Optimized synthesis of (Z)-3-hexen-1-yl caproate using germinated rapeseed lipase in organic solvent

Article abstract of DOI:10.1016/j.molcatb.2010.09.011

(Z)-3-hexen-1-yl esters are important green top-note components of food flavors and fragrances. Effects of various process conditions on (Z)-3-hexen-1-yl caproate synthesis employing germinated rapeseed lipase acetone powder in organic solvent were investigated. Rapeseed lipase catalyzed ester formation more efficiently with non-polar compared to polar solvents despite high enzyme stability in both types of solvents. Maximum ester yield (90%) was obtained when 0.125 M (Z)-3-hexen-1-ol and caproic acid were reacted at 25 °C for 48 h in the presence of 50 g/L enzyme in heptane. Enzyme showed little sensitivity towards a_w with optimum yield at 0.45, while added water did not affect ester yield. Esterification reduced by increasing molecular sieves (>0.0125%, w/v). The highest yields of caproic acid were obtained with isoamyl alcohol (93%) followed by butanol and (Z)-3-hexen-1-o1 (88%) respectively reflecting the enzyme specificity for straight and branched chain alcohols. Secondary alcohols showed low reactivity, while tertiary alcohol had either very low reactivity or not esterified at all. A good relationship has been found between ester synthesis and the solvent polarity (log P value); while no correlation for the effect of solvents on residual enzyme activity was observed. It may be concluded that germinated rapeseed lipase is a promising biocatalyst for the synthesis of valuable green flavor note compound. The enzyme also showed a wide range of temperature stability (5-50 °C).

Full text of DOI:10.1016/j.molcatb.2010.09.011

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 59–65
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Optimized synthesis of (Z)-3-hexen-1-yl caproate using germinated rapeseed
lipase in organic solvent
Muhammad Liaquat∗
Laboratory of Food Biochemistry and Nutrition, Department of Food Science, University of Leeds, Leeds LS2 9JT, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 March 2010
Received in revised form 17 August 2010
Accepted 27 September 2010
(Z)-3-hexen-1-yl esters are important green top-note components of food flavors and fragrances. Effects
of various process conditions on (Z)-3-hexen-1-yl caproate synthesis employing germinated rapeseed
lipase acetone powder in organic solvent were investigated. Rapeseed lipase catalyzed ester formation
more efficiently with non-polar compared to polar solvents despite high enzyme stability in both types of
solvents. Maximum ester yield (90%) was obtained when 0.125 M (Z)-3-hexen-1-ol and caproic acid were
reacted at 25 ◦C for 48 h in the presence of 50 g/L enzyme in heptane. Enzyme showed little sensitivity
towards aw with optimum yield at 0.45, while added water did not affect ester yield. Esterification reduced
by increasing molecular sieves (>0.0125%, w/v). The highest yields of caproic acid were obtained with
isoamyl alcohol (93%) followed by butanol and (Z)-3-hexen-1-o1 (88%) respectively reflecting the enzyme
specificity for straight and branched chain alcohols. Secondary alcohols showed low reactivity, while
tertiary alcohol had either very low reactivity or not esterified at all. A good relationship has been found
between ester synthesis and the solvent polarity (log P value); while no correlation for the effect of
solvents on residual enzyme activity was observed. It may be concluded that germinated rapeseed lipase
is a promising biocatalyst for the synthesis of valuable green flavor note compound. The enzyme also
showed a wide range of temperature stability (5–50 ◦C).
Keywords:
Plant lipases
Flavor
Ester synthesis
Organic phase biocatalysis
(Z)-3-hexen-1-yl caproate
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
acetate, butyrate, and caproate can be synthesized using micro-
bial lipases suspended in non-aqueous solvents [7–22]. Fungal
Derivatives of (Z)-3-hexen-1-ol including C6 aldehydes and
acyl esters impart green-flavor notes and a character of freshness
to foods and fragrances. Within higher plants, (Z)-3-hexen-1-
ol is formed by the lipoxygenase catalyzed oxidation of linoleic
acid to form a lipid hydroperoxide. This intermediate is cleaved
by hydroperoxide lyase forming (Z)-3-hexen-1-al, (E)-2-hexenal
and hexanal. The aldehydes can be reduced using NADH-linked
alcohol dehydrogenase to form (Z)-3-hexen-1-ol, (E)-2-hexen-
1-ol and hexan-1-ol. These compounds possess the aroma of
freshly cut grass. The production of “leaf alcohol” green notes
using the lipoxygenase reaction has been achieved on an indus-
trial scale [1,2]. Currently 5–10 metric tons of green flavor
notes are produced annually at price of 2500–6000 $US kg−1
[3,4].
