a triglycer-
ide mixture.
Lipases are a specific class of esterases responsible for the
hydrolytic cleavage of carboxyl esters such as triacylglycerols
and are used to concentrate unsaturated fatty acids, which are
known to possess pharmacological effects on animal and
human nutrition (1 ). The majority of the animal kingdom
lacks essential enzymes required for synthesizing them in
sufficient quantity from the precursor molecules. Therefore,
they require unsaturated fatty acids in high concentration for
their survival, and therefore, the preparation of unsaturated
fatty acid concentrates as food supplements is an emerging
research area (2 ). Among several techniques, the use of lipases
to concentrate a specific class of fatty acids is highly preferred
by virtue of their high specificity and mild reaction conditions
(3, 4). There are reports of lipases purified from animals,
plants, and microorganisms (5, 6). The vast majority of
microbial lipases reported in the literature are of bacterial
origin. The bacterial lipases were proved to be valuable by
virtue of their higher stability compared with animal or plant
lipases (7 ). In recent years, a number of bacteria produc-
ing thermophilic and alkalophilic lipases have been purified
and characterized (8 ). The bacterial genus Pseudomonas was
*Author to whom correspondence should be addressed (tele-
phone +91-484-2394867; fax 0091-0484-2394909; e-mail kajal_
iari@yahoo.com).
© 2009 American Chemical Society
Published on Web 3/26/2009
pubs.acs.org/JAFC

3860
J. Agric. Food Chem., Vol. 57, No. 9, 2009
Chakraborty and Paulraj
MATERIALS AND METHODS
(10 mM, pH 8) was dissolved in isopropanol to obtain a final
volume of 3 mL, to which the enzyme (10 μL) was added. The
reaction mixture was incubated at 45 °C for 20 min, and the
reaction was terminated by adding chilled acetone/ethanol
(1:1 v/v). The aliquot was clarified by centrifugation (5000g, 10
min) to furnish a clear supernatant. The absorbance of the
supernatant containing released 4-nitrophenol (4-NP) was mea-
sured at 410 nm (A410). One activity unit of lipase (LU) was
defined as the micromolar concentration of 4-NP released from
hydrolysis of 4-NPP/ mL/min by 1 mL of enzyme under standard
assay conditions. The protein concentration was determined by
measuring the absorbance at 590 nm (Varian Cary 50 Conc.)
using bovine serum albumin (20-150 μg) as standard (15 ).
Chemicals and Reagents. All solvents used for sample pre-
paration were of analytical grade (E-Merck, Darmstadt, Ger-
many). Anion exchanger Amberlite IRA 410 (Cl- form), bovine
serum albumin, and phenylmethanesulfonyl fluoride (PMSF)
were procured from HiMedia (Mumbai, India); Sephadex
G-100 and other chemicals were from Sisco Research Labora-
tories (Mumbai, India). P. fluorescens MTCC 2421 culture was
procured from Institute of Microbial Technology, Chandigarh,
India. Electrophoresis grade acrylamide, bisacrylamide, med-
ium-range molecular marker proteins, and Coomassie brilliant
blue R-250 were procured from Bangalore Genei (Bangalore,
India).
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophor-
esis (SDS-PAGE). SDS-PAGE of the purified enzyme solu-
tions (20 μL) was carried out using 0.1% w/v sodium dodecyl
sulfate on a 12% polyacrylamide gel (with 6% stacking gel) (16 )
on a vertical slab midi-gel apparatus (model PS-500; Bangalore
Genei). After migration, the gels were fixed with acetic acid/
methanol (7% v/v), and the protein bands were visualized by
alternative staining (with Coomassie brilliant blue R-250, 0.1%
w/v) and destaining (with acetic acid/methanol, 14% v/v). The
apparent molecular mass of the purified lipase was determined
with reference to the medium-range markers (14.4-94.0 kDa,
Bangalore Genei). The active lipase fractions were identified by
analytical SDS-PAGE of proteins using 12% gels stained with
Coomassie brilliant blue R-250. The gels were incubated over-
night in the refolding buffer (20 mM Tris-HCl, pH 8.0, 10 mM
CaCl2, 4 °C) and were transferred to a solution of Tris-HCl
(100 mM, pH 8.0) containing β-naphthyl acetate and fast blue
RR (17 ). The gels were rinsed with water and stored in acetic
acid (7% v/v) (18 ) to develop a dark yellow color. To further
validate the approximate molecular weight of the purified lipase,
gel filtration chromatography with Sephadex G-75 was used.
