Lipase-mediated synthesis of dodecyl oleate and oleyl oleate in aqueous foams

Article abstract of DOI:10.1007/s11746-000-0097-2

Engineering media for optimal product yield in enzyme-catalyzed reactions is an important strategy. We report here synthesis of dodecyl oleate and oleyl oleate by lipase (Candida rugosa) in solvent-free substrate foams. Ester formation was characterized with respect to enzyme concentration, pH, temperature, and substrate concentration. The kinetics of ester formation suggest that the formation of ester was 80% complete in 2 h. The pH and temperature optima of lipase suggest that the behavior of lipase in substrate foams was similar to its behavior in water or in organic solvents. The denaturing effect of foams on enzyme was evaluated. Rapid loss in activity (>70% in 1 h) was observed in the presence of oleic acid and dodecanol. The large surface areas generated in aqueous foams offer better accessibility of substrate to lipase for esterification.

Full text of DOI:10.1007/s11746-000-0097-2

Lipase-Mediated Synthesis of Dodecyl Oleate
and Oleyl Oleate in Aqueous Foams
N. Madhusudhana Rao* and V.M. Shanmugam
Centre for Cellular and Molecular Biology, Hyderabad, India 500 007
ABSTRACT: Engineering media for optimal product yield in ence the reaction rate (10). Minimizing the contact of bulk
enzyme-catalyzed reactions is an important strategy. We report nonpolar phase with the enzyme is important so as to main-
here synthesis of dodecyl oleate and oleyl oleate by lipase (Can-
dida rugosa) in solvent-free substrate foams. Ester formation was
characterized with respect to enzyme concentration, pH, tem-
perature, and substrate concentration. The kinetics of ester for-
substrates as media for conducting lipase-mediated reactions
mation suggest that the formation of ester was 80% complete in
tain water associated with the enzyme and also to keep the
equilibrium toward esterification (1).
Earlier we reported the utility of aqueous foams of lipase
(
11,12). The principal advantage of foam media is that they
2
h. The pH and temperature optima of lipase suggest that the
offer substantial contact area between substrates and enzyme
13). Foams are colloidal dispersions of discontinuous gas
phase in a continuous, albeit tiny, water phase. Foam, being
behavior of lipase in substrate foams was similar to its behavior
in water or in organic solvents. The denaturing effect of foams
on enzyme was evaluated. Rapid loss in activity (>70% in 1 h)
(
was observed in the presence of oleic acid and dodecanol. The highly dynamic, offers thorough mixing of the components,
large surface areas generated in aqueous foams offer better ac- which consequently reduces several mass action limitations
cessibility of substrate to lipase for esterification.
(14). Foams or air–water interfaces have been used to con-
duct lipase reactions (13,15,16). We have demonstrated that
the surface activity of the substrates was positively correlated
with the reaction rate (11). The higher the surface activity of
the substrates, the better was the yield of the esters. Foams
were also shown to support esterification of glycerol by fatty
Paper no. J9475 in JAOCS 77, 605–608 (June 2000).
