Partial purification of Nigella sativa L. seed lipase and its application in transesterification reactions

Article abstract of DOI:10.1007/s11746-003-0648-6

Nigella sativa L. seed lipase isolated from defatted seeds was partially purified and used as catalyst in transesterification reactions. Purification of an ammonium sulfate-precipitated sample (at 35% saturation, Nigella PL) by DEAE ion-exchange chromatography increased the specific activity from 13.9 to 156.7 U/mg protein. Nigella PL and Nigella CPL (the partially purified enzyme sample obtained by DEAE ion-exchange chromatography) catalyzed the transesterification of vinyl acetate with octanol, with racemic sulcatol (6-methyl-5-hepten-2-ol), and with racemic trans-sobrerol (trans-p-menth-6-ene-2,8-diol) in different organic solvents. Both activity and enantioselectivity of the enzyme samples used for these biotransformations were affected by the nature of the organic solvent.

Full text of DOI:10.1007/s11746-003-0648-6

Partial Purification of Nigella sativa L. Seed Lipase and
Its Application in Transesterification Reactions
Melek Tutera, Francesco Secundob, Sergio Rivab, H. Ays¸e Aksoya,*, and Guldem Ustuna
aIstanbul Technical University, Chemical Engineering Department, 80626, Maslak, Istanbul, Turkey, and
bIstituto di Chimica del Riconoscimento Molecolare, C.N.R., 20131, Milano, Italy
ABSTRACT: Nigella sativa L. seed lipase isolated from defatted
seeds was partially purified and used as catalyst in transesterifica-
tion reactions. Purification of an ammonium sulfate-precipitated
sample (at 35% saturation, Nigella PL) by DEAE ion-exchange
chromatography increased the specific activity from 13.9 to
156.7 U/mg protein. Nigella PL and Nigella CPL (the partially pu-
rified enzyme sample obtained by DEAE ion-exchange chroma-
tography) catalyzed the transesterification of vinyl acetate with
octanol, with racemic sulcatol (6-methyl-5-hepten-2-ol), and with
racemic trans-sobrerol (trans-p-menth-6-ene-2,8-diol) in different
organic solvents. Both activity and enantioselectivity of the en-
zyme samples used for these biotransformations were affected by
the nature of the organic solvent.
Crude and immobilized N. sativa L. lipases are effective
biocatalysts for the hydrolysis and glycerolysis of TAG and
for the esterification of FFA (5,10–12). A crude lipase sample
from N. sativa L. seeds was immobilized by adsorption on
Celite 535 from phosphate buffer solutions at pH values vary-
ing from 5.0 to 8.0 at 25°C, and pH 6 was established to be
optimal for adsorption (12).
The goals of this study were to obtain crude and partially
purified samples of lipase(s) and to investigate the efficiency
of these enzymatic preparations for transesterification reac-
tions in organic solvents.
Paper no. J10368 in JAOCS 80, 43–48 (January 2003).
EXPERIMENTAL PROCEDURES
KEY WORDS: Enantioselectivity, enzymatic transesterification,
Nigella sativa L. seed lipase, purification, sobrerol, sulcatol.
Materials. Nigella sativa L. seeds from the Denizli region of
Turkey were purchased locally. The proximate compositions
of the seeds, determined according to standard AOCS meth-
ods (13), were 9.4% moisture, 4% ash, 35.4% oil, 23.3% pro-
tein, 6.7% crude fiber, and 21.2% carbohydrates.
Lipases, which catalyze not only the hydrolysis of TAG but
also their synthesis from glycerol and FFA, can be used in the
production of detergents, foods, pharmaceuticals, and other
synthetic organic materials (1).
All chemicals were purchased from Aldrich (Milwaukee,
WI). The solvents used in the reaction were of analytical grade.
Determination of lipase activity and protein content. Two
methods were applied for the determination of lipase activity:
(i) Spectrophotometric assay. A spectrophotometric assay, simi-
lar to that employed by Bornscheuer et al. (14) and based on the
use of p-nitrophenyl laurate (pNPL) as a substrate, was found to
be suitable for a quick analysis of both supernatant liquids and
pellets. One unit of lipase activity was defined as the amount of
enzyme that liberated 1 µmol of p-nitrophenol/min from pNPL.
