Enzymatic esterification in aqueous miniemulsions

Article abstract of DOI:10.1002/chem.200801691

Monoesters of various linear carboxylic acids (C7-C12) with ω-phenyl-labeled primary alcohols (C1-C5) were synthesized in aqueous miniemulsions with various lipases as biocatalysts. The reactants were dispersed in an aqueous solution of a nonionic surfact

Full text of DOI:10.1002/chem.200801691

DOI: 10.1002/chem.200801691
Enzymatic Esterification in Aqueous Miniemulsions
Eugen M. Aschenbrenner,[a, b] Clemens K. Weiss,*[a, b] and Katharina Landfester[a, b]
Abstract: Monoesters of various linear
carboxylic acids (C7–C12) with w-
phenyl-labeled primary alcohols (C1–
C5) were synthesized in aqueous mini-
emulsions with various lipases as bio-
catalysts. The reactants were dispersed
in an aqueous solution of a nonionic
surfactant to form long-term stable
miniemulsions. The esterification of all
of the systems could be catalyzed by
the applied enzyme and yielded signifi-
cant conversions of about 90%. The
hydrophilicity of the reactants and the
specificity of the enzyme toward the
substrates determine the reaction ve-
locity and the final conversion. As a
model system the reaction of nonanoic
acid and 3-phenylpropanol was exten-
sively studied. Among various lipases,
Lipase PS was determined to be the
most effective, and for this reaction the
parameters were optimized. A maxi-
mum conversion of 80% could be ob-
tained in less than one hour of reaction
time. In comparison with an acid-cata-
lyzed esterification performed in mini-
AHCTUNGTRENGNeUN mulsion with the same reaction pa-
rameters, the enzyme-catalyzed reac-
tion showed a significantly faster con-
version. The reactions proved that the
application of the miniemulsion tech-
nique enables efficient direct enzyme-
catalyzed esterification reactions from
carboxylic acids and alcohols in the
presence of large amounts of water.
Keywords: enzyme
catalysis
·
esterification · lipases · miniemul-
sion
Introduction
Still, considering economic and environmental issues, the
employment of water as a solvent for organic reactions
would be desirable. Water is nontoxic, cheap, and environ-
mentally benign. Additionally, the reactants do not have to
be dried before the reaction, thus drying agents, time, and
energy can be saved. These reasons, only briefly mentioned,
are reviewed extensively elsewhere.[10–12]
Despite the chemical obstacles, several groups have al-
ready shown that dehydrative esterification reactions, for
the synthesis of esters and polyesters, can be conducted in
water. It acts as a continuous phase of an emulsion contain-
ing hydrophobic droplets and not as a solvent.[13–15] Thus,
several requirements have to be met by the reactants: For
the formation of an emulsion without the addition of further
organic solvents, the starting materials have to be sufficient-
ly immiscible with water, and liquid at the temperature at
which the experiment is performed. For the stabilization of
the emulsion, a surfactant is required; for the reaction the
Dehydration reactions, for example, esterification reactions,
are of key interest to the synthesis of basic organic chemi-
cals as well as the production of polymers.[1–3] The water
generated during the reaction has to be removed from the
locus of the reaction to shift the equilibrium to the product
side. In the presence of water, the equilibrium is displaced
to the reactant side and hydrolysis of the desired products
takes place. Dehydrative esterification from a carboxylic
acid and an alcohol needs to be catalyzed to perform the re-
action in reasonable time under ambient conditions. Acid
catalyst systems or specialized enzymes can be employed for
these reactions. The generated water can be removed by
evaporation, by conducting the reaction in vacuum,[4–7] or by
adding water-binding agents, such as zeolites or salts.[8,9]
[a] E. M. Aschenbrenner, Dr. C. K. Weiss, Prof. Dr. K. Landfester
Max-Planck-Institute for Polymer Research
Ackermannweg 10, 55128 Mainz (Germany)
Fax : (+49)6131-379100
catalyst. As the catalyst, being
a Lewis or Brønsted
acid[11,16,17] or an enzyme,[18] is generally water-soluble, the
reaction takes place at the interface between the hydropho-
bic droplets and the aqueous phase.[19] Thus, it is convenient
to combine the stabilizing and catalyzing properties in one
molecule (“CATASURF”).
E-mail: weiss@mpip-mainz.mpg.de
[b] E. M. Aschenbrenner, Dr. C. K. Weiss, Prof. Dr. K. Landfester
Organic Chemistry III
(Macromolecular Chemistry and Organic Materials)
University of Ulm
Albert-Einstein-Allee11, 89081 Ulm (Germany)
Regarding enzymes, two types are capable of catalyzing
hydrolysis and esterification reactions: esterases and lipas-
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FULL PAPER
es.[20] Esterases usually act on water-soluble reactants,
whereas lipases are specialized toward hydrophobic sub-
strates, present as droplets in an aqueous phase. Thus, most
lipases exhibit their catalytic abilities at the interface be-
tween the hydrophobic substrates and the aqueous continu-
ous phase.[21–23] Because the catalysis takes place at the inter-
face of the emulsion droplets, a large interfacial area is de-
sirable for a fast conversion. Obviously, the interfacial area
has to be present during the whole course of the reaction,
thus requiring a highly stable emulsion. Therefore, dehydra-
tive esterification reactions catalyzed by an interfacially
active lipase benefit from a large interfacial area and long-
term stable emulsions.