KEY WORDS: Esterification, foams, inactivation, lipases.
The ability of lipases to catalyze reactions in organic media acids (13,15,16). The rates and the extents of ester formation
has been extensively studied (1–3). The mild operating con- in foams were comparable to reported values obtained using
ditions and excellent stereoselectivity of enzyme-catalyzed other reaction media (11). Oleyl glyceride synthesis was also
reactions make them attractive to organic chemists (4). The supported in foam reactions and the reaction went to 90%
ease of controlling the direction of lipase reactions by alter- completion in 15 h (12). Others have reported similar glyc-
ing the composition of the reactants has led to a variety of re- eride synthesis in foam reactors, using either glycerol and lau-
action media. The essential components of these reactions are ric acid (13) or glycerol and stearic acid (15). Yeh and Gulari
enzyme, water, organic solvent, and substrates. The immisci- (13) used glycerol at a concentration of 5.6 M, whereas we
bilities of various components result in multiphasic reaction have used 100 mM glycerol (12). In addition, in our foam re-
media with several advantages and disadvantages associated actions we have maintained the height of the foam by pre-
with each medium. A wealth of information is available on venting evaporative loss of water by pumping air, whose rel-
the adoption of strategies, such as the use of biphasic, water- ative humidity was adjusted. Pinha-Melo et al. (16) demon-
in-oil emulsions and solvent-free reaction media, wherein the strated 50% glyceride synthesis in 7 min in oleic acid
objectives were to solubilize the substrates and the products, monolayers in a Langmuir trough with glycerol (>99.5%
improve the mass action, and keep the enzyme from losing pure) as subphase using cutinase enzyme. Monomolecular
activity (5–7). Several innovative techniques were tested to films formed in a Langmuir trough are similar to substrate
make enzymes more soluble, stable, or reusable in organic foams since the disposition of enzyme and substrate is simi-
media, by modifying the surface of the enzyme preparations lar. Glyceride synthesis was also shown to be dependent on
(8,9). Mass action limitations are of primary concern in media surface pressure in such a system. Another important differ-
design since the contact area of enzyme–substrate will have ence between foams and other reaction geometries is the con-
direct bearing on the reaction yield and kinetics. Further, par- tent of water in the system. Based on chemical equilibrium
titioning coefficients of the substrates and the products be- considerations, the presence of water in the reaction corre-
tween polar and nonpolar phases and interface would influ- lates negatively with ester formation (1). The role of water in
the equilibrium of a reaction occurring in a multiphasic
*
To whom correspondence should be addressed at Centre for Cellular and
medium is not clear. Using water activity as a descriptor, sig-
nificant negative correlation was shown between reaction
Molecular Biology, Uppal Rd., Hyderabad 500 007, India.
E-mail: Madhu@ccmb.ap.nic.in
Copyright © 2000 by AOCS Press
605
JAOCS, Vol. 77, no. 6 (2000)

