Low-temperature enzymatic hydrolysis resolution in mini-emulsion media

Article abstract of DOI:10.1515/chempap-2015-0032

A low-temperature mini-emulsion medium for the enzymatic resolution of 1-phenylethanol is described for the first time. The enzymatic hydrolysis resolution of 1-phenylethyl esters with different chain-lengths in the presence of Candida antarctica lipase B in mini-emulsion media was shown to be significantly controlled by temperature. In this system, the direct effect of temperature on the mini-emulsion size was observed. For the longer 1-phenylethyl ester, 1-phenylethyl dodecanoate, the enzymatic resolution was promoted exclusively at low temperatures. The preparative mini-emulsion enzymatic reaction of 1-phenylethyl dodecanoate at 4°C afforded the isolation of (R)-phenylethanol with a yield of 36 % with an ee of 99 %. (S)-Phenylethanol was isolated with a 51 % yield with an ee of 79 %.

Full text of DOI:10.1515/chempap-2015-0032

Chemical Papers 69 (6) 810–816 (2015)
DOI: 10.1515/chempap-2015-0032
ORIGINAL PAPER
Low-temperature enzymatic hydrolysis resolution
in mini-emulsion media
a
a
aNuno M. T. Louren¸co*, Sara C. Matias, Margarida C. Altas, a,bLuis P. Fonseca
aIBB – Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, bDepartment of
Bioengineering, Instituto Superior Técnico, Av. Rovisco Pais, 1, 1049-001 Lisboa, Portugal
Received 9 September 2014; Revised 31 October 2014; Accepted 12 November 2014
A low-temperature mini-emulsion medium for the enzymatic resolution of 1-phenylethanol is
described for the first time. The enzymatic hydrolysis resolution of 1-phenylethyl esters with different
chain-lengths in the presence of Candida antarctica lipase B in mini-emulsion media was shown to
be significantly controlled by temperature. In this system, the direct effect of temperature on the
mini-emulsion size was observed. For the longer 1-phenylethyl ester, 1-phenylethyl dodecanoate, the
enzymatic resolution was promoted exclusively at low temperatures. The preparative mini-emulsion
enzymatic reaction of 1-phenylethyl dodecanoate at 4◦C afforded the isolation of (R)-phenylethanol
with a yield of 36 % with an ee of 99 %. (S)-Phenylethanol was isolated with a 51 % yield with an
ee of 79 %.
c
ꢀ 2014 Institute of Chemistry, Slovak Academy of Sciences
Keywords: mini-emulsion, enzymatic kinetic resolution, secondary alcohols, low temperature
Introduction
vent have been reported for different organic reactions.
In line with the use of water as a reaction medium,
mini-emulsion systems have recently been exploited.
Mini-emulsions are characterised by a two-phase sys-
tem in which stable nanodroplets, between 20 nm and
500 nm in size, of an organic phase are dispersed in
a second continuous aqueous phase (Landfester, 2006;
Solans et al., 2005). These nanodroplets are stabilised
against coalescence by the addition of appropriate sur-
factants, which provide either electrostatic or steric
stabilisation (Landfester, 2006). These features render
mini-emulsions attractive media for different chemi-
cal reactions, e.g. dehydration reactions (Manabe et
al., 2001; de Barros et al., 2010; Aschenbrenner et
al., 2009). Kobayashi and co-workers showed that di-
rect esterification in water in a mini-emulsion medium
was possible using p-dodecylbenzenesulphonic acid as
a surfactant-type Brønsted acid (Manabe et al., 2001).
With reference to enantioselective enzymatic reactions
in mini-emulsion systems, to the best of our knowl-
edge, only one approach has been developed to date.
Landfester and co-workers described the enzymatic
The enzymatic kinetic resolution (EKR) of sec-
ondary alcohols via transesterification or hydrolysis
is a technique used extensively in the preparation of
enantiomerically pure secondary alcohols (Yun et al.,
2003; Louren¸co & Afonso, 2007; de Souza et al., 2009).
Since lipases are active in a non-aqueous medium,
attention has been focused on transesterification as
an alternative to hydrolysis (Zaks & Klibanov, 1988).