(ii) pH-stat assay. A pH-stat method described by Peled and
Krenz (15) was used, employing tributyrin as the substrate. One
unit of lipase activity was defined as the amount of enzyme that
liberated 1 µmol of butyric acid/min from tributyrin.
Protein content was determined according to the Bradford
method using the Bio-Rad protein assay kit (Bio-Rad Labora-
tories, Milano, Italy), reading the absorbance at a wavelength
of 595 nm.
Preparation of the acetonic powder. A method similar to the
one developed by Afolabi et al. (16) was applied for the prepa-
ration of acetonic powder. Air-dried seeds (100 g) and 200 mL
of cooled acetone (4°C) were homogenized in a blender for 3.5
min. The resulting mixture was transferred to a 1000-mL beaker
that was cooled using a salt/ice mixture (–16°C). The container
was rinsed with acetone (150 mL) and the rinsings were
added to the original mixture, then stirred for 1 min. The sus-
Lipases can be obtained from mammals, yeast, bacteria, and
higher plants. To date, most industrial lipases have been pro-
duced from fungal sources. These enzymes are usually well
characterized, but lipases from higher plants generally have not
yet been investigated in detail despite the fact that they may dis-
play different properties that could be exploited for industrial or
technical purposes. Lipases are present in oil seeds, such as rape
(Brassica napus), mustard (Sinapis alba), and cereals (2,3).
Usually these enzymes are absent in dormant seed and are pro-
duced during seed germination. However, Nigella sativa L, also
known as black cumin or fennel flower, contains active lipases
even in its dormant seeds (3–5).
FA selectivity of lipases has been utilized to enrich for FA
(or of their derivatives) from naturally occurring mixtures (6).
More recently, lipases have been used as biocatalysts for the
enzymatic resolution of racemic substances to produce pure
enantiomeric drugs (7,8).
Nigella sativa L. is a member of the Ranunculaceae family
(9). Its capsule-shaped fruit contains the black-colored, oval, and
three-faced seeds. Owing to their aromatic nature, Nigella sativa
L. seeds are used as condiment or spice in cooking.
*To whom correspondence should be addressed. E-mail: aksoyha@itu.edu.tr
Copyright © 2003 by AOCS Press
43
JAOCS, Vol. 80, no. 1 (2003)

44
M. TUTER ET AL.
pension containing low-density material was decanted and fil- Triton. The detergent was used to avoid nonspecific interactions
tered by vacuum filtration through Schleicher & Schuell between the lipase protein and the DEAE-resin, interactions that
white band filter paper. The seed residue was washed twice might be responsible for the wide spreading of activity in the
with 200 mL acetone in the beaker, followed by filtration on eluted fractions observed when the first protocol was applied.
the previously obtained filter cake. The grayish filter cake was
In the third protocol the pellets were dissolved in buffer A
transferred to a 1000-mL beaker that was cooled with a and directly loaded on the DEAE column without dialysis.
salt/ice mixture and then washed twice with 150-mL portions The column was then developed as described in the second
of acetone and twice with 100-mL portions. The combined protocol.
acetone fractions were vacuum-filtered to leave a light gray-
Transesterification reactions. The partially purified lipase
ish powder. The powder was then air dried and stored at 4°C. samples were tested for their abilities to catalyze transesteri-
The yield of acetone powder was approximately 20% w/w fication reactions in different organic solvents (17,18). Vinyl
from the original seeds.
A sample of 200 mg of acetone powder showed a lipase ther n-octanol (to evaluate enzyme activity) or racemic sulca-
activity of 5.5 units/g using the pH-stat method. tol (6-methyl-5-hepten-2-ol) and racemic trans-sobrerol
acetate was the acyl donor, and the acceptor alcohols were ei-
Preparation of defatted seeds and isolation of lipase(s). To (trans-p-menth-6-ene-2,8-diol) (to evaluate enzyme enantios-
obtain the defatted seeds, four sets of extractions were con- electivity).