6
06
N.M. RAO AND V.M. SHANMUGAM
rates and water activity with a few enzymes (17). However,
Synthesis of lipase substrates. p-Nitrophenyloleate, oleyl
several studies used water content or water activity as a vari- oleate, and dodecyl oleate were synthesized using DCC as a
able and demonstrated an absence of such correlations catalyst in dichloromethane, and the products were separated
(
18,19). Reported high rates of ester formation in foams by using silica gel column chromatography. Purity of esters was
ourselves and others are intriguing and our effort is to under- confirmed on thin-layer chromatography and by proton nu-
stand the influence of water on esterification in foam.
Air–water interfaces are discontinuities in phases and have
clear magnetic resonance.
Lipase activity. Lipase activity was calculated based on hy-
denaturing effects on proteins (19). In foams the lipase is ex- drolysis of p-nitrophenyl oleate/Triton X-100 co-micelles at
posed continuously to an air–water interface which may cause room temperature (20). The reaction was monitored by
denaturation of lipase and thereby decrease enzyme activity. We following the formation of p-nitrophenol, as an increase in
studied the stability of lipase in foam reactions using absorbance at 410 nm. The reaction consisted of 4 µmol of
p-nitrophenyl oleate as substrate (20). In this report we have p-nitrophenyl oleate, 0.8% (wt/vol) of Triton X-100, and 50
studied the formation of dodecyl oleate and oleyl oleate forma- µmol of phosphate buffer in 1 mL (pH 7.2). Protein content
tion catalyzed by lipase from Candida rugosa in foam reactions. was estimated by the method of Lowry et al. (21) using serum
albumin as standard.
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Chemicals. Oleic acid, oleyl alcohol, dodecanol, p-nitrophe-
nol, Triton X-100, N,N′-dicyclohexyl carbodiimide (DCC), Figure 1A shows data on the time course of dodecyl oleate and
and lipase (EC 3.1.1.3) from C. rugosa (Type VII, 950 units oleyl oleate formation by C. rugosa lipase at a concentration of
per mg of solid) were purchased from Sigma Chemical Co. 0.4 mg/mL in foams. The reaction went to >80% conversion
(St. Louis, MO).
with dodecyl alcohol as substrate and to >90% conversion with
Foam reaction. Foam reactions were performed as de- oleoyl alcohol as substrate in 4 h. Beyond 8 h the height of the
scribed earlier (11,12). In brief, foam reactions were con- foam column was unstable. The kinetics and the extent of ester
ducted in double-jacketed glass columns (2.5″ × 10″ column formation were similar with both oleyl alcohol and dodecanol.
fitted with a sintered glass joint at the bottom) by passing com- In identical substrate and reaction conditions, in heptane and in
pressed air into a mix of the reaction components at room tem- buffers (stirred but not foamed) dodecyl oleate formation was
perature (25°C). The air was preequilibrated with water to give less than 10% in 6 h. Ester formation was dependent on the li-
9
0% relative humidity to prevent drying of the foam due to pase concentration in the medium (Fig. 1B). The ester forma-
water evaporation. In a typical reaction, lipase (0.4 mg/mL for tion in foam reactions was higher compared to similar reactions
dodecanol and 1 mg/mL for oleyl alcohol) was mixed with performed in either solvent-free reactions or in hexane (22,23).
5
00 µmol alcohol and 500 µmol oleic acid in 50 µmol Tris- A yield of 60% octyl oleate was achieved in solvent-free
HCl (pH 7.2) in a 5-mL volume. During the reaction the foam medium after 4 h of reaction catalyzed by lipase from Rhi-
was maintained at a constant height (10 ± 2 cm). The reaction zomucor miehei covalently linked to a graft copolymer (22). In
was stopped by adding 5 mL of dichloromethane, the aqueous reactions catalyzed by surfactant-coated lipase in hexane, 50%
phase was extracted once more with dichloromethane, and the dodecyl laurate was formed in 10 h (23). The influence of water
extracts were pooled. The substrates and products were quan- on reaction kinetics was tested in both reactions, and an in-
titated by separating them on a gas chromatograph (GC) using
an HP-1 (methyl silicone gum as immobile phase) column on
an HP 5890 Series II Plus GC (Hewlett-Packard, Wilmington,
DE) using a 100–200°C temperature ramp. By using known
amounts of synthesized dodecyl oleate and oleyl oleate, GC
standard graphs (peak area vs. amount) were constructed and
used to calculate the extent of conversion. The temperature of
the reaction was maintained by connecting the foam column
to a water bath. The pH of the reaction was adjusted with
buffers: acetate in the range of 4–7 and Tris in the range of
7–9. Lipase-catalyzed reactions in organic solvents were con-
ducted in heptane. Enzyme (0.4 mg/mL) and both the sub-
strates (100 µmol each) were added to 1 mL of heptane in
screw-capped tubes and shaken. Aliquots were directly in- FIG. 1. Formation of dodecyl oleate and oleyl oleate by lipase in foams.
(
A) Time course of formation of oleyl oleate (●) and dodecyl oleate (●)
jected into the GC for quantitation. Reactions in buffer were
conducted by shaking (but not to the extent that would allow
foam formation) enzyme and the substrates in 1 mL of 10 mM
Tris-HCl (pH 7.2) buffer. The extraction and analyses were
similar to foam reactions.
by lipase at 1 and 0.4 mg/mL concentration, respectively. Dodecyl for-
mation was also followed, under similar conditions of concentration
and temperature, in heptane (●) and in buffer (●). In both cases the re-
action was stirred and not foamed. (B) Esterification of dodecylalcohol
with oleic acid as a function of lipase concentration.
JAOCS, Vol. 77, no. 6 (2000)