The elution volume of the lipase was compared with those of
known protein standards (horse heart cytochrome C, 12.4 kDa;
soybean trypsin inhibitor, 20.1 kDa; bovine serum albumin, 66
kDa; β-phosphorylase, 97.4 kDa), and the molecular masses of
standard proteins were plotted against their respective ratios of
elution volume to column void volume (Ve/V0) to draw the
calibration curve, the column void volume (V0) being the elution
volume of blue dextran (2000 kDa).
Preparation of Crude Lipase from P. fluorescens MTCC
2421. P. fluorescens MTCC 2421 was grown on tributyrin slants
(0.3% meat extract, 0.5% peptone, 0.9% NaCl, 0.25% tributyr-
in, and 0.001% CaCl2 2H2O) and inoculated in an Erlenmeyer
3
flask containing nutrient broth (50 mL). The content was then
incubated at 37 °C for 24 h under shaking (150 rpm) to raise the
inoculum for the enzyme production. The seed culture (100 mL)
(1% v/v) of P. fluorescens MTCC 2421 was grown in a broth
containing (g L-1) NaNO3, 3; K2HPO4, 0.1; MgSO4 7H2O, 0.5;
3
KCl, 0.5; FeSO4 7H2O, 0.05; and yeast extract, 5.0, and cod
3
liver oil (1%, v/v) at 37 °C in a 1000 mL Erlenmeyer flask with
shaking (150 rpm) for up to 72 h. The culture broth was
harvested at different time intervals (0, 5, 10, 24, 29, 34, 48,
53, 58, and 72 h) to determine the optimum time for maximum
lipase production. Cell growth was measured by recording the
OD of the cultures of different time intervals at 600 nm. The
increase in optical density at 600 nm was considered to be an
indication of the increase in cellular growth of the bacteria. The
crude broth was clarified by centrifugation (10000g for 20 min at
4 °C, Superspin R-V/FM Plasto Crafts, Mumbai, India) to
obtain a supernatant that was filtered (0.2 μm). This concen-
trated filtrate was referred to as the crude lipase.
Purification of Lipase from P. fluorescens MTCC 2421.
The crude lipase thus obtained was purified to homogeneity
by ammonium sulfate precipitation, Amberlite IRA 410
(Cl- form) anion exchange, and Sephadex G-100 gel exclusion
chromatography.
Ammonium Sulfate Precipitation. Solid ammonium sulfate
was added to the crude enzyme extract in increments of 5% until
70% saturation (w/v) with stirring and allowed to stand over-
night to obtain the precipitate, which was thereafter harvested
by centrifugation (10000g, 30 min, 4 °C) (3 ). The supernatant
was discarded to obtain the pellet that was dissolved in Tris-HCl
buffer (50 mM, pH 8.0), dialyzed against the same buffer for 18 h
to remove residual salts, and assayed for lipase activity.
Chromatographic Purification of the Lipase. The enzyme
concentrate obtained from dialysis was loaded on an Amberlite
IRA 410 (Cl- form) column (1.5 Â 15 cm) pre-equilibrated with
Tris-HCl buffer (50 mM, pH 8.0), and the bound proteins were
eluted in the same buffer with a linear gradient of NaCl (0-0.5
M). The active fractions were pooled and concentrated by
lyophilization. The lyophilized protein was dissolved in distilled
water (5 mL) and dialyzed against Tris-HCl buffer (50 mM, pH
8.0) to obtain the concentrated protein, which was rechromato-
graphed on a Sephadex G-100 gel exclusion column (2.5 Â 120
cm). The column was equilibrated and eluted with Tris-HCl
buffer (50 mM, pH 8.0) supplemented with CaCl2 (1.0 mM). The
eluant fractions showing lipase activity were pooled together
and analyzed for lipase activity. The active enzyme fractions
were stored at 4 °C until use for polyacrylamide gel electrophor-
esis and biochemical characterization.