ducted separately using approximately 25 g of untreated
The enzymatic samples tested in the experiments were ob-
seeds. Specifically, the seeds were homogenized in a blender, tained as follows: (i) Nigella LS supernatant, obtained from
then the contained oil was extracted with hexane for 6 h in a the extraction of defatted seeds with distilled water contain-
Soxhlet extraction apparatus equipped with a cooling system. ing 1% vol/vol Triton, was lyophilized. (ii) Nigella PL:
The defatted seeds obtained (approximately 16 g each) were Nigella LS was dissolved in potassium phosphate buffer (5
then extracted for 6 h at room temperature with different so- mM, pH 7.5) and the enzyme was precipitated using 35% am-
lutions: potassium phosphate buffer (10 mM, pH 7) contain- monium sulfate. (iii) Nigella CPL sample, eluted from the
ing 1% Triton X-100 (Triton); phosphate buffer containing DEAE-column, was lyophilized and the residual solid was
1% Triton and 10% ethylene glycol; distilled water contain- dissolved in a smaller amount of the same elution buffer, dia-
ing 1% Triton; and distilled water containing 1% Triton and lyzed against potassium phosphate buffer (5 mM, pH 7.5),
10% ethylene glycol. The different mixtures were placed in and lyophilized.
the refrigerator overnight, then the homogenates were filtered
through cheesecloth. The activities of the different super- the transesterification of vinyl acetate with n-octanol was con-
natants and solid residues were determined. ducted in two different organic solvents (toluene and petro-
Transesterification of vinyl acetate with octanol. Initially,
Precipitation of lipase samples from the supernatant with leum ether) to evaluate the activity of the various partially pu-
ammonium sulfate. The solution obtained from the extraction rified samples of N. sativa L. seed lipases. The reactions were
of defatted seeds using water containing 1% Triton was fil- carried out by adding 90 U lipase (evaluated with the tribu-
tered through cheesecloth and centrifuged at 15,000 × g for tyrin assay) to a solution of 30 µL n-octanol and 100 µL vinyl
20 min. The lipase(s) in the supernatant then were precipi- acetate in 870 µL solvent. The reaction mixtures were shaken
tated by adding different amounts of solid ammonium sulfate in an orbital shaker at 250 rpm at 20°C. At scheduled times
at room temperature, with recovery of the solid residues by reaction samples were analyzed by capillary GC.
centrifugation at 15,000 × g for 20 min.
To investigate the influence of water activity (aw) on the
Enzymatic purification by DEAE exchange chromatography. reaction, this transesterification reaction was subsequently in-
The pellets obtained at 35% ammonium sulfate saturation of the vestigated using 40 U of Nigella PL or of Nigella CPL at
supernatant were dissolved in 5 mM potassium phosphate 40°C and 250 rpm. In this study, six different organic solvents
buffer, pH 7.5 (buffer A), and dialyzed against the same buffer (toluene, petroleum ether, methyl t-butyl ether, t-amyl alco-
overnight. The solution was loaded on a DEAE column (15 × hol, acetone, and acetonitrile), octanol, vinyl acetate, and
2.5 cm) previously equilibrated with buffer A. The column was Nigella PL and Nigella CPL were equilibrated in sealed flasks
washed with the same buffer, and the enzymes were eluted in the presence of molecular sieves, adjusting the aw value to
using a linear gradient according to three different protocols.
<0.1. The other reaction conditions were as above. Three of
In the first protocol, the lipase sample was eluted using a the six solvents (toluene, petroleum ether, and t-amyl alco-
two-step gradient. Flow rate was adjusted to 3 mL/min. In the hol) as well as all the other reaction components (vinyl ac-
first step, which lasted 2 h, the initial solution was buffer A etate, octanol, Nigella PL, and Nigella CPL) were also equili-
and the final solution was buffer A containing 0.3 M NaCl brated at aw = 0.3 (incubation overnight in sealed flasks con-
(buffer B). At the end of the first gradient the column was taining a saturated water solution of MgCl2) and at aw = 0.8
washed for 0.5 h with buffer B, and then the second gradient (incubation overnight in sealed flasks containing a saturated
was applied, moving in 2 h from buffer B to a final solution water solution of KCl) (19).
containing buffer A and 1 M NaCl (buffer C).
Transesterification reactions of vinyl acetate with racemic
In the second protocol the sample was eluted in 2.5 h using a sulcatol and racemic trans-sobrerol. (i) Racemic sulcatol
linear gradient from buffer A to buffer C containing also 0.5% (Scheme 1A). Racemic sulcatol (10 µL) and vinyl acetate (100
JAOCS, Vol. 80, no. 1 (2003)

PURIFICATION AND EXPLOITATION OF NIGELLA SATIVA SEED LIPASE
45
(initial time 10 min) to 160°C with a heating rate of 15°C/
min; H2 was the carrier gas. The peaks were identified using
standard mixtures.