By decreasing the droplet size from, for example, 5 mm in
a conventional emulsion to 500 nm, the interfacial area in-
creases by a factor of 100. Droplets in the submicron range
can be found in systems called “miniemulsions”. Direct (oil
in water) miniemulsions are generated by dispersing organic
liquids in water supported by the application of high-shear
forces as ultrasound.[24] The droplets are of a uniform size
and are stabilized against coagulation by the presence of an
ionic or nonionic surfactant. Diffusional degradation (Ost-
wald ripening) of the droplets can be suppressed by the ad-
dition of a costabilizing, extremely hydrophobic agent.[25]
Thus, the size and the composition of the droplets remain
constant, even when chemical reactions are performed in
the miniemulsion droplets.[25]
So far, only dehydrative polycondensations have been
conducted in a miniemulsion. Barrꢁre and Landfester inves-
tigated dodecylbenzenesulfonic acid (DBSA)-catalyzed
polycondensation reactions of several diols and diacids of
different hydrophobicity in miniemulsions.[26] The polymeri-
zation yield was found to be dependent on the hydrophobic-
ity of the monomers. With the most hydrophobic monomers,
yields of nearly 90% were achieved, despite the presence of
an aqueous continuous phase. Moreover, enzymatic ring-
opening polymerization of lactones, which can be described
as a series of transesterification reactions, conducted in a
miniemulsion was reported by Taden et al.[27] The authors
noted a significant decrease in the reaction time relative to
enzymatic synthesis in organic solvents from several
days[12,28–31] to 12 h and less. Herein, the principle of dehy-
drative polyesterification reactions is extended from poly-
mers to low-molecular-weight systems. Based on other earli-
er work,[17,26,27] we can assume that the mechanism for the
esterification in miniemulsions could run as outlined in
Figure 1. The reactants, and eventually the product, form a
miniemulsion droplet of about 500 nm stabilized by the sur-
factant. The enzyme located at the interface of the droplet
and the aqueous continuous phase catalyzes the esterifica-
tion. The water generated during the reaction is expelled
from the hydrophobic droplet and is transported to the
aqueous continuous phase of the miniemulsion (Figure 1).
To study the dehydrative esterification reaction in the
presence of water as a continuous medium, we chose linear
carboxylic acids of different chain lengths with 7–12 carbon
atoms and linear phenyl alkanoles with 1–5 carbon atoms in
Figure 1. Schematic diagram of an esterification in the miniemulsion.
the alkyl chain as alcohol substrates (Scheme 1). The influ-
ence of the different chain lengths on the reaction velocity
and the final conversion has been studied. The phenyl label
Scheme 1. General representation of the enzyme-catalyzed esterification
reaction.
offers the opportunity for easy detection with a UV detector
in an HPLC setup. All of the experiments were preformed
with enzymatic catalysis, thus ensuring high conversions
with short reaction times at low temperatures. Besides the
application of various substrates, the activities of several
nonimmobilized enzymes, the reaction conditions, the tem-
perature, and the pH value of the continuous phase were
evaluated. The hydrolysis of 3-phenylpropyl nonanoate in a
miniemulsion was performed to determine the position of
the equilibrium. The efficiency of the enzyme-catalyzed re-
action in a miniemulsion was shown by the comparison with
the same enzyme-catalyzed esterification in a macroemul-
sion and a homophase and an acid-catalyzed esterification
in a miniemulsion.
Results and Discussion
Carbon-chain length: For the formation of a miniemulsion,
two phases have to be sufficiently insoluble in each other.
To ensure that no phase separation occurs during the reac-
tion, hydrophobic linear carboxylic acids with seven and
more carbon atoms were selected for the reaction with 3-
phenylpropanol. Although the solubility of the reactants in
the aqueous phase is quite high (e.g., heptanoic acid: ca. 2ꢂ
10ꢀ3 molLꢀ1) effective and quick reactions with high conver-
sions were observed. For all of the investigated systems, the
pH was 3.8 before, during, and after the reaction. The mini-
emulsions used for the reactions comprised a 17% hydro-
phobic phase, stabilized by a nonionic surfactant. The non-
ionic surfactant Lutensol AT 50 was applied at 10 wt% with
respect to the dispersed, hydrophobic phase. Although
lipase-catalyzed reactions could be performed in the pres-
ence of an ionic surfactant, such as sodium di(2-ethylhexyl)
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C. K. Weiss et al.
sulfosuccinate (AOT) or hexadecyltrimethylammonium bro-
mide (CTAB),[32] it is known[33] that ionic surfactants can in-
terfere with enzyme activity. Some anionic surfactants, for
example, sodium dodecylsulfate (SDS), are even used to de-
nature and thus deactivate enzymes.[27] Polysorbates (e.g.,
Tween80) could not be used because they are esters them-
selves and thus prone to lipase-catalyzed hydrolysis.
the enzyme towards these substrates (see Figure 2b).
Enzyme activities are expressed in Umgꢀ1, with U (“unit”)
defined as conversion in mmolminꢀ1. The activity of Lipase
PS is highest for the reaction of nonanoic acid with 3-phe-
nylpropanol with about 2.7 Umgꢀ1, followed by decanoic
and dodecanoic acid with both little less than 2 Umgꢀ1. Sur-
prisingly, the lowest activity was exhibited in the reaction of
undecanoic acid with 3-phenylpropanol. As enzymes are
highly specialized catalysts, the results obtained from the ex-
periments summarized in Figure 2 can be explained so that
Lipase PS shows distinct substrate specificity for nonanoic
acid. Still, as observed in nonenzyme-catalyzed systems, the
solubility of the substrates in the aqueous continuous phase
(their hydrophilicity) might also influence the reaction rate
and final conversion[17,26,27] as a concentration gradient of
the reactants in the droplets might occur. The more hydro-
philic substrates will accumulate at the interface between
the droplet and aqueous phase, whereas the hydrophobic re-
actants will “retreat” to the center of the droplet. Moreover,
water can diffuse into the more hydrophilic regions of the
droplets, thus favoring hydrolysis reactions, and hydrophilic
substrates can diffuse into the continuous phase and are no
longer available for esterfication. As the generated ester is
the most hydrophobic component of the system, it will accu-
mulate more in the center of the droplet and will not inter-
fere with the enzyme action at the droplet interface.
