Investigation of steapsin lipase for kinetic resolution of secondary alcohols and synthesis of valuable acetates in non-aqueous reaction medium

Article abstract of DOI:10.1016/j.molcatb.2012.01.009

In present study, the application of steapsin lipase (as a biocatalyst) was investigated for kinetic resolution of secondary alcohols (1-phenyl ethanol and their derivatives) using vinyl acetate as an activated acyl donor. The enzymatic protocol was optimized for various reaction parameters such as effect of the molar ratio, solvent, temperature, time and biocatalyst loading to obtain best reaction conditions. On optimization, developed enzymatic methodology provided considerable enantiomeric excess of the product (up to 92% ee) at 55 °C in n-hexane as a solvent. Furthermore using the developed protocol, synthesis of several industrially important acetates was successfully achieved with excellent yield (up to 99%). During acetate synthesis, the biocatalyst was remarkably reused for eight consecutive recycles without any significant loss in its catalytic activity. This revealed the good potential of steapsin lipase for application in organic solvents.

Full text of DOI:10.1016/j.molcatb.2012.01.009

Journal of Molecular Catalysis B: Enzymatic 77 (2012) 15–23
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Investigation of steapsin lipase for kinetic resolution of secondary alcohols and
synthesis of valuable acetates in non-aqueous reaction medium
Kishor P. Dhakea, Krishna M. Deshmukha, Yogesh S. Wagha, Rekha S. Singhalb, Bhalchandra M. Bhanagea,∗
a Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
b Department of Food Engineering and Technology, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In present study, the application of steapsin lipase (as a biocatalyst) was investigated for kinetic resolu-
tion of secondary alcohols (1-phenyl ethanol and their derivatives) using vinyl acetate as an activated acyl
donor. The enzymatic protocol was optimized for various reaction parameters such as effect of the molar
ratio, solvent, temperature, time and biocatalyst loading to obtain best reaction conditions. On optimiza-
tion, developed enzymatic methodology provided considerable enantiomeric excess of the product (up to
92% ee) at 55 ◦C in n-hexane as a solvent. Furthermore using the developed protocol, synthesis of several
industrially important acetates was successfully achieved with excellent yield (up to 99%). During acetate
synthesis, the biocatalyst was remarkably reused for eight consecutive recycles without any significant
loss in its catalytic activity. This revealed the good potential of steapsin lipase for application in organic
solvents.
Received 20 September 2011
Received in revised form
18 December 2011
Accepted 6 January 2012
Available online 17 January 2012
Keywords:
Steapsin lipase
Kinetic resolution
Acetate ester synthesis
Transesterification
Biocatalysis
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
pancreatic lipase (PPL) and many more are reported for kinetic reso-
lution of racemic 1-phenyl ethanol in various non-aqueous reaction
Biocatalysis has gained a special attention for the synthesis
of several important biomolecules possessing a great application
in pharmaceuticals, foods, textiles, flavour and fragrance indus-
tries [1,2]. Enzymes being highly specific in nature with regard to
remarkable stereo-, regio- and chemo-selectivities, the enzymes
from oxido-reductase and hydrolase family have achieved a special
importance in the synthesis of chiral molecules [3,4]. Enantiomer-
ically pure alcohols are one of the most important building blocks
and chiral auxiliaries for the synthesis of bioactive drug molecules.
In this regard, lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3)
have received an outstanding credit for kinetic resolution of sec-
ondary alcohols and chiral synthesis of several esters and amides
mainly used in flavour and pharmaceutical preparations [1–4].
Among lipases, Candida antartica lipase B has been widely explored
for chiral resolution of alcohols and amines in organic solvents
as well in neoteric solvents like ionic liquids, supercritical flu-
ids, polyethylene glycol or their combinations [5–7]. Several other
lipases like Candida rugosa lipase, Pseudomonas sps. lipase, Mucor
miehei lipase, Amano PS, Amano AK, Bacillus subtilis lipase, porcine
medium [8–11]. However, use of enzymes like lipases has always
been a high cost issue and hence studies in recyclability of lipase for
several recycles would make the enzymatic processes economically
viable. Apart from lipase, esterase is also reported for kinetic reso-
lution of racemic glycerol ester derivatives via selective hydrolysis
[12].
On the other side, increasing demand of flavour and fragrance
compounds in modern society surges the development in chemical
and biocatalytic procedures rather than natural extraction method.
