Lipase-catalyzed esterification of stearic acid with ethanol, and hydrolysis of ethyl stearate, near the critical point in supercritical carbon dioxide

Article abstract of DOI:10.1007/s11746-002-0429-2

The effect of pressure on the lipase-catalyzed reaction in supercritical carbon dioxide (SCCO₂) was investigated for the esterification of stearic acid (SA) with ethanol and the hydrolysis of ethyl stearate (ES) near the critical point, ranging from 6 to 20 MPa in pressure and 35 to 60°C in temperature. The esterification rate of SA began to increase near the critical point and kept increasing steadily with an increase in pressure, reflecting the increase in SA solubility in SCCO₂. The hydrolysis rate of ES showed a maximum at a pressure near the critical point. When the reaction was carried out with an initial overall ES concentration below its solubility limit in SCCO₂, the maximum pressure shifted along the extended line of the gas-liquid equilibrium in the supercritical region in the pressure-temperature phase plane. This seems to be related to the singular behavior of some properties in SCCO₂ along this line reported in the literature. These results show the importance of pressure, as well as temperature, as a parameter to control enzyme reactions in SCCO₂.

Full text of DOI:10.1007/s11746-002-0429-2

Lipase-Catalyzed Esterification of Stearic Acid with
Ethanol, and Hydrolysis of Ethyl Stearate, Near the
Critical Point in Supercritical Carbon Dioxide
Hideki Nakaya, Kozo Nakamura, and Osato Miyawaki*
Department of Applied Biological Chemistry, The University of Tokyo, Tokyo 113-8657, Japan
ABSTRACT: The effect of pressure on the lipase-catalyzed reac- kernel oil (8); glycerolysis of soybean oil (9); synthesis of
tion in supercritical carbon dioxide (SCCO2) was investigated for
the esterification of stearic acid (SA) with ethanol and the hydroly-
sis of ethyl stearate (ES) near the critical point, ranging from 6 to 20
MPa in pressure and 35 to 60°C in temperature. The esterification
rate of SA began to increase near the critical point and kept in-
creasing steadily with an increase in pressure, reflecting the in-
crease in SA solubility in SCCO2. The hydrolysis rate of ES showed
a maximum at a pressure near the critical point. When the reac-
tion was carried out with an initial overall ES concentration below
its solubility limit in SCCO2, the maximum pressure shifted along
the extended line of the gas–liquid equilibrium in the supercritical
oleoyloleate from oleic acid (10–12); acylation of glucose
with lauric acid (13); thioesterification between oleic acid and
butanethiol (14); transesterifications of milk fat with canola
oil (15); tristearin with palm oil (16); palm oil with stearic acid
(SA) (17); triolein with ethyl behenate and behenic acid (18,19);
triolein with SA (20) and propyl acetate with geraniol (21);
and randomization of fat and oils (22).
In spite of the many investigations, lipase reactions in SCCO2
are not fully understood and not optimized well because of in-
sufficient accumulation of solubility data and inconclusive char-
region in the pressure–temperature phase plane. This seems to be acterization of SCCO2 as an enzyme reaction medium compared
related to the singular behavior of some properties in SCCO2 along
this line reported in the literature. These results show the impor-
tance of pressure, as well as temperature, as a parameter to control
enzyme reactions in SCCO2.
with ordinary solvents. In a previous paper, we measured solu-
bilities of behenic acid and ethyl behenate in SCCO2, which
were described well by a combination of the regular solution
model with the Flory–Huggins theory (23,24). The relationship
between the substrate solubility and enzyme reaction behavior
in SCCO2 has been discussed (19,20).
Paper no. J9928, in JAOCS 79, 23–27 (January 2002).
KEY WORDS: Critical point, enzyme-catalyzed, enzyme in su-
In a recent paper, we determined log P value for SCCO2
(25), which is an important index for describing the hy-
drophobicity of solvents. The log P of SCCO2 changed from
0.9 to 2.0 with a change in pressure between 3 and 11.8 MPa
at 50°C. Judging from log P, SCCO2 was rather hydrophilic
as a solvent. With an increase in pressure, however, the hy-
drophilicity of SCCO2 decreased even though the water solu-
bility in SCCO2 increased. This extraordinary behavior may
characterize a unique property of SCCO2 as a medium for en-
zyme reactions. SCCO2 showed better performance as a sol-
vent for lipase-catalyzed esterification of SA with ethanol and
the hydrolysis of ethyl stearate (ES) as compared to benzene
(log P = 2.0) and hexane (log P = 3.5).
With SCCO2 as a solvent for enzyme reactions, pressure,
as well as temperature, is an important process parameter.
In the present paper, the effect of pressure is investigated
for the lipase-catalyzed esterification of SA with ethanol
and hydrolysis of ES in SCCO2, especially near the critical
point.
percritical carbon dioxide, esterification, hydrolysis, lipase.
Since the initial finding (1–3) of the applicability of some en-
zymes in supercritical fluids (SCF), numerous investigations
on enzyme catalysis in SCF have been carried out because of
their unique solvent properties, such as low viscosity and high
diffusivity in comparison with ordinary organic solvents. SCF
have two parameters, temperature and pressure, that control
their physical properties. Among SCF, supercritical carbon
dioxide (SCCO2) has been most frequently used because of
its relatively mild critical temperature (31.1°C) and pressure
(7.38 MPa) (4). In addition, SCCO2 is nontoxic, nonflamma-
ble, and inexpensive, and is therefore expected to be widely
applicable as a solvent for extractions and reactions in the
food and pharmaceutical industries.
Among enzymes active in SCCO2, lipase (triacylglycerol
lipase, EC 3.1.1.3) has been most frequently used because it
catalyzes various types of reactions with wide substrate speci-
ficity. Thus, lipase-catalyzed reactions in SCCO2 have been
carried out for such reactions as hydrolysis of tripalmitin (5)
and canola oil (6); alcoholysis of cod liver oil (7) and palm
MATERIALS AND METHODS
Materials. Immobilized lipase (Lipozyme IM from Rhizomu-
cor miehei; 1,3-specific) with the enzyme adsorbed on a
macroporous anion exchange resin was purchased from Novo
Nordisk (Tokyo, Japan). SA and ES were purchased from
*To whom correspondence should be addressed at Department of Applied
Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan. E-mail: aosato@mail.ecc.u-tokyo.ac.jp
Copyright © 2002 by AOCS Press
23
JAOCS, Vol. 79, no. 1 (2002)