Figure 2a shows the reaction profiles of the lipase-cata-
lyzed reaction of long-chain, linear carboxylic acids with 3-
phenylpropanol at 408C in a miniemulsion. All of the exam-
ined systems show a significant conversion of at least 60%
after 24 h. The reaction profile with the fastest conversion
(rate=0.32 minꢀ1 for nonanoic acid (C9) with 3-phenylpro-
panol) reached its maximum yield of 78% in 6 h. The pro-
files of the reactions of decanoic acid (C10) and dodecanoic
acid (C12) have slightly lower values (C10: 0.22; C12:
0.21 minꢀ1) and reached their maximum yield of 80% after
about 7–8 h. The maximum conversion (80%) of undecanoic
acid (C11) was reached after 47 h, whereas the reaction of
the two shortest chain acids, heptanoic (C7) and octanoic
acid (C8) yielded a maximum conversion of 70% after
60 hours. It is evident from the reaction profiles that the re-
action of nonanoic acid with 3-phenylpropanol is the fastest.
From the initial slopes (first 2 h) and the molar masses of
the acid substrates, it is possible to calculate the activities of
To evaluate the influence of substrate polarity, competi-
tive reactions were performed. With a given alcohol sub-
strate, two or three acids of different chain lengths were
combined in one miniemulsion droplet. The acid with the
shorter chain, which is more soluble in the aqueous phase, is
expected to accumulate at the interface between the organic
and aqueous phases. As the concentration of the longer car-
boxylic acid will be less at the interface, the reaction rate of
the more hydrophobic, longer chain acid is expected to be
less than 50% relative to the noncompetitive reactions.
Equal interfacial concentrations would result in 50% of the
conversion rate observed in the reaction with the individual
substrates. The acid substrates with the highest reaction rate
(i.e., nonanoic acid), the lowest reaction rate and simultane-
ously the most hydrophilic (i.e., heptanoic acid) and most
hydrophobic acid (i.e., dodecanoic acid) were chosen for the
competitive esterification reactions with 3-phenylpropanol.
Nonanoic acid and heptanoic acid, with the highest and
the lowest reaction rates in the single-acid experiments,
were combined and esterified with 3-phenylpropanol. The
conversions of the individual acids and the alcohol, which
represents the total conversion are shown in Figure 3. It is
clearly visible that the reaction rate of nonanoic acid with 3-
phenylpropanol is higher than the one of heptanoic acid
with 3-phenylpropanol (0.12 and 0.08 minꢀ1, respectively).
As expected, the conversion rates are lower than the values
of the reactions with individual substrates (C9: 0.32; C7:
0.10 minꢀ1). The conversion rate of the reaction with non-
anoic acid is only one third of the individual reaction, where-
as the reaction rate of heptanoic acid is only slightly lower
than without the addition of a second substrate to the reac-
Figure 2. a) Conversion versus time of the reaction of 3-phenylpropanol
with linear carboxylic acids with chain lengths of C7–C12 in the presence
of Lipase PS. The conversion data was obtained by HPLC analysis and
confirmed with NMR spectroscopic analysis. The conversion rates were
calculated from the initial slopes: C7: 0.10; C8: 0.15; C9: 0.32; C10: 0.22;
C11: 0.10; C12: 0.21 minꢀ1. The dotted lines are only a guide for the eye.
b) Enzyme activity of the reactions of 3-phenylpropanol with carboxylic
acid substrates with increasing carbon-chain length. The values are calcu-
lated from the initial slopes obtained from the profiles in graph (a).
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Enzyme-Catalyzed Esterification
FULL PAPER
is about 75%, which is slightly below the value observed for
the individual reaction.
Figure 5 shows the reaction profiles of the competitive
esterification of nonanoic and dodecanoic acid with 3-phe-
nylpropanol. The reaction profile for the esterification of
Figure 3. Conversion of acids and total conversion of both acids versus
time for the competitive enzymatic esterification of heptanoic and nona-
noic acid with 3-phenylpropanol (filled symbols), all catalyzed by Lipase
PS. Conversion rates from the initial slopes: C7ind: 0.10; C9ind: 0.32; C7:
0.08; C9: 0.12 minꢀ1. The dotted lines are only a guide for the eye.
tion mixture. This outcome indicates a higher concentration
of the more hydrophilic heptanoic acid at the oil/water inter-
face and thus displacement of the more hydrophobic nona-
noic acid to the core of the droplet. Nevertheless, nonanoic
acid is converted faster than heptanoic acid, thus indicating
a higher specificity of the applied enzyme for nonanoic acid.
The total reaction rate is lower, although the total conver-
sion still reaches 80% after 24 h.
The competitive esterification of dodecanoic acid and
heptanoic acid with 3-phenylpropanol (Figure 4) shows the
same trends as observed in the previous experiment. Both
conversion rates are lower than in the experiments with the
individually applied acid. Whereas the initial conversion
rate of the esterification of dodecanoic acid is only 34% of
the rate of the individual reaction, heptanoic acid is convert-
ed nearly as quickly (81%) as without the presence of do-
decanoic acid. The explanation for this behavior is the same
as for the previous experiment: the concentration of the
heptanoic acid seems to be higher at the interface than the
concentration of the dodecanoic acid. The total conversion
Figure 5. Conversion of acids and total conversion of both acids versus
time for the competitive enzymatic esterification of combined nonanoic
and dodecanoic acid with 3-phenylpropanol (filled symbols), all catalyzed
by Lipase PS. Conversion rates from the initial slopes: C9ind: 0.32; C12ind
:
0.21; C9: 0.19; C12: 0.11 minꢀ1). The dotted lines are only a guide for the
eye.
nonanoic acid with 3-phenylpropanol indicates a faster con-
version than the profile for dodecanoic acid (0.19 and
0.11 minꢀ1, respectively). Because the slopes of both curves
are approximately half of the value obtained from the reac-
tion profiles of the individual esterification reactions, com-
parable concentrations of both acids can be assumed at the
interface between the organic and the aqueous phase. This
finding is a strong indication for a preferred catalysis of the
esterification of nonanoic acid with 3-phenylpropanol. The
total conversion is about 75% after 24 h.