Since the compound of interest (ester) isolated using extraction
procedure is comparatively low and thus makes the process eco-
nomically unviable. Moreover, increasing health consciousness
encourages the development in enzymatic protocols as synthesis
of these esters via biocatalysis endows them a green label [13].
Among various esters, acetates find a special interest as they are
widely used in food and flavour industries. Recently, we reported
the novel immobilization support as a film prepared using blend
of hydroxypropyl methyl cellulose (HPMC) and polyvinyl alcohol
(PVA) for R. oryzae lipase and was studied for the synthesis of several
industrially important esters [14,15].
Steapsin lipase, a very well known digestive enzyme [16] has
not yet been well studied for kinetic resolution of secondary alco-
hols. In past decades, Francalanci and co-workers [17] reported
the kinetic resolution and chiral synthesis of amino alcohol via
∗
Corresponding author. Tel.: +91 22 3361 2601/2222; fax: +91 22 3361 1020.
E-mail addresses: bhalchandra bhanage@yahoo.com,
bm.bhanage@ictmumbai.edu.in (B.M. Bhanage).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2012.01.009

16
K.P. Dhake et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 15–23
OH
O
OAc
OH
R
OH
R
+
+
R
Steapsin
Lipase
+
R
Vinyl acetate
Vinyl acetate
R
O
Kinetic resolution
Acetate synthesis
(R)-3
(S)-1
Fig. 1. Steapsin lipase catalyzed kinetic resolution of sec-alcohol and acetate synthesis.
transesterification followed by alkaline hydrolysis using steapsin
lipase immobilized on celite. Growing awareness in lipase
catalyzed reactions and remarkable development in enzyme immo-
bilization procedures has made the steapsin lipase to be available
as small globules like preparation insoluble in non-polar sol-
vents (commercially made available by Sisco Research Laboratory).
Recently Kumar et al., immobilized steapsin lipase on celite 545 and
studied for ethyl ferulate synthesis using DMSO as an organic sol-
vent [18]. However, from the literature we acquainted that steapsin
lipase does not find a special attention for kinetic resolution appli-
cation, considering which we investigated the fact.
In continuation to our interest on exploration of lipase for
organic reactions [19,20], in present work we describe the first
effort for investigation of steapsin lipase for kinetic resolution of 1-
phenyl ethanol and their derivatives in organic solvent (Fig. 1). The
reaction was optimized to obtain best reaction conditions for the
resolution of secondary alcohols. Further, we employed steapsin
lipase for the synthesis of various industrially important acetates.
tube. After gentle stirring, 2 mmol of vinyl acetate was added and
to this 50 mg of steapsin lipase was added to initiate the reac-
tion. The reaction was placed at 55 1 ◦C in an orbital shaker at
160 rpm speed for a period of 24–48 h. All the experiments were
carried out at least in duplicate and the mean values are herein
reported. Progress of reaction was monitored by TLC/GC analysis
and was then quantitatively analyzed using gas chromatography
technique (Perkin-Elmer, Clarus 400) equipped with flame ioniza-
tion detector (FID) and chiral capillary column CP-Chirasil-Dex CB
Column (Varian capillary column, 25 m × 0.25 mm × 0.25 ␮m). The
column temperature was kept at 80 ◦C for 3 min and then raised
to 250 ◦C for 30 min with a rise of 1 ◦C/min. The temperature of
the injector and detector was maintained at 220 ◦C and 240 ◦C,
respectively. On completion of reaction, the reaction mixture was
filtered and biocatalyst was thoroughly washed 2–3 times with
n-hexane to remove any traces of reactant/product if remained
adhered. The reaction mixture was then evaporated under vacuum
at very low pressure. The oily residue obtained was then subjected
for column chromatography (silica gel, mesh size 60–120) using
pet ether:ethyl acetate (99:1) as eluent to afford pure products.
All the products are well known in literature [22] and were com-
pared with authentic samples and further characterized using FT-IR
2. Experimental
2.1. Enzymes and chemicals
(Perkin-Elmer, Spectrum 100), GC–MS (Shimadzu QP 2010) and 1
H
and 13C NMR spectra recorded on NMR spectrometer (Varian-300)
using TMS as internal standard. The sign of optical rotation was
compared with the results obtained for authentic samples prepared
using CaL B lipase and as well as from the values reported in litera-
ture. The % enantiomeric excess (ee%) was calculated as the ratio of
{[S] − [R]/[S] + [R]} × 100, where [S] and [R] are the concentration of
(S) and (R) enantiomers respectively. The (%) conversion was cal-
culated as [ees/(ees + eep)] × 100. The enantiomeric ratio (E) was
calculated as ln[(1 − c)(1 − ees)]/ln[(1 − c)(1 + ees)].