lipases are preferred for organic phase biocatalysis (OPB) owing to
their ready availability and low cost [23–25]. Lipases from higher
vegetative plants including peanut [24], wheat germ [27], papaya
[28–30], rapeseed [31–34] and nigella sativa seeds [35] have also
been used for OPB. The cost for biocatalyst remains an impor-
tant consideration in OPB. In our previous study [36], various
plant seedlings were evaluated for their ability to catalyze synthe-
sis of flavor esters in organic solvents. Of the systems examined,
germinated rapeseed showed the highest degree of flavor synthe-
sis.
Enzyme activity, stability, and selectivity are generally influ-
enced by the reaction media and temperature. And also one of the
criteria for choosing best biocatalyst is its ability to remain stable in
solvent over wide range of temperature. In this study, the influence
of process variables (reaction time, temperature, moisture con-
tents, water activity, molecular sieves, enzyme load, its reuse, acid
alcohol concentration, organic solvents and various alcohol chain
lengths) on (Z)-3-hexen-1-yl caproate synthesis is described under
the following headings: solvent characteristics, biocatalyst related
factors and substrate. The aim of study is to perform a comprehen-
sive evaluation of (Z)-3-hexen-1-yl caproate synthesis using crude
rapeseed lipase acetone powder in accordance with the scheme
Conversion of (Z)-3-hexen-1-ol to the acyl ester provides a
means to lower its odor threshold and increase its chemical sta-
bility [5,6]. Short chain flavor esters such as (Z)-3-hexen-1-yl
∗
Correspondence author: Flat 10 Kelso Court Belle, Vue Road, Leeds LS3 1HB,
United Kingdom. Tel.: +44 113 2434776.
E-mail address: Liaquat-Leeds@hotmail.co.uk.
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.09.011

60
M. Liaquat / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 59–65
Scheme 1. Synthesis of (Z)-3-hexen-1-yl caproate catalyzed by rape seedlings lipase in organic solvent via esterification.
below and to determine the optimal conditions for (Z)-3-hexen-1-
yl caproate synthesis (Scheme 1).
2. Materials and methods
2.1. Materials and chemicals
Analar grade chemicals, acids, alcohols, organic solvents (HPLC
grade), were obtained from Sigma–Aldrich Co., Ltd. (Poole,
England). Hexane and heptane were obtained from Fisher (Lough-
borough, UK). Hexane was dried over molecular sieves (3 A,
8–12 mesh; both from Sigma–Aldrich Co., Ltd.) for at least 24 h prior
to use. Seeds were supplied by Nickerson Seeds Ltd., Lincoln (UK).
Fig. 1. Gas chromatogram for the analysis of samples obtained from the esterifi-
cation of caproic acid with (Z)-3-hexen-1-ol after 48 h at 40 ◦C: (Z)-3-hexen-1ol
(A), internal standard (B), (Z)-3-hexen-1-yl caproate (C), caproic acid (D). For gas
chromatographic conditions see Section 2.
2.2. Acetone powder preparation from rape seedlings
Dry whole rapeseeds were surface sterilized by soaking in 0.1%
sodium hypochlorite solution for 30 s, rinsed thoroughly with run-
ning tap water and soaked for 24 h at 26 ◦C (designated as day 1st) in
a dark incubator. Germination was achieved by placing rapeseed on
moist filter paper towels, on top of moist perlite (Silvaperl graded
horticultural) in shallow plastics trays, and then covering with
perforated aluminium foil. Samples of seedlings were withdrawn
on day 4 after germinating for further processing. In preliminary
studies, lipase activity reached to a maximum at 4–6 days after
germination [19]. Germinated rapeseed was washed with distilled
water three times, equilibrated in a refrigerator at 4 ◦C for 10 min,
cut into small pieces, and then homogenized with 5 volumes of cold
acetone (−18 ◦C or less) for 1 min. The resulting solid was recovered
by vacuum filtration using a Buchner funnel, fitted with a Whatman
No. I filter paper. Rapeseed lipase acetone powder was washed with
4-volumes of cold acetone and air dried under a hood for 10 h. The
light greyish powder was kept in sealed bottles at −20 ◦C until used.