This is clearly associated with the insolubility of most
organic substrates in aqueous media. The use of or-
ganic solvents represents a major advantage for ho-
mogenisation of the reaction mixture and consequent
outcomes. Today, the need for more sustainable and
“green” synthetic pathways is not compatible with
the use of large amounts of organic solvents (Dunn,
2012). As a consequence, reactions in water have at-
tracted the attention of different researchers due to
water being a cheap safe, and environmentally benign
solvent (Lindstro¨m, 2007; Simon & Li, 2012). A num-
ber of examples relating to the use of water as a sol-
*Corresponding author, e-mail: nmtl@tecnico.ulisboa.pt
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pase from Candida rugosa (powder, hydrolytic activ-
ity of 0.99 U mg−1) and porcine liver esterase (pow-
der, hydrolytic activity of 24.2 U mg−1) were pur-
chased from Sigma. 1-Phenylethyl dodecanoate was
purchased from Solchemar (Portugal). 1-Phenylethyl
acetate (I ) and 1-phenylethanol were purchased from
Sigma–Aldrich and Fluka, respectively. All the other
reagents were obtained commercially and used as re-
ceived, unless stated otherwise. The mini-emulsions
were prepared using a SONOPLUS HD 3200 series
(Bandelin electronic, Germany). The mini-emulsion
average droplet sizes were analysed using a Zeta-
sizer Nano ZS (Malvern Instrument Co., UK). 1H
(400 MHz) and 13C (100 MHz) NMR spectra were
recorded on a Bruker AMX 400 spectrometer us-
ing tetramethylsilane as a reference. Gas liquid chro-
matography (GLC) was carried out on a Shimadzu
GC-2010 plus instrument (chiral capillary column,
CP-Chirasil-DexCB, 25 m × 0.25 mm × 0.25 m;
He as carrier gas; 250◦C injector; 250◦C detector;
linear velocity: 28.1 cm s−1; flow through column:
0.92 mL min−1; split ratio: 100; oven programme A:
20 min at 120◦C; ramp: 10◦C min−1 up to 180◦C,
5 min; retention time (tR in min): 1-phenylethyl ac-
etate: tR (S) = 7.00; tR (R) = 7.83; 1-phenylethanol:
tR (R) = 10.13; tR (S) = 11.06; 1-phenylethyl bu-
tyrate: tR (S) = 15.42; tR (R) = 16.10; oven program
B: 50 min at 120◦C; ramp: 1◦C min−1 up to 180◦C, 25
min; 1-phenylethyl hexanoate: tR (S) = 46.31; tR (R)
= 48.32; 1-phenylethyl dodecanoate: tR (S) = 125.16;
tR (R) = 125.56.
Fig. 1. Mini-emulsion methodology for enzymatic resolution of
1-phenylethanol.
hydrolysis of amino esters under a mini-emulsion sys-
tem stabilised by the use of Lutensol AT50 as surfac-
tant (Gr¨oger et al., 2006). This method permitted the
preparation of α-amino acids, as well as β-amino acids
in high substrate concentrations from 500 to less than
800 g L−1 with enantiomeric excess (ee) higher than
99 %.
In ongoing efforts toward developing more sustain-
able enzymatic resolutions methodologies (Monteiro
et al., 2010; Lourenc¸o et al., 2007, 2010; Louren¸co &
Afonso, 2007; Garcia et al., 2004), the attractive fea-
tures of mini-emulsions were exploited for the resolu-
tion of 1-phenylethanol by enzymatic hydrolysis catal-
ysed by Candida antarctica lipase B (CALB). The sur-
factant and the organic substrate in water form the
stable nanodroplets that are in contact with lipase,
which has the ability to operate at the oil-water inter-
face (Schmid & Verger, 1998) and selectively hydrol-
yses one of the enantiomers (Fig. 1).
Enzyme activity assay
The enzyme activity rates were investigated by
means of a spectrophotometric assay carried out on
a Hitachi U-2000 spectrophotometer. The substrate
p-NPB (20 L) was delivered from concentrated stock
solutions in pure acetonitrile (35 mM). Activity as-
says were performed in 0.1 M phosphate buffer of pH
7.5 (970 L) at 37◦C, initiated by the addition of en-
zyme (10 L). The release of p-nitrophenol was moni-
tored spectrophotometrically at 400 nm (ε400 = 18400
M−1 cm−1). The non-enzymatic hydrolysis of the sub-
strate was taken into account and subtracted from the
enzyme-catalysed hydrolysis. Enzyme activity was lin-
ear in respect of the amount of enzyme within the
range used in these assays. The assays were recorded
in duplicate. Please refer to the supplementary infor-
mation in electronic form.