A
B
Analysis of transesterification products of racemic sulcatol
and racemic trans sobrerol. Both the conversion of sulcatol and
trans-sobrerol into the corresponding esters and the eeP were
determined by GC using a chiral column (CP-Cyclodextrin-β-
2,3,6-M-19 column, 50 m × 0.25 mm i.d., Chrompack). The
conditions for the analysis of the transesterification products
of racemic sulcatol were: 90°C (initial time 15 min) to 130°C
with a heating rate of 1.5°C/min; H2 was the carrier gas (21).
The reaction mixtures of trans-sobrerol were analyzed using
the same column under the following conditions: 105°C (ini-
tial time 10 min) to 140°C with a heating rate of 0.5°C/min;
H2 was the carrier gas (22).
Nigella sativa lipase(s)
organic solvent
Nigella sativa
lipase(s)
organic solvent
SCHEME 1
µL) were dissolved in petroleum ether (890 µL) and subjected
to the action of different preparations of N. sativa L. seed en-
zymes (approximately 350 U enzyme/10 µL racemic sulca-
tol), the reaction mixtures being shaken in an orbital shaker
at 250 rpm at 20°C.
The degree of conversion of sulcatol into its acetate and
the enantiomeric excess of the product (eeP) were calculated
using the following formulas:
RESULTS AND DISCUSSION
Preparation of defatted seeds and isolation of lipase samples.
Supernatants were obtained from the extraction of defatted
seeds using different solutions: (i) phosphate buffer/1% Tri-
ton (solution I); (ii) phosphate buffer/1% Triton/10% ethyl-
ene glycol (solution II); (iii) distilled water/1% Triton (solu-
tion III); (iv) distilled water/1% Triton/10% ethylene glycol
(solution IV). All supernatants showed higher activities (de-
termined using the pH-stat method) than the solid residues,
increasing from 0.02–0.03 U/mg solid (defatted seeds) to
57.5, 63.9, 93.9, and 99.5 U/mL for solutions I, II, III, and IV,
respectively. Total activities obtained using solutions I, II, III,
or IV were 2731, 3035, 4599, or 4826 U, respectively. Better
results were obtained with the extraction carried out in dis-
tilled water containing both Triton and glycol; however, for
practical reasons, the subsequent purification experiments
were carried out using solution III (no ethylene glycol added),
which gave similar results. Accordingly, the supernatant ob-
tained by extracting the defatted seeds with solution III was
lyophilized and used as a catalyst for transesterification reac-
tions (designed as Nigella LS).
[S]ester + [R]ester
([S]alcohol + [R]alcohol )/0.75 + [S]ester + [R]ester
conversion =
[1]
)
where [R]ester, [S]ester, [R]alcohol, and [S]alcohol were the GC
peak areas of the respective enantiomers [(R) or (S)] (see
below). The value 0.75 represents the combustion ratio of sul-
catol over its acetate using a FID detector.
[R]ester – [S]ester
eeP
=
[2]
[R]ester + [S]ester
The enantiomeric ratio (E), a parameter that indicates the
enantioselectivity of the enzymes in kinetic resolutions, was
calculated from eeP and the conversion degree (c), obtained
from the same chromatographic analysis, using the following
formula (20):
Precipitation of lipase(s) samples from the supernatant
with ammonium sulfate. Table 1 shows the activities, the total
lipase activity units, the total protein contents, and the spe-
cific activities measured both in the solids and in the super-
natants obtained at 35, 40, or 50% (NH4)2SO4 saturation of
solution III.
E = ln[1 – c(1 + eeP)]/ln[1 – c(1 – eeP)]
[3]
(ii) Racemic trans-sobrerol (Scheme 1B). In a typical ex-
As the activities of the pellets obtained at 40 or 50% am-
periment, racemic trans-sobrerol (20 mg) and vinyl acetate monium sulfate saturation were similar or only slightly higher
(100 µL) were dissolved in a solvent (900 µL) and subjected than those obtained at 35% saturation, it was decided to use
to the action of different preparations of N. sativa L. seed en- the latter concentration of (NH4)2SO4 for the precipitation of
zymes (approximately 350 U enzyme/20 mg racemic trans- the crude lipase(s).
sobrerol), the reaction mixtures being shaken in an orbital
shaker at 250 rpm and at 40°C.