GLC was connected to an integrator (Hewlett Packard 3395 inte-
grator) which recorded the peak areas and retention times in a
chromatogram.
2.4.1. Product identification, yield and reaction time
The identity of ester products is possible to achieve either
(a) using the peak retention times obtained with standard com-
pounds (Fig. 1) or (b) interpretation of fragmentation patterns in
a GC–MS analysis or comparing these with a library of standard
esters (Fig. 2). Ester yield was determined by quantitative analysis
using a calibration graph of peak area versus concentration. Stan-
dard concentrations of acids, alcohol or ester (0.0125–0.25 M) were
diluted with n-hexane and 0.2 ␮L was subjected to GLC analysis.
Injections were repeated twice for each vial. Reaction time-course
was determined by monitoring the formation of ester at differ-
ent time intervals. Percent ester yield was defined as molar ester
2.3. Esterification method
Rapeseed acetone powder (50 g/L) was suspended in organic
solvents together with 0.25 M of acid and 0.25 M of alcohol. The
typical reaction volume was 5 mL. Esterification was performed
with shaking at 100 rpm at a temperature of 40 ◦C for 48 h. There-
after, 0.1 mL of reaction mixture was withdrawn at known intervals,
centrifuged (1300 × g for 5 min) to remove suspended matter, and
stored at −10 ◦C until analyzed (usually within 24 h) as described
below. All experiments were performed in duplicate. Synthesis was
also carried out without enzyme.
2.4. Ester analysis
Routine analysis of reactants and products was conducted by
a GLC instrument (Model 5160 Carlo Erba) equipped with a BP-
20 fused silica capillary column (SGE, UK, 25 m × 0.32 mm ID; film
thickness 1 ␮m), and a flame ionization detector. The carrier gas
was helium (2 mL/min, split ratio 1:15). The GLC oven temperature
was maintained at 50 ◦C for 2 min and then increased to 210 ◦C
at a rate of 15 ◦C/min and held for 4 min. The injector tempera-
ture was fixed at 250 ◦C and detector temperature at 240 ◦C. The
Fig. 2. GC–MS spectra of peak C identified as (Z)-3-hexen-1-yl caproate and its
confirmation in this study. (A) Synthesized and (B) library.

M. Liaquat / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 59–65
61
formed × 100/molar acid added. The identity of different esters
100
80
60
40
20
0
was established by GC–MS analysis (Carlo Erba GC Model 4200,
Kratos MS 80 RFA). The GS–MS instrument was equipped with a
38 m × 0.32 ␮m × 0.5 ␮m film thickness BP-20 column (SGE, UK);
elution was performed as described above for conventional GLC
analysis. Injections (0.2 ␮L) were made on column. Mass spectra
were recorded with the ion source energy of 70 eV.
2.5. Effect of solvents
(Z)-3-hexen-1-yl caproate
The reaction of (Z)-3-hexen-1-ol with caproic acid was exam-
ined using seven common organic solvents of varying hydropho-
bicity; dioxane, acetonitrile, tetrahydrofuran (THF), diethyl ether,
toluene, hexane or heptane. All other reaction conditions were as
described above.
0
10
20
30
40
50
60
70
80
Reaction time (h)
Fig. 3. Time course of (Z)-3-hexen-1-yl caproate synthesis. The reaction mixtures
consisted of 0.25 M caproic acid, and0.25 M butanolor (Z)-3-hexen-1-ol and 50 g/L of
rapeseed lipase acetone powder was at 40 ◦C. All experiments were done in duplicate
and the values reported are mean of two determinations.