Experimental
Materials and instrumentation
Candida antarctica lipase B (liquid formulation,
hydrolytic activity 0.24 U L−1) was donated by Novo
Nordisk Bioindustrial (Spain). A lyophilised form of
this lipase (solid, hydrolytic activity of 5.60 U mg−1
)
was obtained from the liquid formulation. Amano li-
pase AK from Pseudomonas fluorecens (powder, hy-
drolytic activity of 0.86 U mg−1), Amano lipase PS
from Burkholderia cepacia (powder, hydrolytic ac-
tivity of 0.62 U mg−1), Amano lipase AYS from
Candida rugosa (powder, hydrolytic activity of 0.36
U mg−1) were donated by Amano Enzyme (UK). Li-
Preparation of 1-phenylethyl butanoate (II)
and 1-phenylethyl hexanoate (III)
A mixture of 1-phenylethanol (26.46 mmol, 2.5 g),
Et2O (25 mL) and pyridine (24.55 mmol, 1.98 mL)
was stirred for 5 min followed by the addition of the
corresponding acid anhydride (24.55 mmol) (butyric
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anhydride or hexanoic anhydride for the preparation
of II or III, respectively) at ambient temperature and
subsequent heating under reflux for 6 h. After cooling
to ambient temperature, the reaction was quenched
by the addition of 1 M aqueous HCl (25 mL) and the
product was extracted using Et2O (2 × 25 mL). The
combined organic layers were washed with saturated
sodium bicarbonate solution (25 mL) and the aqueous
phase was extracted with Et2O (2 × 25 mL). The com-
bined organic layers were dried with Na2SO4, filtered
and the solvent was removed under reduced pressure.
The product was purified by flash chromatography on
a silica gel column using hexane/EtOAc (ϕr = 9 : 1) as
the eluent to afford II (68 % yield) or III (62 % yield),
emulsion was added at 4◦C and the reaction initiated
by adding the biocatalyst (CALB L) (120 U, 500 L).
The reaction mixture was stirred for 96 h maintain-
ing the pH at 8.2 by the addition of 0.1 M NaOH. A
white precipitate of dodecanoic acid was formed dur-
ing the reaction. The reaction mixture was frozen and
extracted using Et2O (3 × 30 mL), the organic lay-
ers were combined, dried with MgSO4, filtered and
the solvent was evaporated. (R)-1-Phenylethanol was
distilled from the reaction mixture at 60◦C and 20
Pa, yielding 363.2 mg (36 %) with an ee > 99 %. Af-
ter distillation, the reaction mixture containing (S)-
1-phenylethyl dodecanoate was chemically hydrolysed
with a 4 M non-aqueous solution of KOH in MeOH at
60◦C for 1 h. The reaction mixture was concentrated
under reduced pressure, Et2O (15 mL) was added and
the solvent was removed from the suspension by evap-
oration. Et2O (30 mL) was added to the suspension
and the mixture was stirred for 10 min and filtered.
The combined organic layers were washed with water
(2 × 30 mL), dried with MgSO4, filtered and the sol-
vents were evaporated to afford (S)-1-phenylethanol
(515.1 mg, 51 % yield, ee = 79 %).
1
respectively. For II: H NMR (CDCl3, 400 MHz), δ:
0.94 (3H, t, J = 7.4 Hz), 1.54 (3H, d, J = 6.6 Hz),
1.67 (2H, q, J = 7.4 Hz), 2.31 (2H, t, J = 6.6 Hz),
5.91 (1H, q, J = 6.6 Hz), 7.30 (5H, m); 13C NMR
(CDCl3, 100 MHz), δ: 13.77, 18.60, 22.41, 36.65, 72.13,
126.18, 127.91, 128.59, 142.00, 173.04; for III: 1H NMR
(CDCl3, 400 MHz), δ: 0.88 (3H, t, J = 6.8 Hz), 1.30
(4H, m), 1.53 (3H, d, J = 6.6 Hz), 1.63 (2H, m), 2.32
(2H, dt, J = 7.5 Hz, 0.8 Hz), 5.90 (1H, q, J = 6.6 Hz),
7.30 (5H, m); 13C NMR (CDCl3, 100 MHz), δ: 14.03,
22.40, 22.45, 24.79, 31.40, 34.73, 72.14, 126.19, 127.91,
128.60, 142.00, 173.24. These data are in accordance
with those already published (Gnanaprakasam et al.,
2010).