Finally, all of the previously used acids (i.e., heptanoic,
nonanoic, and dodecanoic acid) were combined in the drop-
let phase and esterified with 3-phenylpropanol (Figure 6).
As already observed in the previously described competitive
reactions, the conversion profile of the esterification of
nonanoic acid with 3-phenylpropanol shows the fastest con-
version. The curves of the reactions of dodecanoic and hep-
tanoic acid have almost the same shape. With three (hypo-
thetically equally active) acid substrates present, a conver-
sion rate of one third of the reactions of the individual acids
can be assumed. Indeed, the conversion rate of the reaction
of nonanoic acid shows 34% of the value of the reaction of
the individual acid. Regarding the other acids, deviations
from the hypothetical value can be found. Whereas the con-
version rate of heptanoic acid is 67% of the value for the in-
dividual reaction, the conversion rate for dodecanoic acid
can be calculated as only 27% of the value for the individu-
al reaction.
Figure 4. Conversion of acids and total conversion of both acids versus
time for the competitive enzymatic esterification of combined heptanoic
and dodecanoic acid with 3-phenylpropanol (filled symbols), all catalyzed
Despite the significant differences in the reaction rates
and the conversions of the acids, the total conversion of the
alcohol substrate almost reaches the values of the reactions
of the individual acids after 24 h. The low reaction rate of
by Lipase PS. Conversion rates from the initial slopes: C7ind: 0.10; C12ind
:
0.21; C7: 0.08; C12: 0.07 minꢀ1. The dotted lines are only a guide for the
eye.
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C. K. Weiss et al.
Figure 6. Conversion of acids and total conversion of all acids versus time
for the competitive enzymatic esterification of combined heptanoic, non-
anoic, and dodecanoic acid with 3-phenylpropanol (filled symbols), all
Figure 7. Hydrolysis and esterification of 3-phenylpropyl nonanoate cata-
lyzed by Lipase PS versus time. The dotted lines are only a guide for the
eye.
catalyzed by Lipase PS. Conversion rates from the initial slopes: C7ind
:
0.10; C9ind: 0.32; C12ind: 0.21; C7: 0.06; C9: 0.11; C12: 0.06 minꢀ1. The
dotted lines are only a guide for the eye.
obtained under the applied conditions with an equimolar
ratio of the reactants.
heptanoic acid and the fact that dodecanoic acid is not read-
ily available for reaction at the droplet interface decreases
the total yield to 75%. Regarding the reaction profiles, a
plateau is not visible, thus indicating that the reaction has
not obtained equilibrium state after 24 h of reaction time
and that 80% conversion can be obtained.
The interfacial concentration seems to be directly con-
nected to the chain length/hydrophilicity of the acid sub-
strates. Higher hydrophilicity leads to higher interfacial con-
centration and vice versa. On the other hand, all of the re-
sults show a very clear preference for the reaction of nona-
noic acid with 3-phenylpropanol over the other acid sub-
strates applied in the experiments. Thus, this system was
used for further experiments.
Substrates applied in excess: For an evaluation of potential
substrate inhibition, esterification experiments were con-
ducted with an excess (ten- and fivefold) of either nonanoic
acid or 3-phenylpropanol under at same conditions as the
experiments without substrate excess. The reaction profiles
of these experiments are shown in Figure 8 in comparison to
the reaction profile of the enzyme-catalyzed esterification of
equimolar reactants. The profiles obtained from the reac-
tions with an excess of alcohol substrate show faster conver-
sion than the reactions with equimolar reactants or an
excess of acid.
Competitive enzymatic reactions in a homophase were
carried out as well to eliminate the interface and the effect
of hydrophilicity/hydrophobicity so that the substrate prefer-
ence of Lipase PS could be investigated. Because the reac-
tions had extremely low conversion rates, no preference
could be observed. After five days, the conversion of all
three acids (C7, C9, and C12) was about 7% without any
significant difference. This observation confirms the advant-
age of the heterophase for enzymatic ester synthesis.
Equilibrium: To determine the equilibrium of the catalyzed
esterification/hydrolysis reaction, pure ester 3-phenylpropyl
nonanoate was hydrolyzed enzymatically with Lipase PS.
The ester was miniemulsified and hydrolyzed under the
same conditions as the corresponding esterification reaction
(see Figure 7 for the reaction profile). The hydrolysis profile
initially shows a rapid decrease in ester content, and an
equilibrium with an ester content of about 80% is reached
after 3 h. The esterification reaction reaches a maximum
conversion of slightly less than 80% after about six hours
(see also Figure 2). This outcome means that the equilibrium
is successfully shifted toward the products of the esterifica-
tion reaction in the presence of large amounts of water. We
can expect 80% as the maximum conversion, which can be
Figure 8. Reaction profiles of the Lipase PS-catalyzed esterification reac-
tions of nonanoic acid with 3-phenylpropanol with an excess of alcohol
(circles) or acid (squares) compared to the reaction with an equimolar
amount of reactants. The reaction rates: the equimolar reaction: 0.32;
excess of alcohol tenfold: 0.70; fivefold: 0.66; excess of acid tenfold:
0.22; fivefold: 0.25 minꢀ1. The dotted lines are only a guide for the eye.
A maximum conversion of slightly more than 80% is ob-
tained after 4 h with a fivefold excess of alcohol, whereas
the application of a tenfold excess of 3-phenylpropanol re-
sults in yields of nearly 90% after 4 h. Compared to the ex-
periment with equimolar reactants, the values are increased
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Enzyme-Catalyzed Esterification
FULL PAPER
by 20 and 30% conversion, respectively. The esterification
performed with an excess of acid substrate yielded a maxi-
mum conversion of nearly 90% after 5 h. The conversion
rates are lower (tenfold excess: 0.22 minꢀ1; fivefold excess:
0.25 minꢀ1) than for esterification with an excess of alcohol
(tenfold excess: 0.70 minꢀ1, fivefold excess: 0.66 minꢀ1) and
slightly lower than for the reaction with an equimolar
amount of substrates (0.32 minꢀ1). It can be concluded that
an excess of either substrate shifts the equilibrium towards
the product side. The maximum conversion increases from
slightly less than 80% to nearly 90%. An excess of alcohol
significantly increases the reaction velocity as the maximum
conversion can be obtained in less than 4 h. The conversion
rate of the reactions with an excess of the acid substrate
slightly decreases relative to the equimolar application of
the reactants.