2.6. Effect of added moisture and water activity (aw)
Varying amounts of distilled water (0–30%, v/v) was added to
the reaction medium containing rapeseed lipase acetone pow-
der (50 g/L), alcohol and acid. Ester synthesis was performed as
above. To examine the influence of aw on ester yield, first reactants,
enzyme, and organic solvents were equilibrated with standard
saturated salt solutions at room temperature (21 ◦C) in separate
desiccators for 7 days as reported by Chamouleau et al. [53]. The
salt standards were MgCl2 (aw = 0.33), Mg (NO3)2 (aw = 0.55), NaCl
(aw = 0.75), KCl (aw = 0.86), ZnSO4·7H2O (aw = 0.90), and molecular
sieves (aw = 0.04). Reaction was started by mixing the two sepa-
rately equilibrated phases as described above.
rapeseed lipase was recovered by centrifugation, washed with
excess organic solvent and dried under nitrogen. The dried enzyme
powder was then added to a fresh mixture of reactants and the
yield of (Z)-3-hexen-1-yl caproate analyzed by GLC.
2.11. Determination of lipase residual activity
Lipase was assayed using the 4-methylumbelliferyl heptanoate
(4-MUH) fluorimetric assay. The assay mixture (3 mL) comprised
Tris–HCl buffer (0.1 M, pH 8), 25 ␮L of the 4-MUH (0.01 M in
99–100% ethanol), and 200 ␮L of lipase solution. The reaction was
stopped with the addition of 1 M HCl (0.5 mL) after 30 min and
fluorescence was recorded with fluorescence spectrophotometer
(Perkin-Elmer, LS-203) at ꢀex = 330 and ꢀem = 450 nm. Blank assays
were performed by adding HCl to the reaction mixture before
enzyme. A calibration graph for 4-MU determination was con-
structed from fluorescence measurements recorded with 3–70 ␮L
(or 7.5–175 nmol) of 4-MU stock per 3 mL of assay mixture. A unit
of lipase activity was expressed as mole 4-MU produced/min/mL
of enzyme solution.
2.7. Effect of reaction temperature
Ester synthesis was performed at 0–80 ◦C. For temperature
below 40 ◦C a thermo controller (refrigeration unit) was used. Reac-
tion temperatures above 40 ◦C were maintained using an oil bath
filled with Dow Corning Silicon Oil.
2.8. Effect of substrate concentration
Effect of increasing the concentration of one of the substrates
was evaluated, while keeping the other constant. (Z)-3-hexen-1-ol
concentrations of 0.625 M, 0.125 M, 0.25 M, 0.4 M, 0.5 M and 1 M
were reacted with a fixed 0.25 M concentration of caproic acid.
In the reverse study, (Z)-3-hexen-1-ol concentration was fixed at
0.25 M and caproic acid concentration was varied at 0.0625 M,
0.125 M, 0.25 M, 0.4 M, 0.5 M and 1 M. The organic solvent phase
was hexane at a reaction temperature of 40 ◦C.
3. Results and discussion
The time scale for (Z)-3-hexen-1-yl caproate synthesis is pre-
sented in Fig. 3. With increasing reaction time, an increase in ester
yield was observed. (Z)-3-hexen-1-yl caproate synthesis reached
completion in 48 h at 40 ◦C. For the baseline study, product yield
was 90% with no further increase beyond 48 h. Non availability of
the either of substrates to the enzyme could be the one major rea-
sons, because after 48 h, as 90% of the caproic acid was converted to
ester (results not shown). This high conversion was quite surprising
since no attempts were made to prevent water accumulation in the
reaction mixture. This is a considerably faster reaction compared
to reaction of butyl carpylate catalyzed by the psychrotrophic Pseu-
domonas fluorescens P38 lipase which reached an equilibrium after
96 h at 20 ◦C with a final molar conversion of 75% [54]. In our case,
organic phase biocatalysis at 40 ◦C is expected to be associated with
a higher rate of reaction and low organic solvent phase viscosity.
Reaction time and product yield are two important process end-
points in this study. A short reaction time reduces overall process
cost, decreases substrate inventory, and reduces the requirement
for energy. The time of reaction is dependent on kinetic factors such
as, enzyme specific activity, amount of biocatalyst used, concen-
trations of co-substrates, reaction temperature, choice of organic
solvent, and the degree of stirring, shaking or sonication that affects
mass transfer limitations also affects the reaction rate [38,39].
2.9. Enzyme selectivity for alcohols
Caproic acid was reacted with 13 common alcohols in addition
to (Z)-3-hexen-1-ol; ethanol, propanol, 2-propanol, butanol, tert-
butanol, pentanol, isopentanol, hexanol, tert-hexanol, heptanol, 3-
heptanol, octanol, and geraniol. The reactions were performed with
hexane as solvent as described above.