Effect of pH and Temperature on Lipase Activity. The pH
profiling of the purified lipase was performed at different pH
values (from 2 to 12, with 1.0 difference). The enzyme (5 μL) was
added with the reaction mixture containing 4-NPP as substrate
and Tris-HCl buffer (50 mM, 50 μL) as reaction medium at each
of the above pH values, and the reaction mixture was incubated
at 40 °C for 20 min. The residual lipase activity was assayed
under standard assay conditions (14 ). The optimum tempera-
ture for lipase activity was determined by incubating the assay
mixture over different temperatures (20-90 °C for 20 min at
5 °C intervals) in Tris-HCl buffer (50 mM, pH 8.0), and the
resulting enzyme activity was assayed under standard assay
conditions (14 ). The thermostability of lipase was tested by
preincubating the enzyme (0.2 mL) at four different tempera-
tures (35-50 °C with 5 °C differences) for 0-120 min. The
enzyme (5 μL) was sampled at intervals of 15 min and assayed
for residual lipase activity. The effect of alkali metal cations
Ca2+ and Mg2+ and polyhydric alcohols (ethylene glycol,
propylene glycol, glycerol, and sorbitol), individually and in
combination, on the thermostability of lipase was also studied
by incubating lipase (50 μL) at 40 °C for various durations
(0-120 min).
Substrate Specificity and Kinetic Parameters of Lipase
from P. fluorescens MTCC 2421. To study the substrate
specificity of the enzyme, each of the five fatty acid triglycerides,
namely, glyceryl esters of tributyrate (C4:0), trilaurate (C12:0),
Lipase Activity Assay. The lipase activity of the enzyme was
estimated spectrophotometrically (Varian Cary 50 Conc.) using
4-nitrophenyl palmitate (4-NPP) as substrate following an
established procedure with modification (14 ). Briefly, a reaction
mixture consisting of 4-NPP stock solution (20 mM, 100 μL)
in Tris-HCl buffer (50 mM, pH 8.0, 1.0 mL) containing CaCl2
tripalmitate (C16:0), tristearate (C18:0), and trioleate (C18:1n9
)
(50 mM, 100 μL), were included in the reaction mixture

Article
J. Agric. Food Chem., Vol. 57, No. 9, 2009 3861
containing isopropanol, Tris-HCl buffer (20 mL, 50 mM), and
lipase (10 μL) maintained at pH 8.0. The reaction mixture was
incubated for various durations (5-60 min) at 40 °C under
shaking to determine the optimum incubation time. Samples
were withdrawn at regular intervals to determine the residual
lipase activity following the hydrolysis of substrates at 40 °C.
The affinity of the purified lipase toward different fatty acid
triglycerides was studied by determining the maximum velocity
for the reaction (Vmax) of lipase and Michaelis-Menten con-
stant (KM) using Lineweaver-Burk double-reciprocal plots.
Briefly, assays with purified lipase (100 μL) were performed in
Tris-HCl buffer (pH 8.0) at 40 °C with increasing concentration
of fatty acid triglycerides (10-50 mM) to calculate KM and Vmax
following the reported literature (7 ).
Effects of Metal Ions and Enzyme Modulators on Lipase
Activity and Stability. To evaluate the effect of metal ions
(Na+, K+, Ca2+, Mg2+, Cu2+, Mn2+, Co2+, and Fe2+) and
enzyme modulators for protein secondary structure (urea,
2-mercaptoethanol, phenylmethanesulfonyl fluoride, and ethy-
lenediaminetetraacetic acid) on the lipase activity, stock solu-
tions (100 mM) of the metal ion and enzyme modulators were
prepared in Tris-HCl buffer (100 mM, pH 8.0), and serial
dilutions were made to arrive at different concentrations ran-
ging from 0.001 to 50 mM. The purified enzyme (0.20 mg/mL,
50 μL) was added to the solutions containing substrate and
inhibitors (100 μL) at different concentrations, and the residual
lipase activity was assayed after 20 min of incubation at 40 °C
under standard assay condition (14 ).