The steapsin lipase (immobilized form, readily disperse in
water/polar solvents, small globules with activity ≥20 U/mg)
was purchased from Sisco Research Laboratories, Mumbai, India
whereas Candida antartica lipase B immobilized on acrylic resin
(with ≥10,000 U/g, recombinant, and expressed in Aspergillus
oryzae) was purchased from Sigma–Aldrich, India. The (R)-menthol
(ee 99%) was purchased from Sigma–Aldrich, India. The various
substrates, chemicals and solvents used were purchased from firms
of repute with their highest purity available and were used without
any further treatment.
2.4. General procedure for steapsin lipase catalyzed acetate
synthesis
2.2. Synthesis of racemic phenyl ethanol derivatives
The substituted racemic phenyl ethanol derivatives were syn-
thesized from corresponding acetophenone derivatives namely
p-chloro acetophenone, p-methyl acetophenone using sodium
borohydride (NaBH4) as a reducing reagent with reported method
in literature [21]. The authentic standards of (S)-(−)-1-phenyl
ethanol and (R)-(+)-1-phenyl ethyl acetate were synthesized using
CaL B lipase. The enantiomeric compounds were then purified using
column chromatography and the purity was then examined by chi-
ral gas chromatography. The sign of optical rotation were recorded
using the digital polarimeter (JASCO, DIP-370) and were compared
with reported literature values. The similar protocol was followed
for preparation of authentic samples of other racemic secondary
alcohols.
The 1 mmol of primary alcohol was added to 3 mL of n-hexane
in 10 mL glass stoppered tube. On gentle stirring, 5 mmol of vinyl
acetate and 70 mg of steapsin lipase were subsequently added.
The reaction tubes were then placed at 55 1 ◦C in an orbital
shaker at 160 rpm speed for a period of 24–72 h. All the experi-
ments were carried out at least in duplicate and the mean values
are herein reported. The progress of reaction was monitored by
TLC/GC analysis and was quantitatively analyzed using gas chro-
matography technique (Perkin-Elmer, Clarus 400) equipped with
flame ionization detector (FID) and a capillary column (Elite-1,
30 m × 0.32 mm × 0.25 ␮m). The column temperature was kept
at 80 ◦C for 3 min and then raised to 250 ◦C for 30 min with a
rise of 10 ◦C/min. Temperature of the injector and detector was
maintained at 220 ◦C and 240 ◦C, respectively. After completion of
reaction, the reaction mixture was filtered and the biocatalyst was
thoroughly washed 2–3 times with n-hexane to remove traces of
reactant/product if remained adhered. The reaction mixture was
then evaporated under vacuum at very low pressure. The oily
residue obtained was then subjected for column chromatography
2.3. General procedure for steapsin lipase catalyzed kinetic
resolution of secondary alcohols
In typical experimental procedure, 0.5 mmol of secondary alco-
hol and 3 mL of n-hexane were added to 10 mL glass stoppered

K.P. Dhake et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 15–23
17
(silica gel, mesh size 60–120) using pet ether:ethyl acetate (99:1)
as eluent to afford pure products. All the products are well known
in literature [14,23] and were compared with authentic samples
and further characterized using FT-IR (Perkin-Elmer, Spectrum
100), GC–MS (Shimadzu QP 2010) and 1H and 13C NMR spectra
recorded on NMR spectrometer (Varian-300) using TMS as internal
standard.
be the best solvent for present reaction, whereas other solvents
like toluene and cyclohexane hampered the eep to a certain extent
(Fig. 2b). With regards to E value we found the optimum E value
for n-hexane while for other solvents it decreased from 66 to 20,
and hence n-hexane was the best suitable solvent for the present
system. The similar observation was made for kinetic resolution of
secondary alcohols with other sources of lipase by several research
groups [10,24].