2.10. Lipase stability and reuse
Enzyme stability was determined by incubation with various
organic solvents for 24 h followed by drying under nitrogen. The
recovered powders were re-suspended with a known volume of
distilled water, centrifuged and assayed for remaining lipase activ-
ity (see below). To examine the effect of co-substrates on enzyme
stability, lipase residual activity was determined following 48 h
ester synthesis reaction. Enzyme stability was also evaluated in
terms of the degree of re-use. After a 48 h ester synthesis episode,

62
M. Liaquat / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 59–65
Table 1
A
100
80
60
40
20
0
Effect of solvent choice on the yield of (Z)-3-hexen-1-yl caproate.
Organic solvent
Log P
Ester yield (%)
1,4-Dioxane
Acetonitrile
Tetrahydrofuran
Diethyl ether
Toluene
−1.1
−0.33
0.49
0.85
2.5
0.1 1.7
1.0 0.0
2.0 0.0
7.9 0.0
36.6 0.7
48.3%a
Hexane
Heptane
3.5
4.0
86.5 0.6
a
See text for details.
(Z)-3-hexen-1-yl caproate
10
3.1. Solvent factors
0.1
1
100
Added water (%)
3.1.1. Effect of solvent on ester synthesis
The effect of different organic solvents, with log P values rang-
ing from −1.1 to 4.0, on (Z)-3-hexen-1-yl caproate yield is shown in
Table 1; P is the partition coefficient for solvent molecules between
water and octanol [37,38]. Clearly, (Z)-3-hexen-1-yl caproate yield
was high for non-polar solvents with log P > 2.5. A yield of 86%
was obtained in n-heptane followed by hexane (48.3%) after 48 h
at 40 ◦C. In agreement with earlier reports [39,40] hydrophilic
solvents (log P < 2.0) were associated with a low yield of ester.
Hydrophilic solvents such as dioxane (log P = −1.1), acetonitrile
(log P = −0.33), and tetrahydrofuran (log P = 0.48) remove a layer of
adsorbed water from enzyme particles leading to inactivation [41].
By contrast, non-polar solvents such as hexane (log P = 3.5), and n-
heptane (log P = 4.0) preserve the micro aqueous layer surrounding
biocatalyst particles and so help to retain enzymatic activity [41,42]
leading to high product yields.
100
B
90
80
70
60
50
40
30
20
10
0
(Z)-3-hexen-1- yl caproate
0
0.2
0.4
0.6
0.8
1
Water activity (a )
w
100
C
90
80
70
60
50
40
30
20
10
0
3.1.2. Effect of added water and water activity (aw)
A minimum amount of water may be essential to produce a
degree of enzyme flexibility necessary for catalysis [56]. The yield of
(Z)-3-hexen-1-yl caproate (94–97%) was not affected by the addi-
tion of water to the organic solvent phase over the range 0.125–30%
(w/w) (biocatalyst) (Fig. 4A). Analysis of lipase powders used in
this study showed that these contained 9–9.5% (w/w) water mea-
sured by oven drying to a constant weight at 105 ◦C overnight. The
total amount of water present in the current reaction system was
0.48–1.95% (v/v) or 0.27–1.08 M expressed with respect to the net
solvent volume.
(Z)-3-hexen-1-yl caproate
0
0.025
0.05
0.075
0.1
0.125
Compared to this, P38 lipase activity was optimum at an organic
phase water concentration of 0.25% (v/v) to catalyze the synthe-
sis of butyl caprylate. At a higher or lower water concentration
the yield of ester decreased [54]. The optimum amount of water
required for organic phase biocatalysis may depend on factors such
as the type of organic phase and choice of enzyme [37,56]. Com-
pared to the nominal moisture content of 0.48% (w/v) used for
the present baseline studies, previous investigators found 0.1–0.6%
(v/v) of added water to be compatible with ester synthesis [43,44].
However, >0.6% (v/v) added water encouraged ester hydrolysis and
decreased yield. Inappropriately high moisture levels also reduce
the efficiency of OPB due to increased diffusion limitations, inter-
facial inactivation of enzymes and decreased enzyme stability. In
this study, adding 20% (w/w) of water led to agglomeration of lipase
powder though this did not seem to affect ester synthesis adversely.