It seems that excessive acid has a slight inhibitory effect
on Lipase PS. Acid inhibition has already been observed by
Daneshfar et al.[34] in the lipase-catalyzed reaction (Candida
antarctica) of 2-ethylhexanol with 2-ethylhexanoic acid in n-
heptane. As no change in pH value was observed during all
the reactions with excessive substrates, the influence of the
pH value on the reaction rates could be excluded.
Figure 9. a) Enzyme-catalyzed reaction of nonanoic acid with 3-phenyl-
propanol with enzymes of different origins. The reaction rates: Chiro-
zyme L-5: 0.14; Rhizopus arrhizus lipase: 0.09; Esterase 009: 0.15;
Lipase PS: 0.32 minꢀ1. The dotted lines are only a guide for the eye.
b) Calculated activities for the different enzymes in the esterification re-
action of nonanoic acid with 3-phenylpropanol. RAL=Rhizopus arrhizus
lipase.
Influence of the enzyme: Besides Lipase PS, several other
enzymes were evaluated for their ability to catalyze the re-
action of nonanoic acid and 3-phenylpropanol in a mini-
ACHTUNGTRENNUNGemulsion. The enzyme preparation was used at a constant
concentration of 1 mgmLꢀ1 (see Figure 9 for the results of
the reactions). With the exception of Esterase 009, only li-
pases were applied. Besides the enzymes listed in Figure 9,
Lipase G and a lipase from hog pancreas were also applied.
Both enzymes led to immediate destabilization of the mini-
Although all of the activities of the enzymes provided by
the suppliers are given as 20–30 Umgꢀ1 against olive oil
(Lipase PS, rhizopus arrhizus lipase, and Esterase 009) or
glyceroltributyrin (Chirazyme L-5), significant differences in
the activity toward the examined system were found (see
Figure 2 for a comparison). With the exception of the reac-
tion catalyzed with rhizopus arrhizus lipase, the initial
slopes were calculated from the conversion values obtained
during the first 2 h of the reaction. Because the miniemul-
sion with added rhizopus arrhizus lipase was destabilized
after 2 h, only the conversion values obtained during the
first hour were used for the calculation. Lipase PS shows the
highest activity for the esterification of nonanoic acid with
3-phenylpropanol, and the activities of the other enzymes
are around 1 Umgꢀ1, which is about one third of the activity
of Lipase PS (Figure 9b). Lipase PS is obviously the most
efficient enzyme of for the esterification of nonanoic acid
with 3-phenylpropanol in a miniemulsion at 408C and
pH 3.8, at which the highest reaction rate was observed.
Enzyme activity is determined by several factors, such as
pH value and ionic strength of the solvent and temperature
of the environment. Figure 10 and Figure 11 show a plot of
conversion versus time for the Lipase PS-catalyzed esterifi-
cation of nonanoic acid with 3-phenylpropanol in a mini-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
arated after about 2 h. The other enzyme preparations did
not affect the stability of the miniemulsion during the
course of the reaction. Even several weeks after prepara-
tion, the emulsions were still stable. There are two reasons
for the destabilization of the miniemulsion: As some en-
zymes can strongly interact with surfactants,[32] the surfactant
in such a system is no longer available for the stabilization
of the miniemulsion droplets. In addition to the actual
enzyme, the preparations consist of different impurities that
might also adsorb the surfactant and thus interfere with the
stabilization of the dispersed phase.
Figure 9a shows that with the exception of rhizopus arrhi-
zus lipase (RAL) all the enzymes yield a conversion of 80%
after at least 24 h. Whereas the reaction catalyzed with
Lipase PS reaches maximum conversion after 5 h, the Ester-
ase 009-catalyzed reaction shows a conversion of 50% and
the Chirazyme L-5-catalyzed reaction only 30%. The maxi-
mum conversion of the reaction catalyzed by rhizopus arrhi-
zus lipase is only approximately 20%. In the two-phase
system formed after the phase separation (2 h) of the mini-
AHCTUNGTREGeNNUN mulsion with variation of the reaction temperature
A
(Figure 10) and the pH value of the continuous phase
corded.
(Figure 11). Three temperatures (i.e., 30, 40, and 508C) and
Chem. Eur. J. 2009, 15, 2434 – 2444
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2439

C. K. Weiss et al.
two pH values (i.e., pH 3.8 and 6.8) were chosen. Regarding
Figure 10, all of the three curves give a maximum conver-
sion of about 80%. Although the differences are not signifi-
Figure 11. Reaction profiles of the Lipase PS-catalyzed esterification of
nonanoic acid with 3-phenylpropanol in miniemulsions with continuous
phases of pH 3.8 and 6.8, respectively. Conversion rates at pH 6.8: with
Lutensol AT50: 0.16 minꢀ1
; ; at
without Lutensol AT50: 0.19 minꢀ1
pH 3.8: 0.32 minꢀ1. The dotted lines are only a guide for the eye.
Figure 10. Influence of temperature on the reaction rate of the Lipase
PS-catalyzed esterification of nonanoic acid with 3-phenylpropanol. The
dotted lines are only a guide for the eye.
tant Lutensol AT50 decreases the enzyme activity. The sur-
factant, which is, like the lipase, located at the interface be-
tween the organic hydrophobic droplet and the aqueous
continuous phase, might interfere with the active site of the
enzyme, block the reactivity, or adsorb on the enzyme, alter
the conformation of the active site, and thus decrease the
enzyme activity. The system needs to be stabilized at pH 3.8
by the application of a surfactant. The carboxylic acid is pro-
tonated at this pH value and cannot act as a surfactant.