Figure 1. Time course dependent lipase activity and cell growth curve of a
cultureof P. fluorescens MTCC 2421. Cultureconditions are described in the
text. Extracellular activity of lipase was assayed in the growth medium at
various intervals using 4-nitrophenyl palmitate as substrate. Bacterial cell
growth was monitored by measuring the absorbance at 600 nm.
the molecular sieve or to be inactivated by accompanying
protease. The homogeneity of the purified lipase was
checked by the presence of a single band corresponding to
an apparent molecular mass of 65.3 kDa on SDS-PAGE gels,
suggesting it to be a homomeric protein (Figure 2C). How-
ever, one band in SDS-PAGE is insufficient to conclude that
the enzyme is monomeric; it could be an oligomer consisting
of identical subunits. Gel filtration chromatography using
Sephadex G-75 was also used to determine the approximate
molecular weight of the lipase as 66.5 kDa. Activity staining
confirmed the presence of the purified lipase. The principal
theory of activity staining lies in the function of Ca2+ ion
that is essential for lipase refolding in its native form (17 ).
Bacterial lipases were reported to possess a characteris-
tic R-helix and β-sheet, and the conformational change
of lipases containing R,β-esterase fold in the presence of
Ca2+, indicating the structural transition from non-native to
native conformation (22 ).
Effect of pH on Lipase Activity. The effect of pH on lipase
activity with 4-NPP as substrate was examined at various
pH values at 40 °C. The enzyme was active in the range of
7.0-9.0, and the optimum activity was exhibited at pH 8.0
(Figure 3A). The lipase retained 80.74% of its initial activity
at a pH of 9.0, and at still higher pH (pH 10.0) the enzyme
lost 64.16% of its original activity. This high activity at pH
8.0 makes the lipase applicable at alkaline pH conditions.
Significant reduction in lipase activity was apparent in the
acidic range. For example, the enzyme retained only 27.44
and 18.89% of the maximum activity at pH 6.0 and 7.0,
respectively. These findings are in agreement with the results
obtained for the purified lipase from Pseudomonas fragi,
which had an optimum for lipase activity at pH 9.0 (23 ). An
optimal pH of 8.6-8.7 for the exolipase purified from
P. fragi NRRL B-25 was also reported (24 ). Industrial
demand for lipases active at alkaline pH is essential for
running bioprocesses, for their use to concentrate target
polyunsaturated fatty acids, and other biotechnological
applications.
Statistical Analyses. Data were expressed as mean ( stan-
dard deviation of three different experiments (n = 3) and
subjected to one-way analysis of variance (ANOVA) using SPSS
(ver. 10.0) software. A significance level of 95% (p = 0.05) was
used throughout.
RESULTS AND DISCUSSION
Time Course Dependent Lipase Activity of a Culture of P.
fluorescens MTCC 2421. The lipase-producing P. fluorescens
MTCC 2421 was detected using rhodamine B-triolein agar
plates. The time course of lipase production followed at
37 °C with typical cell growth is shown in Figure 1. The
lipase activity appeared to couple to growth. The lipase
activity was observed to start soon after incubation, and
the crude enzyme extract obtained after 48 h of growth in the
culture medium exhibited highest activity (11.85 LU/mL) at
the beginning of the stationary phase. A decrease in lipase
activity was apparent during the late stationary phase (after
53 h of incubation) presumably due to the presence of
proteases in the culture medium. The lipase activities were
found to be significantly reduced after 58 h of cultivation
(9.15 LU/mL) (Figure 1). These results are in agreement with
earlier results (7, 19), which reported maximum lipase activ-
ities at the onset of the stationary phase of bacterial growth.
Purification of P. fluorescens MTCC 2421 Lipase and
Molecular Mass Studies. The results of the purification
profile of the extracellular lipase secreted by P. fluorescens
MTCC 2421 are summarized in Table 1 and illustrated in
Figure 2A,B. The enzyme was purified 4.30-fold over the
crude extract by ammonium sulfate precipitation with
35.18% yield. The enzyme solution, obtained after anion
exchange chromatography, was purified 24.78-fold with
13.04% recovery. Further purification by gel exclusion
chromatography on Sephadex G-100 resulted in fractions
with 184.37-fold purity and 6.27% yield (Table 1). The
recovery of the lipase activity was marginally lower in this
study, although not significantly, than those reported for the
purification of other Pseudomonas lipases (8-12%) (20, 21).