24
H. NAKAYA ET AL.
Sigma (St. Louis, MO). Ethanol (99.5%), acetonitrile, tetra-
hydrofuran, dichloromethane of high-performance liquid
chromatography (HPLC) grade, and acetic acid (99.7%) were
purchased from Kanto Chemical Co. (Tokyo, Japan). Liquid
CO2 (99.99%) was from Suzuki Shokan Co. (Tokyo, Japan).
Lipase-catalyzed reactions in pressurized CO2. The en-
zyme reaction was carried out in pressurized CO2 by batch-
wise operation. Immobilized lipase (0.045 g) with water con-
tent adjusted by addition of water, a magnetic stirring bar, and
substrates for the enzyme reaction were placed separately at
the bottom of a high-pressure reaction vessel (40 mL in vol-
ume), which was submerged in a water bath (8,9). Prior to
starting the reaction, pressurized CO2 was passed through a
molecular sieve column and then flushed into the reactor. The
reaction was started by mixing the enzyme and the substrates
in the reactor with the magnetic stirring bar. The reaction was
stopped after 30 min by rapid cooling of the reactor with a dry
ice–acetone mixture to solidify the reaction mixture inside.
The apparent reaction rate was calculated from the increase
in product concentration during the reaction.
Analytical procedure. Concentrations of substrates and
water were calculated from the initial loading and the reactor
volume. When the initial molar ratio of ES and CO2 was kept
constant ([ES]/[CO2] = 1:1835.3, mol/mol) in the process
with pressure varied, the initial loading of ES was adjusted
depending on the density of CO2, which was obtained from
the web site of the National Institute of Standards and Tech-
nology (http://webbook.nist.gov/chemistry/fluid/) and based
on Reference 26. The solidified reaction mixture, after subli-
mation of CO2, was extracted with 10 mL of diethylether. The
extract was filtered through a disc membrane filter (5.0 µm fil-
ter unit; Millipore Co., Tokyo, Japan) resistant to organic sol-
vent, and then diethylether was evaporated in a rotary evapora-
tor. The residue was dissolved in HPLC eluent and subjected to
HPLC analysis. The HPLC system was composed of a pump
(PU980; Jasco Co., Tokyo, Japan), a refractive index detector
(Shodex SE-51; Showa Denko Co., Tokyo, Japan), and a col-
umn (Inertsil ODS 2 column, 4.6 × 250 mm; GL Science,
Tokyo, Japan) held at 37°C. A mixed solution of acetonitrile,
tetrahydrofuran, dichloromethane, and acetic acid (70:20:20:0.8,
by vol) was used as HPLC eluent at a flow rate of 0.9 mL/min.
Calculation of solubilities. The solubilities of SA, water,
and ES in SCCO2 were calculated from our previous data
(24), data by Wiebe and Gaddy (27), and data by Bharath et
al. (28), respectively.
Pressure (MPa)
FIG. 1. Effects of pressure and temperature on the reaction rate of lipase-
catalyzed esterification of stearic acid (SA) with ethanol (EtOH) in SCCO2
at 50°C. Initial overall concentrations of SA, EtOH, and water were 22.6,
415, and 9.17 mM, respectively. The reaction time was 30 min.
that the overall SA concentration in the present experiment
was much higher than its solubility limit. In Figure 2, effects
of pressure and temperature on the calculated solubility of SA
are shown. The calculated solubility of SA began to increase
near the critical point with an increase in pressure. This in-
crease in the solubility of SA corresponded well to the in-
crease in the esterification rate of SA shown in Figure 1. The
SCCO2 phase was the only reaction phase because the undis-
solved SA phase was solid (melting point = 69.6°C). There-
fore, the esterification of SA seemed to have been limited by
the solubility of SA.
RESULTS AND DISCUSSION
Effect of pressure on the esterification rate. The effect of pres-
sure on the lipase-catalyzed esterification rate of SA with
ethanol in SCCO2 was measured between 35 and 60°C (Fig.
1). At all the temperatures tested, the esterification rate
steadily increased with an increase in pressure.
As shown in Figure 1, the initial amounts of SA and
ethanol were 22.6 and 415 mM, respectively. In SCCO2, the
solubility of ethanol is high but that of SA is very limited so
Pressure (MPa)
FIG. 2. Solubility of SA as a function of pressure at various tempera-
tures. See Figure 1 for abbreviations.
JAOCS, Vol. 79, no. 1 (2002)