Thus, no comparison between a Lutensol AT50-stabilized
system and a system without an additional surfactant is pos-
sible in an acidic medium.
The miniemulsion droplets provide a large and defined in-
terfacial area. Thus, there must be a certain amount of
enzyme that covers the entire interface provided. Until this
enzyme concentration is reached, an increase in the conver-
sion rate should be observed. As soon as the entire interface
is covered by the enzyme, no further effect should be ob-
servable. To determine this critical value, increasing
amounts of enzyme were added to the miniemulsion that
contained nonanoic acid and 3-phenylpropanol. The reac-
tion profiles and the relative conversions are shown in
Figure 12, in which it is clearly visible that the conversion
rate is increased after the addition of a higher amount of
enzyme that catalyzes the esterification. Whereas the maxi-
mum conversion (ꢁ80%) of the reaction catalyzed with
0.5 mgmLꢀ1 of the enzyme is reached after more than 8 h, it
is reached after only 40 min after applying 10 mgmLꢀ1 of
the enzyme. An almost linear relation can be obtained by
plotting the relative conversions (minꢀ1) versus the concen-
tration of the enzyme that catalyzes the reaction.
cant, the initial slopes of the reaction profiles change slightly
with the reaction temperature. The value calculated for the
reactions increases from 30 to 40 and 508C (0.26, 0.27, and
0.28 minꢀ1, respectively). These results indicate that the ac-
tivity of the enzyme for the given system is only very slightly
dependent on the reaction temperature in the range 30–
508C. As mentioned above, the enzyme activity is generally
dependent on the pH value of the reaction environment,
which is the continuous phase in the case of the miniemul-
sion system. Significant amounts of the carboxylic acid will
be deprotonated at pH >5. The carboxy anion itself is a sur-
factant that can stabilize the droplets containing the reac-
tants. It was thus possible to prepare a stable miniemulsion
from nonanoic acid and 3-phenylpropanol by using an aque-
ous phase of pH 6.8 without the addition of further surfac-
tant. To be able to compare the results with those obtained
from reactions in miniemulsions stabilized with Lutensol
AT50, the esterification reactions at pH 6.8 were performed
in miniemulsions with and without the addition of Lutensol
AT50.
The reaction profiles over 24 h are shown in Figure 11.
Comparing the reaction performed at pH 3.8 with the
esterification reactions performed at pH 6.8, it can be no-
ticed that the reaction profile of the first-mentioned reaction
shows a higher conversion rate than the other profiles. Re-
garding the maximum conversions, the values are 5% (no
Lutensol AT50) and 8% (Lutensol AT50) lower for the
esterification reactions performed at higher pH values.
Among the reactions conducted at pH 6.8, the profile of the
reaction performed in droplets stabilized exclusively by the
nonanoate species shows the faster conversion.
Before the addition of enzyme the z-average size of the
droplets is 500 nm. Assuming that this value remains unal-
tered during the course of the reaction, a total interfacial
area of 0.9 m2 per mL of emulsion can be calculated. With
an assumed protein content of 20% of the enzyme prepara-
tion and a molar mass of the enzyme of M=30000 gmolꢀ1,
the area available for one enzyme molecule can be calculat-
ed to be 22 nm2 for an enzyme content of 10 mgmLꢀ1. As
Usually, the enzyme shows the highest activity at a
pH value between 7 and 8 (lipolysis of olive oil). The reac-
tion performed at pH 3.8 has the highest velocity. Regarding
the reactions at pH 6.8, the presence of the nonionic surfac-
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Enzyme-Catalyzed Esterification
FULL PAPER
Figure 13. a) Reaction profiles of Lipase PS-catalyzed esterification reac-
tions of nonanoic acid with w-phenyl-labeled linear alcohols from benzyl
alcohol (C1) to 5-phenylpentanol (C5). Reaction rates: C1: 0.31; C2:
0.20; C3: 0.32; C4: 0.42; C5: 0.40 minꢀ1. The dotted lines are only a guide
for the eye. b) Enzyme activity of the reactions of nonanoic acid with w-
phenyl alcohol substrates with increasing carbon-chain length. The values
are calculated from the initial slopes obtained from the plots shown on
the left.
Figure 12. a) Reaction profile of the esterification of nonanoic acid with
3-phenylpropanol with different amounts of Lipase PS (0.5–10.0 mgmLꢀ1
with respect to the emulsion). The dotted lines are only a guide for the
eye. b) Conversion per minute calculated from the initial slopes of the
plot of conversion versus time. The dotted line is the linear fit of the data
points.
the curve in Figure 12b does not show any sign of satura-
tion, it can be assumed that further enzyme molecules can
be brought to the interface for a further acceleration of the
conversion. It can be expected that as soon as the complete
interfacial area provided by the droplets is completely cov-
ered with enzyme no further increase in the reaction veloci-
ty will be observed.
the reaction profiles indicate a faster reaction. The maxi-
mum conversion of the reaction of nonanoic acid with
benzyl alcohol and 2-phenylethanol is only 70%. The maxi-
mum conversion of the reactions of the other alcohols can
be found to be 80%. At first glance, the reaction velocities
are directly related to the hydrophobicity of the alcohol sub-
strate and thus to the stability of the miniemulsion. Alcohols
with a longer carbon chain are less soluble in the aqueous
phase of the miniemulsion, thus there will be a decrease in
the amount of diffusion from the particles, which increases
the stability of the droplets. Regarding the propanol, buta-
nol, and pentanol derivatives, the statement is valid. Accord-
ing to this hypothesis, benzyl alcohol should be the substrate
with the lowest conversion rate because it is the alcohol
with the highest solubility in the aqueous phase. In contra-
diction to this hypothesis, fast conversion and very high ac-
tivity of the enzyme toward the esterification of benzyl alco-
hol with nonanoic acid can be found, thus indicating a dis-
tinct specificity of Lipase PS toward this alcohol substrate.