Enzymatic purification by DEAE ion-exchange chromatog-
raphy. The pellet samples obtained by precipitation at 35%
Chromatographic analysis of the products of transesterifi- (NH4)2SO4 saturation were used for further purification by
cation of vinyl acetate with octanol. The reaction products DEAE ion-exchange chromatography (described in the Exper-
were analyzed using a capillary methyl silicone column (HP-1 imental Procedures section). Total activity, total protein con-
Crosslinked Methyl Silicone Gum, 20 m × 0.32 mm i.d., tent, and the specific activity of the partially purified enzyme
Hewlett-Packard). The conditions for the analysis were: 35°C solution which was obtained using the first ion-exchange chro-
JAOCS, Vol. 80, no. 1 (2003)

46
M. TUTER ET AL.
TABLE 1
Activity and Protein Content of Samples Obtained by Extraction of Defatted Seeds Using Water Containing 1% vol/vol Triton (solution III)
Followed by Precipitation with Ammonium Sulfate
Total lipase
activity (units)
Total
protein (mg)
Specific activity
(U/mg protein)
Procedure
Sample
Activity
c
c
c
c
Cheese cloth
Filtration
Centrifugation
35% (NH4)2SO4 precipitation
Solid residue
Filtrate
Supernatant
Pellet
Supernatant
Pellet
Supernatant
Pellet
0.03a
65.45b
61.24b
101.48a
2.70b
534
458
3797
1015
58
1364
87
1342
42
583
73
81
78
70
69
39
7.0
13.9
0.7
17.4
1.2
40% (NH4)2SO4 precipitation
50% (NH4)2SO4 precipitation
136.60a
4.35b
134.19a
2.44b
19.6
1.1
Supernatant
aU/mg solid.
bU/mL solution.
cThe protein content could not be determined because of the turbidity of the filtrate.
matography protocol (see Experimental Procedures section)
The influence of different aw values on this transesterifica-
were determined as 2,092 U, 19.9 mg, and 105.1 U/mg protein, tion reaction is shown in Table 3. It is clear that an increase
respectively. The eluate obtained by DEAE chromatography of aw was accompanied by a decrease in the initial rate of
with the second protocol contained 134.4 mg protein and transesterification. The effect was more evident with hy-
14,776 total U, with a specific activity of 109.9 U/mg protein. drophobic solvents such as toluene and petroleum ether.
The eluate obtained with the third DEAE chromatography
(ii) Transesterification reactions of vinyl acetate with
protocol had a total activity, total protein content, and specific racemic sulcatol and racemic trans-sobrerol. For racemic sul-
activity of 18,477 U, 117.9 mg, and 156.7 U/mg protein, re- catol, transesterification reactions were performed in differ-
spectively.
ent organic solvents. As shown in Table 4, the nature of the
This third chromatographic protocol allowed an 11-fold solvent influenced enzyme enantioselectivity (21–24), best
increase of the specific activity of the ammonium sulfate pre- results being obtained in acetone (E = 4.1) (20).
cipitated enzyme that moved from an initial value of 13.9
The data obtained in the kinetic resolution of racemic
U/mg (see Table 1) to a final value of 156.7 U/mg after the trans-sobrerol are reported in Table 5. In this case too, the na-
DEAE-ion exchange chromatography. However, as might be ture of the solvent affected the enantioselectivity, best results
expected, the recovered material was still a complex mixture being obtained using neat vinyl acetate. Additionally, enzy-
of proteins (shown by SDS-PAGE gel electrophoresis) con- matic activity and enantioselectivity in transesterification was
taining more than one hydrolase (shown by native gel elec- increased by increasing the enzyme purity (Nigella CPL was
trophoresis; data not shown). Nevertheless, this purification better than Nigella PL).
protocol (oil extraction with hexane, enzyme extraction with
The high activity of these enzymatic preparations in apo-
water containing 1% Triton, 35% (NH4)2SO4 precipitation, lar solvents may make them suitable to catalyze selective
DEAE ion-exchange chromatography) was repeated several transesterification of complex TAG mixtures. Future work
times and proved to be highly reproducible in terms of the re- will focus on this specific target as well as on the purification
covered total and specific activity.