2.5. Recyclability study of steapsin lipase
Enzymes being protein in nature are sensitive to higher tem-
perature and thus effect of temperature was deliberated. We found
that as temperature of the reaction was increased from 35 ◦C to
55 ◦C, eep also increased where the optimum eep was observed at
55 ◦C while further increase or decrease in temperature lowers the
eep (Fig. 2c). Also, we recognized that the E value was optimum for
55 ◦C. The observed results are in acceptance to the literature data,
where Xia et al. reported that increase in temperature increases the
conversion of the reaction while further increase in temperature
above a certain level decreases the eep of the reaction [10]. How-
ever, the optimum temperature for every different lipase catalyzed
reaction is variable and thus optimization of reaction parameters
is crucial to obtain the best result for the respective enzymatic
protocol.
The steapsin lipase was recycled for four consecutive recycles
for kinetic resolution of 1-phenyl ethanol whereas eight consec-
utive recycles were studied for the synthesis of isoamyl acetate
following the experimental procedure discussed in Sections 2.3
and 2.4 respectively. On completion of reaction, the reaction mix-
ture was filtered and the biocatalyst was thoroughly washed
2–3 times with n-hexane to remove traces of reactant/product
if remained adhered. The biocatalyst was dried at 42–45 ◦C for
10–12 h and then used for next recycle. All the experiments
were carried out at least in duplicate and the mean values are
reported.
2.6. Morphological analysis of the steapsin lipase biocatalyst
In order to obtain the optimum yield of desired (R)-ester, the bio-
catalyst loading was studied ranging from 30 mg to 90 mg (Fig. 2d).
It was found that 50–70 mg of lipase concentration was sufficient,
however 70 mg was considered for further study as the other sub-
stituted secondary alcohol would rather require higher biocatalyst
loading. The more interesting results were obtained with 90 mg of
biocatalyst loading where ees was increased than eep, this strat-
egy could be applied for one’s interest in isolation of either (S)
or (R) enantiomer. As the amount of biocatalyst loading increases
it tends to lose its selectivity for (R)-ester and hence the E value
declines. Thus, amount of biocatalyst loading is quite important
parameter to be considered during the optimization study. A con-
trol reaction in absence of enzyme was also performed, however we
observed no product formation thus elucidating that steapsin lipase
was essential to catalyze the reaction. Most of the time, the optimal
performance of biocatalyst depends on the water activity of enzyme
[26]. The solvents (i.e. n-hexane, toluene and cyclohexane) used for
the present study were of hydrous grade, thus contained trace % of
water which we believed was quite sufficient to maintain the essen-
tial water content for lipase activity. This assumption was further
confirmed by controlling the water content of the reaction medium
The photographic image of steapsin lipase (before use and
after eight recycle) versus black background was captured using
SONY Camera (12.1 megapixels, 4× zoom). To further elucidate
the detailed physical appearance of beads, the SEM analysis (FEI,
Quanta 200) of steapsin lipase before use and at the end of eight
recycle was studied. This study was performed so as to investigate
any breakage or major damage if occurred to the steapsin lipase
globules due to more number of recycles.
3. Results and discussion
3.1. Application of steapsin lipase for kinetic resolution
Lipase catalyzed kinetic resolution of 1-phenyl ethanol is the
most widely studied model substrate in literature [6–11], and thus
was used to investigate the catalytic behaviour of steapsin lipase.
The effect of various optimization parameters like molar ratio, sol-
vent, temperature, biocatalyst loading and time was studied. The
molar ratio has always played a vital role in transesterification reac-
tion and hence was firstly considered [14]. The different molar ratio
was studied ranging from 1:1 to 1:6 of alcohol to vinyl acetate.
During the study, we observed that for molar ratio of 1:4 (alco-
hol: vinyl acetate) the eep (enantiomeric excess of product (R)-3)
was optimum whereas further increase in molar ratio decreases
eep (Fig. 2a). Also we observed that as the molar ratio increases
from 1:1 to 1:4, the E value has also increased (from 13 to 66)
while further increase in the molar ratio led to noticeable decrease
in the E value (from 66 to 21) and hence molar ratio of 1:4 was
selected for further study. Similar results were obtained by de Souza
et al. for kinetic resolution of 1-phenyl ethanol using lipase Amano
PS, where increase in certain molar ratio increased the conversion
while further increased in molar ratio decreased the conversion
[24]. During the study, we observed that the eep was optimum
at 24 h of reaction time. The maximum E value was obtained at
this time period while at lower time no considerable E value was
observed, may be due to lower conversion (data not shown). From
our previous studies [14,20], we observed that use of polar sol-
vents has adverse effect on lipase activity as they have tendency to
strip out the essential water from the surface of catalytic site while
such was not the case with non-polar solvents. The non-polar sol-
vent like n-hexane, toluene and cyclohexane were screened for the
present study. The n-hexane (log P value = 3.5) [25] was found to
˚
via drying agent like molecular sieves. Molecular sieves (4 A) are
most widely reported for such experiments [27]. The study was per-
˚
formed by addition of the molecular sieves (4 A) ranging from 50 mg
to 200 mg to make the reaction system anhydrous (Fig. 2e). During
the study, we observed that as the concentration of the molecular
sieves increases (above 50 mg) there is marginal decrease in the eep
while the E value had drastically decreased (up to 27). This signi-
fies that as the medium is dehydrated the steapsin lipase loses its
selectivity, might be due to the removal of the essential water from
the active site of enzyme. The water molecules present near the
active site of enzyme are vital to maintain the three-dimensional
structure of the protein, while loss of this essential water may lead
to distortion in the protein conformation and thus finally loss in its
catalytic activity [27].