LIPASE REACTIONS IN SUPERCRITICAL CO2
25
Effect of pressure on the hydrolysis rate. On the contrary, reaction rate increased with an increase in overall ES concentra-
the hydrolysis rate of ES was affected by solubilities and con- tion even when the ES concentration was higher than its solubil-
centrations of water and ES and also by the dilution effect of ity limit. In a separate experiment, the occurrence of the lipase-
water and ES on the mole fraction basis with an increase in catalyzed hydrolysis of ES was confirmed to take place in the
pressure by addition of CO2. In addition, the reaction might ES phase without SCCO2. Therefore, the increase in the hydrol-
have occurred not only in the SCCO2 phase but also in the ysis rate of ES in Figure 3 with an increase in the ES concentra-
undissolved liquid ES phase depending on the solubility and tion must have been affected by the reaction in the ES phase. In-
the initial overall concentration of ES.
terestingly, a sharp maximum was observed at A1 in Figure 3
Figure 3 shows the effects of initial overall concentrations (A), at which the pressure was below the critical point of CO2.
of water, varied from 9.17 to 31 mM, and ES, varied from 2.4 This would be a mixed result of reactions in the SCCO2 phase
to 22.6 mM, on the hydrolysis rate of ES at 50°C. With in- and the ES phase. A similar maximum in the reaction rate below
creases in initial concentrations of water and ES, the reaction the critical pressure was also reported in the lipase-catalyzed
rate increased. Maxima in the hydrolysis rate, indicated by transesterification of triolein with SA in SCCO2 (20). When the
the marks A1, A2, B1, etc., were observed in all the experi- reaction occurred only in the SCCO2 phase (B2, B3, C2, C3 in
mental conditions tested. With an increase in ES concentra- Fig. 3), the maximal pressure converged to almost the same
tion, the maximal pressure shifted low and the shape of the pressure at about 10 MPa, which is higher than the critical pres-
maximum became sharper.
sure (7.38 MPa).
The initial concentration of water was lower than its solubil-
Effect of temperature on the pressure at maximum in hydrol-
ity limit at all the maxima in Figure 3. The initial overall con- ysis rate. It has been shown that the pressures yielding max-
centration of ES, however, was higher than its solubility limit at ima in the hydrolysis rate of ES were about 10 MPa at 50°C
some of the maxima in Figure 3, which were distinguished by when the ES concentration was below its solubility limit. The
parentheses at the mark of maxima. In these cases with ES over effect of variations in temperature on the pressure at maximal
its solubility limit, sharp maxima were observed, and the reac- hydrolysis was investigated with ES and water concentrations
tion rate increased with an increase in ES concentration. This fixed at 8.0 and 24 mM, respectively. As shown in Figure 4,
strongly suggests the possibility of the reaction in the undis- sharp and temperature-dependent maxima were observed in
solved ES phase because had the reaction occurred only in the the reaction rate near the critical point. The pressure at maxi-
SCCO2 phase, the reaction rate would have been limited only mal activity increased with an increase in temperature from
by the solubility of ES in SCCO2 and the excessive ES might 40 to 60°C. At these peaks, concentrations of ES and water
have no relation to the reaction rate. In Figure 3, however, the were under their solubility limit.
Ethyl stearate:
22.6 mM
Ethyl stearate:
22.6 mM
Ethyl stearate:
22.6 mM
8.0 mM
2.4 mM
15.2 mM
8.0 mM
8.0 mM
2.4 mM
Pressure (MPa)
Pressure (MPa)
Pressure (MPa)
FIG. 3. Effect of pressure on the apparent reaction rate of lipase-catalyzed hydrolysis of ethyl stearate (ES) in 30 min at
various initial overall concentrations of ES and water at 50°C: (A), [water] = 9.17 mM; (B), [water] = 24 mM; (C), [water]
= 31 mM. The reaction time was 30 min. Maxima in the reaction rate are marked by A1, A2, etc. Parentheses at the
mark of maxima show that the initial ES overall concentration was higher than its solubility limit.
JAOCS, Vol. 79, no. 1 (2002)