Transesterification reactions. (i) Transesterification of
vinyl acetate with octanol. The experimental data showed that
all the enzymatic samples were able to catalyze this transes-
terification reaction in toluene and in petroleum ether (con-
version: 99.9%, reaction time: 24 h), the initial rates being
higher with the more purified samples. As shown in Figure 1,
Nigella PL and Nigella CPL gave similar results. Transesteri-
fication activity was higher in petroleum ether than in toluene.
The effect of the solvent on the transesterification reactions
conducted at controlled low water activity (aw < 0.1) using
Nigella PL and Nigella CPL is shown in Table 2. As expected,
transesterification activities were higher in the solvents pos-
FIG. 1. Transesterifica-
tion of vinyl acetate
with n-octanol in
sessing higher log P values (that is, the more hydrophobic
ones, where P represents the partition coefficient). The per-
formances of the two enzymatic preparations, Nigella PL and
Nigella CPL, were again similar, and petroleum ether was the
best solvent.
toluene and in petro-
leum ether using dif-
ferent samples of
Nigella sativa lipase
(90 U) at 20°C.
Nigella LS
Nigella PL
Enzyme samples
Nigella CPL
JAOCS, Vol. 80, no. 1 (2003)

PURIFICATION AND EXPLOITATION OF NIGELLA SATIVA SEED LIPASE
47
TABLE 2
Enzymatic Transesterificationa of Vinyl Acetate with Octanol at aw < 0.1
Conversion at
Initial rate
Enzyme
Solvent
Log Pe
(µmol/min)b
0.5 h
24 h
Nigella PLc
Nigella CPLd
Nigella PL
Nigella CPL
Nigella PL
Nigella CPL
Nigella PL
Nigella CPL
Nigella PL
Nigella CPL
Nigella PL
Nigella CPL
Petroleum ether
Petroleum ether
Toluene
3.50
6.95
4.70
3.11
1.35
2.68
1.96
0.39
0.44
0.38
0.46
0.21
0.21
62.4
51.6
18.8
12.9
17.5
20.3
3.2
3.2
2.7
3.6
2.2
99.8
99.8
99.6
99.99
99.8
99.9
21.5
11.2
24.1
35.4
34.6
55.2
2.50
1.38
Toluene
Methyl t-butyl ether
Methyl t-butyl ether
t-Amyl alcohol
t-Amyl alcohol
Acetone
1.30
–0.23
–0.33
Acetone
Acetonitrile
Acetonitrile
2.4
aReactions were performed with 30 µL n-octanol and 100 µL vinyl acetate in 870 µL solvent using 40 U enzyme at 40°C.
bDetermined after 5 min. aw, water activity.
c35% (NH4)2SO4 precipitate.
dSample obtained by DEAE-ion exchange chromatography.
eLog P, the logarithm of the partition coefficient of a given compound in the octanol/water two-phase system.
TABLE 3
Enzymatic Transesterification of Vinyl Acetate with Octanol at Different aw Valuesa
Conversion at
Initial rate
Enzyme
Solvent
aw
(µmol/min)b
0.5 h
24 h
Nigella PL
Nigella PL
Nigella PL
Nigella CPL
Nigella CPL
Nigella CPL
Nigella PL
Nigella PL
Nigella PL
Nigella CPL
Nigella CPL
Nigella CPL
Nigella PL
Nigella PL
Nigella PL
Nigella CPL
Nigella CPL
Nigella CPL
Toluene
Toluene
Toluene
Toluene
Toluene
<0.1
0.3
0.8
<0.1
0.3
0.8
<0.1
0.3
0.8
<0.1
0.3
0.8
<0.1
0.3
0.8
<0.1
0.3
0.8
3.11
0.92
0.07
1.35
0.21
0.13
6.95
1.53
0.39
4.70
0.85
0.33
0.39
0.45
0.24
0.44
0.33
0.04
18.8
7.3
1.1
12.9
3.6
1.4
62.4
34.7
5.4
51.6
11.9
2.5
3.2
5.0
4.1
3.2
2.9
0.8
99.6
99.8
99.1
99.9
99.9
14.3
99.8
99.9
85.1
99.8
99.9
17.7
21.5
39.9
44.9
11.2
27.6
21.3
Toluene
Petroleum ether
Petroleum ether
Petroleum ether
Petroleum ether
Petroleum ether
Petroleum ether
t-Amyl alcohol
t-Amyl alcohol
t-Amyl alcohol
t-Amyl alcohol
t-Amyl alcohol
t-Amyl alcohol
aReactions were performed with 30 µL n-octanol and 100 µL vinyl acetate in 870 µL solvent using 40 U enzyme at 40°C.
bDetermined after 5 min. aw, water activity.