Influence of the alcohol substrate: Because the enzyme has
specific activities toward the acid substrates, the same be-
havior can be expected regarding the alcohol component.
Further w-phenyl alcohols with a chain length of C1–C5
(benzyl alcohol to 5-phenylpentanol) were selected for the
esterification with nonanoic acid. The reaction profiles are
shown in Figure 13.
As observed with the different carboxylic acids, the solu-
bilities of the alcohols in the aqueous continuous medium
decreases with increasing carbon chain length, which is ex-
pected to increase the stability of the miniemulsion and thus
favor an efficient reaction. Even with the very water-soluble
benzyl alcohol (0.37 molLꢀ1), high ester yields can be ob-
tained. This finding underlines the robustness of the applied
miniemulsion system.
Control experiments: acid-catalyzed esterification in a mini-
emulsion and enzyme-catalyzed reaction in homophase and
in a macroemulsion: For comparison, the esterification reac-
tions were conducted with acid catalysis in a miniemulsion
and with enzyme catalysis in a homophase (only substrates
As shown in Figure 13a, the esterification reaction of
nonanoic acid with 2-phenylethanol (C2) has the lowest con-
version rate. With the increasing chain length of the alcohol,
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2441

C. K. Weiss et al.
without solvent) and a macroemulsion. We applied the same
“CATASURF” (i.e., DBSA) for the acid-catalyzed esterifi-
cation of nonanoic acid and 3-phenylpropanol in a mini-
ACHTUNGTRENNUNGemulsion as Manabe et al. used in their macroemulsion
system.[17] The plot of conversion versus time was compared
with the enzyme-catalyzed reaction of the same educts
(Figure 14). Bearing in mind that the acid catalyst was ap-
plied as a 2 wt% solution, it can easily be calculated that
CATASURF was used at
a concentration of about
17 mgmLꢀ1. Relative to the amount of enzyme (1 mgmLꢀ1),
this concentration is nearly 20 times the concentration of
catalyst in the miniemulsion.
Figure 15. Reaction profiles of Lipase PS-catalyzed esterification of nona-
noic acid with 3-phenylpropanol in macroemulsion relative to miniemul-
sion. The dotted lines are only a guide for the eye.
sites and leading to higher conversion rates. The enzyme-
catalyzed reaction in homophase was even slower. Although
the maximum conversion of 80% could be obtained, signifi-
cant amounts of products could only be detected after sever-
al days.
The presented data show that an acid-catalyzed esterifica-
tion is possible in miniemulsion; however, it is obvious from
the reaction profiles that the enzyme catalysis is superior to
the conventional acid catalysis.
Figure 14. Comparison of conversion rates of DBSA and Lipase PS-cata-
lyzed esterification of nonanoic acid with 3-phenylpropanol. Reaction
rates: “CATASURF”: 0.14; enzyme catalyzed: 0.32 minꢀ1. The dotted
lines are only a guide for the eye.
Conclusion
Regarding the reaction profiles (Figure 14) of the DBSA
(CATASURF) and the enzyme-catalyzed reaction, the con-
version rate of the enzyme-catalyzed reaction is significantly
higher than the rate of the acid-catalyzed reaction. Whereas
the enzyme-catalyzed reaction reaches maximum conversion
of about 80% after 5 h, the DBSA-catalyzed reaction reach-
es only about 35% conversion after this time. Even after
24 h, only about 60% conversion can be detected. Still, com-
pared with the conventional emulsion system, employed by
the above-mentioned authors, the reaction is faster. Manabe
et al. reported that the maximum conversions of most of the
esterification reactions were reached after more than 48 h.
Because the catalysis takes place at the interface between
the organic reactant droplet and the aqueous continuous
phase, it can be assumed that the increase in interfacial area
from macroemulsion to miniemulsion increases the rate of
the catalyzed reaction, which is consistent with the data ob-
tained herein and the data obtained from Manabe et al.[16]
Figure 15 shows the reaction profiles of nonanoic acid with
3-phenylpropanol in a macroemulsion in comparison to the
same system in a miniemulsion.
To summarize, it was shown that it is very suitable to per-
form enzyme-catalyzed esterification reactions in an aque-
ous miniemulsion, as shown for several linear carboxylic
acids with chain lengths of 7–12 carbon atoms with different
w-phenylalkanoles with a linear chain of 1–5 carbon atoms.
The experimental setup enables the reactions to take place
in the presence of large amounts of water, which is not pos-
sible with a conventional esterification setup in homogene-
ous solution. All of the investigated reactions of linear car-
boxylic acids (C7–C12) esterified with 3-phenylpropanol
show more than 60% conversion, in case of excessive acid
or alcohol, even 90% conversion after 24 h. Esterification
reactions of nonanoic, decanoic, and dodecanoic acid with
3-phenylpropanol reach their maximum conversion after
less than 10 h. Competitive esterification reactions of hepta-
noic, nonanoic, and dodecanoic acid with 3-phenylpropanol
indicate that the hydrophilicity of the acid is crucial for its
location within the droplets and exhibit a distinct specificity
of Lipase PS toward the esterification of nonanoic acid over
the other acids investigated.