26
H. NAKAYA ET AL.
At pressures higher than the maxima, reaction rates steadily
decreased with an increase in pressure (Fig. 4). This decrease in
the reaction rate might be affected by the dilution of ES on a
molar fraction basis by the addition of CO2 to increase pressure
(29). Therefore, in a further set of studies the initial molar ratio
of ES and CO2 was kept constant ([ES]/[CO2] = 1:1835.3,
mol/mol) across the pressure range. As shown in Figure 5, sharp
maxima in the hydrolysis rate of ES were also observed, and
pressures at maxima shifted upward with an increase in reaction
temperature. After reaching maxima, the reaction rate once de-
creased down to a minimum, then increased again.
Although the reaction behaviors in the higher pressure
range are different between Figures 4 and 5, pressures at max-
ima were not different. Those pressures at maxima are plot-
ted in the pressure–temperature diagram of CO2 in Figure 6.
It turned out that pressures at maxima shifted along the ex-
tended line of the gas–liquid equilibrium of CO2. In the liter-
ature (30), it has been reported that correlation lengths and
density fluctuations in SCCO2 form a ridge along this ex-
tended line, and some properties in SCCO2, such as solubility
rate and rate constants of some reactions, showed singular be-
havior on this extended line. This suggests the mechanism of
the unusual reaction behavior near the critical point in SCCO2
in the present case. With singularities described above, the in-
termolecular interaction between substrates and the enzyme
would have increased to maximize the reaction rate.
In the near-critical region, the existence of solute–solute ag-
gregation was theoretically proved in extremely dilute super-
critical mixtures (31). Ellington and Brennecke (32) observed
that the rate constant for the uncatalyzed esterification of
phthalic anhydride with methanol at 97.5 bar was 30 times
higher than that at 166.5 bar in SCCO2, suggesting the increased
local concentration of the methanol around the phthalic anhy-
dride near the critical point. Ikushima et al. (33) also observed a
Pressure (MPa)
FIG. 5. Effect of pressure on the apparent rate of lipase-catalyzed hy-
drolysis of ES in pressurized CO2 at various temperatures. Initial molar
ratio of ES and CO2 was kept constant at 1:1835.3. The reaction time
was 30 min. See Figure 3 for abbreviation.
sharp maximum near the critical point in the lipase-catalyzed es-
terification rate of n-valeric acid and citronellol when pressure
was varied. They suggested that the formation of the lipase–sub-
strate complex would be facilitated by an increase in the elec-
tron-accepting power of SCCO2 at the maximum. They also re-
ported the conformational change of lipase to trigger the enzy-
matic activity in the pressure range near the critical point (34).
The results in Figure 6 and discussions above show that
the singular point in the hydrolysis rate exists along the ex-
tended line of the gas–liquid equilibrium in the pressure–tem-
Maximum pressures in Fig. 5
Maximum pressures in Fig. 4
Critical point
Gas–liquid
equilibrium
Triple point
Pressure (MPa)
FIG. 4. Effect of pressure on lipase-catalyzed hydrolysis rate of ES in
Temperature (K)
pressurized CO2 at various temperatures. Initial concentrations of ES FIG. 6. Pressures at maxima in ES hydrolysis rate in Figures 4 and 5 plot-
and water were fixed at 8.0 and 24 mM, respectively. The reaction time ted in pressure–temperature phase diagram of CO2. See Figure 3 for ab-
was 30 min. See Figure 3 for abbreviation.
breviation.
JAOCS, Vol. 79, no. 1 (2002)

LIPASE REACTIONS IN SUPERCRITICAL CO2
27
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at that point. This will be of practical importance in the selec-
tion of operating conditions for enzyme reactions in SCCO2
such as for the lipase-catalyzed transesterification and hydrol-
ysis of triglycerides.
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[Received March 20, 2001; accepted November 4, 2001]
JAOCS, Vol. 79, no. 1 (2002)