As well as the initial moisture content, aw also rises during OPB due
to water produced during esterification [38].
In further investigations of water-relations, all reaction com-
ponents were pre-equilibrated to known aw values using
standardized saturated salt solution. Under such circumstances
ester synthesis was maximum at aw = 0.45 declining slightly at
aw > 0.53 (Fig. 4B). Overall, rapeseed acetone powder lipase prepa-
rations showed little sensitivity to aw. Data in Fig. 4B suggests that
rape seedling lipase belongs to a group of lipase that functions
Molecular sieves (%w/v)
Fig. 4. Moisture–activity relations for ester synthesis: effect of added water (A),
water activity aw (B) or addition of molecular sieves (C) on the synthesis of (Z)-3-
hexen-1-yl caproate. The reaction mixture consisted of 0.25 M of butyric or caproic
acid with 0.25 M butanol or (Z)-3-hexen-1-ol in 5 mL of hexane at 40 ◦C in the
presence of rape seedling acetone powder (50 g/L) for 48 h.
better at a medium aw. Most lipases fall into to second category
[45]. Lipases from different sources vary widely in their depen-
dence on aw in non-aqueous solvents [43,44]. Low, medium and
high water activity dependent lipases work best at aw 0.19, 0.60 and
0.75 respectively. Wheat germ belongs to last type with optimum
water activity of 0.90.
Moisture effect was also examined by adding 0–0.125% (w/v) of
desiccant (molecular sieves 3A) directly to the reaction system. At
low levels of addition (<0.0125%, w/v), the yield of (Z)-3-hexen-1-yl
caproate ranged from 74 to 84%. Further addition of molecu-
lar sieves (>0.0125%, w/v) decreased product yield by about 20%
(Fig. 4C). The effect of added desiccants is two fold. At low levels of
addition, molecular sieves increase product yield by sequestering
water thereby shifting KEQ (reaction equilibrium) towards increase
synthesis. However, high amounts of molecular sieves reduce the
yield of ester by absorption. Torres and Otero [46] and also Omar

M. Liaquat / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 59–65
63
100
90
80
70
60
50
40
30
20
10
0
(Z)-3-hexen-1-yl caproate
0
10
20
30
40
50
60
70
80
90
Temperature ( oC)
Fig. 6. Effect of temperature on the synthesis of (Z)-3-473 hexen-1-yl caproate.
The reaction mixture consisted on 0.25 M of caproic acids and (Z)-3-hexen-1-ol in
5 mL of hexane at 40 ◦C in the presence of 50 g/L of acetone powder from day 4 of
germinating rape seedling.
Fig. 5. Effect of enzyme amount on the yield of (Z)-3-hexen-1-yl caproate. The reac-
tion mixture consists of 0.25 M of (Z)-3-hexen-1-ol and 0.25 M caproic acid in 5 mL
of hexane containing different amounts of rape seedling acetone powder at 40 ◦C
for 48 h.
of P38 lipase samples after heating at 10–60 ◦C for 48 h was also
determined [54]. Lipase dissolved in water was found to be least
stable. In comparison, the dry P38 lipase was stable until 40 ◦C but
became increasingly inactivated at higher temperatures.
et al. [42] reported a reduction in the yield of esters in the pres-
ence of desiccant due to the absorption of co-substrates particularly
acids. Controlling the amount of desiccant employed is necessary
to achieve a balance between the opposing effects of the molecular
sieves.
The effect of different organic solvents on the stability of
rapeseed acetone powder lipase is likely to be different from obser-
vations using purified proteins suspended in pure organic solvents
[38–42]. In such idealized systems, biocatalyst stability is generally
higher for high log P solvents accounting for the high product yield
normally associated with non-polar solvents. Crude acetone pow-
der preparations also contain considerable amounts of starch which
is likely to affect the outcome of these studies. Finally, regardless of
the destabilizing effect of co-substrates, rapeseed acetone powder
lipase could be reused. The yield of (Z)-3-hexen-1-yl caproate was
87%, 62% and 48% for three successive uses.