This might be due to the tendency of the enzyme to adsorb on
Effect of Temperature on Activity of Lipase. The effect of
temperature on lipase activity is shown in Figure 3B. The
lipase was active in the temperature range of 35-50 °C, with
maximal activity at 40 °C (376.19 LU/mg). The lipase
activity was found to be reduced to 50.71% of its initial
value at 30 °C, and at 20 °C, it lost over 93% of its initial
activity. At higher temperature (50 °C) the enzyme lost

3862
Table 1. Summary of Various Steps Involved in the Lipase Purification Scheme from P. fluorescens MTCC 2421 Culture Broth (48 h, 37 °C)
step
volume (mL) total activity (LU)b total protein (mg)c specific activity (LU/mg) yield (%) purification (-fold)
crude culture broth (48 h, 37 °C)a
J. Agric. Food Chem., Vol. 57, No. 9, 2009
Chakraborty and Paulraj
500
50
5926.18
2169.33
1225.48
839.59
2576.20
219.64
21.58
2.30
9.84
100
35.18
13.04
6.27
1
4.30
(NH4)2SO4 precipitation
anion exchange chromatography [Tris-HCl buffer (50 mM, pH 8.0)]
gel exclusion chromatography [Tris-HCl buffer (50 mM, pH 8.0)]
10
57.25
24.78
10
1.98
424.04
184.37
a P. fluorescens MTCC 2421 was grown in selective broth (see Materials and Methods), and the harvested broth was clarified to obtain a supernatant that was termed the crude
lipase. The crude lipase was purified by ammonium sulfate salt precipitation, Amberlite IRA 410 (Cl- form) anion exchange, and Sephadex G-100 gel exclusion chromatography;
b The lipase activity was assayed by spectrophotometric method by monitoring 4-nitrophenyl palmitate hydrolysis. c Total protein was estimated according to the Bradford method.
Figure 3. Effect of (A) pH and (B) temperature on the lipase activity. The
experiments were conducted in triplicate, and standard errors are reported.
The advantages of running bioprocesses at elevated tem-
peratures and favorable changes in most physical properties
of lipids at increased temperature determine the increased
interest in new thermostable enzymes for new applica-
Figure 2. Chromatographic purification profile of the lipase on (A) anion
exchanger (Amberlite IRA 410 Cl- form) and (B) Sephadex G-100 gel
exclusion column. (C) SDS-PAGE of the lipase on 12% resolving gel: lane 1,
molecular mass markers [phosphorylase β (97.4 kDa), bovine serum
albumin (66.0 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (29.0
kDa), soybean trypsin inhibitor (20.1 kDa), and R-lactalbumin (14.3 kDa)];
lane 2, crude culture broth supernatant (20 μg); lane 3, proteins (20 μg) after
anion exchange chromatography; lane 4, purified lipase (10 μg) after gel
exclusion chromatography (a homomeric band corresponding to a molecular
mass of about 65.3 kDa represented the purified lipase); lane 5, proteins
(20 μg) after electrolytic precipitation.
tions (27 ).
Thermostability of Lipase. The thermostability of the
lipase was examined by measuring the residual activity for
0-120 min at four different temperatures (35-50 °C with
5 °C difference) at pH 8.0 (Figure 4A). The enzyme was found
to be stable at 35-45 °C with >75% of the residual activity
after 30 min of incubation. After incubation for 45 min, the
enzyme was stable at 35-45 °C with residual activity grea-
ter than 60% of its initial activity. At higher temperatures
(50 °C), the lipase retained 68.1% of its maximum activity
after 30 min of incubation and 41.5% after 1 h of incubation
(Figure 4A). The enzyme exhibited half-lives (t1/2) of 70 min
at 40 °C and 62 min at 45 °C. A lipase in thermophilic Bacillus
sp. was found to have a t1/2 of 30 min at 65 °C (28 ), whereas a
thermostable lipase from B. thermocatenulatus and B. stear-
othermophilus recorded t1/2 of 30 min at 60-62 °C (29 ). The
higher stability of the lipase in alkaline pH and at high
43.76% of the maximal activity, and the lipase activity
dropped off rapidly above 55 °C, with only 13% of the
activity remaining at 60 °C (Figure 3B). These findings are
in agreement with those reported for the lipase from Pseu-
domonas sp., with optimal activity at 40-45 °C (25, 26).