Results and discussion
As stated above, the use of mini-emulsion media for
enzymatic catalysis was motivated by the possibility
of circumventing the insolubility of most organic sub-
strates in aqueous media without the use of organic
solvents. One significant aspect of the mini-emulsion
formation is the surfactant selection. The addition of
an appropriate surfactant, which provides either elec-
trostatic or steric stabilisation to the droplets, is cru-
cial to stabilising mini-emulsions against coalescence.
The current work uses the well-known and benign an-
ionic soap, sodium dodecanoate. Taking as a start-
ing point the sodium dodecanoate critical micelle con-
centration (cmc) of 7.15–30 mM (Merta et al., 2000;
Akhter & Al-Alawi, 2000), the investigation started
by using 50 mM sodium dodecanoate at pH 8.5.
The studies began by screening the influence of 1-
phenylethyl esters chain-length on enantioselective hy-
drolysis catalysed by Candida antarctica lipase B at
25◦C in a mini-emulsion medium. Four substrates, 1-
phenylethyl acetate (I ), 1-phenylethyl butanoate (II ),
1-phenylethyl hexanoate (III ) and 1-phenylethyl do-
decanoate (IV ) were investigated (Fig. 2, Table 1).
A pronounced effect of the substrates’ chain-length
in the enzymatic reaction was noted. The increasing
chain-length led to a dramatic decrease in the conver-
sion from 48 % to 4 % after 6 h (acetate and dode-
canoate, respectively, entries 3 and 12, Table 1). With
regard to the enantioselective hydrolysis, the enan-
tiomeric excess follows the same trend and ee from
92 % to 5 % were obtained for (S)-enantiomer (en-
tries 3 and 12, Table 1).
General procedure for enzymatic hydrolysis
kinetic resolution of 1-phenylethyl esters in
mini-emulsion
A mixture of 1-phenylethyl ester (1.64 mmol),
50 mM aqueous solution of sodium dodecanoate (pH =
8.5, 2.5 g) and hexadecane (0.11 mmol, 32.0 L) was
stirred at ambient temperature for 1 h. Next, the mix-
ture was sonicated with an ultrasonic tip MS72 (2 min,
5 s on, 5 s off, 78 W, 52 % amplitude, three times) un-
der cooling (ice bath). The mini-emulsion was added
at different temperatures 4◦C, 25◦C and 40◦C and the
reaction initiated by adding the biocatalyst (CALB)
(24 U, 100 L), then the reaction mixture was stirred
for 48 h. Aliquots of 250 L were collected at differ-
ent times, acidified with 1 M HCl and extracted using
Et2O (3 × 2 mL). The combined organic layers were
dried with MgSO4, filtered and analysed by GLC.
Preparative enzymatic kinetic resolution of 1-
phenylethanol in mini-emulsion
A mixture of 1-phenylethyl dodecanoate (8.2 mmol,
2.5 g), 12.5 mM aqueous solution of sodium dode-
canoate (12.5 g, pH = 8.5) and hexadecane (0.554
mmol, 0.162 mL) was stirred at ambient temperature
for 1 h. Next, the mixture was sonicated with an ul-
trasonic tip (2 min, 5 s on, 5 s off, 98 W, 60 % ampli-
tude, four times) under cooling (ice bath). The mini-
In addition, the enzymatic hydrolysis of 1-phenyl-
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Table 1. Mini-emulsion enzymatic hydrolysis of 1-phenylethyl esters I–IV using CALB as biocatalyst
Entrya
Substrate
Time/h
Convb/%
ee (R)-Vc/%
ee (S)-I–IVc/%
1
2
3
4
5
6
7
8
9
I
I
I
II
II
II
III
III
III
IV
IV
IV
1
3
6
1
3
6
1
3
6
1
3
6
40
48
48
28
34
35
27
26
27
1
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
67
91
92
40
52
53
36
36
37
2
10
11
12
2
4
3
5
a) Mini-emulsion system was composed of 50 mM aqueous sodium dodecanoate solution (2.5 g, pH = 8.5), 1-phenylethyl ester (I–IV,
1.64 mmol), hexadecane (0.11 mmol, 32.4 L) and CALB (24 U, 100 L); b) Conv = conversion = ees/(ees + eep), determined by
GLC; c) determined by GLC; enantiomeric ratio (E) calculated according to Chen et al. (1982) was > 200 for all entries.