3.2. Biocatalyst factors
3.2.1. Effect of enzyme concentration
The amount of lipase is a crucial economic factor for any biocon-
version process. The yield of (Z)-3-hexen-1-yl caproate increased
with increasing amount of biocatalyst (Fig. 5). However, no signif-
icant increase in yield was observed with lipase loadings of more
than 50–60 g/L (Fig. 5). At low additions of biocatalyst, ester syn-
thesis did not reach equilibrium within the 48 h reaction time. The
concentrations of lipase reported are often too high to meet indus-
trial requirements [57]. For P38 lipase, a concentration of 20 g/L has
been reported [54].
3.2.3. Effect of reaction temperature
The temperature dependence of ester synthesis is shown in
Fig. 6. (Z)-3-hexen-1-yl caproate formed over a broad tempera-
ture range of 5–50 ◦C, with the maximum yield at 25–50 ◦C. A
low temperature sensitivity is observed in this study which sug-
gests that (Z)-3-hexen-1-yl caproate could be produced at ambient
temperatures (25–30 ◦C), without stringent temperature control;
this feature can be expected to lead to considerable savings in
energy cost for ester production. Compared to the present results,
the optimum temperature for ester synthesis using papaya (Car-
ica papaya) lipase was 63 ◦C [30]. Temperature optimum for butyl
caprylate synthesis, using Pseudomonas P38 lipase was 20 ◦C [54].
The decrease in ester synthesis above 20 ◦C was associated with
lipase inactivation at higher temperature. In general, thermosta-
bility within an organic solvent is achieved if the enzyme is
intrinsically rigid, or if the environment (e.g. low water activity)
prohibits enzyme flexibility. Enzymes that are stable/rigid within
organic solvent phases are also likely to exhibit low specific activ-
ity at low temperatures. On the other hand, high specific activity
within solvents may be associated with a highly flexible and rela-
tively heat-labile enzymes [55].
3.2.2. Rape seedlings lipase stability in organic solvents
The stability of rapeseed lipase acetone powder was influenced
by exposure to solvent choice (Table 2). No significant relation
was observed between log P values and activity retention. When
suspended with pure solvents, high residual lipase activities were
observed with THF (log P = 0.47), diethyl ether (log P = 0.85), hexane
(log P = 3.5) and heptane (log P = 4.0) but toluene (log P = 2.5) was
deactivating. It has been reported previously that log P values is
not always a good parameter to predict the toxic effects of solvent
in enzyme reactions at high temperature [47,48]. Residual activity
Table 2
Effect of organic solvent on rapeseed lipase stability.
Solvent
Log P
Residual activity (%)a
0.00
1,4-Dioxane
Acetonitrile
THF
Diethyl ether
Toluene
Pentane
Hexane
Heptane
−1.1
−0.3
0.49
0.85
2.5
3.0
3.5
4.0
5
63 0.00
89 0.05
85 0.08
40 0.02
77 0.00
72 0.06
81 0.04
3.3. Substrate factors
3.3.1. Effect of alcohol structure
The effect of alcohol structure on ester synthesis was examined
by considering chain length, position of the hydroxyl groups, degree
of branching, presence of unsaturated bonds and geometric iso-
merism (Table 3). Rapeseed lipase showed selectivity for C3:0–C6:0
Untreated control has 100% activity. THF, tetrahydrofuran.
a
Rape seedling acetone powder (50 g/L) was exposed to different solvents for
24 h at 25 ◦C, dried under nitrogen gas and residual activity was determined by
fluorimetric assay.

64
M. Liaquat / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 59–65
Table 3
ity at high alcohol concentrations might be due to its dehydrating
effects to rape seed lipase stability.
Effect of alcohol structure on the synthesis of caproic acid esters catalyzed by rape-
seed lipase.
Alcohols have been shown to inhibit lipases from Bacillus
licheniformis [26], M. miehei [40], Candida antarctica [51], Candida
cylinracea [52], and goat pregastric lipase [49]. The inactivating
effect of different alcohol declines with their log P value which
increases with chain length. Methanol is more inactivating com-
pared to long chain alcohols such as butanol and octanol. The
inactivating effects of acid co-substrates are related to their pKa
value which increases (acidity decreases) with chain length. Dur-
ing OPB using lipases, high concentrations of butyric acid were
tolerated [23,40,34,52] but not acetic acid [5,6,25].