Article
J. Agric. Food Chem., Vol. 57, No. 9, 2009 3863
the hydrated Ca2+/ Mg2+ as CaCl2 2H2O and MgCl2
2
3
3
H2O), which are liberated when the ions become bound to
the protein. The free water molecules and -OH groups, in
turn, hydrate the catalytic site of the enzyme, resulting in
hindered denaturation. The catalytic triads of most lipases
are constructed by negatively charged carboxylate side-chain
groups of aspartyl, serine, and glutamyl residues, which are
“cross-linked” by the Ca2+/ Mg2+ bridge, and the enzyme-
Ca2+/ Mg2+ complex should, therefore, be more rigid and
hence more stable (31 ). Significant increase in thermostabil-
ity in the presence of sorbitol/Ca2+ combination has valu-
able practical importance in the design of reaction media to
enrich specific unsaturated fatty acids.
The catalytic activity of the lipase was found to be en-
hanced by 3% in the presence of K+ at 30 mM (Figure 5A).
Na+ does not have any activating effect on this lipase,
although at higher concentration (50 mM) significant ( p =
0.05) inhibition of this lipase (38%) was apparent. It is well-
known that nearly one-third of enzymes require the presence
of metal ions as tightly bound metal ion cofactors for
catalytic activity. The alkaline earth metals, Ca2+ and
Mg2+, were found to enhance the lipase activity by 10 and
5%, respectively, at 40 mM. Lower concentrations (0.001-5
mM) of these metals were found to reduce lipase activity,
although not significantly (Figure 5B). About 90 and 86%
residual lipase activities were apparent after incubation with
0.001 mM Ca2+ and Mg2+, respectively, thus leading to the
conclusion that these alkaline earth metals have a direct
effect on the lipase activity. Among other divalent metal
cations, the lipase activity was found to be significantly ( p =
0.05) blocked by Cu2+ and Fe2+ (42 and 48%, respectively)
at 10 mM. Using 10-50 mM metal ions, inhibitory effects
were greater. The inhibitory activity of the transition metal
cations at high concentrations may be due to direct inhibi-
tion of the catalytic site and/or formation of complexes
between metal ions and ionized fatty acids (32 ). Extracellu-
lar lipases from Pseudomonas sp. were reported to be in-
hibited by Cu2+ at higher concentration (33 ). However, this
lipase was unaffected by most of the metal ions tested at
lower concentration (0.1-0.001 mM), and the relative activ-
ity was recorded to be more than 70%.
Figure 4. Effects of divalent ions and polyhydric alcohols on the lipase
thermostability: (A) thermostability profile of the lipase at different tempera-
tures (35-50 °C) at 0-120 min; (B) influence of polyhydric alcohols on
lipase thermostability for 0-120 min at 40 °C; (C) influence of Ca and Mg
`
vis-a-vis Caand Mgcombined withsorbitol onthermostabilityofthe lipasefor
0-120 min at 40 °C.
temperature indicates its potential utility in industrial appli-
cation.
Effect of Polyhydric Alcohols and Metal Ions on Lipase
Activity and Stability. Sorbitol, a polyhydric alcohol with
five hydroxyl groups, was found to be an effective stabilizer
of the lipase. About 95.3% of the residual activity was
apparent after 45 min of incubation with sorbitol, and after
2 h, it retained 81.5% of the initial activity. Polyhydric
alcohols with fewer numbers of free hydroxyl groups such
as glycerol (three -OH groups), propylene glycol, and
ethylene glycol (two -OH groups) had a detrimental effect
on the activity of the lipase apparently due to lower interac-
tion with negatively charged groups in the active site (30 ).
Lipase when incubated with glycerol retained 86.9% of the
residual activity as compared to 71.3 and 58.6% activity
when incubated for 1 h with propylene and ethylene glycol,
respectively (Figure 4B). Lipase incubated with Ca2+/sorbi-
tol induced a synergistic effect as indicated by the signifi-
cantly higher residual lipase activity (100%) after 30 min
of incubation, as compared to 90.6% when incubated with
Ca2+ alone ( p = 0.05). The Mg2+/sorbitol combination
also exhibited significant increase in lipase activity (96.3%)
after 30 min of incubation as compared to 80.6% in the
control (Figure 4C). Enzyme stabilization at high tempera-
tures by Ca2+/ Mg2+ ions and sorbitol might be due to the
free -OH groups (in sorbitol) and water molecules (bound to
Effect of Enzyme Modulators on Lipase Activity. The lipase
was found to retain 58% of its initial activity after incubation
with 0.1 mM EDTA and 69% by using 0.001 mM of the same
modulator. As reported earlier, a concentration as low as 1
mM EDTA was found to inhibit the lipase activity (33, 34).