Furthermore to investigate the general applicability of devel-
oped methodology, the kinetic resolution of substituted racemic
1-phenyl ethanol derivatives was studied (Table 1, entries 1–3).
The (%) conversion was good for 1-phenyl ethanol (in 24 h) and
para-methyl-1-phenyl ethanol (in 36 h) whereas for para-chloro-
1-phenyl ethanol (in 48 h) the conversion has decreased with
marginal low enantioselectivity. The presence of halogen like chlo-
rine, an electron withdrawing group might have hindering effect
on lipase activity and thus have led to lower conversion in case of

18
K.P. Dhake et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 15–23
Fig. 2. Effect of various reaction parameters on steapsin lipase catalyzed kinetic resolution of secondary alcohol. Reaction conditions: rac-secondary alcohol (0.5 mmol),
agitation speed (160 rpm), time (24 h). Conversion (%) = [ees/(ees + eep)] × 100. ee (%) = [%(S) − %(R)]/[%(S) + % (R)] × 100. E = ln[(1 − c)(1 − ees)]/ln[(1 − c)(1 + ees)].
para-chloro-1-phenyl ethanol. The similar effect was observed by
Xia et al. for the wheat germ lipase catalyzed kinetic resolution of
para-chloro-1-phenyl ethanol where the conversion was too low,
however the selectivity reported was against the Kazlauskas route
[10]. Moreover, to make the biocatalytic protocol more economical
the recyclability of steapsin lipase was studied for four consecutive
recycles (Fig. 3). We observed that as recycle runs are increased the
enantiomeric excess of product eep has decreased although con-
version (%) remained the same. Also, the decline in E value for the
third and fourth recycle is observed.
3.2. Application of steapsin lipase for acetate synthesis
Apart from its kinetic resolution behaviour study, we employed
the steapsin lipase for the synthesis of industrially important
acetates using primary alcohols and vinyl acetate as an activated
acyl donor. In order to obtain the best optimized reaction con-
ditions, the transesterification reaction of isoamyl alcohol with
vinyl acetate was selected as a model reaction. During optimiza-
tion study, we observed that the optimized reaction conditions
were very similar to the protocol developed for kinetic resolution

K.P. Dhake et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 15–23
19
Table 1
Steapsin lipase catalyzed kinetic resolution of rac-secondary alcohols.a
Entry
Substrate rac-alcohol
Product (R)-3
Time (h)
24
Conv. (%)b
48 0.5
ees (%)c (S)-1
eep (%)c (R)-3
Ed
OH
OAc
1
85 1.1 (95 1.2)
92 1.4 (91 1.4)
66
OH
OAc
OAc
2
36
48
52 0.5
92 0.7 (92 0.8)
86 1.6 (95 1.5)
40
08
Me
Cl
Me
Cl
OH
3
26 0.3
25 0.9 (94 1.8)
71 1.5 (30 1.6)
a
Reaction conditions: rac-secondary alcohol (0.5 mmol), vinyl acetate (2 mmol), n-hexane (3 mL), steapsin lipase (70 mg), temperature (55 ◦C), agitation speed (160 rpm).
Conversion (%) = [ees/(ees + eep)] × 100.
b
c
ee (%) = [%(S) − %(R)]/[%(S) + %(R)] × 100. The GC yields are mentioned in parentheses.
