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B.S. Demir, S.S. Tükel / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 123–128
and was maintained and grown in inorganic medium as reported
by Ihena [19].
Purity was analyzed by SDS-PAGE and native polyacrylamide
gel electrophoresis (native PAGE) using the method of Laemmli
[22] on 12.5% polyacrylamide gel on a vertical mini gel apparatus
(Bio-Rad) at 150 V for 1 h. Reference proteins for molecular mass
determination (Fermentas SM0431) were lysozyme (14.4 kDa),
-lactoglobulin (18.4 kDa), REase Bsp98I (25 kDa), lactate dehy-
drogenase (35 kDa), ovalbumin (45 kDa), bovine serum albumin
(66.2 kDa), -galactosidase (116 kDa). Protein bands were visual-
ized by silver staining.
2.2. Lipase activity assay
Lipase activity was measured with a modified spectrophoto-
metric assay with p-NPP as substrate [20]. The substrate solution
(0.1 mM) was prepared by dissolving p-NPP in ethanol. 50 mM
phosphate buffer (pH 6.5) was used as reaction buffer. 0.5 ml of
enzyme or blank solution (50 mM phosphate buffer pH 6.5) was
added into 0.5 ml of reaction buffer and then 1 ml of substrate solu-
tion was added to the reaction medium. The enzyme–substrate
mixture or blank–substrate mixture was incubated at 45 ◦C for
5 min with stirring. The reaction was stopped by adding 2 ml of
0.25 M Na2CO3 solution. The molar extinction coefficient of p-
nitrophenol (p-NP) was estimated as 5.5 × 103 cm2 mol−1 from the
absorbance measured at 410 nm of standard solutions of p-NP. One
enzyme unit (U) was defined as the lipase activity that liberated
1 mol equivalent of p-NP per mililiter per minute under the stan-
dard assay conditions.
2.5. Isoelectric focusing
Isoelectric focusing was performed in a Protean IEF cell system
(Bio-Rad) following the manufacturer’s instructions. Isoelectric
points of reference proteins for determination of pI were phyco-
cyanin (4.45, 4.65, 4.75), -lactoglobulin B (5.1), bovine carbonic
anhydrase (6.0), human carbonic anhydrase (6.5), equine myo-
globin (6.8, 7.0), human hemoglobin A (7.1), human hemoglobin
C (7.5), lentil lectin (7.8, 8.0, 8.2) and cytochrome c (9.6). Gels were
stained with Coomassie Brilliant Blue R250.
2.3. Purification of lipase
2.6. Determination of protein
2.3.1. Step 1: preparation of cell free extract and ammonium
sulphate precipitation
Protein was assayed by the method of Lowry et al. [23].
Before ammonium sulphate precipitation, the harvested cells
were washed three times with 10 mM pH 7.0 phosphate buffer at
room temperature, and 15 g wet sample suspended in 45 ml of son-
icating buffer (0.01 M pH 7.0 phosphate buffer containing 0.1 mM
EDTA). Cell free extracts were prepared by sonicating the suspen-
sion at 149 m amplitude using cycles of 20 s on and 10 s off. The
sonicate was centrifuged at 12,000 rpm for 30 min, at 4 ◦C and the
supernatant was used for lipase as crude extract.
The ammonium sulphate precipitation of cell free extract was
carried out at a range of 20–90% (w/v) saturation at 4 ◦C [18]. Pre-
cipitation was allowed for 1 h at 4 ◦C and followed by centrifugation
at 12,000 rpm for 30 min. The precipitate was dissolved in 50 mM
pH 7.0 phosphate buffer. Dialysis of this sample was carried out
using Sigma cellulose tubing (mol.wt. cut off: 12–14 kDa) for 24 h
with three changes in the buffer at 4 ◦C.
2.7. Determination of optimal pH, temperature, buffer
concentration, and kinetic constants
The optimal pH of the lipase was determined by measuring
the activity in a pH range of 5–8 in the following buffers: 50 mM
sodium acetate buffer (5.0–5.5), 50 mM sodium citrate buffer (pH
6.0), 50 mM potassium phosphate buffer (6.5–8.0). Lipase activity
of samples in each buffer was measured using the standard assay
as described previously, and buffer concentrations of 25, 50, 75 and
100 mM at pH 6.5 were also tested.
The optimal temperature for the purified lipase was determined
by measuring the activity at various temperatures in the range of
25–70 ◦C at pH 6.5 in 50 mM phosphate buffer.
The activity assays were carried out in different p-NPP concen-
trations (0.066–0.331 mM) to determine maximum reaction rate
(Vmax) and Michaelis–Menten constant (Km) of S. platensis lipase.
Turnover numbers (kcat) of lipase was calculated from the equation:
2.3.2. Step 2: DEAE-Sepharose column chromatography
The clear sample obtained in the previous step was loaded on a
DEAE-Sepharose column (1.2 cm × 10 cm) previously equilibrated
with 50 mM phosphate buffer (pH 7). After washing with two bed
volumes of the equilibration buffer, elution was performed with a
linear gradient of 0–1.0 M NaCl in the same buffer at a flow rate of
60 ml/min [3]. Fractions showing lipase activity were pooled and
analyzed.
Vmax
kcat
=
[E]T
where [E]T is the total amount of enzyme in reaction medium.
Catalytic efficiency (kcat/Km) of lipase was also calculated. The acti-
vation energy (Ea) was estimated by using the Arrhenius equation.
The partially purified enzyme was applied to a Sepharose-6B
fast flow column (1.2 cm × 20 cm) which was pre-equilibrated with
50 mM phosphate buffer (pH 7) and eluted with the same buffer
at a flow rate of 30 ml/min [21]. The fractions were collected and
analyzed.
2.8. Determination of thermal and storage stabilities
An estimation of the thermal stability was performed by
measuring the residual activity of purified lipase exposed to tem-
peratures of 25, 35, 45 and 55 ◦C. Samples were taken at 2, 8, 16,
Table 1
Summary of the results of purification procedures of the lipase from S. platensis.
Purification steps
Volume
(ml)
Activity
(U/ml)
Protein
(mg/ml)
Total protein
(mg)
Total activity
(U)
Specific activity
(U/mg prot)
Yield (%)
Fold
purification
Crude enzyme solution
(NH4)2SO4 precipitation
DEAE-Sepharose
60
14
48
9
0.23
0.92
0.26
0.45
2.03
0.13
0.02
0.01
121.80
1.82
0.96
13.80
12.88
12.48
4.05
0.12
7.08
13.00
45.00
100.00
93.33
90.43
29.35
1
59
108
375
Sepharose-6B
0.09