TABLE 4
TABLE 5
Enantiomeric Ratio (E) of Kinetic Resolution of Racemic Sulcatol
Enantiomeric Ratio (E) of Kinetic Resolution of Racemic
trans-Sobrerol Catalyzed by Nigella sativa L. Seed Lipases
(Nigella PL and Nigella CPL)a
Catalyzed by Nigella PLa
Conversion
(%)b
eeP
E
Enzyme
Solvent
eeP
Conversionb (%)
E
Solvent
Petroleum ether
Toluene
t-Butyl methyl ether
Acetone
10.4
19.9
16.8
4.3
0.464
0.496
0.496
0.602
2.9
3.3
3.3
4.1
Nigella PL
Nigella PL
Nigella PL
Nigella CPL
Nigella CPL
Nigella CPL
Methyl t-butyl ether
Vinyl acetate
t-Amyl alcohol
Methyl t-butyl ether
Vinyl acetate
15.0
15.3
49.0
38.7
67.3
68.1
23.5
15.3
5.0
36.9
31.6
7.3
1.4
1.4
3.0
2.8
6.9
5.6
aReactions were performed with 10 µL racemic sulcatol (6-methyl-5-hepten-
2-ol) and 100 µL vinyl acetate in 890 µL solvent using approximately 350 U
enzyme/10 µL substrate at room temperatures.
t-Amyl alcohol
aReactions were performed with 20 mg racemic trans-sobrerol (trans-p-menth-
6-ene-2,8-diol) and 100 µL vinyl acetate in 900 µL solvent using approxi-
mately 350 U enzyme/20 mg substrate at 40°C.
bReaction time, 2 h. eeP, enantiomeric excess of the product.
bReaction time, 5 d. For abbreviation see Table 4.
JAOCS, Vol. 80, no. 1 (2003)

48
M. TUTER ET AL.
11. Dandik, L., and H.A. Aksoy, Applications of Nigella sativa
Seed Lipase in Oleochemical Reactions, Enzyme Microb. Tech.
19:277–281 (1996).
to homogeneity of this/these lipases(s) and their biochemical
characterization.
12. Akova, A., and Ustun, G., Activity and Adsorption of Lipase
from Nigella sativa Seeds on Celite at Different pH Values,
Biotechnol. Lett. 22:355–359 (2000).
13. AOCS, Official Methods and Recommended Practices of the
American Oil Chemists’ Society, 3rd ed., American Oil Chemists’
Society, Champaign, 1971.
14. Bornscheuer U., O.W. Reif, R. Lausch, R. Freitag, T. Scheper,
F.N. Kolisis, and U. Menge, Lipase of Pseudomonas cepacia
for Biotechnological Purposes: Purification, Crystallization and
Characterization, Biochim. Biophys. Acta 1201:55–60 (1994).
15. Peled, N., and M.C. Krenz, A New Assay of Microbial Lipases
with Emulsified Trioleoyl Glycerol, Anal. Biochem. 112:219–
222 (1981).
16. Afolabi, O.A., M.A. Ologunde, W.A. Anderson, J.S. Read, M.D.
Dacosta, F.A. Epps, and F.O. Ayorinde, The Use of Lipase (ace-
tone powder) from Vernonia galemensis in Fatty Acids Analysis
of Seed Oils, J. Chem. Technol. Biotechnol. 52:41–46 (1991).
17. Klibanov, A.M., Improving Enzymes by Using Them in Or-
ganic Solvents, Nature 409:241–246 (2001).
ACKNOWLEDGMENT
We are grateful to CNR (Consiglio Nazionale delle Ricerche, Italy)
and TUBITAK (Turkish Scientific and Technical Research Center)
for financial support of this joint research project. The facilities of
the laborotorium of the Chemical Engineering Department, Istanbul
Technical University, Turkey are also acknowledged.