Table 2. Mini-emulsion enzymatic hydrolysis of I using different commercial biocatalysts
Entrya
Lipase
Conv/%
ee (R)-V/%
ee (S)-I/%
E
1
2
3
4
5
6
7
8
Candida antarctica lipase B
Porcine liver esterase
48
< 1
4
37
27
2
> 99
–
> 99
> 99
> 99
54
92
–
4
59
37
2
> 200
–
> 200
> 200
> 200
3.4
Amano lipase AK from Pseudomonas fluorecens
Amano lipase PS from Burkholderia cepacia
Lipase PS Amano SD from Burkholderia cepacia
Lipase from Candida rugosa
Amano lipase AYS from Candida rugosa
Candida antarctica lipase B (lyophilised)
2
48
73
> 99
1
92
6.5
> 200
a) For mini-emulsion system composition and determination of Conv, ee and E, see Table 1; hydrolysis time was 6 h for all entries.
Fig. 2. Mini-emulsion enzymatic hydrolysis of 1-phenylethyl esters I–IV. Reaction conditions: i) mini-emulsion, CALB, 25◦C, 1–6 h
(see Table 1); ii) mini-emulsion, different lipases, 25◦C, 6 h (see Table 2); iii) mini-emulsion, CALB, different temperature,
6 h (see Table 3); iv) mini-emulsion, CALB, 4◦C, different time (see Table 4).
ethyl acetate with other commercially available en-
zymes was performed. Of the seven enzymes screened
(see Experimental), CALB was shown to be the more
efficient biocatalyst (entries 1 and 8, Table 2). Either
the liquid formulation or the lyophilised enzyme af-
forded the enzymatic resolution with 48 % conversion,
> 99 % ee for (R)-enantiomer and 92 % ee for (S)-
enantiomer. Porcine liver esterase, Amano lipase AYS
and lipase from Candida rugosa afforded very low con-
versions (< 2 %) (entries 2, 3, 6, 7, Table 2). Amano
lipase PS and lipase PS Amano SD from Burkholde-
ria cepacia afforded moderate results with conversion
in the range of 27–37 % (entries 4 and 5, Table 2).
These results appear to indicate that Candida antarc-
tica lipase B, unlike others, is suitable for use in a
mini-emulsion system.
To assess the effect of temperature on the biotrans-
formation, the reaction was performed at three differ-
ent temperatures (40◦C, 25◦C and 4◦C) in the pres-
ence of CALB (Table 3). It was found that, for sub-
strates I–III, the reactivity was not very sensitive to
the temperature. Only minor changes were observed
at different temperatures. Surprisingly, the reactivity
was effectively sensitive to the temperature for IV and
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Table 3. Effect of temperature on mini-emulsion enzymatic hydrolysis of 1-phenylethyl esters I–IV using CALB as biocatalyst
Entrya
Substrate
T/◦C
Conv/%
ee (R)-V/%
ee (S)-I–IV/%
Mini-emulsion sizeb/nm
1
2
3
4
5
6
7
8
9
I
I
I
II
40
25
4
25
4
25
4
40
25
4
47
48
47
35
36
27
26
< 1
4
> 99
> 99
> 99
> 99
> 99
> 99
> 99
–
90
92
87
53
55
37
35
–
–
173
152
206
167
298
287
–
II
III
III
IV
IV
IV
> 99
> 99
5
38
359
302
10
29
a) For mini-emulsion system composition and determination of Conv, ee and E, see Table 1; E was > 200 for all entries (except for
entry 8); b) average diameter determined prior to addition of enzyme.
Table 4. Mini-emulsion enzymatic hydrolysis of IV using CALB as biocatalyst at 4◦C
Entrya
Surfactant/mM
Time/h
Conv/%
ee (R)-V/%
ee (S)-IV/%
E
1
2
3
4
50
50
12.5
12.5
24
48
24
48
37 (29)
40 (29)
41
> 99 (> 99)
> 99 (> 99)
> 99
58 (42)
66 (41)
71
> 200
> 200
> 200
> 200
44
> 99
78
a) Mini-emulsion system was composed of 50 mM or 12.5 mM aqueous sodium dodecanoate solution (2.5 g, pH = 8.5), 1-phenylethyl
ester IV (1.64 mmol), hexadecane (0.11 mmol, 32.4 L) and CALB (24 U, 100 L) with 0.1 M NaOH titration (values obtained
under the same conditions without 0.1 M NaOH titration are given in brackets); for determination of Conv, ee and E, see Table 1.
conversion increased to 29 % at 4◦C (from 4 % at 25◦C
and 1 % 40◦C) after 6 h (entries 10, 9 and 8, respec-
tively, Table 3). These results clearly show that the
reaction is driven by the decrease in temperature.