Alcohol
Ester yield (%)
Methanol
1-Propanol
43
82
35
88
0
71
93
60
2
88
36
53
29
49
2-Propanol (isopropanol)
1-Butanol
2-Methyl-2-propanol (tert-butanol)
1-Pentanol (amyl alcohol)
3-Methyl-1-butanol (isoamyl alcohol)
Hexanol
2-Methyl-2-pentanol (tert-hexyl alcohol)
(Z)-3-hexen-1-ol
(E)-2-hexen-1-ol
1-Heptanol
3-Heptanol
1-Octanol
4. Conclusions
This study shows that (Z)-hexen-1-yl caproate can be produced
in quantitative yields using rapeseed lipase (50 g/L) suspended in
hexane or heptane as organic solvent under mild reaction condi-
tions (40 ◦C, 48 h). Ester synthesis is dependent on organic solvent
characteristics, biocatalyst factors and substrate factors. The cost
of biocatalyst remains an important consideration in OPB as puri-
fied enzymes are expensive. Crude seedling powder is potentially
inexpensive alternative form of biocatalyst for OPB. Procedures
for preparing acetone powder are simple, making it quite suit-
able for synthesis of green note flavor ester. Moreover, the impact
of the amount of enzyme is crucial and rape seedlings acetone
powder can be reused without stringent control of moisture at a
broad range of temperature (5–50 ◦C) in non-polar solvents. This
work illustrates the possibility of using acetone powder lipase from
germinated rapeseeds for low temperature biocatalysis. This is
particularly important because, commercially, use of ambient tem-
perature (25–30 ◦C) is economical. Low temperature OPB might in
future have applications in the preparation of heat-sensitive, high
value, products. The present data is highly encouraging suggest
that optimization studies employing pilot scale studies are war-
ranted. Finally, certain limitations of the current discussion should
be highlighted chief amongst which is the realization that rapeseed
acetone powder could conceivably contain more than one lipases
species. The substrate selectivity demonstrated here, applies to
caproic acid as the co-substrate which may change when different
acid substrates are considered.
Reaction was carried out at 40 ◦C for 48 h in 5 mL of hexane containing 0.25 M of
various alcohols and 0.25 M caproic acid, and 50 g/L of rape seeding lipase. Results
are average of two independent determinations with highest errors on mean was
less than 10%.
alcohols whilst the yield of ester decreased for C7:0–C8:0 alcohols.
Primary alcohols (1-propanol and 1-butanol) were more effec-
tively esterified compared with secondary or tertiary alcohols
(2-propanol, t-butanol or t-hexanol). Yields were higher for (Z)-
3-hexen-1-o1 compared to either hexan-1-ol or (E-) 3-hexen-1-ol.
The cis/trans selectivity may be due to shape differences between
cis- and trans-isomers. Previous studies show that lipases from
Mucor miehei, Aspergillus, Candida rugosa, and Rhizopus arrhizus [25]
and also goat pregastric lipase [49] have selectivity for C4:0–C6:0
alcohols. The purified lipase from oilseed rape (Brassica napus L. cv
Ceres) did not esterify secondary alcohols [50] and papaya lipase
was equally reactive with isoamyl alcohol and pentanol [30].
3.3.2. Effect of substrate concentration
The goal is to increase the concentration of reactants in hexane
without causing adverse effects on the reaction rate or the yield.
The concentration of acid or alcohol used in this study affected the
yield of (Z)-3-hexen-1-yl caproate (Fig. 7). When the concentration
of caproic acid or (Z)-3-hexen-1-ol was increased (0.06–1.00 M)
whilst the other co-substrate was fixed at 0.25 M, product yield
increased until the caproic acid and alcohol concentration reached
0.125 M. Further increasing the concentration resulted in a decrease
synthesis. The loss of effectiveness resulting from an increase of
substrate concentration may be due either to a substrate inhibition
or, more likely, to a modification of the polarity of the medium.
Effect was more pronounced for alcohol. Loss of synthetic activ-
Acknowledgements
I would like to thank the Pakistan Government for financial
sponsorship to complete the study. I am also grateful to Dr RKO
Apenten for his help, invaluable input and suggestions.
100
90
80
70
60
50
40
30
20
10
0
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