E = ln[(1 − c)(1 − ees)]/ln[(1 − c)(1 + ees)].
d
of secondary alcohols (data not shown). The time course of acetate
n-butyl acetate (found in fruits like red delicious apple), n-octyl
acetate (fruity orange) were synthesized with remarkable yield
(99%) within 24–48 h (Table 2, entries 1–2). While the branched
aliphatic acetates such as isoamyl acetate (pear, banana flavour)
one of the most widely used flavour compound in food industries
and 2-ethyl-hexan-1-ol acetate were synthesized with excellent
yield within 24–30 h (Table 2, entries 3–4). The 1, 4 butane diol
was also subjected for the developed methodology and was found
to provide di-acetate as a product in 24 h (Table 2, entry 5). One
of the industrially valuable acetate i.e. citronellol acetate was syn-
thesized with 94% yield using the developed enzymatic protocol
(Table 2, entry 6). The substrates like 2-ethyl-hexan-1-ol and cit-
ronellol used in the present study were of racemic type and we
didn’t observe any significant chiral resolution of these compounds
during the study, whereas the chiral (R)-menthol (ee 99%) was used
for synthesis of its acetate derivative. The alicyclic alcohols like
cyclopentanol, cyclohexanol, (R)-menthol were also studied and
found to provide appreciable yield (57–77%) of desired product
(Table 2, entries 7–9). The R-menthol is a naturally occurring enan-
tiomercially pure compound, and its acetyl derivative finds a great
application in food and flavour industries. The kinetic resolution of
(R/S)-menthol was studied by Zhou et al., using Novozyme 435 in
synthesis was then studied for model reaction with defined inter-
val period (Fig. 4). It was observed that the yield of isoamyl acetate
increases as reaction time increased where maximum yield (99%)
was obtained within 24 h. In present reaction system, the forma-
tion of acetate mainly depends on the three types of reaction going
on i.e. firstly hydrolysis of vinyl acetate, then formation of desired
product (i.e. iso amyl acetate) and thirdly hydrolysis of the product
formed [28]. However, even on prolonged period (of 36 h), the yield
of desired product was not altered.
Further to explore the potential of steapsin lipase as biocatalyst,
the obtained optimized conditions were employed for transesteri-
fication of several aliphatic, alicyclic, allylic and aromatic alcohols
using vinyl acetate as an acyl donor (Table 2). Contentedly, steapsin
lipase was found to be a suitable biocatalyst for transesterification
reaction providing various valuable acetates [29,30] with remark-
able yields within 24–72 h. The straight chain aliphatic acetates like
Fig. 3. Recyclability study of steapsin lipase for kinetic resolution of 1-phenyl
ethanol. Reaction conditions: rac-secondary alcohol (0.5 mmol), vinyl acetate
(2 mmol), n-hexane (3 mL), steapsin lipase (70 mg), temperature (55 ◦C), agi-
tation speed (160 rpm), time (24 h). Conversion (%) = [ees/(ees + eep)] × 100. ee
(%) = [%(S) − %(R)]/[%(S) + %(R)] × 100. E = ln[(1 − c)(1 − ees)]/ln[(1 − c)(1 + ees)].
Fig. 4. Time course for lipase catalyzed isoamyl acetate synthesis. Reaction condi-
tions: isoamyl alcohol (1 mmol), vinyl acetate (5 mmol), n-hexane (3 mL), steapsin
lipase (70 mg), temperature (55 ◦C), agitation speed (160 rpm). Yields based on GC
analysis.

20
K.P. Dhake et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 15–23
Table 2
Steapsin lipase catalyzed acetate synthesis.a
Entry
1
Substrate (alcohol)
Product (acetate)
Time (h)
24
Yield (%)b
99 0.3
O
OH
O
O
OH
2
48
99 0.2
6
O
6
O
O
OH
3
4
24
30
99 0.3
99 0.4
OH
O
O
O
OH
O
5
6
O
24
24
99 0.2
94 1.1
HO
O
O
O
OH
O
OH
OH
7
8
48
48
74 1.7
O
O
77 1.4
O
OH
O
9
72
57 1.9
O
O
10
11
24
24
99 0.3
OH
O
O
99 0.3
HO
O
O
O
OH
O
O
12
24
99 0.4
O
13
14
24
24
99 0.3
O
OH
O
O
O
OH
O
99 0.5

K.P. Dhake et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 15–23
21
Table 2 (Continued)
Entry
Substrate (alcohol)
Product (acetate)
Time (h)
48
Yield (%)b
O
OH
O
15
16
99 0.3
MeO
MeO
O
OH
O
48
48
48
99 0.2
Cl
Cl
O
OH
O
17
18
99 0.3
OMe
OMe
O
OH
O
99 0.3
O
OH
O
O
19
20
48
99 0.6
NO2
NO2
O
OH
60
60
81 1.3
24 1.8
OH
O
21
O
a
Reaction conditions: alcohol (1 mmol), vinyl acetate (5 mmol), steapsin lipase (70 mg), temperature (55 ◦C), agitation speed (160 rpm).