Although the homophase reaction and the reaction in a
macroemulsion of nonanoic acid with 3-phenylpropanol cat-
alyzed by Lipase PS reached the conversion of the reaction
conducted in a miniemulsion, the velocities were significant-
ly slower. The same reaction, performed with acid catalysis
in a miniemulsion is faster than this reaction performed in
The reaction profiles show a slower conversion in macro-
ACHTUNGTRENNUNG
emulsion (0.18 minꢀ1), which is 56% of the rate of the mini-
emulsion (0.32 minꢀ1). The final conversion in both reactions
is almost 80%. A higher interfacial area offers more space
for the adsorption of enzyme, thus generating more reaction
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Chem. Eur. J. 2009, 15, 2434 – 2444

Enzyme-Catalyzed Esterification
FULL PAPER
HPLC analysis: After certain periods of time (see general procedure), a
sample of the reaction miniemulsion (10 mL) was injected into acetoni-
trile/water 90:10 (1 mL). An aliquot (20 mL) was injected into the HPLC
setup with an autosampler Ti Series 1050 (Hewlett Packard). The eluent
was acetonitrile/water 90:10 with a flow rate of 0.5 mLminꢀ1. The column
used was a RP C-18 (nucleosil 120–5 C180) from Macherey–Nagel. The
fractions were detected using a UV detector UVD 170U (Dionex) with
variable wavelengths. The conversions were calculated from the respec-
tive peak areas [see Eq. (1)]. All the calculated reaction data is subject
to an error of approximately 5% attributed to weighting and chromato-
gram-peak integration errors.
an emulsion but still slower than the enzymatically catalyzed
reaction.
High ester yields can be obtained after short reaction
times under mild conditions in the presence of large
amounts of water with the presented strategy. Additionally,
the robustness of the system underlines the high potential of
enzyme-catalyzed esterification in a miniemulsion for envi-
ronmentally friendly organic chemistry.
NMR spectroscopic analysis: Samples were withdrawn at the same time
as for HPLC analysis. A sample of the miniemulsion (30 mL) was diluted
with [D6]DMSO (0.5 mL). The measurements were performed on a
Bruker DRX 400 NMR spectrometer at 400.13 MHz. The conversions
were calculated [see Eq. (1)] from the integrals of the peaks of the hydro-
gen atoms bound to the a-carbon atom of the acid and the ester at d=
2.28 and 2.18 ppm, respectively:
Experimental Section
Materials: Heptanoic acid (99%), nonanoic acid (>99%), undecanoic
acid (>98%), dodecanoic acid (99%), benzyl alcohol (>99%), 2-phenyl-
ethanol (99%), and 4-phenylbutanol were purchased from Merck. Octa-
noic acid (99%) was obtained from Riedel-de Haꢃn and decanoic acid
from Acros. 3-Phenylpropanol (98%), 5-phenylpentanol (99%), and
hexadecane (HD; 99%) were purchased from Aldrich. 4-Dodecylbenze-
nesulfonic acid (DBSA; 90%) was purchased from Fluka. Lutensol
AT50 (poly(ethyleneoxide)hexadecyl ether) was a gift from BASF. The
following enzymes were used: Chirazyme L-5 (Candida antarctica) pro-
vided by Roche and Lipase G (Penicillum cammemberti) and Lipase PS
(Burkholderia cepacia) provided by Amano. Lipase from hog pancreas
and lipase from Rhizopus arrhizus were provided by Fluka and Esterase
009 (recombinant Aspergillus oryzae) from Jꢄlich Chiral Solutions
GmbH.
Conversion ¼ ½peak area ðesterÞꢂ=½ðpeak area ðesterÞ þ peak area ðacidÞꢂ
ð1Þ
The reaction rates were calculated from the data points acquired during
the first 2 h of the experiments, if not otherwise stated in the discussion.
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Enzyme-catalyzed reaction in miniemulsion: An equimolar solution (5 g)
of the carboxylic acid and alcohol components with hexadecane (4 wt%)
was added to an aqueous solution of Lutensol AT50 (25 g, 2 wt%). This
two-phase system was stirred for 1 h. The macroemulsion was subse-
quently sonicated with a Branson sonifier 450D (1/4-inch tip) for 2 min.
Ultrasound was applied in 5-s pulses with 5-s pauses. The vessel was
cooled in an ice bath. From the miniemulsion formed, aliquots (20 mL)
were transferred to a screwcap glass vial containing the enzyme prepara-
tion. The pH value of the reaction mixture was 3.8, except for the reac-
tion without the addition of Lutensol AT50 (see below). If not otherwise
stated, a 20-mg aliquot of the enzyme preparation was added after soni-
cation to initiate the reaction. The reaction vessels were placed into a
PLS 4ꢂ4 thermoshaker (Advanced ChemTech) at a fixed temperature
and a speed of 400 rpm for 24 h. The samples were taken after 20 min,
40 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 24 h.
Variation of the pH value of the continuous phase: The miniemulsion
was prepared from 0.1m NaOH solution without Lutensol AT50 by using
the generated carboxy anion as a surfactant. Another miniemulsion was
prepared using basic Lutensol AT50 solution (2 wt% Lutensol AT50 in
0.1m NaOH solution). Both miniemulsions were used as described.
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added to an equimolar homophasic mixture of 3-phenylpropanol (2.3 g,
17.1 mmol) and nonanoic acid (2.7 g, 17.1 mmol). The mixture was agitat-
ed at 408C. Samples were withdrawn after selected periods of time.
Acid-catalyzed reaction in
a miniemulsion: Nonanoic acid (2.7 g,
17.1 mmol), 3-phenylpropanol (2.3 g, 17.1 mmol), and hexadecane
(200 mg) were emulsified as described. Aqueous DBSA (2 wt%) was
used as the surfactant. The miniemulsion was agitated in the thermoshak-
er at 408C and 400 rpm. Samples were taken after 30 min, 1 h, 2.5 h, 4 h,
5 h, 6 h, and 24 h.
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Hydrolysis of 3-phenylpropyl nonanoate: The miniemulsion of 3-phenyl-
propyl nonanoate (2.5 g, 8.9 mmol) and hexadecane (100 mg) was pre-
pared with aqueous Lutensol AT50 (12.5 g, 2 wt%) as described. The
miniemulsion (10 mL) was poured into a screwcap glass vial containing
Lipase PS (10 mg) and put into the thermoshaker at 408C and 400 rpm
for 24 h.
Chem. Eur. J. 2009, 15, 2434 – 2444
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Received: August 14, 2008
Revised: November 11, 2008
Published online: January 20, 2009
2444
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Chem. Eur. J. 2009, 15, 2434 – 2444