SYNTHESIS OF ESTERS IN FOAMS BY LIPASES
607
crease in water content in the reaction was found to decrease from R. miehei showed a pH optimum between 5 and 8 in cat-
the rate of ester formation. However, the effects were less com- alyzing glyceride formation in foams (12). Temperature de-
pared to expectations based on reaction equilibrium considera- pendence of dodecyl oleate was studied in the range of 15 to
tions. Recently, a mixture of cholesterol/docosahexaenoic acid 50°C. Beyond 40°C the activity fell very sharply. We plotted
(3:1 mol%), 30% water, and 3,000 units/g of lipase was stirred the data in the range of 15–35°C using the van’t Hoff relation
at 40°C for 24 h, and the esterification extent attained was and estimated the activation energy (E ) to be 24.4 kJ/mol
a
8
9.5% (24). In foam, the esterification occurs at the air/water (Fig. 3B). This value of E is in the range of other E values
a a
interface. The lipase substrates and products occupy the determined for other esterification reactions catalyzed by li-
air–water interface. Lipases are interfacially active, and the re- pase (25). The loss in enzyme activity beyond 40°C may be
action rate primarily would depend on the interaction between due to a combination of such features as enzyme inactivation
the lipase and the substrate in the monolayer (4). Foam being a and changes in the partitioning terms of the substrates to the
biphasic medium, the partitioning coefficients and surface ac- interface. At higher temperatures it was difficult to maintain
tivities of the substrates and the products would play an impor- the foam height, probably because the stability of the foam
tant role. Pinho-Melo et al. (16) have shown that the surface may depend sharply on temperature (14).
pressure applied to monomolecular films acts as a physical se-
lectivity factor in glyceride synthesis catalyzed by cutinase.
Activity of lipase in foams. Air–water interfaces are known
to have denaturing effects on proteins (19). The structure of
The apparent K of dodecanol for ester formation was esti- soluble proteins is primarily due to isotropic interactions of
m
mated by conducting foam reactions at various dodecanol con- protein surface groups with the surrounding water molecules.
centrations and a fixed oleic acid concentration (100 mM). Ap- However, when proteins are exposed to phase discontinuities
parent K for dodecanol was calculated to be 50 mM based on such as air–water interfaces, the structure, and consequently
m
Lineweaver-Burk transformation of the data (Fig. 2). At the function, suffers. In foams, lipase is continuously exposed
dodecanol concentrations more than 100 mM, ester formation to the interface throughout the reaction period. We tested the
was inhibited. This may be partly due to the surface denatura- activity of lipase, after exposure to foams for various times.
tion of lipase by dodecanol (see below). In hexane, the K for At each time point an aliquot of the sample was briefly spun
m
lauric acid in dodecyl ester formation was 10 mM (23). Glyc- and the water phase, containing the lipase, was kept in the
erol showed an apparent K of approximately 5 M in a foam cold. The enzyme activity and the protein content were then
m
reaction, which may be due to the low surface activity of glyc- determined as given in the Materials and Methods section.
erol compared to fatty acids (12). The observed ester yield The loss in lipase activity was very sharp (nearly 80%) even
would be a sum of ester formation and ester hydrolysis in after 30 min of exposure to foam (Fig. 4A). After 30 min of
foams, since foam reaction would allow ester hydrolysis to exposure to heptane the enzyme lost only 20% of its activity.
proceed simultaneously with ester formation.
This suggests that the loss of activity was rapid and that inac-
pH and temperature dependence of dodecyl oleate forma- tivation may partly be responsible for the decrease in esterifi-
tion by lipase. To understand whether dodecyl oleate forma- cation seen in Figure 1A. To verify this we have compared
tion in foams by lipase is similar to other reactions catalyzed percent conversion in two reactions after 1 h: in one reaction
by lipase, we studied its formation at various pH values and (5 mL), 4 mg of lipase was used and in another reaction (5
temperatures. Between pH 6–9 lipase-mediated formation of
both dodecyl oleate and oleyl oleate was insensitive to pH
changes. Below pH 6 and above pH 9 the activity decreased
sharply (Fig. 3A). We did not observe any obvious changes
in foamability of the reactions at various pH values. Lipase
FIG. 3. pH and temperature dependence of esterification catalyzed by
lipase in foams. (A) Oleyl oleate (●) and dodecyl oleate (●) formation
were studied at various pH values at room temperature. Between pH
4
7
–7 acetate buffer was used, and Tris-HCl buffer was used between pH
–10. Comparison of esterification at overlapping pH values in the two
FIG. 2. Dependence of esterification on concentration of dodecyl alco- buffers suggested that there were no buffer-specific effects. (B) Temper-
hol in foams. (A) The oleic acid concentration was fixed at 100 mM in
ature dependence of dodecyl oleate formation in foams by lipase stud-
all the reactions. (B) Lineweaver-Burk transformation of the data. Cal- ied at pH 7.2. The activation energy of dodecyl oleate formation was
culated apparent Km is 50 mM.
calculated to be 24.4 kJ/mol.
JAOCS, Vol. 77, no. 6 (2000)

6
08
N.M. RAO AND V.M. SHANMUGAM
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phenyloleate was used as a substrate to quantitate the enzyme activity.
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1
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1
1
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4
B). Denaturation in the absence of substrates was similar to
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1
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2
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JAOCS, Vol. 77, no. 6 (2000)