Yields based on GC analysis.
b
ionic liquids, however 15% of conversion with 86 eep was obtained
application of enzymes. Hence, the recyclability of steapsin lipase
was studied for isoamyl acetate synthesis under the optimized
reaction conditions (Fig. 5). We observed that the biocatalyst was
capable to furnish appreciable yield of isoamyl acetate even at the
after 24 h [11]. The allyl alcohol also reacted well providing excel-
lent yield of 99% while industrially important cinnamyl acetate
was synthesized efficiently with appreciable yield in 24 h (Table 2,
entries 10–11). The 2-phenoxy derivatives like 2-phenoxy ethanol
and 2-phenoxy benzyl alcohol were compatible for the developed
protocol furnishing excellent yield (Table 2, entries 12–13). Ben-
zyl alcohol and its several derivatives like p-OMe, o-Cl, o-OMe,
o-Me, m-NO2 were investigated for the developed protocol and
found to furnished the corresponding substituted benzyl acetate
with excellent yields (Table 2, entries 14–19). The reaction of phe-
nol derivative like p-cresol was found to be sluggish as good yield of
the desired product was obtained after a long period of 60 h (Table 2,
entry 20), similar was the case with alicyclic alcohols where good
yields were obtained for (R)-menthol after 72 h. The 2-napthol was
not so compatible substrate as poor yield of desired product was
obtained even after a prolonged reaction time (Table 2, entry 21).
The lower yields obtain for p-cresol, (R)-menthol and 2-napthol
may be due to the steric hindrance effect.
The feasibility of biocatalytic methodology sounds superior only
when they are economical for application. In contribution to this,
recyclability of enzyme is one of the vital parameter for large scale
Fig. 5. Recyclability study of steapsin lipase for acetate synthesis. Reaction condi-
tions: isoamyl alcohol (1 mmol), vinyl acetate (5 mmol), n-hexane (3 mL), steapsin
lipase (70 mg), temperature (55 ◦C), agitation speed (160 rpm), time (24 h). Yields
based on GC analysis.

22
K.P. Dhake et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 15–23
Fig. 6. Images of steapsin lipase beads. Photographic image captured from SONY Camera (12.2 megapixel, 4× zoom) before use [a] and after eight recycle [d]. SEM images of
biocatalyst before use [b, c] and after eight recycle [e, f].
end of eight consecutive recycles without any significant loss in its
catalytic activity demonstrating that the biocatalyst was catalyti-
cally and thermally stable.
alcohols (up to 92% ee). In addition, several valuable acetates were
successfully synthesized with good to excellent yield (up to 99%)
using the developed methodology. The recyclability of steapsin
lipase was appreciable for eight consecutive cycles. The present
investigation hence appeals for exhaustive research with respect
to immobilization strategies, genetic engineering protocols to mod-
ify the catalytic properties of the steapsin lipase thus endowing it
as a robust biocatalyst with excellent stereo-selectivity and higher
catalytic activity.
3.3. Morphological study of steapsin lipase
To investigate the occurrence of possible distortion in the physi-
cal appearance of steapsin lipase, the photographic image and SEM
analysis of the biocatalyst before use and after eight recycle was
examined (Fig. 6). The SEM analysis of steapsin lipase revealed no
major structural changes in appearance as the surface of steapsin
lipase globules was observed to remain intact even after eight recy-
cle.
Acknowledgments
The financial assistance provided by UGC-SAP (University Grant
Commission, India) is gratefully acknowledged.
4. Conclusions
Appendix A. Supplementary data
In conclusion, for the first time steapsin lipase as a biocatalyst
was investigated for its kinetic resolution ability. The biocata-
lyst has satisfactorily resolved the racemic mixture of secondary
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2012.01.009.

K.P. Dhake et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 15–23
23
References
[16] R.J. Jandacek, J.A. Whiteside, B.N. Hokombe, R.A. Volpenhein, J.D. Taulbee, Am.