The enzyme exhibited a significant reduction in activity by
PMSF, a thiol, and serine protease inhibitor, thus indicating
an important role of sulfhydryl groups in catalytic mechan-
ism (27 ). The lipase was found to retain 61% of its initial
activity when incubated with 0.001 mM PMSF and 42% by
using 0.1 mM PMSF (Figure 5C). Complete inhibition by
PMSF at higher concentration (>1 mM) might be due to the
modification of an essential serine residue in the active site
(27 ). Thiol inhibitor 2-mercaptoethanol (CH3CH2SH) was
found to inhibit the lipase even at diluted concentration
(0.001 mM), thereby indicating an important role of sulfhy-
dryl groups in the catalytic mechanism. A thermostable
lipase from B. stearothermophilus was reported to be inhib-
ited by 2-mercaptoethanol (25%) at a concentration of 1 mM
(27 ). Urea was found to exhibit least reduction in lipase
activity (84% at 0.001 mM and 78% at 0.1 mM) (Figure 5C).
Fatty Acid Specificity of Lipase from P. fluorescens MTCC
2421. To study the substrate specificity of the lipase, the
hydrolytic kinetic variables of fatty acyl triglycerides with

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J. Agric. Food Chem., Vol. 57, No. 9, 2009
Chakraborty and Paulraj
Figure 6. Lineweaver-Burk double-reciprocal plots for the purified bacter-
ial lipase using glyceryl esters of trioleate, tristearate, tripalmitate, and
trilaurate as substrates. 1/Vmax values (intercept of the straight lines to Y axis
of the plot) were recorded as 1.96, 2.17, 3.13, and 4.35 (mM acid/mg/min)-1
when glycerol trilaurate (C12:0), tripalmitate (C16:0), tristearate (C18:0), and
trioleate (C18:1n9), respectively, were used as substrates toward the lipase.
The corresponding -1/KM values (intercept of the straight lines to X axis)
were found to be 0.043, 0.048, 0.058, and 0. 086 mM-1, respectively, for
these fatty acid triglycerides. The Lineweaver-Burk equation of glyceryl
esters of trilaurate (C12:0), tripalmitate (C16:0), tristearate (C18:0), and trioleate
(C18:1n9) weredetermined as1/V0 = 48.29/[S] + 1.93, 1/V0 = 49.25/[S] + 2.28,
1/V0 = 50.0/[S] + 2.98, and 1/V0 = 49.69/[S] + 4.38, respectively.
Table 2. Substrate Specificity of the Lipase from P. fluorescens MTCC 2421
to Triacylglycerols of Different Fatty Acyl Chain Lengths (C12-18).
substrate (fatty
acyl triglycerides)
relative activitya (%) KM (mM) Vmax (mM/mg/min)
glycerol trioleate (C18:1n9
)
9 ( 0.27
23 ( 0.56
29 ( 1.25
49 ( 1.63
11.6 ( 0.11
17.2 ( 0.38
20.7 ( 1.71
23.3 ( 0.85
0.23 ( 0.04
0.32 ( 0.09
0.46 ( 0.12
0.51 ( 0.07
glycerol tristearate (C18:0
glycerol tripalmitate (C16:0
glycerol trilaurate (C12:0
)
)
)
Figure 5. Effect of metal cations: (A) monovalent, (B) divalent, and
(C) enzyme modulators on the lipase activity. A stock solution (100 mM)
of the metal ion and enzyme modulators were prepared in Tris-HCl buffer
(100 mM, pH 8.0), and serial dilutions were made to arrive at 0.001-50 mM.
a The relative activities were calculated by taking the activity of glyceryl tributyrate
(C4:0) as 100%.