REFERENCES
1. Malcata, F.X., Engineering of/with Lipases, Scope and Strate-
gies, in Engineering of/with Lipases, edited by F.X. Malcata,
Kluwer Academic, Dordrecht, 1996, pp. 1–16.
2. Huang, A.H.C., Plant Lipases, in Lipases, edited by B.
Borgstrom, and H.L. Brokman, Elsevier Science, Amsterdam,
1990, pp. 419– 442.
3. Mukherjee, K.D., Plant Lipases in Lipid Biotransformations, in
Engineering of/with Lipases, edited by F.X. Malcata, Kluwer
Academic, Dordrecht, 1996, pp. 391–401.
4. Dandik, L., and H.A. Aksoy, The Kinetics of Hydrolysis of
Nigella sativa (black cumin) Seed Oil Catalyzed by Native Lipase
in Ground Seed, J. Am. Oil Chem. Soc. 69:1239–1241 (1992).
5. Dandik, L., G. Arioglu, and H.A. Aksoy, The Enzymatic Hy-
drolysis of Used Frying Oil by Native Lipase, Appl. Biochem.
Biotechnol. 42:119–126 (1993).
18. Carrea, G., and S. Riva, Properties and Synthetic Applications
of Enzymes in Organic Solvents, Angew. Chem. Int. Ed. 39:
2226–2254 (2000).
19. Valivety, R.H., P.J. Halling, and A.R. Macrae, Reaction Rate with
Suspended Lipase Catalyst Shows Similar Dependence on Water
Activity in Different Organic Solvents, Biochim. Biophys. Acta
1118:218–222 (1992).
6. Mukherjee, K.D., Lipase-Catalyzed Reactions for the Fraction-
ation of Fatty Acids, in Engineering of/with Lipases, edited by
F.X. Malcata, Kluwer Academic, Dordrecht, 1996, pp. 51–64.
7. Kamiya, N., M. Goto, and F. Nakashio, Surfactant-coated Lipase
Suitable for the Enzymatic Resolution of Menthol as a Biocatalyst
in Organic Media, Biotechnol. Prog. 11:270–275 (1995).
8. Sinisterra, J.V., Synthesis of Chiral Drugs Using Lipases, in En-
gineering of/with Lipases, edited by F.X. Malcata, Kluwer Aca-
demic, Dordrecht, 1996, pp. 73–101.
9. Üstün, G., S.Türkay, and A. Karaali, Nigella sativa Seeds: A
Potential Source for Oil and Oleochemicals, in Proceedings of
the World Conference on Oilseed and Edible Oils Processing,
edited by S.S. Koseoglu, K.C. Rhee, and R.F. Wilson, AOCS
Press, Champaign, 1998, Vol. 2, pp. 155–160.
20. Chen, C.S., Y. Fujimoto, G. Girdaukas, and C.J. Sih, Quantita-
tive Analysis of Biochemical Kinetic Resolutions of Enan-
tiomers, J. Am. Chem. Soc. 104:7294–7299 (1982).
21. Secundo, F., S. Riva, and G. Carrea, Effects of Medium and Reaction
Conditions on the Enantioselectivity of Lipases in Organic Solvents
and Possible Rationales, Tetrahedron: Asymmetry 3:267– 280 (1992).
22. Bovara, R., G. Carrea, L. Ferrara, and S. Riva, Resolution of ( )-
trans-Sobrerol by Lipase PS-Catalyzed Transesterification and Ef-
fects of Organic Solvents on Enantioselectivity, Tetrahedron:
Asymmetry 2:931–938 (1991).
23. Carrea, G., G. Ottolina, and S. Riva, Role of Solvents in the Con-
trol of Enzyme Selectivity in Organic Media, Trends Biotechnol.
13:63–70 (1995).
24. Wescott, C.R., and A.M. Klibanov, The Solvent Dependence of
Enzyme Specificity, Biochim. Biophys. Acta 1206:1–9 (1994).
10. Üstün, G., L. Kent, N. Cekin, and H. Civelekoglu, Investigation
of the Technological Properties of Nigella sativa (black cumin)
Seed Oil, J. Am. Oil Chem. Soc. 67:958–960 (1990).
[Received June 26, 2002; accepted October 9, 2002]
JAOCS, Vol. 80, no. 1 (2003)