J. Clin. Nutr. 45 (1987) 940–945.
[17] F. Francalanci, P. Cesti, W. Cabri, D. Bianchi, T. Martinengo, M. Foa, J. Org. Chem.
52 (1987) 5079–5082.
[18] A. Kumar, S.S. Kanwar, Bioresour. Technol. 102 (2011) 2162–2167.
[19] K.P. Dhake, Z.S. Qureshi, R.S. Singhal, B.M. Bhanage, Tetrahedron Lett. 50 (2009)
2811–2814.
[20] K.P. Dhake, P.J. Tambade, R.S. Singhal, B.M. Bhanage, Tetrahedron Lett. 51 (2010)
4455–4458.
[21] M. Vallin, P.O. Syren, K. Hult, ChemBioChem 11 (2010) 411–416.
[22] M. Paivio, D. Mavrynsky, R. Leino, L.T. Kanerva, Eur. J. Org. Chem. (2011)
1452–1457.
[23] T. Mino, T. Hasegawa, Y. Shirae, M. Sakamoto, T. Fujita, J. Organomet. Chem.
692 (2007) 4389–4396.
[24] R.O.M.A. de Souza, O.A.C. Antunes, W. Kroutil, C.O. Kappe, J. Org. Chem. 74
(2009) 6157–6162.
[25] C. Laane, S. Boeren, K. Vos, C. Veeger, Biotechnol. Bioeng. (1987) 81–87.
[26] H. Hirata, K. Higuchi, T. Yamashina, J. Biotechnol. 14 (1990) 157–167.
[27] S. Gayot, X. Santarelli, D. Coulon, J. Biotechnol. 101 (2003) 29–36.
[28] V. Kasche, Enzyme Microb. Technol. 8 (1986) 4–16.
[29] P. Kraft, K.A.D. Swift, Perspectives in Flavor and Fragrance Research,
VHCA, Verlag Helvetica Chimica Acta AG/WILEY–VCH, GmbH & Co. KGaA,
Zurich/Weinheim, Germany, 2005.
[1] V. Gotor-Fernandez, F. Rebolledo, V. Gotor, in: R.N. Patel (Ed.), Biocatalysis in the
Pharmaceutical and Biotechnology Industry, CRC Press, Boca Raton, FL, 2007,
pp. 203–248.
[2] J.M. Woodley, Trends Biotechnol. 26 (2008) 321–327.
[3] F. Hasan, A. Alishah, A. Hameed, Enzyme Microb. Technol. 39 (2006) 235–251.
[4] D. Acetti, E. Brenna, C. Fuganti, F.G. Gatti, S. Serra, Eur. J. Org. Chem. (2010)
142–151.
[5] P. Lozano, Green Chem. 12 (2010) 555–569.
[6] P. Lozano, T.D. Diego, C. Mira, K. Montague, M. Vaultier, J. Iborra, Green Chem.
11 (2009) 538–542.
[7] A. Paiva, P. Vidinha, M. Angelova, S. Rebocho, S. Barreiros, G. Brunner, J. Super-
crit. Fluid 55 (2011) 963–970.
[8] M. Singh, R.S. Singh, U.C. Banerjee, Process Biochem. 45 (2010) 25–29.
[9] P. Hara, J.P. Mikkola, D.Y. Murzin, L.T. Kanerva, J. Mol. Catal. B: Enzym. 67 (2010)
129–134.
[10] X. Xia, Y.H. Wang, B. Yang, X. Wang, Biotechnol. Lett. 31 (2009) 83–87.
[11] H. Zhou, J. Chen, L. Ye, H. Lin, Y. Yuan, Bioresour. Technol. 102 (2011) 5562–5566.
[12] S. Hamaguchi, T. Ohashi, K. Watanabe. Eur. Pat. Appl. EP 189878 A2 (1986).
[13] V. Athawale, N. Manrekar, M. Athawale, Biotechnol. Prog. 19 (2003) 298–302.
[14] K.P. Dhake, P.J. Tambade, Z.S. Qureshi, R.S. Singhal, B.M. Bhanage, ACS Catal. 1
(2011) 316–322.
[30] D.J. Rowe, Chemistry and Technology of Flavors and Fragrances, first ed., Black-
well Publishing/CRC Press, Boca Raton, FL, USA, 2005.
[15] K.P. Dhake, K.M. Deshmukh, Y.P. Patil, R.S. Singhal, B.M. Bhanage, J. Biotechnol.
156 (2011) 46–51.