It is known that CALB comprises a small, five-
residues α-helix located close to the active centre that
has been identified as a candidate for anchoring the
lipase at the oil–water interface (Martinelle et al.,
1995). However, the ability to operate at the oil–water
interface is directly related to access to the substrate.
It is apparent that only low temperatures effectively
afford the access of 1-phenylethyl dodecanoate to the
lipase.
With the aim of understanding this effect, the
mini-emulsion size was evaluated and correlated to
the reaction extension. The first observation was that
mini-emulsion size increased with the use of longer-
chain 1-phenylethyl esters. Independently of the tem-
perature, for the same 1-phenylethyl esters concen-
tration, a two-fold increase in the mini-emulsion size
was noted from I to IV (Table 3). The second obser-
vation was that, by decreasing the temperature, the
size of the mini-emulsion was slightly reduced. For
the system composed of IV, the mini-emulsion size
reduction is crucial to allow the enzymatic reaction to
proceed. The results indicate that the reactivity can
be controlled by the size of the mini-emulsion and,
consequently, by the temperature. To the best of our
knowledge, this is the first time that a enzymatic re-
action has been recorded as proceeding exclusively at
low temperatures.
In addition to the reactivity observed at low tem-
perature for the mini-emulsion system containing IV,
the highest conversion obtained proved to be far from
ideal (entries 1 and 2, values in brackets, Table 4).
Longer reaction times, together with the 0.1 M NaOH
titration of the dodecanoic acid that is released during
the reaction, were essential for extending the conver-
sion up to 40 % after 48 h, with an ee > 99 % for
(R)-V (entry 2, Table 4).
Seeking more practical reaction conditions, the
amount of surfactant was reduced to 12.5 mM, which
is the value close to sodium laurate cmc (Akhter &
Alawi, 2000) thus reducing the high amounts of 0.1 M
NaOH needed to titrate the excess dodecanoic acid.
Under these conditions, 44 % of (R)-V was converted
with an ee > 99 % after 48 h (E > 200) (entry 4,
Table 4).
The excellent outcome of this reaction system
prompted an attempt to scale up the reaction. A 2.5
grams substrate scale reaction was performed (Fig. 3).
The conversion of (R)-V was 44 % with an ee
of 99 % and (S)-IV with an ee of 77 % after 96 h.
One of the main problems encountered in the use of
mini-emulsions on an industrial scale is separation of
the surfactant from the organic phase. Since mini-
emulsions are extremely stable systems, their desta-
bilisation is often a difficult process. Usually, alco-
hols, salts or acids are used to destabilise the mini-
emulsion system. However, the addition of additives
entails economic and environmental costs, as well as
contamination of the surfactants and/or organic sol-
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system was demonstrated for the first time. The
mini-emulsion temperature control and, consequently,
the mini-emulsion size were shown to be crucial for
the enzymatic resolution development. This prepar-
ative methodology afforded the isolation of both
1-phenylethanol enantiomers with good yields and
enantiomeric excess. This methodology is currently
being applied to other substrates, with the possibil-
ity of recycling the biocatalyst being addressed.
The proposed methodology can contribute to new
opportunities on matters of enzymatic resolution, us-
ing water as a solvent.
Acknowledgements. The authors wish to thank Fundac¸a˜o
para
a Cie¸ncia e Tecnologia (POCI 2010) and FEDER
(SFRH/BPD/41175/2007 and PTDC/QUI-QUI/119210/2010)
for the financial support received. In addition, the authors are
grateful to Novozymes for the generous enzyme gift.
Supplementary data
Fig. 3. Mini-emulsion reaction system for preparative enzy-
matic resolution of IV: (a) prior formation of mini-
emulsion, two distinct phases (upper – organic phase
and lower – aqueous phase); (b) after mini-emulsion for-
mation (single phase – mini-emulsion); (c) at the end
of reaction (two distinct phases: liquid phase – mini-
emulsion and solid phase – sodium dodecanoate).
Supplementary data associated with this article
can be found in the online version of this paper (DOI:
10.1515/chempap-2015-0032).
References
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optical purity (Fig. 4).
Conclusions
In conclusion, the enzymatic kinetic resolution of
1-phenylethanol at low temperature in a mini-emulsion
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