(C18:0), thus indicating its potential use to enrich long-chain
unsaturated fatty acids (viz., triolein). These findings are in
agreement with earlier results, which reported that a lipase
from P. fragi CRDA 037 is most active with fatty acid
triglycerides of intermediate carbon chain lengths such as
tristearine with a higher Vmax value of 13.3 μM/mg/min than
recorded for glycerol trioleate of 11.1 μM/mg/min (7 ). The
unsaturated fatty acids such as oleic acid with a double bond
at the Δ9 position apparently hinder the bacterial lipase from
acting on it, resulting in enrichment of the fatty acid in the
triglyceride fraction. On the contrary, saturated fatty acids
are exposed to the reactive groups at the enzyme active site
apparently due to the absence of an olefinic bond, resulting
in their rapid breakdown (hydrolysis of esteritic linkage)
from the fatty acid triglycerides. Such fatty acid selectivity of
lipases, that is, hydrolysis favoring unsaturates over satu-
rates, may therefore be useful to fractionate highly unsatu-
rated fatty acids from marine oil.
In conclusion, an extracellular alkaline metallolipase from
P. fluorescens MTCC 2421 has been purified by a combina-
tion of precipitation and chromatography. The homogeneity
of the purified lipase was checked by the presence of a single
band corresponding to an apparent molecular mass of 65.3
kDa on SDS-PAGE gels, suggesting it as a homomeric
protein. The purified lipase had optimum activity at 40 °C
and alkaline pH (pH 8.0). A combination of divalent metal
variable chain length were determined. The Lineweaver-
Burk double-reciprocal plot of activity of the P. fluorescens
MTCC 2421 lipase incubated with substrates of variable
fatty acyl chain length is shown in Figure 6. The linearity of
the plot indicates that hydrolysis of triglyceride esters by the
lipase followed Michaelis-Menten kinetics. Incubation of
the lipase with glycerol trioleate (C18:1n9) and glycerol tris-
tearate (C18:0) furnished KM values of 11.6 and 17.2 mM,
respectively, and KM values of 20.7 and 23.3 mM were found
when glycerol tripalmitate (C16:0) and glycerol trilaurate
(C12:0) were employed as substrates (Table 2). The Vmax
values were found to decrease with the increase in acyl chain
length of the triglycerides, with Vmax values of 0.51, 0.46,
0.32, and 0.23 (mM/mg/min)-1 after incubation with glycer-
ol trilaurate (C12:0), glycerol tripalmitate (C16:0), glycerol
tristearate (C18:0), and glycerol trioleate (C18:1n9), respec-
tively, thus indicating that the lipase from P. fluorescens
MTCC 2421 was highly active toward fatty acid triglycerides
having short to medium carbon chain length (<C18) result-
ing in rapid breakdown of ester bonds of glycerol trilaurate
(C12:0), followed by glycerol tripalmitate (C16:0) and glycerol
tristearate (C18:0). The longer chain length unsaturated fatty
acid triglyceride (as in glycerol trioleate) was found to be
hydrolyzed slowly as compared to its saturated homologue

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J. Agric. Food Chem., Vol. 57, No. 9, 2009 3865
cation and polyhydric alcohol, particularly Ca2+/sorbitol,
appeared to play an important role in the structure and
function of the lipase. Ca2+ plays an important role in the
increased activity of lipase, possibly due to the hydration of
the catalytic site and cross-linking by a Ca-ion bridge with
negatively charged amino acyl residues. The lipase appeared
to be a metalloenzyme due to its inhibition by EDTA. The
purified lipase exhibited a lower affinity for medium to long
fatty acyl chain length ester bonds (as in glycerol trioleate)
than short acyl chain fatty acid triglycerides. Because of its
high specificity toward short carbon chain length esters, the
potential application(s) of the lipase from P. fluorescens
MTCC 2421 in the enrichment of unsaturated fatty acids
as triglycerides may be further explored by performing
enzyme-catalyzed hydrolytic reactions of marine oils.
Further optimization of hydrolysis parameters will be taken
up in future studies for obtaining targeted unsaturated fatty
acids from marine sources, which will pave the way for lipase
application on a commercial scale. This study has potential
applications to design the specific reaction medium with
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ACKNOWLEDGMENT
We are thankful to Prof. (Dr.) Mohan Joseph Modayil,
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Received for Review December 8, 2008. Revised manuscript received
March 7, 2009. Accepted March 10, 2009.